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Sample records for deoxynucleotidyl transferase-mediated dutp-biotin

  1. Apoptosis in human hepatocellular carcinoma by terminal deoxynucleotidyl transferase-mediate dUTP-FITC nick end labeling%TUNEL技术原位观察肝细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    李江; 王文亮; 王文勇; 刘斌; 王泊云

    1998-01-01

    目的采用TUNEL技术对福尔马林固定、石蜡包埋的肝组织和培养肝癌细胞进行染色,以探讨肝细胞凋亡的形态特征和分布.方法使用德国宝灵曼公司生产的原位末端标记试剂盒. 石蜡包埋组织经微波处理后,行常规TUNEL染色,观察凋亡细胞的形态和分布.结果 TUNEL阳性信号为黄绿色或黄色荧光,位于胞核,呈小圆形或环行. TUNEL阳性细胞大多在形态学上表现为凋亡的细胞. 对肝组织TUNEL LI的分析显示,肝癌组织中细胞凋亡指数(0.395)比肝硬变(0.075)和癌旁肝组织(0.105)要高,且癌组织恶性程度越高,这种凋亡就越显著.结论改进的TUNEL技术对福尔马林固定、石蜡包埋的难处理组织进行染色是一个有效的方法.

  2. Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

    Science.gov (United States)

    Schmidt, W M; Mueller, M W

    1996-05-01

    Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). By virtue of the homopolymeric ribo-tail, the modified cDNA is anchored to the 3' overhang of a double-stranded DNA-adaptor in a T4 DNA ligase-dependent ligation. PCR amplification, mediated by two sequence-specific primers, yields the desired unique product suitable for cloning and dideoxy-sequencing.

  3. [Cell apoptosis and expression of hypoxia-inducible transcription factor-1 alpha in kidney tissue after severe burn with delayed fluid resuscitation in rats in areas of different altitude].

    Science.gov (United States)

    Jiang, Jiang; Liu, Yi; Zhang, Shi-fan; Cai, Qian; Zhang, Xian-ying; Zhang, Bin; Xiao, Bin

    2008-07-01

    To explore the relationship of cell apoptosis and expression regularity of hypoxia-inducible transcription factor (HIF)-1 alpha after severe burn with delayed fluid resuscitation in areas of different altitude. A total of 240 male Wistar rats, which were raised in areas of different altitude (1,517 and 3,840 meters), were employed as the experimental models [They received a 30% total body surface area (TBSA)III degree scald injury], and then they were randomly divided into 3 groups: delayed fluid resuscitation group (DFR, n=50), immediate fluid resuscitation group (IFR, n=60) and control group (CG, n=10). Renal tissue samples were harvested at 1, 6, 12, 24, 72 and 168 hours after burn, respectively. Cell apoptosis was detected by tissue chip technology and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL). The expression of HIF-1 alpha was assessed by immunohistochemistry and image analysis. With increase in altitude, cellular edema, degeneration, necrosis and disintegration of renal tissue were gradually worsening, the capillaries of renal glomeruli became dilated and engorged, with degeneration and necrosis of endothelial cells, engorgement and edema of renal interstitium, and infiltration of inflammatory cells. Pathological changes in DFR group were more serious than that of IFR group. Cell apoptosis and the expression of HIF-1 alpha were both enhanced, the latter mainly appeared in nuclei of renal cells, and they were more marked at 3,840 meters compared with those at 1,517 meters. They were more marked in experimental groups than in control group, especially so in DFR group (Pkidney cell apoptosis.

  4. Terminal deoxynucleotidyl transferase requires KU80 and XRCC4 to promote N-addition at non-V(D)J chromosomal breaks in non-lymphoid cells.

    Science.gov (United States)

    Boubakour-Azzouz, Imenne; Bertrand, Pascale; Claes, Aurélie; Lopez, Bernard S; Rougeon, François

    2012-09-01

    Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that increases the repertoire of antigen receptors by adding non-templated nucleotides (N-addition) to V(D)J recombination junctions. Despite extensive in vitro studies on TdT catalytic activity, the partners of TdT that enable N-addition remain to be defined. Using an intrachromosomal substrate, we show here that, in Chinese hamter ovary (CHO) cells, ectopic expression of TdT efficiently promotes N-additions at the junction of chromosomal double-strand breaks (DSBs) generated by the meganuclease I-SceI and that the size of the N-additions is comparable with that at V(D)J junctions. Importantly, no N-addition was observed in KU80- or XRCC4-deficient cells. These data show that, in a chromosomal context of non-lymphoid cells, TdT is actually able to promote N-addition at non-V(D)J DSBs, through a process that strictly requires the components of the canonical non-homologous end-joining pathway, KU80 and XRCC4.

  5. Plant glutathione transferase-mediated stress tolerance

    NARCIS (Netherlands)

    Nianiou-Obeidat, Irini; Madesis, Panagiotis; Kissoudis, Christos; Voulgari, Georgia; Chronopoulou, Evangelia; Tsaftaris, Athanasios; Labrou, Nikolaos E.

    2017-01-01

    Plant glutathione transferases (EC 2.5.1.18, GSTs) are an ancient, multimember and diverse enzyme class. Plant GSTs have diverse roles in plant development, endogenous metabolism, stress tolerance, and xenobiotic detoxification. Their study embodies both fundamental aspects and agricultural

  6. Rhizome extracts of Curcuma zedoaria Rosc induce caspase dependant apoptosis via generation of reactive oxygen species in filarial parasite Setaria digitata in vitro.

    Science.gov (United States)

    Senathilake, K S; Karunanayake, E H; Samarakoon, S R; Tennekoon, K H; de Silva, E D

    2016-08-01

    Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01-1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 μg/mL for hexane extract, 191.14 μg/mL for chloroform extract) and adult parasites (IC50 = 77.07 μg/mL for hexane extract, 259.87 μg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST

  7. Effects of the treatment with different fluids on alveolar epithelium barrier in rats with acute lung injury%不同液体治疗对急性肺损伤大鼠肺泡上皮细胞屏障功能的影响

    Institute of Scientific and Technical Information of China (English)

    魏洪霞; 杨毅; 邱海波; 郭涛; 赵明明; 陈秋华

    2009-01-01

    目的 观察不同液体治疗对急性肺损伤(ALI)大鼠肺泡上皮细胞屏障功能的影响.方法 ①与对照组比较,LPS组、NS组肺损伤评分明显升高(P均0.05),且后3组间比较差异也无统计学意义.②与对照组比较,LPS组、NS组肺W/D比值明显升高(P均0.05).⑤各组肺泡上皮细胞凋亡指数(AI)明显高于对照组(P均0.05).结论 胶体液较NS更能改善ALI大鼠肺泡上皮通透性,保护上皮细胞屏障功能.%Objective To observe the effects of different fluids on alveolar epithelium barrier in rats with acute lung injury (ALI). Methods Thirty-six Sprague-Dawley (SD) rats were randomly assigned into six groups with 6 rats in each group. ALI was induced by intravenous injection of lipopolysaccharide(LPS). Rats in all treatment groups were given different fluids and sacrificed after 4 hours. Evans blue dye (EBD) was injected via the femoral vein 30 minutes before death. Tracheobronchial tree was washed with normal saline (NS) after death, and broncho-alveolar lavage fluid (BALF) was collected. Leakage of EBD from blood into BALF (alveolar epithelial permeability) and wet/dry (W/D) ratio were measured. The mRNA expression of surfactant protein-C (SP-C) was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Alveolar epithelium apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL). Lung injury was evaluated by Smith lung injury score. Results ①Lung injury scores in LPS and NS groups were significantly higher than in control group (both P0.05). No significant difference was found among the latter three groups. ②W/D ratio in LPS and NS groups were significantly higher than that in control group (both P0.05). ⑤Apoptosis index (AI) of alveolar epithelial cell in all the treatment groups were significantly higher than that in control group (all P<0.05). Compared with NS group, AI were noticeably lower in ALB and HES groups (both P<0

  8. Decreased apoptosis in advanced-stage/high-grade hepatocellular carcinoma complicating chronic hepatitis C is mediated through the downregulation of p21 ras

    Institute of Scientific and Technical Information of China (English)

    Nahed Baddour; Ebtehal Farrag; Ahmed Zeid; Essam Bedewy; Yousry Taher

    2013-01-01

    Objective and background:Although p21 ras has been reported to be upregulated in hepatocellular carcinoma complicating chronic hepatitis C type Ⅰ,p21 ras has a different role in advanced stages,as it has been found to be downregulated.The goal of this study was to investigate the status of p21 ras in early-stage/low-grade and late-stage/high-grade hepatocellular carcinoma and its possible link to apoptosis.Material and methods:Thirty-five cases each of chronic HCV hepatitis type 4 (group Ⅰ) and cirrhosis with hepatocellular carcinoma (HCC) complicating chronic HCV hepatitis (groups Ⅱ and Ⅲ) were immunohistochemically evaluated using a p21 ras polyclonal antibody.The apoptotic index was determined in histologic sections using the terminal deoxynucleotidyl transferase-mediated d-UTP biotin nick end labeling (TUNEL) assay.Results:Significant differences (P=0.001) were detected in p21 ras protein expression between the three groups.A near 2-fold increase in p21 ras staining was observed in the cirrhotic cases compared to the hepatitis cases,and p21 ras expression was decreased in the HCC group.p21 ras expression correlated with stage (r=0.64,P=0.001) and grade (r=-0.65,P=0.001) in the HCC group and grade in the HCV group (r=0.44,P=0.008).Both p21 ras expression and TUNEL-LI were significantly lower in large HCCs compared to small HCCs (P=0.01 each).The TUNEL values were negatively correlated with stage in the HCC group (r=-0.85,P=0.001).The TUNEL values were also negatively correlated with grade in both the HCV and HCC groups (r=0.89,P=0.001 and r=-0.53,P=0.001,respectively).The p21 ras scores were significantly correlated with the TUNEL-LI values in the HCC group (r=0.63,P=0.001) and HCV group (r=0.88,P=0.001).Conclusions:p21 ras acts as an initiator in HCC complicating type 4 chronic HCV and is downregulated with HCC progression,which most likely promotes tumor cell survival because it facilitates the downregulation of apoptosis with tumor progression.

  9. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using bench top flow cytometer for evaluation of sperm DNA fragmentation in fertility laboratories: protocol, reference values, and quality control.

    Science.gov (United States)

    Sharma, Rakesh; Ahmad, Gulfam; Esteves, Sandro C; Agarwal, Ashok

    2016-02-01

    The purpose of this study is to provide a detailed protocol and quality control steps for measuring sperm DNA fragmentation (SDF) by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay using a new bench top flow cytometer, determine the reference value of SDF, and assess sensitivity, specificity, and distribution of SDF in infertile men and controls with proven and unproven fertility. Semen specimens from 95 controls and 261 infertile men referred to a male infertility testing laboratory were tested for SDF by TUNEL assay using Apo-Direct kit and a bench top flow cytometer. Percentage of cells positive for TUNEL was calculated. Inter- and intraobserver variability was examined. TUNEL cutoff value, sensitivity, specificity, and distribution of different cutoff values in controls and infertile patients were calculated. The reference value of SDF by TUNEL assay was 16.8 % with a specificity of 91.6 % and sensitivity of 32.6 %. The positive and negative predictive values were 91.4 and 33.1 %, respectively. The upper limit of DNA damage in infertile men was significantly higher (68.9 %) than that in the controls (19.6 %). TUNEL assay using flow cytometry is a reproducible and easy method to determine SDF. At a cutoff point of 16.8 %, the test showed high specificity and positive predictive value. The results of this test could identify infertile men whose sperm DNA fragmentation does not contribute to their infertility and confirm that a man who tests positive is likely to be infertile due to elevated sperm DNA fragmentation.

  10. A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

    OpenAIRE

    Domínguez, Fernándo; Moreno Onorato, Francisco Javier; Cejudo Fernández, Francisco Javier

    2003-01-01

    The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl trans...

  11. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality

    OpenAIRE

    TAKEDA, Kumiko; UCHIYAMA, Kyoko; KINUKAWA, Masashi; Tagami, Takahiro; Kaneda, Masahiro; Watanabe, Shinya

    2015-01-01

    Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was d...

  12. CNS wound healing is severely depressed in metallothionein I- and II-deficient mice

    DEFF Research Database (Denmark)

    Penkowa, M; Carrasco, J; Giralt, M

    1999-01-01

    . In contrast to normal mice, at 20 dpl no wound healing had occurred. The rate of apoptosis, as determined by using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, was drastically increased in neurons of ipsilateral cortex of the MT-I+II null mice. Our results demonstrate that MT......-I+II are essential for a normal wound repair in the CNS, and that their deficiency impairs neuronal survival....

  13. Human glutathione S-transferase-mediated glutathione conjugation of curcumin and efflux of these conjugates in caco-2 cells

    NARCIS (Netherlands)

    Usta, M.; Wortelboer, H.M.; Vervoort, J.; Boersma, M.G.; Rietjens, I.M.C.M.; Bladeren, P.J. van; Cnubben, N.H.P.

    2007-01-01

    Curcumin, an α,β-unsaturated carbonyl compound, reacts with glutathione, leading to the formation of two monoglutathionyl curcumin conjugates. In the present study, the structures of both glutathione conjugates of curcumin were identified by LC-MS and one- and two-dimensional 1H NMR analysis, and th

  14. Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus.

    Directory of Open Access Journals (Sweden)

    Yuanwei Zhang

    2016-04-01

    Full Text Available Finely tuned changes in cytosolic free calcium ([Ca2+]c mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS. The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs, a putative proton V-type proton ATPase (Vma5 homolog and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress.

  15. Labeling of double-stranded DNA by ROX-dideoxycytosine triphosphate using terminal deoxynucleotidyl transferase and separation by capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Figeys, D.; Renborg, A.; Dovichi, N.J. (Univ. of Alberta, Edmonton, Alberta (Canada))

    1994-12-01

    Terminal transferase is used to add a single fluorescently labeled dideoxynucleotide to double-stranded DNA prepared by restriction endonuclease action on a bacteriophage. The product is separated by capillary electrophoresis with both hydroxypropylmethylcellulose and non-cross-linked polyacrylamide. The reaction products generate single peaks for each fragment with hydroxypropylmethylcellulose. However, the higher resolution separation produced by non-cross-linked polyacrylamide shows that the product contains two components for each restriction digest fragment. This labeling technique should be useful in restriction fragment length polymorphism studies. 9 refs., 2 figs.

  16. Nuclear DNA fragmentation during cell death of short-lived ray tracheids in the conifer Pinus densiflora.

    Science.gov (United States)

    Nakaba, Satoshi; Kubo, Takafumi; Funada, Ryo

    2011-05-01

    One key event in the programmed cell death is nuclear DNA fragmentation. We investigated the timing of nuclear DNA fragmentation during the cell death of short-lived ray tracheids in Pinus densiflora using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Fluorescence due to TUNEL was detected only in deformed nuclei that lacked obvious chromatin in ray tracheids that were adjacent to ray tracheids that no longer contained nuclei. Our observations revealed that nuclear DNA fragmentation occurred only at the final stage of cell death in ray tracheids in situ.

  17. Ureaplasma urealyticum infection and apoptosis of spermatogenic cells

    Institute of Scientific and Technical Information of China (English)

    Xue-JunSHANG; Yu-FengHUANG

    1999-01-01

    Aim: To study the relationship between ureaplasma urealyticum (UU) infection and apoptosis of human spennato-genie cells. Methods: Spermatogenic cells were observed under light microscope with Wright-Giemsa staining andby means of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling(TUNEL) technique. Results: Apoptotic rate of UU-infected males ( 15.5%±6.8% ) was significantly higherthan that of controls (5.2%±2.3 % ). Conclusion: Apoptosis of spermatogenic cells can be caused by UU in-fection, which provides further evidence for UU-induced male infertility.

  18. Safety evaluation of poly(lactic-co-glycolic acid)/poly(lactic-acid) microspheres through intravitreal injection in rabbits.

    Science.gov (United States)

    Rong, Xianfang; Yuan, Weien; Lu, Yi; Mo, Xiaofen

    2014-01-01

    Poly(lactic-co-glycolic acid) (PLGA) and/or poly(lactic-acid) (PLA) microspheres are important drug delivery systems. This study investigated eye biocompatibility and safety of PLGA/PLA microspheres through intravitreal injection in rabbits. Normal New Zealand rabbits were randomly selected and received intravitreal administration of different doses (low, medium, or high) of PLGA/PLA microspheres and erythropoietin-loaded PLGA/PLA microspheres. The animals were clinically examined and sacrificed at 1, 2, 4, 8, and 12 weeks postadministration, and retinal tissues were prepared for analysis. Retinal reactions to the microspheres were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end staining and glial fibrillary acidic protein immunohistochemistry. Retinal structure changes were assessed by hematoxylin and eosin staining and transmission electron microscopy. Finally, retinal function influences were explored by the electroretinography test. Terminal deoxynucleotidyl transferase-mediated dUTP nick end staining revealed no apoptotic cells in the injected retinas; immunohistochemistry did not detect any increased glial fibrillary acidic protein expression. Hematoxylin and eosin staining and transmission electron microscopy revealed no micro- or ultrastructure changes in the retinas at different time points postintravitreal injection. The electroretinography test showed no significant influence of scotopic or photopic amplitudes. The results demonstrated that PLGA/PLA microspheres did not cause retinal histological changes or functional damage and were biocompatible and safe enough for intravitreal injection in rabbits for controlled drug delivery.

  19. Antitumor Effect of Antisense Ornithine Decarboxylase Adenovirus on Human Lung Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Hui TIAN; Lin LI; Xian-Xi LIU; Yan ZHANG

    2006-01-01

    Ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis, was found to increase in cancer cells, especially lung cancer cells. Some chemotherapeutic agents aimed at decreasing ODC gene expression showed inhibitory effects on cancer cells. In this study, we examined the effects of adenoviral transduced antisense ODC on lung cancer cells. An adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549. The 3-(4,5-me thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the effect on cell growth. Expression of ODC and concentration of polyamines in cells were determined by Western blot analysis and high performance liquid chromatography. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cell apoptosis. The expression of ODC in A-549 cells was reduced to 54%, and that of three polyamines was also decreased through the rAd-ODC/Ex3as treatment. Consequently, cell growth was substantially inhibited and terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3as could lead to cell apoptosis, with apoptosis index of 46%. This study suggests that rAd-ODC/Ex3as has an antitumor effect on the human lung cancer cells.

  20. The contribution of low affinity NGF receptor (p75NGFR to delayed neuronal death after ischemia in the gerbil hippocampus.

    Directory of Open Access Journals (Sweden)

    Bagum MA

    2001-02-01

    Full Text Available The implication of low affinity nerve growth factor receptor (p75NGFR, which is believed to play a pro-apoptotic role, in delayed neuronal death (DND after ischemia in the gerbil hippocampus was investigated. Immunohistochemistry and Western blot analysis revealed that the presence of p75 NGFR immunoreactivity (IR was negligible in the hippocampus of the sham control gerbil but appeared clearly in CA1 neurons 3 and 4 days after 5-min transient ischemia. Terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL positive nuclei appeared when the level of p75NGFR IR increased. Furthermore, almost all TUNEL-positive CA1 neurons also costained for p75NGFR. These results suggest that p75NGFR contributes to DND after ischemia by an apoptotic mechanism.

  1. Protective effects of berberine against amyloid beta-induced toxicity in cultured rat cortical neurons

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Yanjun Zhang; Shuai Du; Mixia Zhang

    2011-01-01

    Berberine, a major constituent of Coptidis rhizoma, exhibits neural protective effects. The present study analyzed the potential protective effect of berberine against amyloid G-induced cytotoxicity in rat cerebral cortical neurons. Alzheimer's disease cell models were treated with 0.5 and 2 μmol/Lberberine for 36 hours to inhibit amyloid G-induced toxicity. Methyl thiazolyl tetrazolium assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining results showed that berberine significantly increased cell viability and reduced cell apoptosis in primary cultured rat cortical neurons. In addition, western blot analysis revealed a protective effect of berberine against amyloid β-induced toxicity in cultured cortical neurons, which coincided with significantly decreased abnormal up-regulation of activated caspase-3. These results showed that berberine exhibited a protective effect against amyloid 13-induced cytotoxicity in cultured rat cortical neurons.

  2. Successful ram semen cryopreservation with lyophilized egg yolk-based extender.

    Science.gov (United States)

    Alcay, Selim; Berk Toker, M; Gokce, Elif; Ustuner, Burcu; Tekin Onder, N; Sagirkaya, Hakan; Nur, Zekariya; Kemal Soylu, M

    2015-10-01

    The aim of this study was to evaluate the effects of lyophilized egg yolk extender on ram semen cryopreservation. Ejaculates with a thick consistency, rapid wave motion (3-5 on a 0-5 scale) and >75% initial motility were pooled. Sperm were diluted to final concentration of 1/5 (semen/extender) in lyophilized egg yolk or fresh egg yolk extenders using two-step dilution method. The equilibrated semen was frozen in 0.25 mL straws. Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) at three time points: after dilution with extender A, equilibration and post-thaw. The results showed that freezing and thawing procedures (dilution, equilibration and thawing) had negative effects on motility (Pram semen.

  3. Inhibition of TYRO3/Akt signaling participates in hypoxic injury in hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Yan-zhen Zhu; Wei Wang; Na Xian; Bing Wu

    2016-01-01

    In this study, we investigated the role of the TYRO3/Akt signaling pathway in hypoxic injury to hippocampal neurons. 3-(4,5-Dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that hypoxia inhibited the proliferation and viability of hippocampal neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that hypoxia induced neuronal apoptosis in a time-dependent manner, with a greater number of apoptotic cells with longer hypoxic exposure. Immunolfuorescence labeling revealed that hypoxia suppressed TYRO3 expression. Western blot assay showed that hypoxia decreased Akt phosphorylation levels in a time-de-pendent manner. Taken together, these ifndings suggest that hypoxia inhibits the proliferation of hippocampal neurons and promotes apoptosis, and that the inhibition of the TYRO3/Akt signaling pathway plays an important role in hypoxia-induced neuronal injury.

  4. Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures

    DEFF Research Database (Denmark)

    Penkowa, M; Molinero, A; Carrasco, J

    2001-01-01

    , the immunoreactivity for inducible nitric oxide synthase, peroxynitrite-induced nitration of proteins and byproducts of fatty acid peroxidation were dramatically increased, as was that for metallothionein I+II, Mn-superoxide dismutase and Cu/Zn-superoxide dismutase. In accordance, a significant neuronal apoptosis...... was caused by kainic acid, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and interleukin-1beta converting enzyme/Caspase-1 stainings. In kainic acid-injected interleukin-6 null mice, reactive astrogliosis and microgliosis were reduced, while......The role of interleukin-6 in hippocampal tissue damage after injection with kainic acid, a rigid glutamate analogue inducing epileptic seizures, has been studied by means of interleukin-6 null mice. At 35mg/kg, kainic acid induced convulsions in both control (75%) and interleukin-6 null (100%) mice...

  5. Dynamic changes in proprotein convertase 2 activity in cortical neurons after ischemia/reperfusion and oxygen-glucose deprivation

    Institute of Scientific and Technical Information of China (English)

    Shuqin Zhan; An Zhou; Chelsea Piper; Tao Yang

    2013-01-01

    In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cultured rat cortical neurons was established. Proprotein convertase 2 activity gradually decreased in the ischemic cortex with increasing duration of reperfusion. In cultured rat cortical neurons, the number of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling-positive neurons significantly increased and proprotein convertase 2 activity also decreased gradually with increasing duration of oxygen-glucose deprivation. These experimental findings indicate that proprotein convertase 2 activity decreases in ischemic rat cortex after reperfusion, as well as in cultured rat cortical neurons after oxygen-glucose deprivation. These changes in enzyme activity may play an important pathological role in brain injury.

  6. Ammonia exposure induces oxidative stress, endoplasmic reticulum stress and apoptosis in hepatopancreas of pacific white shrimp (Litopenaeus vannamei).

    Science.gov (United States)

    Liang, Zhongxiu; Liu, Rui; Zhao, Depeng; Wang, Lingling; Sun, Mingzhe; Wang, Mengqiang; Song, Linsheng

    2016-07-01

    Ammonia is one of major environmental pollutants in the aquatic system that poses a great threat to the survival of shrimp. In the present study, the mRNA expression of endoplasmic reticulum (ER) stress marker and unfolded protein response (UPR) related genes, as well as the change of redox enzyme and apoptosis were investigated in hepatopancreas of the pacific white shrimp, Litopenaeus vannamei after the exposure of 20 mg L(-1) total ammonia nitrogen (TAN). Compared with the control group, the superoxide dismutase (SOD) activity in hepatopancreas decreased significantly (p vannamei after exposure to ammonia by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The results indicated that ammonia exposure could induce oxidative stress, which further caused ER stress and apoptosis in hepatopancreas of L. vannamei.

  7. The Difference of Sensitivity between BXPC-3 and K562 Cells by Treatments with Combination of Indole-3-acetic Acid and Horseradish Peroxidase

    Institute of Scientific and Technical Information of China (English)

    BEN Yali; LIU Deli; ZHU Dali; ZHU Derui; LUO Qin

    2006-01-01

    The difference of sensitivity to indole- 3-acetic acid ( IAA ) combined with horseradish peroxidase (HRP) in K562 and BXPC- 3 cells was investigated. The cell proliferation was determined by MTT assay. The cell cycle and apoptosis of K562 and BXPC- 3 cells were examined by a fluorescence flow cytometer (FCM) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) respectively. The experimental results show that IAA and HRP could inhibit BXPC- 3 cell proliferation greatly compared with K562 cell during the first 48 h . The cell cycle was arrested predominantly at G2/ M phase in K562 and BXPC- 3 cells. The cell apoptosis of K562 and BXPC- 3 was induced by IAA/ HRP. There was a significant difference between the two cell lines since BXPC- 3 cells were more sensitive than K562 cells by treatments with combination of IAA and HRP.

  8. Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

    Institute of Scientific and Technical Information of China (English)

    Yanan Cai; Xiaodong Yuan; Ya Ou; Yanhui Lu

    2011-01-01

    β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro. However, because of the short survival time of the differentiated cells, clinical applications for this technique are limited. As such, we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy. The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended. Taken together, these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death. However, the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

  9. Brain death is associated with endoplasmic reticulum stress and apoptosis in rat liver.

    Science.gov (United States)

    Cao, S; Wang, T; Yan, B; Lu, Y; Zhao, Y; Zhang, S

    2014-12-01

    Cell death pathways initiated by stress on the endoplasmic reticulum (ER) have been implicated in a variety of common diseases, such as ischemia/reperfusion injury, diabetes, heart disease, and neurodegenerative disorders. However, the contribution of ER stress to apoptosis and liver injury after brain death is not known. In the present study, we found that brain death induces a variety of signature ER stress markers, including ER stress-specific X box-binding protein 1 and up-regulation of glucose-regulated protein 78. Furthermore, brain death causes up-regulation of C/EBP homologous protein and caspase-12. Consistent with this, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling assay and transmission electron microscopy confirmed apoptosis in the liver after brain death. Taken together, the present study provides strong evidence supporting the presence and importance of ER stress and response in mediating brain death-induced apoptosis and liver injury.

  10. Apoptosis-mediated seasonal testicular regression in the Japanese Jungle Crow (Corvus macrorhynchos).

    Science.gov (United States)

    Islam, M Nazrul; Tsukahara, N; Sugita, S

    2012-06-01

    The present study investigated effects of apoptosis observed during seasonal testicular regression in Japanese Jungle Crows. The study was conducted during January to June 2008, 2009. Testes from adults captured during non-breeding (January), prebreeding (February to mid-March), main-breeding (late March to early May), transition (mid-May to late May), and post-breeding (June) seasons were analyzed. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Paired-testis volume increased 95-fold from the non-breeding to the main-breeding season (P Crows; however, testis function was terminated rapidly after the breeding season. Furthermore, we concluded, similar to other avian species, Sertoli cell apoptosis followed by massive germ cell death was responsible for rapid testicular regression in Jungle Crows. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Zinc inhibits nuclear factor-kappa B activation and sensitizes prostate cancer cells to cytotoxic agents.

    Science.gov (United States)

    Uzzo, Robert G; Leavis, Paul; Hatch, William; Gabai, Vladimir L; Dulin, Nickolai; Zvartau, Nadezhda; Kolenko, Vladimir M

    2002-11-01

    Prostate carcinogenesis involves transformation of zinc-accumulating normal epithelial cells to malignant cells, which do not accumulate zinc. In this study, we demonstrate by immunoblotting and immunohistochemistry that physiological levels of zinc inhibit activation of nuclear factor (NF)-kappa B transcription factor in PC-3 and DU-145 human prostate cancer cells, reduce expression of NF-kappa B-controlled antiapoptotic protein c-IAP2, and activate c-Jun NH(2)-terminal kinases. Preincubation of PC-3 cells with physiological concentrations of zinc sensitized tumor cells to tumor necrosis factor (TNF)-alpha, and paclitaxel mediated cell death as defined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. These results suggest one possible mechanism for the inhibitory effect of zinc on the development and progression of prostate malignancy and might have important consequences for the prevention and treatment of prostate cancer.

  12. Emphysematous Eosinophilic Lymphangitis in the Ruminal Submucosa of Cattle.

    Science.gov (United States)

    Ohfuji, S

    2015-11-01

    Twenty cattle (14 Holstein-Friesian, 3 Japanese Black, 3 Aberdeen Angus) ranging in age from 3 months to 8 years exhibited, at slaughter, emphysematous thickening of the ruminal submucosa owing to the appearance of numerous, contiguous, small gas bubbles. Microscopic changes in the ruminal submucosa consisted of (1) multiple cystic (emphysematous) lymphangiectasis that was frequently lined or occluded by granulomatous inflammatory infiltrates including macrophages, multinucleate giant cells, and eosinophils; (2) intralymphatic phagocytosis by macrophages and giant cells of eosinophils that showed positive labeling with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay; and (3) an inflammatory infiltrate extending from the area of lymphangitis into surrounding tissue, as well as edema, hemorrhage, fibrin exudation, fibroplasia, or capillary proliferation throughout the lesional submucosa. In addition, 15 (75%) of the cattle had globular leukocyte infiltrates in the mucosal epithelia of the rumen.

  13. Effects of topical human amniotic fluid and human serum in a mouse model of keratoconjunctivitis sicca.

    Science.gov (United States)

    Quinto, Guilherme G; Camacho, Walter; Castro-Combs, Juan; Li, Li; Martins, Suy Anne R; Wittmann, Priscila; Campos, Mauro; Behrens, Ashley

    2012-04-01

    To compare the effects of topical human amniotic fluid (HAF), topical human serum (HS), and topical artificial tears in a mouse model of dry eye. Thirty C57BL/6 mice were divided into 3 treatment groups: HAF, HS, and preservative-free artificial tears. Dry eye was induced by an injection of botulinum toxin B (BTX-B) into the lacrimal gland. Tear production and ocular surface fluorescein staining were evaluated in each mouse at 6 time points during a 4-week period. Goblet cell density was assessed in stained histological sections. Apoptotic keratocytes were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling test assay. A significant decrease in tear production was observed 3 days after BTX-B injection in all groups. At week 1, the HAF and HS groups had improved tear production compared with the control group (P < 0.001 and P = 0.003, respectively). HAF had a significantly improved fluorescein staining score compared with the HS (P = 0.043) and control (P = 0.007) groups at week 2. Goblet cell density was significantly decreased in the control group compared with the HAF and HS groups (P < 0.001). No difference in the amount of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive keratocytes was observed among the groups. HAF was superior to HS and artificial tears for improving corneal staining within 2 weeks of therapy in this induced mouse model of keratoconjunctivitis sicca. Clinical studies are needed to ascertain the benefits of these therapies in patients with ocular surface disorders associated with dry eye.

  14. Safety evaluation of poly(lactic-co-glycolic acid/poly(lactic-acid microspheres through intravitreal injection in rabbits

    Directory of Open Access Journals (Sweden)

    Rong XF

    2014-06-01

    Full Text Available Xianfang Rong,1 Weien Yuan,2 Yi Lu,1 Xiaofen Mo11Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China; 2School of Pharmacy, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaAbstract: Poly(lactic-co-glycolic acid (PLGA and/or poly(lactic-acid (PLA microspheres are important drug delivery systems. This study investigated eye biocompatibility and safety of PLGA/PLA microspheres through intravitreal injection in rabbits. Normal New Zealand rabbits were randomly selected and received intravitreal administration of different doses (low, medium, or high of PLGA/PLA microspheres and erythropoietin-loaded PLGA/PLA microspheres. The animals were clinically examined and sacrificed at 1, 2, 4, 8, and 12 weeks postadministration, and retinal tissues were prepared for analysis. Retinal reactions to the microspheres were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end staining and glial fibrillary acidic protein immunohistochemistry. Retinal structure changes were assessed by hematoxylin and eosin staining and transmission electron microscopy. Finally, retinal function influences were explored by the electroretinography test. Terminal deoxynucleotidyl transferase-mediated dUTP nick end staining revealed no apoptotic cells in the injected retinas; immunohistochemistry did not detect any increased glial fibrillary acidic protein expression. Hematoxylin and eosin staining and transmission electron microscopy revealed no micro- or ultrastructure changes in the retinas at different time points postintravitreal injection. The electroretinography test showed no significant influence of scotopic or photopic amplitudes. The results demonstrated that PLGA/PLA microspheres did not cause retinal histological changes or functional damage and were biocompatible and safe enough for intravitreal injection in rabbits for controlled

  15. A Novel Alkylating Agent, Glufosfamide, Enhances the Activity of Gemcitabine In Vitro, In Vivo

    Directory of Open Access Journals (Sweden)

    W. Steve Ammons

    2007-08-01

    Full Text Available Glufosfamide is an alkylating agent consisting of iphosphoramide mustard conjugated to glucose that is currently included in clinical studies of pancreatic cancer. We studied the effects of glufosfamide, in combination with gemcitabine, on in vitro, in vivo models of pancreatic cancer. In proliferation assays, glufosfamide, gemcitabine inhibited the growth of MiaPaCa- 2, H766t, PANC-1 cells, but the combination of the two agents provided greater effects. Apoptosis of MiaPaCa-2 cells, measured by fluorescence-activated cell sorting, was enhanced by the combination of the two drugs, compared to single-agent treatment. Glufosfamide alone inhibited the growth of red fluorescent proteinexpressing MiaPaCa-2 tumors in an orthotopic nude mouse model in a dose-dependent manner. Combining glufosfamide (30 mg/kg with gemcitabine resulted in enhanced inhibition of tumor growth, significantly prolonged survival. Immunohistochemistry of excised tumors revealed that both glufosfamide, gemcitabine increased levels of apoptosis (measured by terminal deoxynucleotidyl transferase-mediated nick end labeling staining, reduced proliferation (measured by proliferating cell nuclear antigen staining. No effects on microvessel density were observed. These results support the use of the alkylating agent glufosfamide, the DNA synthesis inhibitor gemcitabine, rather than the use of either agent alone, to provide greater benefits, demonstrate that this combination treatment should be useful in the clinical treatment of pancreatic carcinoma.

  16. Region-specific vulnerability to endoplasmic reticulum stress-induced neuronal death in rat brain after status epilepticus

    Indian Academy of Sciences (India)

    Jing Chen; Hu Guo; Guo Zheng; Zhong-Nan Shi

    2013-12-01

    We sought to clarify the involvement and the intra-cerebral distribution variability of C/EBP homologous protein (CHOP), a representative molecule related to endoplasmic reticulum (ER) stress-induced cell death signalling pathways, in neuronal death resulting from status epilepticus in rats. The expression patterns of CHOP and glucose-regulated protein (GRP) 78, a good marker of ER stress, were assessed by Western blotting, real-time PCR, Hoechst and immunohistochemistry in the hippocampus, cortex and striatum on a status epilepticus (SE) model. Double-fluorescent staining of CHOP and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labelling (TUNEL) method were performed to clarify the involvement of CHOP in cell death. SE resulted in a time-dependent increase in the expression of GRP78 and CHOP. The expression of GRP78 protein was increased at 3, 6 and 12 h after SE and no brain region variability was found. The expression of CHOP protein was also increased, reached its peak at 24 h and remained high at 48 h. CHOP protein expression, however, showed brain region variability with highest expression noted in the hippocampus followed by the striatum, and lowest in the cortex. The up-regulation of CHOP occurring at the transcriptional level was demonstrated by real-time PCR. Double fluorescence showed that CHOP expression strongly correlated with neurons undergoing apoptosis. The results indicated that SE compromises the function of the ER and that the hippocampus is more vulnerable than the cortex and the striatum.

  17. Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats.

    Science.gov (United States)

    Roth, Steven; Dreixler, John C; Mathew, Biji; Balyasnikova, Irina; Mann, Jacob R; Boddapoti, Venkat; Xue, Lai; Lesniak, Maciej S

    2016-06-01

    We have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina. Rats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays. Eyes injected with hypoxic BMSC-conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium. The medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect.

  18. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.

    Science.gov (United States)

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-06-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  19. Protective effect of inhalation of hydrogen gas on radiation-induced dermatitis and skin injury in rats.

    Science.gov (United States)

    Watanabe, Sadahiro; Fujita, Masanori; Ishihara, Masayuki; Tachibana, Shoichi; Yamamoto, Yoritsuna; Kaji, Tatsumi; Kawauchi, Toshio; Kanatani, Yasuhiro

    2014-11-01

    The effect of inhalation of hydrogen-containing gas (1.3% hydrogen + 20.8% oxygen + 77.9% nitrogen) (HCG) on radiation-induced dermatitis and on the healing of healing-impaired skin wounds in rats was examined using a rat model of radiation-induced skin injury. An X-ray dose of 20 Gy was irradiated onto the lower part of the back through two holes in a lead shield. Irradiation was performed before or after inhalation of HCG for 2 h. Inhalation of HCG significantly reduced the severity of radiodermatitis and accelerated healing-impaired wound repair. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and 8-hydroxy-2(')-deoxyguanosine (8-OHdG) showed that the proportion of apoptotic keratinocytes and the level of staining in the X-irradiated skin of rats that pre-inhaled HCG were significantly lower than that of rats which did not pre-inhale HCG. Cutaneous full-thickness wounds were then created in the X-irradiated area to examine the time-course of wound healing. X-irradiation significantly increased the time required for wound healing, but the inhalation of HCG prior to the irradiation significantly decreased the delay in wound healing compared with the control and post-inhalation of HCG groups. Therefore, radiation-induced skin injury can potentially be alleviated by the pre-inhalation of HCG.

  20. In Vitro Cytotoxicity of GuttaFlow Bioseal, GuttaFlow 2, AH-Plus and MTA Fillapex.

    Science.gov (United States)

    Saygili, Gokhan; Saygili, Suna; Tuglu, Ibrahim; Davut Capar, Ismail

    2017-01-01

    The aim of the present in vitro study was to evaluate the cytotoxicity of different sealers including GuttaFlow Bioseal, GuttaFlow 2, AH-Plus and MTA Fillapex on L929 murine fibroblasts. Samples of GuttaFlow Bioseal, GuttaFlow 2, AH-Plus and MTA Fillapex were fabricated in Teflon disks of 5 mm diameter and 3 mm thickness. L929 fibroblasts were exposed to the extracts of these materials for 3, 24, 72 and 168 h at 37(°)C with 5% CO2. Cell viability was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The data were analysed by ANOVA. GuttaFlow Bioseal was nontoxic at all experimental time points (P>0.05), whereas MTA Fillapex and AH-Plus were toxic (PMTA Fillapex was more cytotoxic than AH-Plus. There were more apoptotic cells in the MTA Fillapex and AH-Plus groups than in the other groups at 3 h (PMTA Fillapex and AH-Plus. At all experimental time points, there was no significant difference in the cell viability between the GuttaFlow Bioseal group and the control group.

  1. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality.

    Science.gov (United States)

    Takeda, Kumiko; Uchiyama, Kyoko; Kinukawa, Masashi; Tagami, Takahiro; Kaneda, Masahiro; Watanabe, Shinya

    2015-01-01

    Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was determined. Individual differences in semen were detected using the sperm TUNEL index in these bulls (P bulls with a conception rate lower than 10%) and Holstein (n = 34) bulls were analyzed. The average sperm TUNEL index and conception rate resulting from artificial insemination (AI) were 4.7% and 55.7% for Japanese Black, and 4.9% and 39.5% for Holstein, respectively. A weak negative correlation between sperm TUNEL index and conception rate was observed in Holstein bulls (P bulls with more than 10% sperm TUNEL index were studied, and these samples showed low sperm viability. However, semen resulting in a very low conception rate did not have a high sperm TUNEL index. Although it would be difficult to predict a low conception rate resulting from AI using the sperm TUNEL index alone, the index can be used as an additional parameter to provide a more comprehensive description of semen quality.

  2. Protective effect of daidzin against D-galactosamine and lipopolysaccharide-induced hepatic failure in mice.

    Science.gov (United States)

    Kim, Sung-Hwa; Heo, Jeong-Haing; Kim, Yeong Shik; Kang, Sam Sik; Choi, Jae Sue; Lee, Sun-Mee

    2009-05-01

    This study examined the effects of daidzin, a major isoflavone from Puerariae Radix, on D-galactosamine (D-GalN) and lipopolysaccharide (LPS)-induced liver failure. Mice were given an intraperitoneal injection of daidzin (25, 50, 100 and 200 mg/kg) 1 h before receiving an injection of D-GalN (700 mg/kg)/LPS (10 microg/kg). Daidzin markedly reduced the elevated serum aminotransferase activity and the levels of lipid peroxidation and tumor necrosis factor-alpha. The glutathione content was lower in the D-GalN/LPS group, which was attenuated by daidzin. The daidzin pretreatment attenuated the swollen mitochondria observed in the d-GalN/LPS group. Daidzin attenuated the apoptosis of hepatocytes, which was confirmed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and a caspase-3 assay. Overall, these results suggest that the liver protection of daidzin is due to reduced oxidative stress and its antiapoptotic activity.

  3. Study on Relationship between the Thickness of Tongue Fur and the Expressions of Apoptosis-related Genes of the Tongue Epithelial Cells in Patients with Diseases of the Digestive System

    Institute of Scientific and Technical Information of China (English)

    Wu Zhengzhi; Li Ming; Zhang Yongfeng; Chen Manyin

    2007-01-01

    To investigate the relationship between the thickness of tongue fur, apoptosis of the tongue fur epithelial cells and expressions of apoptosis-related genes in diseases of the digestive system,apoptosis-related genes TGF-β3, fas mRNA and protein products were detected with terminal deoxynucleotidyl transferase-mediated deoxyurine triphosphate (d-UTP) nick-end labeling(TUNEL)technique, in situ hybridization, immunohistochemical methods, and image analysis technique,respectively. Results indicated that compared with the normal tongue fur, over-expression of fas gene was found in the peeling fur with an increase in cell apoptosis, while a low-expression of TGF-β3 in the thick fur with a decrease in cell apoptosis. The changes in expression levels of fas and TGF-β3 genes,apoptosis-promoting genes in the tongue fur epithelial cells, had a similar tendency of cell apoptosis level.It is concluded that the changes in expression levels of fas and TGF-β3 are possibly important reasons influencing apoptosis of epithelial cells of tongue fur and leading to changes in thickness of the tongue fur.

  4. Cadmium-induced ectopic apoptosis in zebrafish embryos

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Po Kwok; Cheng, Shuk Han [Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon (Hong Kong)

    2003-02-01

    In this study, we tested the hypothesis that cadmium-induced developmental toxicity was mediated via ectopic occurrence of apoptosis during embryonic development. We employed confocal microscopy to acquire images of whole-mount staining of apoptotic cells in zebrafish embryo exposed to 100 {mu}M cadmium from 5 hours post fertilisation (hpf) to 28 hpf. Three-dimensional reconstruction of the images was performed and the spatial and temporal distributions of apoptotic cells in the embryos were compared. In cadmium-treated embryos with varying degrees of gross developmental malformations, significantly higher numbers of apoptotic cells were detected with this method. In order to detect the precise locations of apoptotic cells, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay in sectioned embryos. In the degenerating neural tube of cadmium-treated embryos apoptotic cells were detected, while in the healthy neural tube of the untreated controls no apoptotic cells were found. We then employed flow cytometry to investigate whether cadmium exposure would affect the dynamics of apoptosis or induce any abnormalities in cell-cycle progression. It appeared that cadmium did not induce cell-cycle arrest. The percentages of apoptotic cells did not differ in the two groups at 13, 16 or 19 hpf. At 28 hpf, however, a significantly higher percentage of apoptotic cells were found in the cadmium-treated group. Exposure to cadmium, therefore, induced ectopic apoptosis at 28 hpf without affecting the dynamics of apoptosis at earlier developmental stages. (orig.)

  5. Neuroprotective effect of penehyclidine hydrochloride on focal cerebral ischemiareperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Cuicui Yu; Junke Wang

    2013-01-01

    Penehyclidine hydrochloride can promote microcirculation and reduce vascular permeability. However, the role of penehyclidine hydrochloride in cerebral ischemia-reperfusion injury remains unclear. In this study, in vivo middle cerebral artery occlusion models were established in experimental rats, and penehyclidine hydrochloride pretreatment was given via intravenous injection prior to model establishment. Tetrazolium chloride, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and immunohistochemical staining showed that, penehyclidine hydrochloride pretreatment markedly attenuated neuronal histopathological changes in the cortex, hippocampus and striatum, reduced infarction size, increased the expression level of Bcl-2, decreased the expression level of caspase-3, and inhibited neuronal apoptosis in rats with cerebral ischemia-reperfusion injury. Xanthine oxidase and thiobarbituric acid chromogenic results showed that penehyclidine hydrochloride upregulated the activity of superoxide dismutase and downregulated the concentration of malondialdehyde in the ischemic cerebral cortex and hippocampus, as well as reduced the concentration of extracellular excitatory amino acids in rats with cerebral ischemia-reperfusion injury. In addition, penehyclidine hydrochloride inhibited the expression level of the NR1 subunit in hippocampal nerve cells in vitro following oxygen-glucose deprivation, as detected by PCR. Experimental findings indicate that penehyclidine hydrochloride attenuates neuronal apoptosis and oxidative stress injury after focal cerebral ischemia-reperfusion, thus exerting a neuroprotective effect.

  6. Advanced glycation end products (AGEs) and their receptor (RAGE) induce apoptosis of periodontal ligament fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li, D.X.; Deng, T.Z.; Lv, J.; Ke, J. [Department of Stomatology, Air Force General Hospital PLA, Haidian District, Beijing (China)

    2014-09-19

    Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01) and increased apoptosis (11.31±1.73%, P<0.05). Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.

  7. Protective effect of prostaglandin E₁ on radiation-induced proliferative inhibition and apoptosis in keratinocytes and healing of radiation-induced skin injury in rats.

    Science.gov (United States)

    Takikawa, Megumi; Sumi, Yuki; Tanaka, Yoshihiro; Nambu, Masaki; Doumoto, Takashi; Yanagibayashi, Satoshi; Azuma, Ryuichi; Yamamoto, Naoto; Kishimoto, Satoko; Ishihara, Masayuki; Kiyosawa, Tomoharu

    2012-01-01

    We examined the effects of prostaglandin E₁ (PGE₁) on radiation-induced proliferation inhibition and apoptosis in keratinocytes and healing of radiation-induced skin injury in a rat model. PGE₁ had a protective effect on radiation-induced growth inhibition in keratinocytes in vitro, but not in fibroblasts. Varying concentrations of PGE₁ were subcutaneously administered into the posterior neck region. X-irradiation at a dose of 20 Gy was administrated to the lower part of the back using a lead sheet with two holes 30 min to 1 h before or after the administration of PGE₁. Although X-irradiation induced epilation, minor erosions, or skin ulcers in almost all rats, PGE₁ administration prior to irradiation reduced these irradiation injuries. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling showed that proportions of apoptotic keratinocytes in the X-irradiated skin of PGE₁-administered rats were significantly lower than for those in the skin of rats which did not receive PGE₁. Cutaneous full-thickness defective wounds were then formed in X-irradiated areas to examine the time course of wound healing. Wound healing was significantly delayed because of X-irradiation, but PGE₁ administration prior to irradiation led to a significantly shorter delay in wound healing compared with controls. Decreasing delay in wound healing was correlated with concentration of PGE₁ administrated. Thus, PGE₁-administration may potentially alleviate the radiation-induced skin injury.

  8. Cigarette smoking induces heat shock protein 70 kDa expression and apoptosis in rat brain: Modulation by bacoside A.

    Science.gov (United States)

    Anbarasi, K; Kathirvel, G; Vani, G; Jayaraman, G; Shyamala Devi, C S

    2006-01-01

    Cigarette smoking is associated with the development of several diseases and antioxidants play a major role in the prevention of smoking-related diseases. Apoptosis is suggested as a possible contributing factor in the pathogenesis of smoking-induced toxicity. Therefore the present study was designed to investigate the influence of chronic cigarette smoke exposure on apoptosis and the modulatory effect of bacoside A (triterpenoid saponin isolated from the plant Bacopa monniera) on smoking-induced apoptosis in rat brain. Adult male albino rats of Wistar strain were exposed to cigarette smoke and simultaneously administered with bacoside A (10 mg/kg b.w./day, orally) for a period of 12 weeks. Expression of brain hsp70 was analyzed by Western blotting. Apoptosis was identified by DNA fragmentation, terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick end labeling (TUNEL) staining and transmission electron microscopy. The results showed that exposure to cigarette smoke induced hsp70 expression and apoptosis as characterized by DNA laddering, increased TUNEL-positive cells and ultrastructural apoptotic features in the brain. Administration of bacoside A prevented expression of hsp70 and neuronal apoptosis during cigarette smoking. We speculate that apoptosis may be responsible for the smoking-induced brain damage and bacoside A can protect the brain from the toxic effects of cigarette smoking.

  9. Taurolidine and oxidative stress: a rationale for local treatment of mesothelioma.

    Science.gov (United States)

    Aceto, N; Bertino, P; Barbone, D; Tassi, G; Manzo, L; Porta, C; Mutti, L; Gaudino, G

    2009-12-01

    Malignant mesothelioma is an asbestos-related, aggressive tumour, resistant to most anticancer therapies. Akt is a key mediator of mesothelioma cell survival and chemoresistance. This study aimed to clarify the mechanism by which taurolidine (TN), a known synthetic compound with antimicrobial and antineoplastic properties, leads to mesothelioma cell death. Apoptosis was studied by annexin V binding, cell cycle analysis, caspase-8 activation, poly(ADP-ribose) polymerase (PARP) cleavage and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL). Oxidative stress was measured by nitrite production and DNA oxidative damage. Protein expression and phosphorylation were evaluated by immunoprecipitation and immunoblotting. TN induces cell death of mesothelioma cells, but not of non-neoplastic human mesothelial cells. After TN treatment of mesothelioma cells, Akt but not extracellular signal-regulated kinase (Erk) 1/2 activity is inhibited a in time- and dose-dependent manner. Protein phosphatase (PP)1alpha and PP2A are activated several hours after drug addition. Apoptosis induced by TN is driven by oxidative stress and cell exposure to sulfydryl donors, such as glutathione monoethylester and l-N-acetylcysteine, significantly reduced pro-apoptotic effects and Akt inhibition. Conversely, expression of constitutively activated Akt did not affect cytoxicity elicited by TN, which retained its ability to inhibit the kinase. TN induces mesothelioma cell death via oxidative stress, accompanied by inhibition of Akt signalling. This provides a promising molecular rationale for TN as local treatment of malignant mesothelioma.

  10. Experimental studies on ultralow frequency pulsed gradient magnetic field inducing apoptosis of cancer cell and inhibiting growth of cancer cell

    Institute of Scientific and Technical Information of China (English)

    曾繁清; 郑从义; 张新晨; 李宗山; 李朝阳; 王川婴; 张新松; 黄晓玲; 张沪生

    2002-01-01

    The morphology characteristics of cell apoptosis of the malignant tumour cells in magnetic field-treated mouse was observed for the first time. The apoptotic cancer cell contracted, became rounder and divorced from adjacent cells; the heterochromatin condensed and coagulated together along the inner side of the nuclear membrane; the endoplasmic reticulums(ER) expanded and fused with the cellular membrane; many apoptotic bodies which were packed by the cellular membrane appeared and were devoured by some lymphocytes and plasma. Apoptosis of cancer cells was detected by terminal deoxynucleotidyl transferase mediated in situ nick end labeling(TUNEL). It was found that the number of apoptosis cancer cells of the sample treated by the magnetic field is more than that of the control sample. The growth of malignant tumour in mice was inhibited and the ability of immune cell to dissolve cancer cells was improved by ultralow frequency(ULF) pulsed gradient magnetic field; the nuclei DNA contents decreased, indicating that magnetic field can block DNA replication and inhibit mitosis of cancer cells. It was suggested that magnetic field could inhibit the metabolism of cancer cell, lower its malignancy, and restrain its rapid and heteromorphic growth. Since ULF pulsed gradient magnetic field can induce apoptosis of cancer cells and inhibit the growth of malignant tumour, it could be used as a new method to treat cancer.

  11. Studies on apoptosis of spermatogenic cells in normal fertile men treated with supraphysiological doses of testosterone undecanoate

    Institute of Scientific and Technical Information of China (English)

    Yi-FengGE; Yu-FengHUANG; Gui-YuaaZHANG; Xinghaiwang; jianpingxu

    1999-01-01

    Aim: To study the anli-spermatogenic mechanism of supra-physiological doses of testosterone undecanoate (TU).Methods: Twenty fertile adult men received four intranmscular injections of TU at monthly intervals, 1000 nag uponadmission and 500 mg for the subsequent injections. The apoptotic germ cells in the semen were studied under light mi-croscope with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and Wright-Giem-sa staining methods. Results: After treatment, the sperm density and the number of spenmtogenic cells in the semenwere significantly decreased ( P <0.01), while the apoptotic ratios of spemaatccytes and spextmtids increased significantly(P<0.01) as compared with the pretreatment levels. Apoptosis was found to be augmented in the whole series of castoffspenmatogenic cells. Conclusion: Besides its suppressive effect on spextmtogenesis through a negative feed-bookn mechmaism, TU enhances apoptosis of spenmtogenic cells, which may be an additional mechanism of its anti-spenmto-genic activity.

  12. THE ROLES OF bcl-2 GENE FAMILY IN THE PULMONARY ARTERY REMODELING OF HYPOXIA PULMONARY HYPERTENSION IN RATS

    Institute of Scientific and Technical Information of China (English)

    杨成; 王胜发; 梁桃; 王巨; 王凯; 王柏春

    2001-01-01

    Objective. To investigate the roles of apoptosis in the pulmonary artery remodeling of pulmonary hypertension secondary to hypoxia and illustrate the relative genes expression.Methods. Thirty rats were divided into hypoxia group(10%O2, 8h/d) and normal control group. On the 15th day of hypoxia, pulmonary artery pressure and right ventricular hypertrophy index were measured and pulmonary artery vessels were studied by light microscope. Then terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)technique was used to detect nucleosomal DNA fragmentation of apoptotic cells.In situ hybridization and RT-PCR were used to detect the expression level of bcl-2 and bax.``Results. The pulmonary artery pressure and right ventricular hypertrophy index of hypoxia group were increased significantly, the pulmonary artery wall of hypoxic group become incrassate than control group. Apoptotic cells can be found in lung with hypoxia or without hypoxia. Compared with control group, apoptotic index of hypoxic group decreased significantly. Through the methods of in situ hybridization and RT-PCR, we found the expression of bcl-2 increased whereas bax decreased significantly in the hypoxic group.``Conclusion. The alternation in bcl-2 and bax expression induced by hypoxia play an important role in the pulmonary artery remodeling which is the main pathologic change of pulmonary hypertension secondary to hypoxia.

  13. Rolipram stimulates angiogenesis and attenuates neuronal apoptosis through the cAMP/cAMP-responsive element binding protein pathway following ischemic stroke in rats.

    Science.gov (United States)

    Hu, Shouye; Cao, Qingwen; Xu, Peng; Ji, Wenchen; Wang, Gang; Zhang, Yuelin

    2016-03-01

    Rolipram, a phosphodiesterase-4 inhibitor, can activate the cyclic adenosine monophosphate (cAMP)/cAMP-responsive element binding protein (CREB) pathway to facilitate functional recovery following ischemic stroke. However, to date, the effects of rolipram on angiogenesis and cerebral ischemia-induced neuronal apoptosis are yet to be fully elucidated. In this study, the aim was to reveal the effect of rolipram on the angiogenesis and neuronal apoptosis following brain cerebral ischemia. Rat models of ischemic stroke were established following transient middle cerebral artery occlusion and rolipram was administered for three, seven and 14 days. The results were examined using behavioral tests, triphenyl tetrazolium chloride staining, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to evaluate the effects of rolipram therapy on functional outcome, angiogenesis and apoptosis. Western blot analysis was used to show the phosphorylated- (p-)CREB protein level in the ischemic hemisphere. The rolipram treatment group exhibited a marked reduction in infarct size and modified neurological severity score compared with the vehicle group, and rolipram treatment significantly promoted the microvessel density in the ischemic boundary region and increased p-CREB protein levels in the ischemic hemisphere. Furthermore, a significant reduction in the number of TUNEL-positive cells was observed in the rolipram group compared with the vehicle group. These findings suggest that rolipram has the ability to attenuate cerebral ischemic injury, stimulate angiogenesis and reduce neuronal apoptosis though the cAMP/CREB pathway.

  14. Effect of cholecystokinin on learning and memory, neuronal proliferation and apoptosis in the rat hippocampus

    Science.gov (United States)

    Reisi, Parham; Ghaedamini, Ali Reza; Golbidi, Mohammad; Shabrang, Moloud; Arabpoor, Zohreh; Rashidi, Bahman

    2015-01-01

    Background: Cholecystokinin (CCK) has roles in learning and memory, but the cellular mechanism is poorly understood. This study investigated the effect of CCK on spatial learning and memory, neuronal proliferation and apoptosis in the hippocampus in rats. Materials and Methods: Experimental groups were control and CCK. The rats received CKK octapeptide sulfated (CCK-8S, 1.6 μg/kg, i.p.) for 14 days. Spatial learning and memory were tested by Morris water maze and finally immunohistochemical study was performed; neurogenesis by Ki-67 method and apoptosis by Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) assay in hippocampal dentate gyrus (DG). Results: Cholecystokinin increased Ki-67 positive cells and reduced TUNEL positive cells in the granular layer of hippocampal DG. CCK failed to have a significant effect on spatial learning and memory. Conclusion: Results indicate neuroprotective and proliferative effects of CCK in the hippocampus; however, other factors are probably involved until the newly born neurons achieve necessary integrity for behavioral changes. PMID:26623402

  15. Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the “ladder pattern” revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner.The number of TUNEL-positive cells was dramatically increased to 33.6±1.2% from 2.8±0.3% by treat ment with heparin in different concentrations (10~40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2,bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations.These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.

  16. Differential routes of carboplatin administration influence lymphocyte apoptosis in retroperitoneal lymph nodes.

    Science.gov (United States)

    Huang, Yong-Wen; Zeng, Zheng; Li, Su; Liu, Ji-Hong

    2012-12-01

    We aimed to investigate carboplatin distribution in retroperitoneal lymph nodes and its effect on lymphocyte apoptosis following intravenous (IV), intra-arterial (IA), and retroperitoneal (RP) administration. Sixty-three healthy female canines were randomly assigned as IV, IA, or RP administration of carboplatin. At 0.5, 1, 2, 4, 8, 24, and 72 h after carboplatin treatment, retroperitoneal lymph nodes (n = 6 at each time point) were collected and high-performance liquid chromatography was employed to measure the carboplatin content. The differences in carboplatin pharmacokinetics of the three administration routes were compared. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was carried out to measure the lymphocyte apoptosis of the retroperitoneal lymphocytes. The peak concentration of carboplatin in plasma following IV administration was the highest among all approaches; as to the peak time, RP administration was longer than the other two administrations. Concentration for carboplatin in the retroperitoneal lymph node was highest following IA administration at early time points, but at higher time points, concentration was significantly higher following RP administration. Penetration of carboplatin into the retroperitoneal space was higher following RP administration. Following RP administration, the level of apoptotic lymphocytes in the retroperitoneal lymph nodes was significantly greater than either IV or IA. Following RP administration of carboplatin, the concentration, area under the curve of carboplatin and the number of apoptotic lymphocytes were significantly higher than those following IV and IA administration. This suggests that RP administration of carboplatin is beneficial for the treatment of retroperitoneal lymph node metastasis.

  17. Ulinastatin suppresses endoplasmic reticulum stress and apoptosis in the hippocampus of rats with acute paraquat poisoning

    Directory of Open Access Journals (Sweden)

    Hai-feng Li

    2015-01-01

    Full Text Available Lung injury is the main manifestation of paraquat poisoning. Few studies have addressed brain damage after paraquat poisoning. Ulinastatin is a protease inhibitor that can effectively stabilize lysosomal membranes, prevent cell damage, and reduce the production of free radicals. This study assumed that ulinastatin would exert these effects on brain tissues that had been poisoned with paraquat. Rat models of paraquat poisoning were intraperitoneally injected with ulinastatin. Simultaneously, rats in the control group were administered normal saline. Hematoxylin-eosin staining showed that most hippocampal cells were contracted and nucleoli had disappeared in the paraquat group. Fewer cells in the hippocampus were concentrated and nucleoli had disappeared in the ulinastatin group. Western blot assay showed that expressions of GRP78 and cleaved-caspase-3 were significantly lower in the ulinastatin group than in the paraquat group. Immunohistochemical findings showed that CHOP immunoreactivity was significantly lower in the ulinastatin group than in the paraquat group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining showed that the number of apoptotic cells was reduced in the paraquat and ulinastatin groups. These data confirmed that endoplasmic reticular stress can be induced by acute paraquat poisoning. Ulinastatin can effectively inhibit this stress as well as cell apoptosis, thereby exerting a neuroprotective effect.

  18. Aqueous extract of Cordyceps alleviates cerebral ischemia-induced short-term memory impairment in gerbils.

    Science.gov (United States)

    Lee, Sang-Hak; Ko, Il-Gyu; Kim, Sung-Eun; Hwang, Lakkyong; Jin, Jun-Jang; Choi, Hyun-Hee; Kim, Chang-Ju

    2016-04-01

    Cerebral ischemia is caused by reduced cerebral blood flow due to a transient or permanent cerebral artery occlusion. Ischemic injury in the brain leads to neuronal cell death, and eventually causes neurological impairments. Cordyceps, the name given to the fungi on insects, has abundant useful natural products with various biological activities. Cordyceps is known to have nephroprotective, hepatoprotective, anti-inflammatory, antioxidative, and antiapoptotic effects. We investigated the effects of Cordyceps on short-term memory, neuronal apoptosis, and cell proliferation in the hippocampal dentate gyrus following transient global ischemia in gerbils. For this study, a step-down avoidance test, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, immunohistochemistry for caspase-3 and 5-bromo-2'-de-oxyuridine, and western blot for Bax, Bcl-2, brain-derived neurotrophic factor (BDNF), and tyrosin kinase B were performed. In the present study, Cordyceps alleviated cerebral ischemia-induced short-term memory impairment. Cordyceps showed therapeutic effects through inhibiting cerebral ischemia-induced apoptosis in the hippocampus. Cordyceps suppressed cerebral ischemia-induced cell proliferation in the hippocampal dentate gyrus due to the reduced apoptotic neuronal cell death. Cordyceps treatment also enhanced BDNF and TrkB expressions in the hippocampus of ischemic gerbils. It can be suggested that Cordyceps overcomes cerebral ischemia-induced neuronal apoptosis, thus facilitates recovery following cerebral ischemia injury.

  19. Inhibitory effects of oleoylethanolamide (OEA) on H₂O₂-induced human umbilical vein endothelial cell (HUVEC) injury and apolipoprotein E knockout (ApoE-/-) atherosclerotic mice.

    Science.gov (United States)

    Ma, Li; Guo, Xiaobing; Chen, Wei

    2015-01-01

    Atherosclerosis (AS) is initiated by vascular endothelial cell injury, which is induced by lipid and protein oxidation. Oleoylethanolamide (OEA), a dietary fat-derived lipid, has shown atheroprotective effect. In vitro studies demonstrated that OEA showed cytoprotective effects on H2O2-induced primary cultured human umbilical vein endothelial cell (HUVEC) injury model. Further investigation of the cytoprotective effects of OEA demonstrated that OEA exerted its function by scavenging for reactive oxygen species, as well as increasing anti-oxidative enzymes, reducing lipid peroxidation, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and apoptosis-related proteins expression. The in vivo study using an ApoE-/- mouse model fed with high-fat diet for 8 weeks showed that OEA (10 mg/kg/day, i.g.) administration reduced blood lipid levels, prevented endothelial cell damage and inhibited early AS plaque formation. In conclusion, our results suggested that OEA exerted a pharmacological effect on ameliorating atherosclerotic plaque formation through the inhibition of oxidative stress-induced endothelial cell injury and therefore OEA can be a potential candidate drug for anti-atherosclerosis.

  20. Expression of NF-кB in Schwann cells and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in rats

    Institute of Scientific and Technical Information of China (English)

    WANG Yong-tang; LU Xiu-min; YU Ying; YANG Yan-hong; GAO Jie

    2007-01-01

    Objective:To explore the expression of nuclear factor-kappa B (NF-кB) in Schwann cells (SCs) and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in adult rats. Methods:Thirty-six adult Sprague-Dawley (SD) rats were divided randomly into normal control group (n=6),and sciatic nerves crushing group (n=30),and the later was further equally randomized into 5 nerves were examined by immunohistochemistry staining,and the apoptosis of motor neurons in spinalcord of lumbar 4 to lumbar 6(L4-L6)was investigated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay.Both were quantitated by image analysis.Results:In the normal control group (P<0.05,P<0.01).At 1 d after sciatic nerves crushing,the expression of served in the time-course on motor neuron apoptosis after sciatic nerves injury.Correlation analyses refollowing sciatic nerves injury(r=0.976 0,P<0.01).Conclusion:After injury of sciatic nerves,the presence and up-regulation of NF-κB in SCs may be involved in motor neuron apoptosis in L4-L6 spinal cord.

  1. Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with PRP-containing fragmin/protamine microparticles (PRP&F/P MPs).

    Science.gov (United States)

    Takikawa, Megumi; Ishihara, Masayuki; Takabayashi, Yuki; Sumi, Yuki; Takikawa, Makoto; Yoshida, Ryuichi; Nakamura, Shingo; Hattori, Hidemi; Yanagibayashi, Satoshi; Yamamoto, Naoto; Kiyosawa, Tomoharu

    2015-04-13

    The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.

  2. Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injur y

    Institute of Scientific and Technical Information of China (English)

    Guo-zhu Sun; Fen-fei Gao; Zong-mao Zhao; Hai Sun; Wei Xu; Li-wei Wu; Yong-chang He

    2016-01-01

    Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endo-plasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of lfuid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediat-ed dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These ifndings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury.

  3. Genome-wide mapping of DNA strand breaks.

    Directory of Open Access Journals (Sweden)

    Frédéric Leduc

    Full Text Available Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP, uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

  4. Preterm as compared with full-term neonatal calves are characterized by morphological and functional immaturity of the small intestine.

    Science.gov (United States)

    Bittrich, S; Philipona, C; Hammon, H M; Romé, V; Guilloteau, P; Blum, J W

    2004-06-01

    Intestinal diseases in neonatal calves may be due to morphological and functional immaturity. We have studied histomorphology, crypt cell proliferation rates (based on incorporation of 5-bromo-2'-deoxyuridine into DNA), presence of apoptotic cells (based on terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling), and brush border enzyme activities in preterm calves (277 d of gestation), euthanized on d 1 (P0) or 8 (P8), and in full-term calves (290 d of gestation), euthanized on d 1 (F0) or 8 (F8). Vacuolated epithelial cells were present in ileum of P0 and F0 but not in P8 and F8. During the first 8 d, villus sizes, crypt depths, and proliferation rates of crypt cells in the small intestine of preterm calves did not significantly change. In contrast, in full-term calves during the first 8 d, villus sizes in jejunum decreased, crypt depths increased in small intestine and colon, and crypt cell proliferation increased in duodenum and jejunum. Submucosal thickness in jejunum was highest in P0, but in ileum it increased with gestational age and feeding. Gestational age x feeding interactions indicated increased activities of aminopeptidase N and reduced lactase activities only in F8 and reduced dipeptidylpeptidase IV activities only in P8. In conclusion, in preterm calves the small intestinal epithelium was immature and brush border enzyme activities differed in part from those in full-term calves.

  5. Bilobalide inhibits 6-OHDA-induced activation of NF-κB and loss of dopaminergic neurons in rat substantia nigra

    Institute of Scientific and Technical Information of China (English)

    Ling-yun LI; Xi-lin ZHAO; Xi-feng FEI; Zhen-lun GU; Zheng-hong QIN; Zhong-qin LIANG

    2008-01-01

    Aim: To investigate the offect of bilobalide on the activation of NF-κB, and apoptasis of dopaminergic neurons induced by 6-hydroxydopamine (6-OHDA). Methods: A rat model of Parkinson's disease was produced with a unilateral infu-sion of 6-OHDA (8 μg) into the substantia nigra par compact. Bilobalide was administered 5, 10, and 20 mg/kg (ip) once a day for 7 d, starting 6 d prior to the 6-OHDA infusion. The rats were subjected to locomotor activity and rotational behavior testing 2 or 3 weeks after the 6-OHDA infusion. The expressions of tyrosine hydroxylase (TH) and NF-κB p65 were examined by immunofluorescence. The loss of dopaminergic neurons was detected by Nissl's staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to iden-tify apoptosis. ResUltS; Tho behavioral changes due to 6-OHDA were signifi-cantly restored by bilobalide pretreatment. Bilobalide inhibited the 6-OHDA-induced loss of TH-positive neurons, decreased the activation of NF-κB, and protected dopaminergic neurons from apoptosis remarkably. Conclusion: NF-κB activation contributes to the 6-OHDA-induced loss of dopaminergic neurons, and the inhibition of the NF-κB pathway is likely to be involved in the neuroprotective effect of bilobalide.

  6. Effects of Ethyl Pyruvate on Myocardial Apoptosis and Expression of Bcl-2 and Bax Proteins after Ischemia-reperfusion in Rats

    Institute of Scientific and Technical Information of China (English)

    Jialong GUO; Kailun ZHANG; Yanmei JI; Xionggang JIANG; Shunqing ZUO

    2008-01-01

    In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups (n=8 in each group): control group was perfused for 120min. In the I/R group, after 30min stabilization the injury was induced by 30min global ischemia followed by 60min reperfusion. Ethyl pyruvate (EP) group was set up with the same protocol as I/R group except that it was supplied with 2mmol/L EP 15min before ischemia and throughout reperfusion. Myocardial malonaldehyde (MDA) content Was measured. Myocardial apoptotic index (AI) was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group (P<0.05). These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.

  7. MG132 Inhibits Myocardial Ischemia-reperfusion Injury by Regulating Apoptotic Pathway

    Institute of Scientific and Technical Information of China (English)

    Dai Cuilian; Luo Kailiang; Chen Zhangrong

    2007-01-01

    Objectives To administrated proteasome inhibitor-MG-132 prior to reperfusion in rat myocardial ischemia-reperfusion model to determine whether MG-132 could reduce myocytic apoptosis. Methods and results MG-132 (0.75 mg/kg in 2 ml DMSO) injection 5 min prior to reperfusion resulted significant reduction of myocardial reperfusion injury. This effect was accompanied by reduced polymorphonuclear neutrophils(PMN) infiltration in myocardial region surrounding the myocardial infarct, reduced apoptosis in cardiac myocytes, reduced NF-κB activation, as determined by electron microscopy, histology, immunohistochemistry, the terminal deoxynucleotidyl transferase-mediated nick endlabeling (TUNEL) method, reverse transcription-polymerase chain reaction. Functional effects of MG-132 on PMN accumulation, activation of nuclear factor kappa B(p65 mRNA and protein levels ), and apoptosis were characterized in rat myocardial tissue. MG132 time-dependently inhibited myocardial p65 mRNA expression and reduced myocardial apoptotic index (AI) after reperfusion for 2 h, 6 h and 24 h ( P<0.01 ). Moreover, MG-132 time-dependently decreased Bax protein levels, while increased Bcl-2 protein levels in ischemic and reperfused myocardium ( P<0.05 ), its effect peaked after reperfusion for 24 h. Conclusions Our results demonstrate that MG-132 reduced myocardial reperfusion injury by inhibiting myosytic apoptotic cell death and blocking activation of NF-κB, down-regulating Bax expression and up-regulating Bcl-2 expression as well as elevating Bcl-2/Bax ratio.

  8. Hydroxysafflor Yellow A protects spinal cords from ischemia/reperfusion injury in rabbits

    Directory of Open Access Journals (Sweden)

    Shan Le-qun

    2010-08-01

    Full Text Available Abstract Background Hydroxysafflor Yellow A (HSYA, which is one of the most important active ingredients of the Chinese herb Carthamus tinctorius L, is widely used in the treatment of cerebrovascular and cardiovascular diseases. However, the potential protective effect of HSYA in spinal cord ischemia/reperfusion (I/R injury is still unknown. Methods Thirty-nine rabbits were randomly divided into three groups: sham group, I/R group and HSYA group. All animals were sacrificed after neurological evaluation with modified Tarlov criteria at the 48th hour after reperfusion, and the spinal cord segments (L4-6 were harvested for histopathological examination, biochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL staining. Results Neurological outcomes in HSYA group were slightly improved compared with those in I/R group. Histopathological analysis revealed that HSYA treatment attenuated I/R induced necrosis in spinal cords. Similarly, alleviated oxidative stress was indicated by decreased malondialdehyde (MDA level and increased superoxide dismutase (SOD activity after HSYA treatment. Moreover, as seen from TUNEL results, HSYA also protected neurons from I/R-induced apoptosis in rabbits. Conclusions These findings suggest that HSYA may protect spinal cords from I/R injury by alleviating oxidative stress and reducing neuronal apoptosis in rabbits.

  9. Keratinocyte Apoptosis is Decreased in Psoriatic Epidermis

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    Fatma Eskioğlu

    2009-12-01

    Full Text Available Background and Design: Abnormal differentiation and hyperproliferation of keratinocytes are the hallmarks of psoriasis vulgaris. Although psoriasis vulgaris is generally accepted as a disease of decreased keratinocyte apoptosis, the results are contradictory. The aim of the current study is to investigate whether decreased keratinocyte apoptosis contributes to the formation of a thickened epidermis as increased keratinocyte proliferation. Material and Method: Forty-three untreated psoriasis vulgaris patients and 20 healthy control subjects were included into the study. Biopsy specimens taken from the enrollee were evaluated by immunohistochemical staining for Ki-67 expressions to show the proliferation of keratinocytes and by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL method to show the apoptotic keratinocytes. Results: Apoptotic index (percentage of the TUNEL positive cells was significantly lower in psoriatic epidermis (0.33±0.64 than in normal epidermis (0.75±0.85; whereas Ki-67 index (percentage of positively staining cells for Ki-67 was significantly higher in psoriatic epidermis (30.86±10.49 than in normal epidermis (11.65±2.98, (p=0.021 and p=0.00; respectively. Conclusion: Decreased keratinocyte apoptosis also contribute to increased epidermal thickness in psoriasis as well as increased keratinocyte proliferation.

  10. Caspase-3 and survivin expression in pediatric neuroblastoma and their roles in apoptosis

    Institute of Scientific and Technical Information of China (English)

    王家祥; 郑树

    2004-01-01

    Background Neuroblastoma, one of the common tumors in children, possesses the feature of natural regression that might be related to apoptosis caspase-3 and survivin are believed to respectively induce and inhibit apoptosis. We investigated the expression of caspase-3 and survivin in pediatric neuroblastoma and the role that these genes played in apoptosis.Methods The expression of caspase-3 and survivin in pediatric neuroblastoma tissue samples was detected using in situ hybridization, ter mintuesal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical staining. The role that these genes played in apoptosis was then evaluated.Results A converse correlation was observed between the expression of survivin and caspase-3. When survivin was expressed at high levels in neuroblastoma samples, caspase-3 expression was downregulated, and the apoptotic index decreased simultaneously.Conclusion There is a converse correlation between the expression of caspase-3 and the expression of survivin in neuroblastoma cells, indicating that caspase-3 might induce apoptosis, and survivin may inhibit this process.

  11. Comparison of apoptosis between adult worms of Schistosoma japonicum from susceptible (BALB/c mice) and less-susceptible (Wistar rats) hosts.

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    Wang, Tao; Guo, Xiaoyong; Hong, Yang; Han, Hongxiao; Cao, Xiaodan; Han, Yanhui; Zhang, Min; Wu, Miaoli; Fu, Zhiqiang; Lu, Ke; Li, Hao; Zhao, Zhixin; Lin, Jiaojiao

    2016-10-30

    Schistosomiasis remains a serious public health concern in China. BALB/c mice are susceptible to Schistosoma japonicum infection, whereas the Wistar rats are less susceptible. Apoptosis phenomenon was observed in 42d adult worms of S. japonicum from both rats and mice at the morphologic, DNA, cellular, and gene levels by transmission electron microscopy (TEM), fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, fluorescein isothiocyanate-annexin-V/propidium iodide staining flow cytometry (FCM) analysis, and real-time PCR. The results showed that the apoptotic state in worms from two different susceptible hosts was diverse. Several classical hallmarks of apoptosis, including cell shrinkage, chromatin condensation and lunate marginalization, splitting of the nucleoli, nuclear shrinkage and apoptotic body formation were observed by TEM. TUNEL analysis showed that there were much more apoptosis spots in adult worms from rats than those from mice. Statistical analysis revealed that the degree of apoptosis and percentage of necrotic cells in adult worms from Wistar rats were significantly greater (Pworms from Wistar rats, as compared to those from BALB/c mice. The results obtained in this study collectively demonstrated that differential development of adult S. japonicum in less-susceptible rats and susceptible mice was significantly associated with apoptosis in the worm, and provided valuable information to guide further investigations of the mechanisms governing apoptosis and host interactions in schistosome infection.

  12. Protective role of NecroX-5 against neomycin-induced hair cell damage in zebrafish.

    Science.gov (United States)

    Song, Jae-Jun; Chang, Jiwon; Choi, Jungim; Im, Gi Jung; Chae, Sung Won; Lee, Seung Hoon; Kwon, Soon-Young; Jung, Hak Hyun; Chung, Ah-Young; Park, Hae-Chul; Choi, June

    2014-02-01

    NecroX-5, one of the derivatives of NecroX series compounds, is a mitochondrial reactive oxygen species and reactive nitrogen species scavenger that inhibits cell death against various kinds of oxidative stresses. The objective of the present study was to evaluate the effects of NecroX-5 on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five days post-fertilization, zebrafish larvae were exposed to 125 μM neomycin and one of the following NecroX-5 concentrations for 1 h: 10, 25, 50, and 75 μM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy (n = 10). The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Ultrastructural changes were evaluated using scanning electron microscopy. NecroX-5 decreased neomycin-induced hair cell loss in the neuromasts (NecroX-5 50 μM: 13.4 ± 2.0 cells, 125 μM neomycin only: 8.1 ± 1.2 cells; n = 10, P neomycin and 50 μM NecroX-5. NecroX-5 decreased apoptosis and mitochondrial damage. In conclusion, NecroX-5 attenuated neomycin-induced hair cell loss in zebrafish.

  13. The Extract of Aster Koraiensis Prevents Retinal Pericyte Apoptosis in Diabetic Rats and Its Active Compound, Chlorogenic Acid Inhibits AGE Formation and AGE/RAGE Interaction

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    Junghyun Kim

    2016-09-01

    Full Text Available Retinal capillary cell loss is a hallmark of early diabetic retinal changes. Advanced glycation end products (AGEs are believed to contribute to retinal microvascular cell loss in diabetic retinopathy. In this study, the protective effects of Aster koraiensis extract (AKE against damage to retinal vascular cells were investigated in streptozotocin (STZ-induced diabetic rats. To examine this issue further, AGE accumulation, nuclear factor-kappaB (NF-κB and inducible nitric oxide synthase (iNOS were investigated using retinal trypsin digests from streptozotocin-induced diabetic rats. In the diabetic rats, TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling-positive retinal microvascular cells were markedly increased. Immunohistochemical studies revealed that AGEs were accumulated within the retinal microvascular cells, and this accumulation paralleled the activation of NF-κB and the expression of iNOS in the diabetic rats. However, AKE prevented retinal microvascular cell apoptosis through the inhibition of AGE accumulation and NF-κB activation. Moreover, to determine the active compounds of AKE, two major compounds, chlorogenic acid and 3,5-di-O-caffeoylquinic acid, were tested in an in vitro assay. Among these compounds, chlorogenic acid significantly reduced AGE formation as well as AGE/RAGE (receptor for AGEs binding activity. These results suggest that AKE, particularly chlorogenic acid, is useful in inhibiting AGE accumulation in retinal vessels and exerts a preventive effect against the injuries of diabetic retinal vascular cells.

  14. Obesity potentiates the growth and dissemination of pancreatic cancer.

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    Zyromski, Nicholas J; Mathur, Abhishek; Pitt, Henry A; Wade, Terrence E; Wang, Sue; Nakshatri, Poornima; Swartz-Basile, Deborah A; Nakshatri, Harikrishna

    2009-08-01

    Obesity is an independent risk factor for pancreatic cancer development and progression, although the mechanisms underlying this association are completely unknown. The aim of the current study was to investigate the influence of obesity on pancreatic cancer growth using a novel in vivo model. Lean (C57BL/6 J) and obese (Lep(Db) and Lep(Ob)) mice were inoculated with murine pancreatic cancer cells (PAN02), and studied after 5 weeks of tumor growth. Tumor histology was evaluated by hematoxylin and eosin staining, cellular proliferation was assessed by 5-bromodeoxyuridine, and apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Serum adiponectin, leptin, and insulin levels were assayed. Obese mice developed larger tumors, and a significantly greater number of mice developed metastases; mortality was also greater in obese mice. Tumor apoptosis did not differ among strains, but tumors from both obese strains had greater proliferation relative to those growing in lean animals. Serum adiponectin concentration correlated negatively and serum insulin concentration correlated positively with tumor proliferation. Intratumoral adipocyte mass in tumors from both obese strains was significantly greater than that in tumors of lean mice. Data from this novel in vivo model suggest that the altered adipokine milieu and insulin resistance observed in obesity may lead directly to changes in tumor microenvironment, thereby promoting pancreatic cancer growth and dissemination.

  15. Therapeutic Effects of Microbubbles Added to Combined High-Intensity Focused Ultrasound and Chemotherapy in a Pancreatic Cancer Xenograft Model

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    Yu, Mi Hye [Department of Radiology, Konkuk University Medical Center, Seoul 05030 (Korea, Republic of); Lee, Jae Young [Department of Radiology, Seoul National University Hospital, Seoul 03080 (Korea, Republic of); Kim, Hae Ri [Department of Pre-Dentistry, Gangneung-Wonju National University College of Dentistry, Gangneung 25457 (Korea, Republic of); Kim, Bo Ram; Park, Eun-Joo; Kim, Hoe Suk; Han, Joon Koo [Department of Radiology, Seoul National University Hospital, Seoul 03080 (Korea, Republic of); Choi, Byung Ihn [Department of Radiology, Chung-Ang University Hospital, Seoul 06973 (Korea, Republic of)

    2016-11-01

    To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy. A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice. The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences. High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.

  16. Leaf-shape remodeling: programmed cell death in fistular leaves of Allium fistulosum.

    Science.gov (United States)

    Ni, Xi-Lu; Su, Hui; Zhou, Ya-fu; Wang, Feng-Hua; Liu, Wen-Zhe

    2015-03-01

    Some species of Allium in Liliaceae have fistular leaves. The fistular lamina of Allium fistulosum undergoes a process from solid to hollow during development. The aims were to reveal the process of fistular leaf formation involved in programmed cell death (PCD) and to compare the cytological events in the execution of cell death to those in the unusual leaf perforations or plant aerenchyma formation. In this study, light and transmission electron microscopy were used to characterize the development of fistular leaves and cytological events. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays and gel electrophoresis were used to determine nuclear DNA cleavage during the PCD. The cavity arises in the leaf blade by degradation of specialized cells, the designated pre-cavity cells, in the center of the leaves. Nuclei of cells within the pre-cavity site become TUNEL-positive, indicating that DNA cleavage is an early event. Gel electrophoresis revealed that DNA internucleosomal cleavage occurred resulting in a characteristic DNA ladder. Ultrastructural analysis of cells at the different stages showed disrupted vacuoles, misshapen nuclei with condensed chromatin, degraded cytoplasm and organelles and emergence of secondary vacuoles. The cell walls degraded last, and residue of degraded cell walls aggregated together. These results revealed that PCD plays a critical role in the development of A. fistulosum fistular leaves. The continuous cavity in A. fistulosum leaves resemble the aerenchyma in the pith of some gramineous plants to improve gas exchange.

  17. Heme oxygenase-1 inhibits neuropathic pain in rats with diabetic mellitus

    Institute of Scientific and Technical Information of China (English)

    Qian Kong; Kang Liu; Lingxi Wu; Long Wang

    2012-01-01

    A diabetes mellitus model was established through single intraperitoneal injection of streptozotocin into rats.Seven days later,model rats were intraperitoneally administered zinc protoporphyrin,a heme oxygenase-1 inducer,and cobalt protoporphyrin,a heme oxygenase-1 inhibitor,once every two days,for 5 successive weeks.After administration,the paw withdrawal mechanical threshold of diabetic mellitus rats significantly decreased,the myelin sheath of the sciatic nerve thickened or showed vacuole defects,the number of spinal dorsal horn neurons reduced,some neurons degenerated and were necrotic,and heme oxygenase-1 was visible in the cytoplasm of spinal dorsal horn neurons.Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling demonstrated that the number of apoptotic neurons increased,which could be inhibited by cobalt protoporphyrin,however,zinc protoporphyrin led to an opposite effect.Our experimental findings indicate that heme oxygenase-1 attenuates neuropathic pain in diabetic mellitus rats through amelioration of peripheral neuropathy and inhibition of spinal dorsal horn neuron apoptosis.

  18. Hyperlipidemia intensifies cerulein-induced acute pancreatitis associated with activation of protein kinase C in rats

    Institute of Scientific and Technical Information of China (English)

    Ya-Jun Wang; Jia-Bang Sun; Fei Li; Shu-Wen Zhang

    2006-01-01

    AIM: To investigate the effects of hyperlipidemia on acute pancreatitis (AP) and the possible mechanisms.METHODS: Rat models of hyperlipidemia and AP were established by Triton WR1339 and cerulein respectively.Human albumin was used to treat AP complicated by hyperlipidemia. In each group, we compared the histological score, volume of ascites, ratio of pancreatic wet/dry weight, serum amylase (AMY) and pancreatic acinar cell apoptosis. The level of protein kinase C (PKC) membrane translocation in pancreatic tissue was detected by Western blot.RESULTS: In the hyperlipidemia model established by Triton WR1339, triglyceride (TG) increased remarkably and reached its peak 6 h after injection, and most rats developed mild acute pancreatitis. Histological score, volume of ascites, ratio of wet/dry weight and serum AMY in AP animals with hyperlipidemia were obviously higher than those in AP animals (P <0.05) and decreased after albumin therapy but not significantly (P > 0.05). Apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) increased in AP animals with hyperlipidemia and did not change distinctly after albumin therapy. PKC membrane translocation level increased in AP animals with hyperlipidemia and decreased remarkably after albumin therapy (P < 0.05).CONCLUSION: Hyperlipidemia may induce AP or intensify pancreatic injury. Albumin therapy can not alleviate pancreatic lesion effectively. PKC activation may be one mechanism by which AP is intensified by hyperlipidemia.

  19. Chlorella vulgaris triggers apoptosis in hepatocarcinogenesis-induced rats

    Institute of Scientific and Technical Information of China (English)

    Emey Suhana MOHD AZAMAI; Suhaniza SULAIMAN; Shafina Hanim MOHD HABIB; Mee Lee LOOI; Srijit DAS; Nor Aini ABDUL HAMID; Wan Zurinah WANG NGAH; Yasmin Anum MOHD YUSOF

    2009-01-01

    Chlorella vulgaris (CV) has been reported to have antioxidant and anticancer properties. We evaluated the effect of CV on apoptotic regulator protein expression in liver cancer-induced rats. Male Wistar rats (200-250 g) were divided into eight groups: control group (normal diet), CDE group (choline deficient diet supplemented with ethionine in drinking water to induce hepatocarcinogenesis), CV groups with three different doses of CV (50, 150, and 300 mg/kg body weight), and CDE groups treated with different doses of CV (50, 150, and 300 mg/kg body weight). Rats were sacrificed at various weeks and liver tissues were embedded in paraffin blocks for immunohistochemistry studies. CV, at increasing doses, decreased the expression of anti-apoptotic protein, Bcl-2, but increased the expression of pro-apoptotic protein, caspase 8, in CDE rats, which was correlated with decreased hepatoctyes proliferation and increased apoptosis as determined by bromodeoxy-uridine (BrdU) labeling and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay, respectively. Our study shows that CV has definite chemopreventive effect by inducing apoptosis via decreasing the expression of Bcl-2 and increasing the expression of caspase 8 in hepatocarcinogenesis-induced rats.

  20. Ocular Lesions in Red-Tailed Hawks ( Buteo jamaicensis) With Naturally Acquired West Nile Disease.

    Science.gov (United States)

    Wünschmann, A; Armién, A G; Khatri, M; Martinez, L C; Willette, M; Glaser, A; Alvarez, J; Redig, P

    2017-03-01

    Ocular lesions are common in red-tailed hawks with West Nile (WN) disease. These lesions consist of pectenitis, choroidal or retinal inflammation, or retinal necrosis, but detailed investigation of the ocular lesions is lacking. Postmortem examination of the eyes of 16 red-tailed hawks with naturally acquired WN disease and 3 red-tailed hawks without WN disease was performed using histopathology, immunohistochemistry for West Nile virus (WNV) antigen, glial fibrillary acid protein, cleaved caspase-3, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Retinal lesions were classified as type I or type II lesions. Type I lesions were characterized by lymphoplasmacytic infiltrates in the subjacent choroid with degeneration limited to the outer retina (type Ia lesion) or with degeneration and necrosis of the outer retina or outer and inner retina (type Ib lesion) while retinal collapse, atrophy, and scarring were hallmarks of type II lesions. Type II retinal lesions were associated with a more pronounced choroiditis. Although not statistically significant, WNV antigen tended to be present in larger quantity in type Ib lesions. Type I lesions are considered acute while type II lesions are chronic. The development of retinal lesions was associated with the presence of an inflammatory infiltrate in the choroid. A breakdown of the blood-retina barrier is suspected to be the main route of infection of the retina. Within the retina, virus appeared to spread via both neuronal and Müller cell processes.

  1. Cytotoxic Effects of 2-Bromopropane on Embryonic Development in Mouse Blastocysts

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    Wen-Hsiung Chan

    2010-02-01

    Full Text Available 2-Bromopropane (2-BP, an alternative to ozone-depleting solvents, is used as a cleaning solvent. Here, we examined the cytotoxic effects of 2-bromopropane (2-BP on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Mouse blastocysts were incubated in medium with or without 2-BP (2.5, 5 or 10 μM for 24 h. Cell proliferation and growth were investigated with dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL analysis, and implantation and post-implantation development of embryos were assessed using in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 5 or 10 μM 2-BP displayed significantly increased apoptosis, and decreased inner cell mass (ICM and trophectoderm (TE cell number. Additionally, the implantation success rates of 2-BP-pretreated blastocysts were lower than those of untreated controls. In vitro treatment with 5 or 10 μM 2-BP was associated with increased resorption of postimplantation embryos, and decreased placental and fetal weights. Our results collectively indicate that in vitro exposure to 2-BP induces apoptosis, suppresses implantation rates after transfer to host mice, and retards early postimplantation development.

  2. Adequate ovarian follicular status does not prevent the decrease in pregnancy rates associated with high sperm DNA fragmentation.

    Science.gov (United States)

    Frydman, Nelly; Prisant, Nadia; Hesters, Laetitia; Frydman, René; Tachdjian, Gérard; Cohen-Bacrie, Paul; Fanchin, Rénato

    2008-01-01

    Potential reparation of sperm DNA fragmentation in the oocyte may disturb any relationship between DNA-damaged sperm and the implantation ability of resulting embryos. To rule out this factor, we analyzed the consequences of sperm DNA fragmentation on IVF-ET outcome in women with healthy ovarian function. Prospective study. Teaching hospital, France. All 117 women were <38 years old, who combined normal serum day-3 FSH and inhibin B levels with an adequate response to controlled ovarian hyperstimulation. The DNA fragmentation rate was determined in the raw sperm used for conventional IVF by flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Cycles were sorted into two groups according to whether DNA fragmentation exceeded (high fragmentation [HF], n = 52) or did not exceed (low fragmentation [LF], n = 65) the 50th percentile of values (35%). D2 embryo quality and implantation and ongoing pregnancy rates. Patients' characteristics, raw semen parameters, fertilization rates, and embryology data were similar in HF and LF groups. Clinical (37.5% vs. 62.5%) and ongoing (23.5% vs. 57.8%) pregnancy rates per ET and implantation rates (24.5% vs. 42.4%) were lower in the HF group than in the LF group. High sperm DNA fragmentation spares fertilization and top embryo morphology rates but is associated with decreased IVF-ET outcome.

  3. Arsenic trioxide treatment of rabbit liver VX-2 carcinoma via hepatic arterial cannulation-induced apoptosis and decreased levels of survivin in the tumor tissue.

    Science.gov (United States)

    Li, Hong; Gong, Jian; Jiang, Xuyuan; Shao, Haibo

    2013-02-01

    To investigate the role of tumor apoptosis-inhibitory protein survivin in arsenic trioxide-induced apoptosis in VX-2 carcinoma in the rabbit liver by means of transcatheter arterial chemoembolization. Sixteen rabbits with 32 implanted hepatic VX-2 tumors were randomly divided into two groups. The experimental group received 2 mg of arsenic trioxide and 1 mL of ultra-fluid lipiodol co-injected via hepatic arterial cannulation and the control group received only 1 mL of lipiodol. Animals were sacrificed 3 weeks after trans-catheterial arterial chemoembolization. Tumor tissue and tumor-peripheral tissue were collected for analysis. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling staining was used to assess tumor cells apoptosis. Immunohistochemistry was used to assess the presence of survivin protein. Reverse transcription polymerase chain reaction was used to determine the expression of survivin gene. The number of apoptotic cells significantly increased in the tumor tissue (5.20 ± 0.60%) compared to tumor-peripheral tissue (1.29 ± 0.42%) of the arsenic trioxide-treated group. Survivin expression levels in the tumor tissue were significantly reduced in arsenic trioxide-treated group (7.68 ± 0.65) compared to the control group (35.30 ± 4.63). Transcatheter arterial chemoembolization with arsenic trioxide induced apoptosis of VX-2 carcinoma, in which tumor apoptosis-inhibitory protein survivin may have played a role.

  4. Occurrence of complement protein C3 in dying pyramidal neurons in rat hippocampus after systemic administration of kainic acid.

    Science.gov (United States)

    Morita, Hiroyuki; Suzuki, Katsuaki; Mori, Norio; Yasuhara, Osamu

    2006-11-27

    To evaluate the roles of complement in kainic acid (KA)-induced neuronal damages, the immunohistochemical localization of the complement protein C3 was examined in rat hippocampus after systemic KA injection. The immunoreactivity for C3 was found in glial cells in control rats, and such glial cells were increased in number after KA injection. Our confocal study showed that C3-positive glial cells were microglia. Three to seven days after KA, C3 immunoreactivity appeared in CA1 and CA3 pyramidal neurons. Double staining for C3 combined with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling showed that occurrence of C3 immunoreactivity in neurons coincided well with that of DNA fragmentation. Western blot analysis and RT-PCR experiments suggested local synthesis of C3 by brain cells. Our results suggest that C3 contributes greatly to neuronal death after systemic KA administration, and that microglia and neurons are the local source of C3 in KA-induced brain injury.

  5. Korean Red Ginseng water extract inhibits COX-2 expression by suppressing p38 in acrolein-treated human endothelial cells

    Directory of Open Access Journals (Sweden)

    Seung Eun Lee

    2014-01-01

    Full Text Available Cigarette smoke is considered a major risk factor for vascular diseases. There are many toxic compounds in cigarette smoke, including acrolein and other α,β-unsaturated aldehydes, which are regarded as mediators of inflammation and vascular dysfunction. Furthermore, recent studies have revealed that acrolein, an α,β-unsaturated aldehyde in cigarette smoke, induces inflammatory mediator expression, which is known to be related to vascular diseases. In this study, we investigated whether Korean Red Ginseng (KRG water extract suppressed acrolein-induced cyclooxygenase (COX-2 expression in human umbilical vein endothelial cells (HUVECs. Acrolein-induced COX-2 expression was accompanied by increased levels of phosphorylated p38 in HUVECs and KRG inhibited COX-2 expression in HUVECs. These results suggest that KRG suppresses acrolein-induced COX-2 expression via inhibition of the p38 mitogen-activated protein kinase signaling pathway. In addition, KRG exhibited an inhibitory effect on acrolein-induced apoptosis, as demonstrated by annexin V–propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Consistent with these results, KRG may exert a vasculoprotective effect through inhibition of COX-2 expression in acrolein-stimulated human endothelial cells.

  6. A potential oral anticancer drug candidate, Moringa oleifera leaf extract, induces the apoptosis of human hepatocellular carcinoma cells.

    Science.gov (United States)

    Jung, Il Lae; Lee, Ju Hye; Kang, Se Chan

    2015-09-01

    It has previously been reported that cold water-extracts of Moringa oleifera leaf have anticancer activity against various human cancer cell lines, including non-small cell lung cancer. In the present study, the anticancer activity of M. oleifera leaf extracts was investigated in human hepatocellular carcinoma HepG2 cells. By the analysis of apoptotic signals, including the induction of caspase or poly(ADP-ribose) polymerase cleavage, and the Annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, it was demonstrated that M. oleifera leaf extracts induce the apoptosis of HepG2 cells. In the hollow fiber assay, oral administration of the leaf extracts significantly reduced (44-52%) the proliferation of the HepG2 cells and A549 non-small cell lung cancer cells. These results support the potential of soluble extracts of M. oleifera leaf as orally administered therapeutics for the treatment of human liver and lung cancers.

  7. A histopathological study of premature and mature infants with pontosubicular neuron necrosis: neuronal cell death in perinatal brain damage.

    Science.gov (United States)

    Takizawa, Yuji; Takashima, Sachio; Itoh, Masayuki

    2006-06-20

    Perinatal hypoxic-ischemic brain damage is a major cause of neuronal and behavior deficits, in which the onset of injury can be before, at or after birth, and the effects may be delayed. Pontosubicular neuron necrosis (PSN) is one of perinatal hypoxic-ischemic brain injury and its pathological peculiarity is neuronal apoptosis. In this study, we investigated whether apoptotic cascade of PSN used a caspase-pathway or not, and whether hypoglycemia activated apoptosis or not. Sections of the pons of PSN with and without hypoglycemia were stained using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and immunohistochemistry for glial fibrillary acidic protein (GFAP), Bcl-2, Bcl-x and activated caspase 3. Additionally, we performed immunoblot analysis of Bcl-2, Bcl-x and activated caspase 3. TUNEL-positive cell was closely associated with the presence of karyorrhexis. Under combination of karyorrhectic and TUNEL-positive cells, number of apoptotic cells in premature brains was significantly more than in mature brains. Hypoxic-ischemic brain injury was considered to easily lead to apoptosis in premature infants. Moreover, as this pathophysiology, caspase-pathway activation contributed to neuronal death from caspase-immunoexpression analyses. PSN with hypoglycemia showed large number of apoptotic cells and higher expression of activated caspase 3. The result may be more severe with the background of hypoglycemia and prematurity complicated by hypoxia and/or ischemia.

  8. Apoptosis of transgenic cloned and recloned bovine blastocysts

    Institute of Scientific and Technical Information of China (English)

    Guojie Sun; Rong Li; Yunping Dai; Haiping Wang; Lili Wang; Ying Liu; Fangrong Ding; Hengxi Wei; Ning Li

    2009-01-01

    Apoptosis plays an important role in preimplantation embryonic development. Investigating mechanisms of apoptosis can provide useful information for obtaining high-quality embryos and help to improve cloning efficiency. Here, we investigated the incidence of blastomere apoptosis in transgenic blastocysts generated by somatic cell nuclear transfer (SCNT) and recloning using a terminal deoxy-nucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. Transgenic recloned embryos were the second generation SCNT embryos derived from the somatic cells of a transgenic SCNT calf. The blastocyst rate of transgenic SCNT embryos was lower than that of nontransgenic SCNT embryos. The incidence of apoptosis in transgenic SCNT embryos was higher than that of nontrans-genie SCNT embryos. The blastocyst rate and the incidence of apoptosis in transgenic recloned embryos were similar to nontransgenic SCNT embryos. The process of donor cell transfection and drug selection may decrease the developmental capacity of transgenic SCNT embryos. Serial cloning did not influence the developmental capacity of transgenic recloned embryos.

  9. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chen-Ming [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Wang, Shih-Wei [Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (China); Lee, Tzong-Huei [Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan (China); Tzeng, Wen-Pei [Graduate Institute of Sports and Health, National Changhua University of Education, Changhua, Taiwan (China); Hsiao, Che-Jen [School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Liu, Shih-Chia [Department of Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan (China); Tang, Chih-Hsin, E-mail: chtang@mail.cmu.edu.tw [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)

    2013-10-15

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  10. dNTP deficiency induced by HU via inhibiting ribonucleotide reductase affects neural tube development.

    Science.gov (United States)

    Guan, Zhen; Wang, Xiuwei; Dong, Yanting; Xu, Lin; Zhu, Zhiqiang; Wang, Jianhua; Zhang, Ting; Niu, Bo

    2015-02-03

    Exposure to environmental toxic chemicals in utero during the neural tube development period can cause developmental disorders. To evaluate the disruption of neural tube development programming, the murine neural tube defects (NTDs) model was induced by interrupting folate metabolism using methotrexate in our previous study. The present study aimed to examine the effects of dNTP deficiency induced by hydroxyurea (HU), a specific ribonucleotide reductase (RNR) inhibitor, during murine neural tube development. Pregnant C57BL/6J mice were intraperitoneally injected with various doses of HU on gestation day (GD) 7.5, and the embryos were checked on GD 11.5. RNR activity and deoxynucleoside triphosphate (dNTP) levels were measured in the optimal dose. Additionally, DNA damage was examined by comet analysis and terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) assay. Cellular behaviors in NTDs embryos were evaluated with phosphorylation of histone H3 (PH-3) and caspase-3 using immunohistochemistry and western blot analysis. The results showed that NTDs were observed mostly with HU treatment at an optimal dose of 225 mg/kg b/w. RNR activity was inhibited and dNTP levels were decreased in HU-treated embryos with NTDs. Additionally, increased DNA damage, decreased proliferation, and increased caspase-3 were significant in NTDs embryos compared to the controls. Results indicated that HU induced murine NTDs model by disturbing dNTP metabolism and further led to the abnormal cell balance between proliferation and apoptosis.

  11. Effects of cinnamon (Cinnamomum zeylanicum) bark oil on testicular antioxidant values, apoptotic germ cell and sperm quality.

    Science.gov (United States)

    Yüce, A; Türk, G; Çeribaşi, S; Sönmez, M; Çiftçi, M; Güvenç, M

    2013-08-01

    Cinnamon and its contents have multifactorial properties such as antioxidant, anti-inflammatory and antidiabetic. Male infertility is one of the major health problems in life. The aim of this study was to investigate the effects of long-term cinnamon bark oil (CBO) ingestion on testicular antioxidant values, apoptotic germ cell and sperm quality of adult rats. Twelve male healthy Wistar rats were divided into two groups, each group containing six rats. While olive oil was given to control group, 100 mg kg(-1)  CBO was administered to the other group by gavage daily for 10 weeks. Body and reproductive organ weights, sperm characteristics, testicular lipid peroxidation and antioxidant enzyme activities, and testicular apoptosis via terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method were examined. A significant decrease in malondialdehyde level and marked increases in reduced glutathione level, glutathione peroxidase and catalase activities were observed in rats treated with CBO compared with the control group. CBO consumption provided a significant increase in weights of testes and epididymides, epididymal sperm concentration, sperm motility and diameter of seminiferous tubules when compared with the control group. However, CBO consumption tended to decrease the abnormal sperm rate and apoptotic germ cell count, but it did not reach statistical significance. It is concluded that CBO has improvement effect on testicular oxidant-antioxidant balance and sperm quality, and its consumption may be useful for asthenozoospermic men.

  12. Time-course and molecular mechanism of hepatotoxicity induced by 1,3-dichloro-2-propanol in rats.

    Science.gov (United States)

    Lee, In-Chul; Ko, Je-Won; Lee, Sang-Min; Kim, Sung-Hwan; Shin, In-Sik; Moon, Og-Sung; Yoon, Won-Kee; Kim, Hyoung-Chin; Kim, Jong-Choon

    2015-07-01

    This study investigated the time-course of 1,3-dichloro-2-propanol (1,3-DCP)-induced hepatotoxicity and the molecular mechanism of its oxidative stress and apoptotic changes in rats. Thirty-six male rats were randomly assigned to six groups of six rats each and were administered a single oral dose of 1,3-DCP (90 mg/kg) or its vehicle. 1,3-DCP caused acute hepatic damage, as evidenced by marked increases in serum aminotransferase, alkaline phosphatase, and histopathological alterations. These functional and histopathological changes in the liver peaked at 12h after administration and then decreased progressively. Oxidative stress indices were increased significantly at 6h, peaked at 12h, and then decreased progressively. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)- and caspase-3-positive cells increased after 6h, peaked at 12 and 24h, and then decreased. The protein levels of phosphorylated mitogen-activated protein kinases (MAPKs) including p-Erk1/2 and p-JNK showed a similar trend to the numbers of TUNEL- and caspase-3-positive cells. These results indicate that 1,3-DCP increases oxidative stress, nuclear translocation of Nrf2, and expression of Nrf2-targeted genes, followed by increased functional and histopathological alterations in the liver. The increase in hepatocellular apoptosis induced by 1,3-DCP may be related to oxidative stress-mediated MAPK activation.

  13. The effects of erdosteine, N-acetylcysteine, and vitamin E on nicotine-induced apoptosis of hippocampal neural cells.

    Science.gov (United States)

    Demiralay, Rezan; Gürsan, Nesrin; Erdem, Havva

    2008-08-01

    This study investigated the frequency of apoptosis in rat hippocampal neural cells after intraperitoneal nicotine injection, examining the roles of the inflammatory markers myeloperoxidase (MPO) and tumor necrosis factor alpha (TNF-alpha) in nicotine-induced brain damage and the protective effects of three known antioxidant agents, N-acetylcysteine (NAC), erdosteine, and vitamin E. Female Wistar rats were divided into seven groups, each composed of nine rats: 2 negative control groups, 2 positive control groups, one erdosteine-treated group (500 mg/kg), one NAC-treated group (500 mg/kg), and one vitamin E-treated group (500 mg/kg). Nicotine was intraperitoneally injected at a dosage of 0.6 mg/kg for 21 days. Following nicotine injection, the antioxidants were administered orally; treatment was continued until the rats were killed. Apoptosis level in hippocampal neural cells was determined by using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling) method. Staining of cytoplasmic TNF-alpha in hippocampal neural cells and hippocampus MPO activity were evaluated by immunohistochemistry. Nicotine administration had no effect on local TNF-alpha production, or hippocampal MPO activity. The treatments with erdosteine, NAC and vitamin E significantly reduced the rate of nicotine-induced hippocampal neural cell apoptosis. This findings suggest that erdosteine and NAC can be as effective as vitamin E in protecting against nicotine-induced hippocampal neural cell apoptosis.

  14. Study on Effect of IH764-3, an Active Principle of Salviae miltiorrhizae, in Inducing Hepatic Stellate Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    赵东强; 姜慧卿; 修贺明; 张晓岚

    2002-01-01

    Objective: To explore the anti-fibrotic mechanism of Salviae miltiorrhizae from the view of proliferation and apoptosis of hepatic stellate cells (HSC).Methods: IH764-3, an active principle of Salviae miltiorrhizae, was used to intervene in the cultured HSC in vitro. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) method, and the cell apoptosis was examined by electron microscopy, flow cytometer and terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling method (TUNEL).Results: MTT showed that IH764-3 has obvious inhibition on the proliferation of HSC. Specific cell apoptosis figures of HSC, such as chromatin agglutination, were seen under electron microscopy in the IH764-3 treated group. By flow cytometer, it was shown that the HSC apoptosis rate in the IH764-3 treated group was higher than that in the control group, and the apoptosis inducing effect of IH764-3 was dose- and time-dependent. TUNEL analysis showed that the HSC apoptotsis rate was 28.3±1.5% after being incubated for 48 hrs with IH764-3, which was significantly higher than that in the control group (6.7±0.6%, P<0.05).Conclusion: IH764-3 could inhibit the proliferation of HSC and induce its apoptosis. These effects may be one of the anti-fibrotic mechanisms of Salviae miltiorrhizae.

  15. Study on cognition disorder and morphologic change of neurons in hippocampus area following traumatic brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    洪军; 崔建忠; 周云涛; 高俊玲

    2002-01-01

    Objective: To explore the correlation between cognition disorder and morphologic change of hippocampal neurons after traumatic brain injury (TBI).   Methods: Wistar rat models with severe TBI were made by Marmarous method. The histopathological change of the neurons in the hippocampus area were studied with hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated X-dUPT nick end labeling (TUNEL), respectively. The cognitive function was evaluated with the Morris water maze test.   Results: The comprehensive neuronal degeneration and necrosis could be observed in CA2-3 regions of hippocampus at 3 days after injury. Apoptotic positive neurons in CA2-4 regions of hippocampus and dentate gyrus increased in the injured group at 24 hours following TBI. They peaked at 7 days and then declined. Significant impairment of spatial learning and memory was observed after injury in the rats.   Conclusions: The rats have obvious disorders in spatial learning and memory after severe TBI. Meanwhile, delayed neuronal necrosis and apoptosis can be observed in the neurons in the hippocampus area. It suggests that delayed hippocampal cell death may contribute to the functional deficit.

  16. The Relationship between Apoptosis and the Expression of Proliferating Cell Nuclear Antigen and the Clinical Stages in Gastric Carcinoma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5±3.7) % and (49.8±15.9) % respectively, and the rate of AI/PI was 0.13±0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer (P<0.01). With the advanced TNM stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage Ⅱ to Ⅳ (P<0.05). It was suggested that the decreased apoptotic cells and the increased proliferating cells were obviously related to the tumor genesis and tumor progression in gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.

  17. Cisplatin cytotoxicity of auditory cells requires secretions of proinflammatory cytokines via activation of ERK and NF-kappaB.

    Science.gov (United States)

    So, Hongseob; Kim, HyungJin; Lee, Jeong-Han; Park, Channy; Kim, Yunha; Kim, Eunsook; Kim, Jin-Kyung; Yun, Ki-Jung; Lee, Kang-Min; Lee, Haa-Yung; Moon, Sung-Kyun; Lim, David J; Park, Raekil

    2007-09-01

    The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, through the activation of ERK and NF-kappaB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-alpha. Immunohistochemical studies revealed that TNF-alpha expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-kappaB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-alpha, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

  18. Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica.

    Science.gov (United States)

    Carrero, Julio C; Cervantes, Claudia; Moreno-Mendoza, Norma; Saavedra, Emma; Morales-Montor, Jorge; Laclette, Juan P

    2006-02-01

    In vitro exposure of Entamoeba histolytica trophozoites to the sex steroids 17beta-estradiol, progesterone, and dehydrotestosterone had little effect on parasite viability or proliferation. However, treatment with the adrenal steroid dehydroepiandrosterone (DHEA) markedly inhibited parasite proliferation, adherence and motility, and at a certain dose it induced trophozoite lysis. The opposite effect on proliferation was found when the trophozoites were exposed to cortisol. Moreover, DHEA decreased while cortisol increased the parasite's DNA synthesis determined by 3H-thymidine incorporation. Trophozoite lysis by DHEA appeared to be caused by a necrotic rather than an apoptotic process, as observed in propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assays. A possible mechanisms of action was derived from experiments demonstrating that the activity of a putative 3-hydroxy-3-methyl glutaryl CoA reductase detected in trophozoite extracts was inhibited in the presence of DHEA. Contrary to its in vitro inhibitory effect, in vivo administration of DHEA to infected hamsters resulted in exacerbation of the amebic liver abscesses. These results demonstrated that androgen steroids act directly upon E. histolytica growth and viability, and may shed new light on some age and gender differences in disease progression, as well as finding application in the drug treatment of human amebiasis.

  19. Polymeric micelles encapsulating fisetin improve the therapeutic effect in colon cancer.

    Science.gov (United States)

    Chen, Yishan; Wu, Qinjie; Song, Linjiang; He, Tao; Li, Yuchen; Li, Ling; Su, Weijun; Liu, Lei; Qian, Zhiyong; Gong, Changyang

    2015-01-14

    The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) was discovered to possess antitumor activity, revealing its potential value in future chemotherapy. However, its poor water solubility makes it difficult for intravenous administration. In this study, the monomethyl poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) copolymer was applied to prepare nanoassemblies of fisetin by a self-assembly procedure. The prepared fisetin micelles gained a mean particle size of 22 ± 3 nm, polydisperse index of 0.163 ± 0.032, drug loading of 9.88 ± 0.14%, and encapsulation efficiency of 98.53 ± 0.02%. Compared with free fisetin, fisetin micelles demonstrated a sustained and prolonged in vitro release behavior, as well as enhanced cytotoxicity, cellular uptake, and fisetin-induced apoptosis in CT26 cells. As for in vivo studies, fisetin micelles were more competent for suppressing tumor growth and prolonging survival time than free fisetin in the subcutaneous CT26 tumor model. Furthermore, histological analysis, terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, immunohistochemical detection of Ki-67, and microvessel density detection were conducted, demonstrating that fisetin micelles gained increased tumor apoptosis induction, proliferation suppression, and antiangiogenesis activities. In conclusion, we have successfully produced a MPEG-PCL-based nanocarrier encapsulating fisetin with enhanced antitumor activity.

  20. Abnormal meiotic recombination with complex chromosomal rearrangement in an azoospermic man.

    Science.gov (United States)

    Wang, Liu; Iqbal, Furhan; Li, Guangyuan; Jiang, Xiaohua; Bukhari, Ihtisham; Jiang, Hanwei; Yang, Qingling; Zhong, Liangwen; Zhang, Yuanwei; Hua, Juan; Cooke, Howard J; Shi, Qinghua

    2015-06-01

    Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis and recombination in an azoospermic reciprocal translocation 46, XY, t(5;7;9;13)(5q11;7p11;7p15;9q12;13p12) carrier. Histological examination of the haematoxylin and eosin stained testicular sections revealed reduced germ cells with no spermatids or sperm in the patient. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay showed apoptotic cells in testicular sections of translocation carrier. Immnunofluorescence analysis indicated the presence of an octavalent in all the pachytene spermatocytes analysed in the patient. Meiotic progression was disturbed, as an increase in zygotene (P recombination frequency was observed on 5p, 5q, 7q, 9p and 13q in the translocation carrier compared with the reported controls. A significant reduction in XY recombination frequency was also found in the participants. Our results indicated that complex chromosomal rearrangements can impair synaptic integrity of translocated chromosomes, which may reduce chromosomal recombination on translocated as well as non-translocated chromosomes, a phenomenon commonly known as interchromosomal effect.

  1. Brief left ventricular pressure overload reduces myocardial apoptosis.

    Science.gov (United States)

    Huang, Hsien-Hao; Lai, Chang-Chi; Chiang, Shu-Chiung; Chang, Shi-Chuan; Chang, Chung-Ho; Lin, Jin-Ching; Huang, Cheng-Hsiung

    2015-03-01

    Both apoptosis and necrosis contribute to cell death after myocardial ischemia and reperfusion. We previously reported that brief left ventricular pressure overload (LVPO) decreased myocardial infarct (MI) size. In this study, we investigated whether brief pressure overload reduces apoptosis and the mechanisms involved. MI was induced by a 40-min occlusion of the left anterior descending coronary artery and 3-h reperfusion in male anesthetized Sprague-Dawley rats. Brief LVPO was achieved by two 10-min partial snarings of the ascending aorta, raising the systolic left ventricular pressure 50% above the baseline value. Ischemic preconditioning was elicited by two 10-min coronary artery occlusions and 10-min reperfusions. Brief LVPO and ischemic preconditioning significantly decreased MI size (P Brief pressure overload significantly reduced myocardial apoptosis, as evidenced by the decrease in the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei (P brief pressure overload significantly increased Bcl-2 (P brief pressure overload (P Brief left LVPO significantly reduces myocardial apoptosis. The underlying mechanisms might be related to modulation of Bcl-2 and Bax, inhibition of p53, increased Akt phosphorylation, and suppressed c-Jun N-terminal kinase phosphorylation. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Fenugreek, a naturally occurring edible spice, kills MCF-7 human breast cancer cells via an apoptotic pathway.

    Science.gov (United States)

    Khoja, Kholoud K; Shaf, Gowhar; Hasan, Tarique N; Syed, Naveed Ahmed; Al-Khalifa, Abdrohman S; Al-Assaf, Abdullah H; Alshatwi, Ali A

    2011-01-01

    There is growing use of anticancer complementary and alternative medicines worldwide. Trigonella foenum graecum (Fenugreek) is traditionally applied to treat disorders such as diabetes, high cholesterol, wounds, inflammation, and gastrointestinal ailments. Fenugreek is also reported to have anticancer properties due to its active beneficial chemical constituents. The mechanism of action of several anticancer drugs is based on their ability to induce apoptosis. The objective of the study was to characterize the downstream apoptotic genes targeted by FCE in MCF-7 human immortalized breast cells. FCE effectively killed MCF-7 cells through induction of apoptosis,confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and RT-PCR assays. When cells were exposed to 50 μg/mL FCE for 24 hours, 23.2% apoptotic cells resulted, while a 48-hour exposure to 50 μg/mL caused 73.8% apoptosis. This was associated with increased expression of Caspase 3, 8, 9, p53, Fas, FADD, Bax and Bak in a time-and dose-dependent manner, as determined by real- time quantitative PCR. In summary, the induction of apoptosis by FCE is effected by its ability to increase the expression of pro-apoptotic genes and the spice holds promise for consideration in complementary therapy for breast cancer patients.

  3. Experimental Study on the Expression of HIF-1α and Its Relationship to Apoptosis in Tissues around Cerebral Bleeding Loci

    Institute of Scientific and Technical Information of China (English)

    朱遂强; 唐洲平; 郭守刚; 彭岚; 方思羽; 张苏明

    2004-01-01

    The expression of hypoxia inducible factor-1 alpha (HIF-1α) and its relationship to apoptosis in tissues around cerebral bleeding loci was studied. The expression of HIF-1α and apoptosis in 37 samples of tissues around cerebral bleeding loci and 9 samples of normal cerebral tissues was assessed by immunohistochemical straining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling methods. In 37 tissue samples around cerebral bleeding loci, the positive rate of the HIF-1α expression was 40.6 %. Especially in the patients with amount of bleeding>60 ml, the positive rate (88.9 %) of the HIF-1α expression was significantly higher than those with the amount of bleeding ranging from 30-45 ml or 45-60 ml (P<0.05). The expression of HIF-1α was increased as the amount of bleeding and operative time increased (P<0.05). There existed a positive correlation between HIF-1α labeling index and apoptosis index (n= 12, r= 0.56, P<0.01). These results suggested that the expression of HIF-1α was closely related with the time of hemorrhage and the amount of bleeding, and could induce the apoptosis of neurons.

  4. Comparative studies of mitochondrial proteomics reveal an intimate protein network of male sterility in wheat (Triticum aestivum L.)

    Science.gov (United States)

    Wang, Shuping; Zhang, Gaisheng; Zhang, Yingxin; Song, Qilu; Chen, Zheng; Wang, Junsheng; Guo, Jialin; Niu, Na; Wang, Junwei; Ma, Shoucai

    2015-01-01

    Plant male sterility has often been associated with mitochondrial dysfunction; however, the mechanism in wheat (Triticum aestivum L.) has not been elucidated. This study set out to probe the mechanism of physiological male sterility (PHYMS) induced by the chemical hybridizing agent (CHA)-SQ-1, and cytoplasmic male sterility (CMS) of wheat at the proteomic level. A total of 71 differentially expressed mitochondrial proteins were found to be involved in pollen abortion and further identified by MALDI-TOF/TOF MS (matrix-assisted laser desorption/ionization-time of fight/time of flight mass spectrometry). These proteins were implicated in different cellular responses and metabolic processes, with obvious functional tendencies toward the tricarboxylic acid cycle, the mitochondrial electron transport chain, protein synthesis and degradation, oxidation stress, the cell division cycle, and epigenetics. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways involved in anther abortion and pollen defects. Accordingly, a mitochondria-mediated male sterility protein network in wheat is proposed; this network was further confirmed by physiological data, RT-PCR (real-time PCR), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. The results provide intriguing insights into the metabolic pathway of anther abortion induced by CHA-SQ-1 and also give useful clues to identify the crucial proteins of PHYMS and CMS in wheat. PMID:26136264

  5. DNA fragmentation of spermatozoa and assisted reproduction technology.

    Science.gov (United States)

    Henkel, Ralf; Kierspel, Eva; Hajimohammad, Marjam; Stalf, Thomas; Hoogendijk, Christiaan; Mehnert, Claas; Menkveld, Roelof; Schill, Wolf-Bernhard; Kruger, Thinus F

    2003-01-01

    Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.

  6. Cardioprotective Effect of Aloe vera Biomacromolecules Conjugated with Selenium Trace Element on Myocardial Ischemia-Reperfusion Injury in Rats.

    Science.gov (United States)

    Yang, Yang; Yang, Ming; Ai, Fen; Huang, Congxin

    2017-06-01

    The present study was undertaken to evaluate the cardioprotection potential and underlying molecular mechanism afforded by a selenium (Se) polysaccharide (Se-AVP) from Aloe vera in the ischemia-reperfusion (I/R) model of rats in vivo. Myocardial I/R injury was induced by occluding the left anterior descending coronary artery (LAD) for 30 min followed by 2-h continuous reperfusion. Pretreatment with Se-AVP (100, 200, and 400 mg/kg) attenuated myocardial damage, as evidenced by reduction of the infarct sizes, increase in serum and myocardial endogenous antioxidants (superoxide dismutase (SOD), glutathione peroxidase (GSH), and catalase (CAT)), and decrease in the malondialdehyde (MDA) level in the rats suffering I/R injury. This cardioprotective activity afforded by Se-AVP is further supported by the decreased levels of cardiac marker enzymes creatine kinase (CK) and lactate dehydrogenase (LDH), as well as the rise of myocardial Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in I/R rats. Additionally, cardiomyocytic apoptosis was measured by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining and the result showed that the percent of TUNEL-positive cells in myocardium of Se-AVP-treated groups was lower than I/R rats. In conclusion, we clearly demonstrated that Se-AVP had a protective effect against myocardial I/R injury in rats by augmenting endogenous antioxidants and protecting rat hearts from oxidative stress-induced myocardial apoptosis.

  7. Apoptosis in Granulosa cells during follicular atresia:relationship with steroids and insulin-like growth factors

    Institute of Scientific and Technical Information of China (English)

    Yuan Song YU; Hong Shu SUI; Zheng Bin HAN; Wei LI; Ming Jiu LUO; Jing He TAN

    2004-01-01

    It is well known that during mammalian ovarian follicular development, the majority of follicles undergo atresia at various stages of their development. However, the mechanisms controlling this selection process remain unknown. In this study, we investigated apoptosis in granulosa cells during goat follicular atresia by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The changes in the levels of steroids, insulin-like growth factors (IGFs) and IGF receptors were studied by radioimmunoassay (RIA) and semi-quantitative reverse transcription-PCR. We found that the percentage of apoptotic granulosa cells in the atretic (A) follicles was significantly higher than that in the slightly atretic (SA) and healthy (H) follicles. The level of estradiol and the ratio of estradiol to progesterone in H follicles were significantly higher than those in A follicles. On the other hand, the level of progesterone was not significantly different among these follicle types. We also found that the level of IGF-I in H follicles was higher than in SA and A follicles, whereas the amount of IGF-Ⅱ did not vary significantly. The expression of IGF receptor also decreased in A follicles as compared to that in H and SA follicles. These results suggested that estradiol and IGF-I might be involved in controlling apoptosis in granulosa cells during follicular atresia.

  8. Alterations in the expression of atrial calpains in electrical and structural remodeling during aging and atrial fibrillation.

    Science.gov (United States)

    Xu, Guo-Jun; Gan, Tian-Yi; Tang, Bao-Peng; Chen, Zu-Heng; Mahemuti, Ailiman; Jiang, Tao; Song, Jian-Guo; Guo, Xia; Li, Yao-Dong; Zhou, Xian-Hui; Zhang, Yu; Li, Jin-Xin

    2013-11-01

    The aim of this study was to investigate the correlation between the change in the expression of atrial calpains and electrical, molecular and structural remodeling during aging and atrial fibrillation (AF). Adult and aged canines in sinus rhythm (SR) and with persistent AF (induced by rapid atrial pacing) were investigated. A whole-cell patch clamp was used to measure the L-type Ca2+ current (ICa-L) in cells in the left atrium. The mRNA and protein expression of the L-type calcium channel alc subunit (LVDCCa1c) and calpains were measured by quantitative (q)PCR and western blot analysis. Histopathological and ultrastructural changes were analyzed via light and electron microscopy. The quantity of apoptotic myocytes was determined by a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay. In SR groups, atrial cells of the aged canines exhibited a longer action potential (AP) duration to 90% repolarization (APD90), lower AP plateau potential and peak ICa-L current densities (Pcalpain 1 was increased in the adult and the aged groups with AF (Pcalpain 1. The general pathophysiological alterations in normal aged atria may therefore produce a substrate that is conducive to AF.

  9. Therapeutic effects of microbubble added to combined high-intensity focused ultrasound and chemotherapy in a pancreatic cancer xenograft model

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mi Hye [Dept. of Radiology, Konkuk University Medical Center, Seoul (Korea, Republic of); Lee, Jae Young; Kim, Bo Ram; Park, Eun Joo; Kim, Hoe Suk; Han, Joon Koo [Dept. of Radiology, Seoul National University Hospital, Seoul (Korea, Republic of); Kim, Hae Ri [Dept. of Pre-Dentistry, Gangneung-Wonju National University College of Dentistry, Gangneung (Korea, Republic of); Choi, Byung Ihn [Dept. of Radiology, Chung-Ang University Hospital, Seoul (Korea, Republic of)

    2016-09-15

    To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy. A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice. The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences. High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.

  10. Myofibrillogenesis regulator-1 attenuated hypoxia/reoxygenation-induced apoptosis by inhibiting the PERK/Nrf2 pathway in neonatal rat cardiomyocytes.

    Science.gov (United States)

    Tao, Tian-Qi; Wang, Xiao-Reng; Liu, Mi; Xu, Fei-Fei; Liu, Xiu-Hua

    2015-03-01

    The purpose of this study was to investigate the role of myofibrillogenesis regulator-1 (MR-1) in cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R), through protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. To address this aim, an H/R model of neonatal rat cardiomyocytes was used. MR-1 was overexpressed using an adenoviral vector system and knocked down using MR-1 specific siRNA. Apoptosis was assessed by using Annexin V/PI double staining, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay, and the Bcl-2/Bax ratio. Western blotting was used to detect the protein levels of MR-1, glucose-regulated protein 78 (GRP78), total and phosphorylated PERK, Nrf2, activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), Bcl-2 and Bax. Immunofluorescence staining was used to assess the subcellular location of Nrf2. We found that H/R induced significant apoptosis in neonatal rat cardiomyocytes. MR-1 overexpression attenuated H/R-induced apoptosis, decreased GRP78 (P apoptosis, increased expression of GRP78 and CHOP (P apoptosis through inhibition of the PERK/Nrf2 pathway.

  11. Effect of Wenxin Granule on Ventricular Remodeling and Myocardial Apoptosis in Rats with Myocardial Infarction

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    Aiming Wu

    2013-01-01

    Full Text Available Aim. To determine the effect of a Chinese herbal compound named Wenxin Granule on ventricular remodeling and myocardial apoptosis in rats with myocardial infarction (MI. Methods. Male Sprague-Dawley (SD rats were randomly divided into four groups: the control group, the model group, the metoprolol group, and the Wenxin Granule group (WXKL group with sample size (n of 7 rats in each group. An MI model was established in all rats by occlusion of the left anterior descending coronary artery (the control group was without occlusion. Wenxin Granule (1.35 g/kg/day, metoprolol (12 mg/kg/day, and distilled water (5 mL/kg/day for the control and model groups were administered orally for 4 weeks. Ultrasonic echocardiography was used to examine cardiac structural and functional parameters. Myocardial histopathological changes were observed using haematoxylin and eosin (H&E dyeing. Myocardial apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL staining. Serum angiotensin II (Ang II concentration was measured using the enzyme-linked immunosorbent assay (ELISA. Results. It was found that Wenxin Granule could partially reverse ventricular remodeling, improve heart function, alleviate the histopathological damage, inhibit myocardial apoptosis, and reduce Ang II concentration in rats with MI. Conclusions. The results of the current study suggest that Wenxin Granule may be a potential alternative and complementary medicine for the treatment of MI.

  12. Isorhamnetin attenuates atherosclerosis by inhibiting macrophage apoptosis via PI3K/AKT activation and HO-1 induction.

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    Yun Luo

    Full Text Available Isorhamnetin (Iso is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/- mice fed a high fat diet.Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions.In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction.

  13. Characterization of neuronal cell death in the spiral ganglia of a mouse model of endolymphatic hydrops.

    Science.gov (United States)

    Semaan, Maroun T; Zheng, Qing Y; Han, Fengchan; Zheng, Yuxi; Yu, Heping; Heaphy, John C; Megerian, Cliff A

    2013-04-01

    Spiral ganglion neurons (SGN) in the Phex male mouse, a murine model of postnatal endolymphatic hydrops (ELH) undergo progressive deterioration reminiscent of human and other animal models of ELH with features suggesting apoptosis as an important mechanism. Histologic analysis of the mutant's cochlea demonstrates ELH by postnatal Day (P) 21 and SGN loss by P90. The SGN loss seems to occur in a consistent topographic pattern beginning at the cochlear apex. SGN were counted at P60, P90, and P120. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR, and immunohistochemical analyses of activated caspase-3, caspase-8, and caspase-9 were performed on cochlear sections obtained from mutants and controls. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay (TUNEL) was carried out on 2 mutants and 2 controls. Corrected SGN counts in control mice were greater in the apical turn of the cochleae at P90 and P120, respectively (p animals with ELH. Apoptosis plays an important role in the degeneration of the SGN in the Phex male mouse.

  14. Effect of caffeic acid phenethyl ester on proliferation and apoptosis of hepatic stellate cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Wen-Xing Zhao; Jing Zhao; Chong-Li Liang; Bing Zhao; Rong-Qing Pang; Xing-Hua Pan

    2003-01-01

    AIM: To investigate the role of nuclear factor-κB (NF-κB)inhibitor caffeic acid phenethy1 ester (CAPE) in the proliferation, collagen synthesis and apoptosis of hepatic stellate cells (HSCs) of rats. METHODS: The HSCs from rats were isolated and cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with CAPE. The proliferation and collagen synthesis of HSCs were determined by 3H-TdR and 3H-proline incorporation respectively, and the expression of type Ⅰ, Ⅲ procollagen genes was further explored byin situ hybridization. Apoptosis cell indices (AIs) were examined using terminal deoxynucleotidyl transferase- mediated DIG-dUTP nick end labeling (TUNEL). RESULTS: Tn activated HSC in culture, CAPE significantly inhibited 3H-TdR and 3H-proline incorporation by HSCs at concentrations of 5 μmol/L and 10 μmol/L respectively. CAPE also reduced the type I procollagen gene expression (P<0.05)at higher concentration. Apoptosis of HSC was induced by CAPE and the AIs were time-and dose-dependently increased from 2.82+0.73 % to 7.66±1.25 % at 12 h (P<0.01) and from 3.15±0.88 % to 10.6L±2.88 % at 24 h (P<0.01). CONCLUSION: CAPE inhibits proliferation and collagen synthesis of HSC at lower concentration and induces HSC apoptosis at higher concentration.

  15. Studies on the apoptosis of autonomic neuron in streptozotocin-induced diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Qinglin Lou; Rongwen Bian; Tao Peng; Xiaojun Ouyang; Hui Xia; Liubao Gu; Yongzhen Mo

    2006-01-01

    Objective: The aim of the study was to investigate apoptosis of the autonomic neuron in streptozotocin-induced diabetic rats, and observe the effect of intervention with nerve growth factor (NGF) on the apoptosis. Methods: A total of 29 male Sprague-Dawley rats were divided into three groups, i.e. normal control (NC, n = 12), untreated diabetic (DM, n =9) and diabetic treated with NGF daily of 500 μg/kg for 30 days(DM+NGF, n = 8). The diabetic rat models were produced by intraperitoneal injection with streptozotocin(65 mg/kg). Over 3 months since the diabetes were setup, the superior cervical sympathetic ganglions(SCG) and the celiac ganglions(CG) were removed and fixed with 10% paraformaldchyde. Apoptosis was measured using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling(TUNEL). Apoptotic index (AI) was calculated by computer image analysis system. Results: The AI of SCG and CG in DM and DM+NGF group were significant higher than those of NC group (P < 0.001) respectively. There was no difference of AI of SCG and CG between DM group and DM+NGF group (both P > 0.05). Conclusion: Neuron apoptosis may contribute to the pathophysiology of diabetic autonomic neuropathy and NGF can not prevent the apoptosis of autonomic neuron in diabetic rats.

  16. Anatomical Study on the Multi-Ovule Development and Abortion of Hanfu Apple

    Institute of Scientific and Technical Information of China (English)

    YANG Xu-yuan; MA Huai-yu; LIU Guo-cheng; LÜ De-guo; QIN Si-jun; DU Guo-dong

    2014-01-01

    The terminal flower buds of 6-yr-old Hanfu apple were used to study the ovule development, ovular characteristics, cell death of abortive ovules, and dynamic change of starch grain quantity in the embryo sac with parafifn slices and terminal deoxynucleotidyl transferase-mediated fluorescein deoxyuridine triphosphate nick-end labeling (TUNEL) system. Four anatropous ovules in each ventricle could be observed before lfowering. With the developing of lforal organ, the bulk of normal ovules enlarged in each ventricle, the mature embryo sac differentiated into nucellus, and the egg cell developed into zygote by double fertilization. A large number of starch grains were observed during pollen tube growth and double fertilization, which guaranteed basic nutrient supply in the normal development of ovules. Moreover, abortion phenomenon of runtish ovules emerged at the stages of mature embryo sac, double fertilization and zygote development. The abortion characteristics included deformity of ovule development, degradation of nucellus tissue, separation between funiculus and ovule, abnormality of four-nucleate embryo sac, as well as development interruption of mature embryo sac. TUNEL analysis proved that ovule abortion was programmed cell death.

  17. "The Role ofL-arginine in Control of Apoptosis in Preimplantation Mouse Embryos Cultured in High Glucose Media "

    Directory of Open Access Journals (Sweden)

    Mohammad Barbarestani

    2004-06-01

    Full Text Available Maternal hyperglycemia causes delay in early stages of embryonic growth and development, higher incidence of congenital malformations and spontaneous miscarriage compared with those of non-diabetic conditions. High glucosis tratogenicity seems to be related to reduction of Nitric Oxide production (NO in hyperglycemic condition. In order to test this hypothesis, 2-cell stage embryos of normal mice were cultured with high concentration of glucose (30mM and different concentrations of L-arginine (5,10,20 mM or L-NAME, an NO syntase (NOS inhibitor. In the end of culture, blastocysts were stained by by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL technique and apoptotic cells were detected by using a Fluorescence microscope. Finally the amount of nitrite in the cultured media was assayed by Griess method. The results indicated that high glucose reduces Nitric Oxide production by preimplantation embryos and increases apoptosis of embryonic cells, but 5-20mM of L-arginine significantly increases Nitric Oxide production and decreases apoptosis. On the contrary L-NAME significantly inhibits the development of pre-implantation embryos. In conclusion, this study indicated that reduced nitric oxide production in high glucosis condition is a main factor for embryonic damage, and supplementation of high glucose media with L-arginine has an important role in prevention of high glucosis embryotoxicity

  18. The Vascular-Targeting Fusion Toxin VEGF121/rGel Inhibits the Growth of Orthotopic Human Bladder Carcinoma Tumors

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    Khalid Mohamedali

    2005-10-01

    Full Text Available Vascular endothelial growth factor. (VEGF and its receptors. (FLT-1 and KDR are overexpressed by human bladder cancer cells and tumor endothelial cells, respectively. Strategies that target VEGF receptors hold promise as antlanglogenic therapeutic approaches to bladder cancer. A fusion protein of VEGF121 and the plant toxin gelonin (rGel was constructed, expressed in bacteria, purified to homogeneity. Cytotoxicity experiments of VEGF121/rGel on the highly metastatic 253J B-V human bladder cancer cell line demonstrated that the VEGF121/rGel does not specifically target these cells, whereas Western blot analysis showed no defectable expression of KDR. Treatment with VEGF121/rGel against orthotopically implanted 253J B-V xenografts in nude mice resulted in a significant suppression of bladder tumor growth (-60% inhibition; P < .05 compared to controls. lmmunohistochemistry studies of orthotopic 253J B-V tumors demonstrated that KDR is highly overexpressed in tumor vasculature. Immunofluorescence staining with antibodies to CD-31 (blood vessel endothelium and reel demonstrated a dramatic colocalization of the construct on tumor neovasculature. Treated tumors also displayed an increase in terminal deoxynucleotidyl transferase-mediated dUTPblotin end labeling staining compared to controls. Thus, VEGF121/rGel inhibits the growth of human bladder cancer by cytotoxic effects directed against the tumor vascular supply and has significant potential as a novel antlangiogenic therapeutic against human bladder cancer.

  19. Effect of L-Arginine on Pulmonary Artery Smooth Muscle Cell Apoptosis in Rats with Hypoxic Pulmonary Vascular Structural Remodeling

    Institute of Scientific and Technical Information of China (English)

    Ingrid Karmane SUMOU; Jun-Bao DU; Bing WEI; Chun-Yu ZHANG; Jian-Guang QI; Chao-Shu TANG

    2006-01-01

    This study investigated the effect of L-arginine (L-Arg) on the apoptosis of pulmonary artery smooth muscle cells (PASMC) in rats with hypoxic pulmonary vascular structural remodeling, and its mechanisms. Seventeen Wistar rats were randomly divided into a control group (n=5), a hypoxia group (n=7), and a hypoxia+L-Arg group (n=5). The morphologic changes of lung tissues were observed under optical microscope. Using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling assay, the apoptosis of PASMC was examined. Fas expression in PASMC was examined using immunohistochemistry. The results showed that the percentage of muscularized artery in small pulmonary vessels, and the relative medial thickness and relative medial area of the small and median pulmonary muscularized arteries in the hypoxic group were all significantly increased. Pulmonary vascular structural remodeling developed after hypoxia. Apoptotic smooth muscle cells of the small and median pulmonary arteries in the hypoxia group were significantly less than those in the control group. After 14 d of hypoxia, Fas expression by smooth muscle cells of median and small pulmonary arteries was significantly inhibited. L-Arg significantly inhibited hypoxic pulmonary vascular structural remodeling in association with an augmentation of apoptosis of smooth muscle cells as well as Fas expression in PASMC. These results showed that L-Arg could play an important role in attenuating hypoxic pulmonary vascular structural remodeling by upregulating Fas expression in PASMC, thus promoting the apoptosis of PASMC.

  20. Advanced glycation end products (AGEs and their receptor (RAGE induce apoptosis of periodontal ligament fibroblasts

    Directory of Open Access Journals (Sweden)

    D.X. Li

    2014-12-01

    Full Text Available Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL fibroblasts induced by advanced glycation end products (AGEs and their receptor (RAGE. We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA, bovine serum albumin (BSA alone, or given no treatment (control. Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01 and increased apoptosis (11.31±1.73%, P<0.05. Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.

  1. X-radiation inhibits histone deacetylase 1 and 2, upregulates Axin expression and induces apoptosis in non-small cell lung cancer

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    Han Yang

    2012-10-01

    Full Text Available Abstract Background Histone deacetylase (HDAC plays an important role in the deacetylation of histone, which can alter gene expression patterns and affect cell behavior associated with malignant transformation. The aims of this study were to investigate the relationships between HDAC1, HDAC2, clinicopathologic characteristics, patient prognosis and apoptosis, to clarify the mechanism of upregulation of the Axis inhibitor Axin (an important regulator of the Wnt pathway by X-radiation and to elucidate the effect of siRNA on radiation therapy of non-small cell lung cancer (NSCLC. Methods HDAC1 and HDAC2 expression levels were measured by immunohistochemistry and reverse transcription PCR. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling and fluorescence activated cell sorting. BE1 cells expressing Axin were exposed to 2 Gy of X-radiation. Results Expression of HDAC1 and that of HDAC2 were correlated, and significantly higher in NSCLC tissues than in normal lung tissues (P P  Conclusions X-radiation and siRNA inhibit expression of HDAC1 and HDAC2, weaken the inhibitory effect of HDAC on Axin, upregulate Axin expression and induce apoptosis of lung cancer cells. Inhibition of HDAC1 and HDAC2 is a means of enhancing the radiosensitivity of NSCLC.

  2. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    徐昌杰; 陈昆松; FERGUSONIanB

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple celi death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  3. Programmed cell death features in apple suspension cells under low oxygen culture.

    Science.gov (United States)

    Xu, Chang-jie; Chen, Kun-song; Ferguson, Ian B

    2004-02-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  4. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    XU Chang-jie(徐昌杰); CHEN Kun-song(陈昆松); FERGUSON Ian B.

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  5. Mitochondrial Fission Increases Apoptosis and Decreases Autophagy in Renal Proximal Tubular Epithelial Cells Treated with High Glucose.

    Science.gov (United States)

    Lee, Wen-Chin; Chiu, Chien-Hua; Chen, Jin-Bor; Chen, Chiu-Hua; Chang, Hsueh-Wei

    2016-11-01

    The aim of this study was to examine the effect of mitochondrial morphogenesis changes on apoptosis and autophagy of high-glucose-treated proximal tubular epithelial cells (HK2). Cell viability, apoptosis, and mitochondrial morphogenesis were examined using crystal violet, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and mitotracker staining, respectively. High glucose inhibited cell viability and induced mitochondrial fission in HK2 cells. After depleting mitofusin 1 (MFN1), the MFN1(-) HK2 cells (fission type) became more susceptible to high-glucose-induced apoptosis and mitochondrial fragmentation observed by TUNEL and mitotracker assays. In siMFN2 HK2 cells (fission type), mitochondria were highly fragmented (>80% fission rate) with or without high-glucose treatment; however, siFIS1 (mitochondrial fission protein 1) HK2 cells (fusion type) exhibited little fragmentation (High-glucose treatment induced autophagy, characterized by the formation of autophagosome and microtubule-associated protein light chain 3 (LC3) B-II, as observed by transmission electron microscopy and western blotting, respectively. LC3B-II levels decreased in both MFN1(-) and siMFN2 HK2 cells, but increased in siFIS1 HK2 cells. Moreover, autophagy displays a protective role against high-glucose-induced cell death based on cotreatment with autophagy inhibitors (3-methyladenine and chloroquine). Mitochondrial fission may increase apoptosis and decrease autophagy of high-glucose-treated HK2 cells.

  6. Effects of ultrasound and ultrasound contrast agent on vascular tissue

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    Wood Steven C

    2012-07-01

    Full Text Available Abstract Background Ultrasound (US imaging can be enhanced using gas-filled microbubble contrast agents. Strong echo signals are induced at the tissue-gas interface following microbubble collapse. Applications include assessment of ventricular function and virtual histology. Aim While ultrasound and US contrast agents are widely used, their impact on the physiological response of vascular tissue to vasoactive agents has not been investigated in detail. Methods and results In the present study, rat dorsal aortas were treated with US via a clinical imaging transducer in the presence or absence of the US contrast agent, Optison. Aortas treated with both US and Optison were unable to contract in response to phenylephrine or to relax in the presence of acetylcholine. Histology of the arteries was unremarkable. When the treated aortas were stained for endothelial markers, a distinct loss of endothelium was observed. Importantly, terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling (TUNEL staining of treated aortas demonstrated incipient apoptosis in the endothelium. Conclusions Taken together, these ex vivo results suggest that the combination of US and Optison may alter arterial integrity and promote vascular injury; however, the in vivo interaction of Optison and ultrasound remains an open question.

  7. Effect of Taurine on Acinar Cell Apoptosis and Pancreatic Fibrosis in Dibutyltin Dichloride-induced Chronic Pancreatitis

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    Sawa,Kiminari

    2012-08-01

    Full Text Available The relationship between pancreatic fibrosis and apoptosis of pancreatic acinar cells has not been fully elucidated. We reported that taurine had an anti-fibrotic effect in a dibutyltin dichloride (DBTC-chronic pancreatitis model. However, the effect of taurine on apoptosis of pancreatic acinar cells is still unclear. Therefore, we examined apoptosis in DBTC-chronic pancreatitis and in the AR42J pancreatic acinar cell line with/without taurine. Pancreatic fibrosis was induced by a single administration of DBTC. Rats were fed a taurine-containing diet or a normal diet and were sacrificed at day 5. The AR42J pancreatic acinar cell line was incubated with/without DBTC with taurine chloramines. Apoptosis was determined by using terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL assay. The expression of Bad and Bcl-2 proteins in the AR42J cells lysates was detected by Western blot analysis. The apoptotic index of pancreatic acinar cells in DBTC-administered rats was significantly increased. Taurine treatment inhibited pancreatic fibrosis and apoptosis of acinar cells induced by DBTC. The number of TUNEL-positive cells in the AR42J pancreatic acinar cell lines was significantly increased by the addition of DBTC. Incubation with taurine chloramines ameliorated these changes. In conclusion, taurine inhibits apoptosis of pancreatic acinar cells and pancreatitis in experimental chronic pancreatitis.

  8. High clusterin expression correlates with a poor outcome in stage II colorectal cancers.

    LENUS (Irish Health Repository)

    Kevans, David

    2012-02-01

    The role of clusterin in tumor growth and progression remains unclear. Overexpression of cytoplasmic clusterin has been studied in aggressive colon tumors; however, no correlation between clusterin expression and survival in colorectal cancer has been identified to date. We assessed levels of clusterin expression in a group of stage II colorectal cancer patients to assess its utility as a prognostic marker. The study included 251 patients with stage II colorectal cancer. Tissue microarrays were constructed and immunohistochemistry done and correlated with clinical features and long term outcome. Dual immunofluorescence and confocal microscopy were used with terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling probes and clusterin antibody to assess the degree of co localization. Percentage epithelial cytoplasmic staining was higher in tumor compared with nonadjacent normal mucosa (P < 0.001). Within the stromal compartment, percentage cytoplamic staining and intensity was lower in tumor tissue compared with normal nonadjacent mucosa (P < or = 0.001). Survival was significantly associated with percentage epithelial cytoplasmic staining (P < 0.001), epithelial cytoplasmic staining intensity (P < 0.001), percentage stromal cytoplasmic staining (P = 0.002), and stromal cytoplasmic staining intensity (P < 0.001). Clusterin levels are associated with poor survival in stage II colorectal cancer.

  9. TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes: the effect of microorganisms within human follicular fluid collected during IVF cycles.

    Science.gov (United States)

    Pelzer, Elise S; Harris, Jessica E; Allan, John A; Waterhouse, Mary A; Ross, Tara; Beagley, Kenneth W; Knox, Christine L

    2013-09-01

    Recently we reported the presence of bacteria within follicular fluid. Previous studies have reported that DNA fragmentation in human spermatozoa after in vivo or in vitro incubation with bacteria results in early embryo demise and a reduced rate of ongoing pregnancy, but the effect of bacteria on oocytes is unknown. This study examined the DNA within mouse oocytes after 12 hours' incubation within human follicular fluids (n=5), which were collected from women undergoing in vitro fertilization (IVF) treatment. Each follicular fluid sample was cultured to detect the presence of bacteria. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was used to label DNA fragmentation in ovulated, non-fertilized mouse oocytes following in vitro incubation in human follicular fluid. The bacteria Streptococcus anginosus and Peptoniphilus spp., Lactobacillus gasseri (low-dose), L. gasseri (high-dose), Enterococcus faecalis, or Propionibacterium acnes were detected within the follicular fluids. The most severe DNA fragmentation was observed in oocytes incubated in the follicular fluids containing P. acnes or L. gasseri (high-dose). No DNA fragmentation was observed in the mouse oocytes incubated in the follicular fluid containing low-dose L. gasseri or E. faecalis. Low human oocyte fertilization rates (DNA fragmentation in mouse oocytes following 12h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism.

    Science.gov (United States)

    Ruvolo, Giovanni; Roccheri, Maria Carmela; Brucculeri, Anna Maria; Longobardi, Salvatore; Cittadini, Ettore; Bosco, Liana

    2013-04-01

    An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p15 %), while no significant variation occurred in the patients with DFI values ≤ 15 %. Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 % .

  11. Chromosomal aneuploidies and DNA fragmentation of human spermatozoa from patients exposed to perfluorinated compounds.

    Science.gov (United States)

    Governini, L; Guerranti, C; De Leo, V; Boschi, L; Luddi, A; Gori, M; Orvieto, R; Piomboni, P

    2015-11-01

    This study investigated chromosomal aneuploidies and DNA damage in spermatozoa from male patients contaminated by perfluorinated compounds (PFCs) in whole blood and seminal plasma. Sperm aneuploidy and diploidy rate for chromosomes 18, X and Y were evaluated by FISH; sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling technique coupled to flow cytometry. Our results indicated that PFC contamination was present in 58% of subjects included in the study. A significant increase in alterations of sperm parameters was observed in PFC-positive subjects compared to PFC-negative subjects. As regards the sperm aneuploidy, both disomy and diploidy rates resulted significantly increased in subjects positive for PFC contamination compared to PFC-negative samples. In addition, sperm DNA fragmentation index resulted significantly increased in PFC-contaminated subjects compared to PFC-non-contaminated subjects, with a significant increased level of dimmer DNA fragmentation index. Our results clearly indicate that PFC contamination may detrimentally affect spermatogenesis, disturbing both meiotic segregation and DNA integrity. We could therefore suggest cautions to reduce or eliminate any contact with these compounds because the long-term effects of PFC accumulation in the body are not predictable. © 2014 Blackwell Verlag GmbH.

  12. Mechanical processing of hyperviscous semen specimens can negatively affect sperm DNA fragmentation.

    Science.gov (United States)

    Kussler, Ana Paula S; Pimentel, Anita M; Alcoba, Diego D; Liu, Isabella P; Brum, Ilma Simoni; Capp, Edison; Corleta, Helena V E

    2014-04-01

    The present study compared the DNA fragmentation in human sperm samples with reduced, physiological, and increased viscosity in order to evaluate whether the process used to reduce viscosity (expulsion of semen through a needle and syringe) alters significantly sperm DNA fragmentation. The seminal parameters of semen samples from 123 patients were evaluated and classified according to their viscosity. Samples with increased viscosity were submitted to a process of expulsion of semen through a 10-mL syringe and an 18-gauge (18G) needle to reduce the seminal viscosity. The DNA fragmentation of all samples was analysed using TUNEL assay (Terminal deoxynucleotidyl transferase mediated dUTP Nick-end labelling assay); in samples with increased viscosity, the fragmentation was assessed before and after the process of expulsion with syringe and needle. There was no difference in DNA fragmentation between groups with different viscosity (P = 0.857). A significantly increase in sperm DNA fragmentation after expulsion of hyperviscous semen through the syringe was observed (P = 0.035). There was no difference in DNA fragmentation rate between samples with reduced, increased and physiological viscosities; however, the physical process of expulsion of semen through a syringe and needle increased sperm DNA fragmentation.

  13. Protective effects of astragalus extract against intermittent hypoxia-induced hippocampal neurons impairment in rats

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qiang; GAO Wen-yuan; ZHANG Yun; CHEN Bao-yun; CHEN Zhe; ZHANG Wei-san; MAN Shu-li

    2013-01-01

    Background Intermittent hypoxia is the main pathophysiological cause of the obstructive sleep apnea syndrome.Astragalus shows improvement of spatial learning and memory abilities under intermittent hypoxia.Our study aimed to investigate the protective effect of astragalus against intermittent hypoxia induced-hippocampal neurons impairment in rats and lay the theoretical foundation for the sleep apnea improvement in cognitive function by astragalus.Methods Male Wistar rats were divided into 4 groups:blank control group,normoxia group,intermittent hypoxia group and astragalus treated intermittent hypoxia group.After 6-week treatment,apoptosis of neurons was evaluated by terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay.Furthermore,the expression of HIF-1a was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) at the mRNA level as well as by immunohistochemistry (IHC) and Western blotting at the protein level.Results HPLC analysis indicated that astragaloside Ⅳ,astragaloside Ⅱ and astragaloside Ⅰ were the main compounds in astragals extract.Astragalus extract reduced the apoptosis of hippocampal neurons (P <0.05) and decreased the expression of HIF-1a at both the mRNA and protein levels in hippocampus compared with non-treated groups (P <0.05).Conclusion Astragalus protects against intermittent hypoxia-induced hippocampal neurons impairment in rats.

  14. Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis.

    Science.gov (United States)

    Ji, Qianqian; Zhu, Kongju; Liu, Zhiguo; Song, Zhenwei; Huang, Yuankai; Zhao, Haijing; Chen, Yaosheng; He, Zuyong; Mo, Delin; Cong, Peiqing

    2013-03-15

    Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality.

  15. Peran Kedelai (Glycine max L. dalam Pencegahan Apoptosis pada Cedera Jaringan Hati

    Directory of Open Access Journals (Sweden)

    Maya Tejasari

    2013-02-01

    Full Text Available Abstrak Pada liver injury akibat berbagai sebab, terjadi apoptosis sel yang sangat banyak yang dapat memengaruhi fungsi metabolik hati.  Isoflavon kedelai telah diketahui dapat mencegah apoptosis sel pada folikel ovarium dan osteoblas. Penelitian ini bertujuan untuk mengetahui pengaruh kedelai pada pencegahan apoptosis sel pada jaringan hati mencit yang diinduksi CCl4.  Penelitian dilakukan menggunakan 30 ekor mencit jantan galur DDY berumur 8─10 minggu yang dibagi dalam 6 kelompok perlakuan.  Kelompok 1 merupakan kontrol positif yang hanya diberi makanan pelet standar selama 3 minggu kemudian diberi 0,2 mL larutan CCl4 per oral selama 4 hari. Kelompok 2 merupakan kontrol negatif yang hanya diberi makanan pelet standar dan tidak diberi CCl4, sedangkan kelompok 3─6 merupakan kelompok uji yang selain diberi makanan pelet standar juga diberi kedelai dengan kadar berturut-turut 145,6 mg/hari, 218,4 mg/hari, 291,2 mg/hari dan 364 mg/hari selama 3 minggu kemudian diberi 0,2 mL larutan CCl4 peroral selama 4 hari.  Seluruh kelompok kemudian dikorbankan dan diambil organ hatinya untuk dilakukan pemeriksaan histokimia terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling (TUNEL.  Parameter yang diukur adalah jumlah apoptosis sel pada sayatan jaringan hati mencit menggunakan mikroskop cahaya.  Data disajikan dan dianalisis secara statistik menggunakan uji analysis of varians (ANOVA untuk menganalisis perbedaan antar kelompok. Hasil penelitian memperlihatkan bahwa dari hasil pemeriksaan imunohistokimia TUNEL tampak jumlah sel yang mengalami apoptosis pada kelompok yang diberi kedelai lebih sedikit dibandingkan dengan kelompok yang tidak diberi kedelai. Analisis uji ANOVA antara kelompok tersebut menunjukan perbedaaan yang signifikan dengan nilai p<0,05. Simpulan, bahwa pemberian kedelai dapat mencegah apoptosis sel pada jaringan hati mencit yang diinduksi CCl4. Kata kunci: Apoptosis, CCl4, isoflavon, kedelai, liver injury, TUNEL

  16. The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice.

    Science.gov (United States)

    Uchida, Kenzo; Nakajima, Hideaki; Hirai, Takayuki; Yayama, Takafumi; Chen, Kebing; Guerrero, Alexander Rodriguez; Johnson, William Eustace; Baba, Hisatoshi

    2012-12-15

    The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS

  17. Glucocorticoid effects on adult hippocampal neural progenitor cells%糖皮质激素对成体海马神经祖细胞的影响****☆◆

    Institute of Scientific and Technical Information of China (English)

    于秀军; 李奕; 台立稳; 陈相

    2013-01-01

      背景:体内研究显示,高浓度糖皮质激素可抑制大鼠内源性神经前体细胞增殖。而神经胶质抗原2蛋白聚糖阳性神经祖细胞(NG2细胞)是成熟中枢神经系统中最大的增殖细胞群体,具有多分化潜能。目的:观察糖皮质激素对体外培养的成熟大鼠海马由来的NG2细胞生存和增殖的影响。方法:原代及传代培养成年大鼠海马NG2细胞,以0,0.1,1,10,100μmol浓度糖皮质激素类药物地塞米松干预48 h后,采用乳酸脱氢酶分析法测定细胞活性,原位缺口末端标记技术(即TUNEL法)观察细胞凋亡情况,5’-溴脱氧尿嘧啶核苷掺入法鉴定细胞增殖状况。结果与结论:1,10,100μmol浓度地塞米松干预明显减少NG2细胞数,并显著增加TUNEL阳性细胞率,明显减少 BrdU 阳性细胞率。结果可见高浓度糖皮质激素能抑制 NG2细胞分裂增殖,并诱导细胞凋亡,从而明显减少NG2细胞数。%BACKGROUND:In vivo studies have shown that the high concentration of glucocorticoids can inhibit the proliferation of rat endogenous neural precursor cel s. Neuron-glia antigen 2 proteoglycan-positive neural progenitor cel s are the largest proliferating cel population in the mature central nervous system, which are pluripotent cel s. OBJECTIVE:To investigate the effects of glucocorticoid on the survival and proliferation of the neuron-glia antigen 2 proteoglycan-positive neural progenitor cel s from in vitro cultured adult rat hippocampus. METHODS:Neuron-glia antigen 2 proteoglycan-positive neural progenitor cel s were primary cultured and sub-cultured. The passage 1 cel s were treated with dexamethasone with the concentrations of 0, 0.1, 1, 10 and 100μmol for 48 hours, and then the cel activities were determined by lactate dehydrogenase assay, the apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, and the proliferation of the

  18. The effects of silibin administration for different time periods on mouse liver with Ehrlich ascites carcinoma.

    Science.gov (United States)

    Beydogan, Alisa Bahar; Bolkent, Sema

    2016-06-01

    Ehrlich ascites carcinoma is the one of the animal cancer models having high malignancy and rapid growth resistance. Silibin has reported to be an antioxidant in previous studies. We aimed to investigate the effects of silibin on mouse liver with Ehrlich ascites tumor (EAT) cells in different time periods. Balb/c mice were divided into five groups. Group I (Control): The saline buffer (sb) was injected intraperitoneally (ip) to the mice for 15 days. Group II (Silibin): 150mg/kg silibin was injected ip for 15 days. Group III (Ehrlich): 2×10(5) cells were transferred from the donor mouse to healthy mice on first day. Group IV (Ehrlich+Silibin): Silibin was given between 5th and 15th days to mice inoculated with EAT. Group V (Silibin+Ehrlich): Silibin was injected for 15 days after EAT cells. The liver sections were stained with matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), caspase 3, caspase 8, and proliferating cell nuclear antigen (PCNA) antibodies by the streptavidin-biotin-peroxidase technique. Biochemical analysis and Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method were performed in the liver. Superoxide dismutase levels of liver increased in Ehrlich+Silibin group compared with Ehrlich group. Malondialdehyde levels significantly decreased in Silibin+Ehrlich group compared with Ehrlich+Silibin. MMP-2 and MMP-9 immunopositive cells increased in Silibin+Ehrlich compared with Ehrlich group. Caspase 3 and TUNEL signals significantly increased in Silibin+Ehrlich group compared with Ehrlich group. PCNA positive signals significantly increased in Ehrlich+Silibin group compared with Ehrlich group. According to our findings, we suggest that silibin treatment after EAT cells inoculation has more effective than concurrently EAT and silibin treatment. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  19. Mild maternal iron deficiency anemia induces DPOAE suppression and cochlear hair cell apoptosis by caspase activation in young guinea pigs.

    Science.gov (United States)

    Yu, Fei; Hao, Shuai; Zhao, Yue; Ren, Yahao; Yang, Jun; Sun, Xiance; Chen, Jie

    2014-01-01

    Iron deficiency (ID) anemia (IDA) alters auditory neural normal development in the mammalian cochlea. Previous results suggest that mild maternal IDA during pregnancy and lactation altered the hearing and nervous system development of the young offspring, but the mechanisms underlying the association are incompletely understood. The objective of this study was to evaluate the role of apoptosis in the development of sensory hair cells following mild maternal IDA during pregnancy and lactation. We established a maternal anemia model in female guinea pigs by using a mild iron deficient diet. The offspring were weaned on postnatal day (PND) 9 and then was given the iron sufficient diet. Maternal blood samples were collected on gestational day (GD) 21, GD 42, GD 63 and PND 9, serum level of iron (SI) or hemoglobin (Hb) was measured. Blood samples of pups were collected on PND 9 for SI measurement. On PND 24, pups were examined the distortion product otoacoustic emission (DPOAE) task, and then the cochleae were harvested for assessment of apoptosis by immunohistochemistry of cysteine-aspartic acid proteases 3/9 (caspase-3/9) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and by double immunofluorescence for the colocalization of TUNEL and caspase-3. Blood samples of pups were collected on PND 24 for SI and Hb measurements. Here we show that mild maternal IDA during pregnancy and lactation resulted in hearing impairment, decreased hair cell number, caspase-3/9 activation and increased apoptotic cell number of young guinea pigs. These results indicate a key role for apoptosis in inhibition of hair cell development, caused by mild maternal IDA during pregnancy and lactation.

  20. Antitumor effect of malaria parasite infection in a murine Lewis lung cancer model through induction of innate and adaptive immunity.

    Science.gov (United States)

    Chen, Lili; He, Zhengxiang; Qin, Li; Li, Qinyan; Shi, Xibao; Zhao, Siting; Chen, Ling; Zhong, Nanshan; Chen, Xiaoping

    2011-01-01

    Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8(+) T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a novel strategy or therapeutic vaccine vector for anti-lung cancer

  1. Beneficial or harmful influence of phytosterols on human cells?

    Science.gov (United States)

    Rubis, Blazej; Paszel, Anna; Kaczmarek, Mariusz; Rudzinska, Magdalena; Jelen, Henryk; Rybczynska, Maria

    2008-12-01

    So far, a protective influence of phytosterols on the human organism and atherogenesis has been suggested. Most studies have concentrated on the cytotoxic efficacy of phytosterols on cancer cells. However, there are only a few reports showing their influence on normal cells. The aim of the present study was to determine whether dietary plant sterols and their thermal processing products could influence the viability of normal, abdominal endothelial cells that play a crucial role in atherogenesis. Thus, we studied the effect of rapeseed oil-extract components, beta-sitosterol, cholesterol and their epoxy-derivatives, 5 alpha,6 alpha-epoxy-beta-sitosterol and 5 alpha,6 alpha-epoxycholesterol, on the proliferation and viability of human abdominal aorta endothelial cells HAAE-2 in vitro. We showed strong cytotoxic properties of beta-sitosterol in HAAE-2 cells (half maximal inhibitory concentration (IC50) = 1.99 (SEM 0.56) microm) and, interestingly, a weaker cytotoxic effect of 5 alpha,6 alpha-epoxy-beta-sitosterol (IC50>200 microm). Moreover, we observed a significantly stronger cytotoxic activity of beta-sitosterol than cholesterol (IC50 = 8.99 (SEM 2.74) microm). We also revealed that beta-sitosterol as well as cholesterol caused apoptosis, inducing caspase-3 activity in the cells (60 % increase compared with control cells) that corresponded to the DNA fragmentation analysis in a terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) study. Although absorption of plant sterols is low compared with cholesterol, they can still influence other physiological functions. Since they effectively reduce serum LDL-cholesterol and atherosclerotic risk but also decrease the viability of cancer cells as well as normal cells in a time- and dose-dependent manner in vitro, their influence on other metabolic processes remains to be elucidated.

  2. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of p27(kip¹) and FoxO3a in female rat cerebral cortex and PC12 cells.

    Science.gov (United States)

    Xu, Guangfei; Liu, Jiao; Yoshimoto, Katsuhiko; Chen, Gang; Iwata, Takeo; Mizusawa, Noriko; Duan, Zhiqing; Wan, Chunhua; Jiang, Junkang

    2014-05-02

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxin that alters normal brain development, producing cognitive disability and motor dysfunction. Previous studies in rats have proved that female rats are more sensitive to TCDD lethality than male ones. Recent studies have shown that TCDD induces cell cycle arrest and apoptosis, but the regulatory proteins involved in these processes have yet to be elucidated. In this study, we constructed an acute TCDD injury female rat model, and investigated the effects of TCDD on apoptosis and expression of cell cycle regulators, forkhead box class O 3a (FoxO3a) and p27(kip1), in the central nervous system (CNS). Increased levels of active caspase-3 were observed in the cerebral cortex of female rats treated with TCDD, suggesting that TCDD-induced apoptosis occurs in the CNS. The terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling assay showed that apoptosis primarily occurred in neurons. Furthermore, Western blot analysis, reverse transcription-polymerase chain reaction, and immunohistochemistry showed a significant up-regulation of FoxO3a and p27(kip1) in the cerebral cortex. Immunofluorescent labeling indicated that FoxO3a and p27(kip1) were predominantly localized in apoptotic neurons, but not in astrocytes. In vitro experiments using PC12, a rat neuron-like pheochromocytoma cell line, also revealed that TCDD induced apoptosis and an increase in FoxO3a and p27(kip1) expression. Furthermore, knockdown of FoxO3a expression inhibited p27(kip1) transcription and TCDD-induced apoptosis. Based on our data, induction of FoxO3a may play an important role in TCDD-induced neurotoxicity.

  3. Effects of 1, 25-Dihydroxyvitamin D3 on Experimental Autoimmune Myocarditis in Mice

    Directory of Open Access Journals (Sweden)

    Fen Hu

    2016-05-01

    Full Text Available Background/Aims: Myocarditis is an important inflammatory disease of the heart which causes life-threatening conditions. 1, 25(OH2 D3 has effects on multiple systems and diseases. The present study was aimed to investigate the effect of 1, 25(OH2 D3 on experimental autoimmune myocarditis (EAM, and explored the underlying mechanisms involved. Methods: EAM was induced by immunizing BALB/c mice with cardiac α-myosin heavy chain peptides (MyHC-α. 1, 25(OH2 D3 (1,000 ng/kg once or vehicle was administered intraperitoneally every other day during the entire experiment. On day 21, transthoracic echocardiography was performed and cardiac inflammatory infiltration was detected by hematoxylin and eosin (HE. The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL assay, and Western blots for the expression of protein caspase-3 and cleaved-caspase3 were used to evaluate apoptosis. Transmission electron microscopy and Western blots for the expression of protein Beclin-1, LC3B, and P62 were used to evaluate autophagy. Results: The ratio of heart weight/body weight was significantly reduced in 1, 25(OH2 D3 -treated EAM mice, compared with vehicle -treated ones. 1, 25(OH2 D3 treatment improved cardiac function, diminished cell infiltration in cardiac, suppressed myocardial apoptosis, decreased the number of autophagosomes, and decreased the protein expression of Beclin-1, LC3-II and p62. Conclusions: The present results demonstrated that administration of 1, 25(OH2 D3 decreased EAM severity. 1, 25(OH2 D3 treatment may be a feasible therapeutic approach for EAM.

  4. Propofol Ameliorates Calpain-induced Collapsin Response Mediator Protein-2 Proteolysis in Traumatic Brain Injury in Rats

    Science.gov (United States)

    Yu, Yun; Jian, Min-Yu; Wang, Yun-Zhen; Han, Ru-Quan

    2015-01-01

    Background: Collapsin response mediator protein-2 (CRMP2), a multifunctional cytosolic protein highly expressed in the brain, is degraded by calpain following traumatic brain injury (TBI), possibly inhibiting posttraumatic neurite regeneration. Lipid peroxidation (LP) is involved in triggering postinjury CRMP2 proteolysis. We examined the hypothesis that propofol could attenuate LP, calpain-induced CRMP2 degradation, and brain injury after TBI. Methods: A unilateral moderate controlled cortical impact injury was induced in adult male Sprague-Dawley rats. The animals were randomly divided into seven groups: Sham control group, TBI group, TBI + propofol groups (including propofol 1 h, 2 h, and 4 h groups), TBI + U83836E group and TBI + fat emulsion group. The LP inhibitor U83836E was used as a control to identify that antioxidation partially accounts for the potential neuroprotective effects of propofol. The solvent of propofol, fat emulsion, was used as the vehicle control. Ipsilateral cortex tissues were harvested at 24 h post-TBI. Immunofluorescent staining, Western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling were used to evaluate LP, calpain activity, CRMP2 proteolysis and programmed cell death. The data were statistically analyzed using one-way analysis of variance and a paired t-test. Results: Propofol and U83836E significantly ameliorated the CRMP2 proteolysis. In addition, both propofol and U83836E significantly decreased the ratio of 145-kDa αII-spectrin breakdown products to intact 270-kDa spectrin, the 4-hydroxynonenal expression and programmed cell death in the pericontusional cortex at 24 h after TBI. There was no difference between the TBI group and the fat emulsion group. Conclusions: These results demonstrate that propofol postconditioning alleviates calpain-mediated CRMP2 proteolysis and provides neuroprotective effects following moderate TBI potentially by counteracting LP and reducing calpain activation

  5. Effect of genistein on mouse blastocyst development in vitro

    Institute of Scientific and Technical Information of China (English)

    Wen-hsiung CHAN; Hsiang-yu LU; Nion-heng SHIAO

    2007-01-01

    Aim: To examine the cytotoxic effects of genistein, an isoflavone compound, on early postimplantation embryonic development in vitro. Methods: Mouse blastocysts were incubated in medium with or without genistein (25 or 50 μmol/L) or daidzein (50 μmol/L) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and apoptotic or necrotic cells were visualized by Annexin-V and propidium iodide (PI) staining. Implantation and postimplantation development of embryos were measured by in vitro development analysis. Results: TUNEL staining and Annexin-V/PI staining. showed that genistein dose-dependently increased apoptosis in mouse blastocysts, while daidzein, another soy isoflavone, had no such effect. The pretreatment of the blastocysts with genistein caused fewer cells than the control group and this effect was primary in the inner cell mass. The genistein-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer genistein-pretreated embryos reached the later stages of embryonic development versus the controls, with many of the former embryos dying at relatively early stages of development. In addition, genistein treatment de-creased the development of morulas into blastocysts, and dietary genistein was found to induce cell apoptosis and decrease cell proliferation in an animal assay model of embryogenesis. Conclusions: Our results collectively indicate that genistein treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death in vitro, while dietary genistein appears to negatively affect mouse embryonic development in vivo by inducing cell apoptosis and inhibiting cell proliferation. These novel findings provide important new insights into the effect of genistein

  6. The anti-tumor effect of p53 gene-loaded hydroxyapatite nanoparticles in vitro and in vivo

    Science.gov (United States)

    Zhao, Ruibo; Yang, Xinyan; Chen, Cen; Chen, Kan; Wang, Shibing; Xie, Chungang; Ren, Xiaoyuan; Kong, Xiangdong

    2014-04-01

    This research focused on anti-tumor effect of pEGFP-C1-p53 (p53) gene-loaded hydroxyapatite (HAp) nanoparticles in vitro and in vivo. Four kinds of HAp nanoparticles, spherical HAp nanoparticles (S-HAp, diameter: 50 nm), needle-like HAp nanoparticles (N-HAp, average length: 110 nm and width: 30 nm), rod-like HAp nanoparticles (R-HAp, average length: 100 nm and width: 30 nm), and short-rod-like HAp nanoparticles (SR-HAp, average length: 40 nm and width: 30 nm), were prepared initially. The HAp nanoparticles with or without being modified by PEI (named HAp and HAp-PEI, respectively) have excellent biocompatibility as shown by MTT assay and crystal violet staining tests. Then, the subsequent MTT, Hocehst staining tests, and Western blot showed that the killing effect of p53-loaded HAp-PEI (HAp-PEI-p53) was effective with fair selectivity toward Hep-3B and HuH-7 cells' cell lines. Moreover, HAp-PEI-p53 could inhibit the tumor growth in vivo, and the mechanism of tumor growth inhibition was verified by the hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, P53 protein immunohistochemistry, and transmission electron microscope of the tumor cell in vivo. We found that HAp-PEI-p53 has good anti-cancer effect in vitro and in vivo, especially for the S-HAp-PEI-p53. Tumor metastasis could be suppressed significantly by the S-HAp-PEI-p53 and N-HAp-PEI-p53 treatments by the in vivo imaging system. All these results lead to the conclusion that the particle sizes of HAp ranging from 100 to 200 nm are appropriate for cancer gene therapy and may be widely used in anti-cancer investigation.

  7. Effects of garlicin on apoptosis in rat model of colitis

    Institute of Scientific and Technical Information of China (English)

    Xi-Ming Xu; Jie-Ping Yu; Xiao-Fei He; Jun-Hua Li; Liang-Liang Yu; Hong-Gang Yu

    2005-01-01

    AIM: To investigate the effects of garlicin on apoptosis and expression of bcl-2 and bax in lymphocytes in rat model of ulcerative colitis (UC).METHODS: Healthy adult Sprague-Dawley rats of both sexes, weighing 180±30 g, were employed in the present study. The rat model of UC was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) enema. The experimental animals were randomly divided into garlicin treatment group (including high and low concentration), model control group, and normal control group. Rats in garlicin treatment group and model control group received intracolic garlicin daily at doses of 10.0 and 30.0 mg/kg and equal amount of saline respectively 24 h after colitis model was induced by alcohol and TNBS co-enema. Rats in normal control group received neither alcohol nor only TNBS but only saline enema in this study. On the 28th d of the experiment, rats were executed, the expression of bcl-2 and bax protein was determined immunohistochemically and the apoptotic cells were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method. At the same time, the rat colon mucosal damage index (CMDI) was calculated.RESULTS: In garlicin treatment group, the positive expression of bcl-2 in lymphocytes decreased and the number of apoptotic cells was more than that in model control group, CMDI was lower than that in model control group. The positive expression of bax in lymphocytes had no significant difference.CONCLUSION: Garlicin can protect colonic mucosa against damage in rat model of UC induced by TNBS enema.

  8. Human equilibrative nucleoside transporter 1 and carcinoma of the ampulla of Vater: expression differences in tumour histotypes

    Science.gov (United States)

    Perrone, G.; Morini, S.; Santini, D.; Rabitti, C.; Vincenzi, B.; Alloni, R.; Antinori, A.; Magistrelli, P.; Lai, R.; Cass, C.; Mackey, J.R.; Coppola, R.; Tonini, G.; Onetti Muda, A.

    2010-01-01

    The human equilibrative nucleoside transporter 1 (hENT1) is the major means by which gemcitabine enters human cells; recent evidence exists that hENT1 is expressed in carcinoma of the ampulla of Vater and that it should be considered as a molecular prognostic marker for patients with resected ampullary cancer. Aim of the present study is to evaluate the variations of hENT1 expression in ampullary carcinomas and to correlate such variations with histological subtypes and clinicopathological parameters. Forty-one ampullary carcinomas were histologically classified into intestinal, pancreaticobiliary and unusual types. hENT1 and Ki67 expression were evaluated by immunohistochemistry, and apoptotic cells were identified by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) method. hENT1 overexpression was detected in 63.4% ampullary carcinomas. A significant difference in terms of hENT1 and Ki67 expression was found between intestinal vs. pancreaticobiliary types (P=0.03 and P=0.009 respectively). Moreover, a significant statistical positive correlation was found between apoptotic and proliferative Index (P=0.036), while no significant correlation was found between hENT1 and apoptosis. Our results on hENT1 expression suggest that classification of ampullary carcinoma by morphological subtypes may represent an additional tool in prospective clinical trials aimed at examining treatment efficacy; in addition, data obtained from Ki67 and TUNEL suggest a key role of hENT1 in tumour growth of ampullary carcinoma. PMID:20839414

  9. Impact of neutrophil apoptosis on haemostatic activation in chronic liver disease patients.

    Science.gov (United States)

    Essawy, Faiza M; Bekheet, Iman W; Saleh, Abeya F; Madkour, Mona E; Bayoumi, Emad El-Din A

    2008-09-01

    Recent studies suggest the impact of apoptosis on the mechanisms leading to hypercoagulability. We aimed to clarify the potential role of neutrophil apoptosis in neutropenia and hypercoagulable state encountered in chronic liver disease patients. This study was conducted on 15 normal controls and 45 patients with chronic liver disease classified according to modified Child Pugh classification into, Child A, B and C groups (15 cases each). Haemostatic parameters studied include, prothrombin time, partial thromboplastin time, tissue factor, protein C antigen, protein S antigen, and markers of haemostatic activation [prothrombin fragment 1+2 (F1+2), thrombus precursor protein (TpP) and D-dimer]. Flowcytometric study was done for quantitative assay of neutrophil apoptotic subpopulations to detect the percentage of early and late apoptotic, and necrotic neutrophils using Annexin V-FITC/propidium iodide dye. Semiquantitative assay of apoptotic neutrophils showing DNA fragmentation was performed on neutrophil culture using terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling test. In addition to enzyme-linked immunosorbent assay for soluble Fas (APO-1/CD95) in culture supernatant. The results revealed a rise in the neutrophil apoptotic and necrotic markers with progression of the disease, and they were inversely correlated with the absolute neutrophil count. The apoptotic neutrophil cells showed a significant positive correlation with several haemostatic parameters (tissue factor, prothrombin fragment 1+2, thrombus precursor protein and D-dimer). Regression analysis proved that apoptotic parameters are independent determinants of prothrombotic markers, which further incriminate the apoptotic mechanisms in the hypercoagulable state encountered in this clinical setting.

  10. Neuropeptide Y expression in mouse hippocampus and its role in neuronal excitotoxicity

    Institute of Scientific and Technical Information of China (English)

    Yong-fei WU; Sheng-bin LI

    2005-01-01

    Aim: To investigate neuropeptide Y (NPY) expression in mouse hippocampus within early stages of kainic acid (KA) treatment and to understand its role in neuronal excitotoxicity. Methods: NPY expression in the hippocampus within early stages of KA intraperitoneal (ip) treatment was detected by immunohistochemistry (IHC) and in situ hybridization (ISH) methods. The role of NPY and Y5, Y2 receptors in excitotoxicity was analyzed by terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) assay. Results: Using IHC assay, in granule cell layer of the dentate gyrus (DG), NPY positive signals appeared 4 h after KA injection, reached the peak at 8 h and leveled off at 16 and 24 h. In CA3, no positive signal was found within the first 4 h after KA injection,but strong signal appeared at 16 and 24 h. No noticeable signal was detected in CA1 at all time points after KA injection. Using the ISH method, positive signals were detected at 4, 8, and 16 h in CA3, CA1, and hilus. In DG, much stronger ISH signals were detected at 4 h, but leveled off at 8 and 16 h. TUNEL analysis showed that intracerebroventricularly (icv) infusion of NPY and Y5, Y2 receptor agonists within 8 h after KA insult with proper dose could remarkably rescue pyramidal neurons in CA3 and CA1 from apoptosis. Conclusion: NPY is an important anti-epileptic agent. The preceding elevated expression of NPY in granule cell layer of DG after KA injection might partially explain its different excitotoxicity-induced apoptotic responses in comparison with the pyramidal neurons from CA3 and CA1 regions. NPY can not only reduce neuronal excitability but also prevent excitotoxicity-induced neuronal apoptosis in a time- and doserelated way by activation of Y5 and Y2 receptors.

  11. Expression of fragile histidine triad in primary hepatocellular carcinoma and its relation with cell proliferation and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ke-Jun Nan; Zhi-Ping Ruan; Zhao Jing; Hai-Xia Qin; Hong-Yan Wang; Hui Guo; Rui Xu

    2005-01-01

    AIM: To evaluate the expression of fragile histidine triad (FHIT) gene protein, product of a candidate tumor suppressor,and to investigate the relationship between FHIT, cell apoptosis and proliferation, and pathological features of primary hepatocellular carcinoma (HCC).METHODS: Forty-seven HCC and ten normal liver specimens were collected during surgical operation between 2001and 2003. FHIT and proliferating cell nuclear antigen (PCNA)expression were detected by immunohistochemistry, and apoptotic level was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay on the tissue sections.RESULTS: All normal liver tissues showed a strong expression of FHIT, whereas 28 of 47 (59.6%) carcinomas showed a significant loss or absence of FHIT expression (P = 0.001).The proportion of reduced FHIT expression in those carcinomas at stages Ⅲ-Ⅳ (70.6%) and in those with extrahepatic metastasis (86.7%) showed an increasing trend compared with those at stages Ⅰ-Ⅱ (30.8%, P= 0.013) and those without metastasis (46.9%, P = 0.010) respectively. Apoptotic incidence in advanced TNM stage carcinoma and those with positive FHIT expression was higher than that in early stage carcinoma (P = 0.030) and in those with negative FHIT expression (P = 0.044) respectively. The proliferating potential of hepatocellular carcinoma was associated with FHIT expression (P = 0.016) and the aggressive feature (P = 0.019). Kaplan-Meier analysis demonstrated that the survival time of these 47 patients correlated with TNM stage,FHIT expression and metastasis.CONCLUSION: There is marked loss or absence of FHIT expression, as well as abnormal apoptosis-proliferation balance in HCC. FHIT may play an important role in carcinogenesis and development of HCC.

  12. Influence of Light Emitting Diode-Derived Blue Light Overexposure on Mouse Ocular Surface.

    Science.gov (United States)

    Lee, Hyo Seok; Cui, Lian; Li, Ying; Choi, Ji Suk; Choi, Joo-Hee; Li, Zhengri; Kim, Ga Eon; Choi, Won; Yoon, Kyung Chul

    2016-01-01

    To investigate the influence of overexposure to light emitting diode (LED)-derived light with various wavelengths on mouse ocular surface. LEDs with various wavelengths were used to irradiate C57BL/6 mice at an energy dose of 50 J/cm2, twice a day, for 10 consecutive days. The red, green, and blue groups represented wavelengths of 630 nm, 525 nm, and 410 nm, respectively. The untouched group (UT) was not exposed to LED light and served as the untreated control. Tear volume, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured on days 1, 3, 5, 7, and 10. Levels of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in the cornea and conjunctiva using a multiplex immunobead assay at day 10. Levels of malondialdehyde (MDA) were measured with an enzyme-linked immunosorbent assay. Flow cytometry, 2'7'-dichlorofluorescein diacetate (DCF-DA) assay, histologic analysis, immunohistochemistry with 4-hydroxynonenal, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining were also performed. TBUT of the blue group showed significant decreases at days 7 and 10, compared with the UT and red groups. Corneal fluorescein staining scores significantly increased in the blue group when compared with UT, red, and green groups at days 5, 7, and 10. A significant increase in the corneal levels of IL-1β and IL-6 was observed in the blue group, compared with the other groups. The blue group showed significantly increased reactive oxygen species production in the DCF-DA assay and increased inflammatory T cells in the flow cytometry. A significantly increased TUNEL positive cells was identified in the blue group. Overexposure to blue light with short wavelengths can induce oxidative damage and apoptosis to the cornea, which may manifest as increased ocular surface inflammation and resultant dry eye.

  13. The effects of exposure to electromagnetic field on rat myocardium.

    Science.gov (United States)

    Kiray, Amac; Tayefi, Hamid; Kiray, Muge; Bagriyanik, Husnu Alper; Pekcetin, Cetin; Ergur, Bekir Ugur; Ozogul, Candan

    2013-06-01

    Exposure to electromagnetic fields (EMFs) causes increased adverse effects on biological systems. The aim of this study was to investigate the effects of EMF on heart tissue by biochemical and histomorphological evaluations in EMF-exposed adult rats. In this study, 28 male Wistar rats weighing 200-250 g were used. The rats were divided into two groups: sham group (n = 14) and EMF group (n = 14). Rats in sham group were exposed to same conditions as the EMF group except the exposure to EMF. Rats in EMF group were exposed to a 50-Hz EMF of 3 mT for 4 h/day and 7 days/week for 2 months. After 2 months of exposure, rats were killed; the hearts were excised and evaluated. Determination of oxidative stress parameters was performed spectrophotometrically. To detect apoptotic cells, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and caspase-3 immunohistochemistry were performed. In EMF-exposed group, levels of lipid peroxidation significantly increased and activities of superoxide dismutase and glutathione peroxidase decreased compared with sham group. The number of TUNEL-positive cells and caspase-3 immunoreactivity increased in EMF-exposed rats compared with sham. Under electron microscopy, there were mitochondrial degeneration, reduction in myofibrils, dilated sarcoplasmic reticulum and perinuclear vacuolization in EMF-exposed rats. In conclusion, the results show that the exposure to EMF causes oxidative stress, apoptosis and morphologic damage in myocardium of adult rats. The results of our study indicate that EMF-related changes in rat myocardium could be the result of increased oxidative stress. Further studies are needed to demonstrate whether the exposure to EMF can induce adverse effects on myocardium.

  14. Down-regulation of Sonic hedgehog signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells

    Institute of Scientific and Technical Information of China (English)

    Qiyu Wang; Shuhong Huang; Ling Yang; Ling Zhao; Yuxia Yin; Zhongzhen Liu; Zheyu Chen; Hongwei Zhang

    2008-01-01

    The Sonic hedgehog (SHh) pathway plays a critical role in normal embryogenesis and carcinogenesis, but its function in cancer cells treated with 5-fluorouracil (5-FU) remains unknown. We examined the expression of a subset of SHh signaling pathway genes, including SHh, SMO, PTC1, Su(Fu) and HIP in human hepatocellular carcinoma (HCC) cell lines,Hep3B and HepG2, treated with 5-FU by reverse transcriptionpolymerase chain reaction. Using trypan blue analysis,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, we also detected the apoptosis of Hep3B cells resulting from the transfection of pCS2-Gli1 expression vector combined with 5-FU treatment.The motility of the cells was detected by scratch wound closure assay. The expression and subcellular location of PTC1 protein in Hep3B cells treated by 5-FU were also investigated by Western blot analysis and immunofluorescent microscopy. The results indicated that the expression of SHh pathway target molecules at both messenger RNA and protein levels are evidently down-regulated in Hep3B cells treated with 5-FU. The overexpression of Gli1 restores cell viability and, to some extent, the migration abilities inhibited by 5-FU.Furthermore, 5-FU treatment affects the subcellular localization of PTC1 protein, a key member in SHh signaling pathway. Our data showed that the down-regulation of SHh signaling pathway activity was involved in 5-FU-induced apoptosis and the inhibition of motility in hedgehog-activated HCC cell lines. This implies that the combination of SHh signaling pathway inhibitor and 5-FU-based chemotherapy might represent a more promising strategy against HCC.

  15. Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa.

    Science.gov (United States)

    Domínguez-Rebolledo, A E; Fernández-Santos, M R; García-Alvarez, O; Maroto-Morales, A; Garde, J J; Martínez-Pastor, F

    2009-11-01

    The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 x g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1mM of the antioxidant Trolox, 100 microM of the oxidant Fe (with ascorbate), or both. The 3x4 treatments were incubated at 37 degrees C and assessed each hour up to 3h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4h was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30+/-3.52% to 67.94+/-5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.

  16. Pre- and Posttreatment With Edaravone Protects CA1 Hippocampus and Enhances Neurogenesis in the Subgranular Zone of Dentate Gyrus After Transient Global Cerebral Ischemia in Rats

    Directory of Open Access Journals (Sweden)

    Shan Lei

    2014-11-01

    Full Text Available Edaravone is clinically used for treatment of patients with acute cerebral infarction. However, the effect of double application of edaravone on neurogenesis in the hippocampus following ischemia remains unknown. In the present study, we explored whether pre- and posttreatment of edaravone had any effect on neural stem/progenitor cells (NSPCs in the subgranular zone of hippocampus in a rat model of transient global cerebral ischemia and elucidated the potential mechanism of its effects. Male Sprague-Dawley rats were divided into three groups: sham-operated (n = 15, control (n = 15, and edaravone-treated (n = 15 groups. Newly generated cells were labeled by 5-bromo-2-deoxyuridine. Immunohistochemistry was used to detect neurogenesis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling was used to detect cell apoptosis. Reactive oxygen species (ROS were detected by 2,7-dichlorofluorescien diacetate assay in NSPCs in vitro. Hypoxia-inducible factor-1α (HIF-1α and cleaved caspase-3 proteins were quantified by western blot analysis. Treatment with edaravone significantly increased the number of NSPCs and newly generated neurons in the subgranular zone (p < .05. Treatment with edaravone also decreased apoptosis of NSPCs (p < .01. Furthermore, treatment with edaravone significantly decreased ROS generation and inhibited HIF-1α and cleaved caspase-3 protein expressions. These findings indicate that pre- and posttreatment with edaravone enhances neurogenesis by protecting NSPCs from apoptosis in the hippocampus, which is probably mediated by decreasing ROS generation and inhibiting protein expressions of HIF-1α and cleaved caspase-3 after cerebral ischemia.

  17. Bioflavonoids Effects of Ginger on Glomerular Podocyte Apoptosis in Streptozotocin-Induced Diabetic Rat

    Directory of Open Access Journals (Sweden)

    Hajhosieni Laleh

    2014-04-01

    Full Text Available Objective: Ginger is a strong antioxidant and long-term treatment of streptozotocin (STZ-diabetic animals, and it has been shown to reduce oxidative stress. Prevalence oxidative stress among urban life and changes in antioxidant capacity are considered asplay an important role in the pathogenesis of chronic diabetes mellitus. Materials and Methods: Wistar male rat (n = 40 were divided into three groups, control group (n = 10 and Ginger Quercetin group that received 100 mg/kg (gavage, (n = 10, and diabetic group, which received 55 mg/kg intra peritoneal (IP STZ (n = 20, which was subdivided to two groups of 10; STZ group and treatment group. Treatment group received 55 mg/kg (IP STZ plus100 mg/kg ginger, daily for, 8 weeks, respectively; however, the control group just received an equal volume of distilled water daily (IP. Diabetes was induced by a single (IP injection of STZ (55 mg/kg. Animals were kept in standard condition. In 28 day after inducing diabetic 5 cc blood were collected for total antioxidant capacity, malondialdehyde and oxidized low density lipoprotein levels and kidney tissues of rat in whole groups were removed then prepared for apoptosis analysis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay (TUNEL method. Results: Apoptotic cells significantly decreased in group that has received 100 mg/kg ginger (P < 0.05 in comparison to experimental groups (P < 0.05. Conclusion: Since in our study 100 mg/kg ginger have significantly preventive effect on kidney cells damages by reducing number of apoptotic cells in kidney and hence it seems that using it can be effective for treatment in diabetic rat.

  18. Treadmill exercise ameliorates symptoms of Alzheimer disease through suppressing microglial activation-induced apoptosis in rats

    Science.gov (United States)

    Baek, Seung-Soo; Kim, Sang-Hoon

    2016-01-01

    Alzheimer disease (AD) is a most common form of dementia and eventually causes impairments of learning ability and memory function. In the present study, we investigated the effects of treadmill exercise on the symptoms of AD focusing on the microglial activation-induced apoptosis. AD was made by bilateral intracerebroventricular injection of streptozotocin. The rats in the exercise groups were made to run on a treadmill once a day for 30 min during 4 weeks. The distance and latency in the Morris water maze task and the latency in the step-down avoidance task were increased in the AD rats, in contrast, treadmill exercise shortened these parameters. The numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive and caspase-3-positive cells in the hippocampal dentate gyrus were decreased in the AD rats, in contrast, treadmill exercise suppressed these numbers. Expressions of glial fibrillary acidic protein (GFAP) and cluster of differentiation molecule 11B (CD11b) in the hippocampal dentate gyrus were increased in the AD rats, in contrast, treadmill exercise suppressed GFAP and CD11b expressions. Bax expression was increased and Bcl-2 expression was decreased in the hippocampus of AD rats, in contrast, treadmill exercise decreased Bax expression and increased Bcl-2 expression. The present results demonstrated that treadmill exercise ameliorated AD-induced impairments of spatial learning ability and short-term memory through suppressing apoptosis. The antiapoptotic effect of treadmill exercise might be ascribed to the inhibitory effect of treadmill exercise on microglial activation. PMID:28119873

  19. The neuroprotective role and mechanisms of TERT in neurons with oxygen-glucose deprivation.

    Science.gov (United States)

    Li, J; Qu, Y; Chen, D; Zhang, L; Zhao, F; Luo, L; Pan, L; Hua, J; Mu, D

    2013-11-12

    Telomerase reverse transcriptase (TERT) is reported to protect neurons from apoptosis induced by various stresses including hypoxia-ischemia (HI). However, the mechanisms by which TERT exerts its anti-apoptotic role in neurons with HI injury remain unclear. In this study, we examined the protective role and explored the possible mechanisms of TERT in neurons with HI injury in vitro. Primary cultured neurons were exposed to oxygen and glucose deprivation (OGD) for 3h followed by reperfusion to mimic HI injury in vivo. Plasmids containing TERT antisense, sense nucleotides, or mock were transduced into neurons at 48h before OGD. Expression and distribution of TERT were measured by immunofluorescence labeling and western blot. The expression of cleaved caspase 3 (CC3), Bcl-2 and Bax were detected by western blot. Neuronal apoptosis was measured with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The mitochondrial reactive oxygen species (ROS) were measured by MitoSOX Red staining. Fluorescent probe JC-1 was used to measure the mitochondrial membrane potential (ΔΨm). We found that TERT expression increased at 8h and peaked at 24h in neurons after OGD. CC3 expression and neuronal apoptosis were induced and peaked at 24h after OGD. TERT inhibition significantly increased CC3 expression and neuronal apoptosis after OGD treatment. Additionally, TERT inhibition decreased the expression ratio of Bcl-2/Bax, and enhanced ROS production and ΔΨm dissipation after OGD. These data suggest that TERT plays a neuroprotective role via anti-apoptosis in neurons after OGD. The underlying mechanisms may be associated with regulating Bcl-2/Bax expression ratio, attenuating ROS generation, and increasing mitochondrial membrane potential.

  20. Simultaneous detection and semiquantification of DNA damage in normal and apoptotic cells: triple-immunofluorescent labeling using DAPI, antibodies, and TUNEL.

    Science.gov (United States)

    Agrawal, Anant; Godar, Dianne E

    2012-07-01

    We developed a triple-labeling immunofluorescence technique that simultaneously identifies total DNA (DAPI), DNA damage (antibodies), and dead cells [terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells] and a method that semiquantifies DNA damage in paraffin-embedded tissues. Using this technique in combination with our analysis method, scientists can now simultaneously detect and compare the relative amounts of DNA damage of almost any kind (except single-strand and double-strand breaks), using indirect fluorescent antibody labeling, in both normal and dying cells of different tissues. Simultaneous labeling of DNA damage and dead or TUNEL-positive cells can reduce processing costs and analysis time, and can lead to discoveries concerning how cells die from different DNA damages. We used increasing doses of UV (290 to 400 nm) radiation to create DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts that kill some of the cells in 3-dimensional tissue-engineered skin and vaginal samples. We describe a protocol that reliably detects and semiquantifies DNA damage in both normal and apoptotic cells. We show this triple-labeling immunofluorescence technique and analysis method yields linear UV dose response curves for damage to DNA bases that allows semiquantification of cyclobutane pyrimidine dimers and calculation of its repair rate (T=1 and 24 h), whereas TUNEL allows quantification of the number of apoptotic cells. Scientists can now create beautiful fluorescent pictures that simultaneously detect DNA damage in both normal and apoptotic cells to assess and semiquantify the damage to understand better how different insults lead to the cell's demise.

  1. Is thioredoxin reductase involved in the defense against DNA fragmentation in varicocele?

    Institute of Scientific and Technical Information of China (English)

    Gül (o)zdemirler Erata; Canan Kü(c)ükgergin; Gülsan Aktan; Ates Kadioglu; Müjdat Uysal; Necla Ko(c)ak-Toker

    2013-01-01

    We aimed to investigate the role of thioredoxin reductase (TR) and inducible heat shock protein 70 (iHsp70) and their relationship with sperm quality in varicocele (VAR) patients.Semen samples were obtained from 16 subfertile men diagnosed as VAR and 10 fertile men who applied to the Andrology Laboratory of Istanbul Medical Faculty of Istanbul University.The sperm TR and iHsp 70 expression levels were determined using Western blot analysis.The TR activity of the sperm was assayed spectrophometrically.The sperm quality was evaluated both by conventional sperm analysis and by a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique that assayed DNA-fragmented spermatozoa in semen samples.The percentage of TUNEL-positive spermatozoa in the VAR group (16.3%±5.6%) was higher than that in the fertile group (5.5%± 1.9%).Significant inverse correlations were detected between the percentage of TUNEL-positive cells and both the concentration (r=-0.609; P=0.001) and motility (r=-0.550;P=0.004) of spermatozoa.Both the TR expression and activity were increased significantly in the VAR group (U=22.0; P=0.001 and U=33.5; P=0.012,respectively)as analyzed usingthe Mann-Whitney UWilcoxon rank sum Wtest.Furthermore,significant positive correlations were found between TR expression and activity (r=0.406; P=0.040) and between TR expression and the percentage of TUNEL-positive cells (r=0.665; P=0.001).Sperm iHsp70 expression did not differ between the VAR and fertile groups.In conclusion,increased sperm TR expression might be a defense mechanism against apoptosis in the spermatozoa of men with VAR.

  2. Electroacupuncture preconditioning and postconditioning inhibit apoptosis and neuroinflammation induced by spinal cord ischemia reperfusion injury through enhancing autophagy in rats.

    Science.gov (United States)

    Fang, Bo; Qin, Meiman; Li, Yun; Li, Xiaoqian; Tan, Wenfei; Zhang, Ying; Ma, Hong

    2017-03-06

    Electroacupuncture (EA) has beneficial effects on spinal cord ischemia reperfusion (I/R) injury, but the underlying mechanisms are not fully understood. This study aimed to investigate the role of autophagy in the protection of EA preconditioning and postconditioning against spinal cord I/R injury. For this, spinal cord I/R injury was induced by 14min occlusion of the aortic arch, and rats were treated with EA for 20min before or after the surgery. The expression of autophagy components, light chain 3 and Beclin 1, was assessed by Western blot. The hind-limb motor function was assessed using the Basso-Beattie-Bresnahan (BBB) criteria, and motor neurons in the ventral gray matter were counted by histological examination. The apoptosis of neurocyte was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and matrix metalloproteinase-9 (MMP-9) was also measured using Western blot or enzyme-linked immunosorbent assay (ELISA). Either EA preconditioning or postconditioning enhanced autophagy, and minimized the neuromotor dysfunction and histopathological deficits after spinal cord I/R injury. In addition, EA suppressed I/R-induced apoptosis and increased in the expression of TNF-α, IL-1β, and MMP-9. In contrast, the autophagic inhibitor (3-methyladenine, 3-MA) inhibited the neuroprotective effects of EA. Moreover, 3-MA increased the apoptosis and the expression of TNF-α, IL-1β, and MMP-9. In summary, these findings suggested that EA preconditioning and postconditioning could alleviate spinal cord I/R injury, which was partly mediated by autophagy upregulation-induced inhibition of apoptosis and neuroinflammation.

  3. Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase

    Institute of Scientific and Technical Information of China (English)

    Song Tusheng; Yang Ling; Huang Chen; Liu Liying; Ni Lei; Wang Aiying; Luo Yu

    2007-01-01

    Objective To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods Human leukemia cell line K562 were exposed to indole-3-acetic acid (IAA) at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase(HRP) at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate (DCFH-DA) uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. Results IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9%, which was 5 times that of control(P<0.01). Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion The combination of IAA and HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.

  4. Human thioredoxin exerts cardioprotective effect and attenuates reperfusion injury in rats partially via inhibiting apoptosis

    Institute of Scientific and Technical Information of China (English)

    WU Xiao-wei; TENG Zong-yan; JIANG Li-hong; FAN Ying; ZHANG Yu-hua; LI Xiu-rong; ZHANG Yi-na

    2008-01-01

    Background Thioredoxin is one of the most important redox regulating proteins. Although thioredoxin has been shown to protect cells against different kinds of oxidative stress, the role of thioredoxin in myocardial ischemia and reperfusion injury has not been fully understood. This study was conducted to explore the protective role of human thioredoxin on myocardial ischemia and reperfusion injury and its potential mechanisms.Methods Purified human thioredoxin was injected into adult Wister rats, which were subjected to 30 minutes of myocardial ischemia followed by 2 or 24 hours of reperfusion. We detected 1) the infarct size; 2) the level of malondisldehyde (MDA) in serum; 3) the expression of caspase-9, and cytochrome c in/out of mitochondia by Western blotting; 4) apoptosis by terminal-deoxynucleotidyl transferase mediated nick end labeling ('rUNEL) assay and caspase-3 and its protein by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting; 5) the expression of bcl-2 and bax in cardium by immunohistochemical (IHC) assay.Results Human thioredoxin reduced myocardial ischemia/reperfusion injury as evidenced by significant decrease of myocardial infarct size (P<0.01), notable reduction of myocyte apoptosis (P <0.01), lower systemic oxidative stress level (P <0.01) after reperfusion for 2 hours, and few inflammatory cell infiltration after reperfusion for 24 hours in rats. Furthermore, treatment with human thioredoxin significantly reduced the release of mitochondrial cytochrome C (P<0.05),and inhibited the activity of caspase-9 (P <0.05) and caspase-3 (P <0.01 in mRNA and P <0.05 at protein level).Meanwhile, human thioredoxin markedly increased bcl-2 expression (P <0.05).Conclusions These results strongly suggest that human thioredoxin has cardioprotective effects on myocardial ischemia/reperfusion and its anti-apoptotic role may be mediated by modulating bcl-2 and the mitochondria-dependent apoptotic signaling pathway.

  5. Effect of dexmedetomidine on hippocampal neuron development and BDNF-TrkB signal expression in neonatal rats

    Science.gov (United States)

    Lv, Jie; Ou, Wei; Zou, Xiao-Hua; Yao, Yin; Wu, Jin-Li

    2016-01-01

    The study aimed to explore the effect of dexmedetomidine (DEX) on hippocampal neuron development process and on molecular expression of brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signaling pathway in neonatal rats. The hippocampal neuron cells were isolated from newborn neonatal rats and cultured in vitro. One control group and three treated groups with 1, 10, and 100 μmol/L DEX were used for the study. Cell activity and apoptosis were detected by the MTT and terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate (UTP) nick end labeling assays. The synaptophysin (SYN) and postsynaptic density 95 (PSD95) were detected by quantitative polymerase chain reaction. There was no difference in the viability of neuron cells among the different dose groups of DEX and the control group during days 2–10 (P>0.05). Compared to the control group, there was no significant difference (P>0.05) in the expressions of SYN and PSD95 in the groups treated with 1 and 10 μmol/L DEX, whereas significant difference in the expression was observed in the group treated with 100 μmol/L DEX (PBDNF was significantly upregulated (PTrkB expression among the four groups. The expression of p-N-methyl-D-aspartate receptor increased with an increase in the concentration of DEX; however, only the high dose revealed a significant upregulation compared with the control group. The neuroprotective effect of DEX may be achieved by upregulating the expression of BDNF and phosphorylation level of N-methyl-D-aspartate receptor. PMID:28003751

  6. In vitro antitumor properties of an isolate from leaves of Cassia alata L.

    Science.gov (United States)

    Olarte, Elizabeth Iglesias; Herrera, Annabelle Aliga; Villaseñor, Irene Manese; Jacinto, Sonia Donaldo

    2013-01-01

    Leaf extracts of Cassia alata L (akapulko), traditionally used for treatment of a variety of diseases, were evaluated for their potential antitumor properties in vitro. MTT assays were used to examine the cytotoxic effects of crude extracts on five human cancer cell lines, namely MCF-7, derived from a breast carcinoma, SK-BR-3, another breast carcinoma, T24 a bladder carcinoma, Col 2, a colorectal carcinoma, and A549, a non- small cell lung adenocarcinoma. Hexane extracts showed remarkable cytotoxicity against MCF-7, T24, and Col 2 in a dose-dependent manner. This observation was confirmed by morphological investigation using light microscopy. Further bioassay-directed fractionation of the cytotoxic extract led to the isolation of a TLC-pure isolate labeled as f6l. Isolate f6l was further evaluated using MTT assay and morphological and biochemical investigations, which likewise showed selectivity to MCF-7, T24, and Col 2 cells with IC50 values of 16, 17, and 17 μg/ml, respectively. Isolate f6l, however, showed no cytotoxicity towards the non-cancer Chinese hamster ovarian cell line (CHO-AA8). Cytochemical investigation using DAPI staining and biochemical investigation using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-a method used to detect DNA fragmentation-together with caspase assay, demonstrated apoptotic cell death. Spectral characterization of isolate f6l revealed that it contained polyunsaturated fatty acid esters. Considering the cytotoxicity profile and its mode of action, f6l might represent a new promising compound with potential for development as an anticancer drug with low or no toxicity to non-cancer cells used in this study.

  7. Cytotoxic Effects of GM1 Ganglioside and Amyloid β-Peptide on Mouse Embryonic Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Makoto Yanagisawa

    2010-01-01

    Full Text Available AD (Alzheimer's disease is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aβs (amyloid β-peptides in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between GM1 ganglioside and Aβs. Several reports have documented the bifunctional roles of Aβs in NSCs (neural stem cells, but the precise effects of GM1 and Aβ on NSCs have not yet been clarified. We evaluated the effect of GM1 and Aβ-(1–40 on mouse NECs (neuroepithelial cells, which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either GM1 or Aβ-(1–40. On the contrary, a decreased number of NECs were cultured in the presence of a combination of GM1 and Aβ-(1–40. The exogenously added GM1 and Aβ-(1–40 were confirmed to incorporate into NECs. The Ras–MAPK (mitogen-activated protein kinase pathway, important for cell proliferation, was intact in NECs simultaneously treated with GM1 and Aβ-(1–40, but caspase 3 was activated. NECs treated with GM1 and Aβ-(1–40 were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, an indicator of cell death. It was found that GM1 and Aβ-(1–40 interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aβ-(1–40 and GM1 co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.

  8. Friend or foe? Effect of oral resveratrol on cisplatin ototoxicity.

    Science.gov (United States)

    Olgun, Yüksel; Kırkım, Günay; Kolatan, Efsun; Kıray, Müge; Bagrıyanık, Alper; Olgun, Aybüke; Kızmazoglu, Deniz Cakır; Ellıdokuz, Hülya; Serbetcıoglu, Bulent; Altun, Zekiye; Aktas, Safiye; Yılmaz, Osman; Günerı, Enis Alpin

    2014-03-01

    Our objectives were to study effects of orally administered resveratrol (RV) against cisplatin (CDDP) ototoxicity in different doses and to investigate ultrastructural changes in the cochlea and brainstem. In vivo study using an animal model. Thirty-two male Wistar albino rats were divided into six groups. Baseline distortion product otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) measurements were made. In groups I, II, and III, only saline, RV, and CDDP were given, respectively. Group IV, V, and VI animals were administered 10 mg/kg/day, 1 mg/kg/day, and 0.1 mg/kg/day of RV for 10 days, respectively, before 16 mg/kg CDDP injections were administered on day 11. All animals were sacrificed after repeated DPOAEs and ABR measurements were made on day 14. Cochleas of animals were investigated with transmission electron microscopy. Apoptosis were investigated with caspase-3 activity and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method in the brainstem. In groups IV and V, DPOAEs and ABR findings revealed that oral administration of RV 10 mg/kg/day and 1 mg/kg/day doses before CDDP injection enhanced ototoxicity. In group VI, electomicroscopy revealed better ultrastructural findings than in the cisplatin group; however, these changes were not reflected in the audiological findings accordingly. Our results implied that there were noticeable differences between different oral RV doses used for cisplatin ototoxicity. Especially in higher doses, RV was observed to enhance cisplatin ototoxicity. © 2013 The American Laryngological, Rhinological and Otological Society, Inc.

  9. Antitumor effect of malaria parasite infection in a murine Lewis lung cancer model through induction of innate and adaptive immunity.

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    Lili Chen

    Full Text Available BACKGROUND: Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. METHODOLOGY/PRINCIPAL FINDINGS: Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL staining and decreased Ki-67 expression in tumors. Through natural killer (NK cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8(+ T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. CONCLUSIONS/SIGNIFICANCE: Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria

  10. Hydrogen-rich saline ameliorates the severity of L-arginine-induced acute pancreatitis in rats

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Han; Sun, Yan Ping; Li, Yang [Department of General Surgery, Shanghai Chang Zheng Hospital, Second Military Medical University, Shanghai 200003 (China); Liu, Wen Wu [Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433 (China); Xiang, Hong Gang [Department of General Surgery, Shanghai Chang Zheng Hospital, Second Military Medical University, Shanghai 200003 (China); Fan, Lie Ying [Department of Clinical Laboratory, Shanghai East Hospital, Tong Ji University, Shanghai 200120 (China); Sun, Qiang [Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433 (China); Xu, Xin Yun [Department of General Surgery, Shanghai Chang Zheng Hospital, Second Military Medical University, Shanghai 200003 (China); Cai, Jian Mei [Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433 (China); Ruan, Can Ping; Su, Ning; Yan, Rong Lin [Department of General Surgery, Shanghai Chang Zheng Hospital, Second Military Medical University, Shanghai 200003 (China); Sun, Xue Jun, E-mail: sunxjk@hotmail.com [Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433 (China); Wang, Qiang, E-mail: wang2929@hotmail.com [Department of General Surgery, Shanghai Chang Zheng Hospital, Second Military Medical University, Shanghai 200003 (China)

    2010-03-05

    Molecular hydrogen, which reacts with the hydroxyl radical, has been considered as a novel antioxidant. Here, we evaluated the protective effects of hydrogen-rich saline on the L-arginine (L-Arg)-induced acute pancreatitis (AP). AP was induced in Sprague-Dawley rats by giving two intraperitoneal injections of L-Arg, each at concentrations of 250 mg/100 g body weight, with an interval of 1 h. Hydrogen-rich saline (>0.6 mM, 6 ml/kg) or saline (6 ml/kg) was administered, respectively, via tail vein 15 min after each L-Arg administration. Severity of AP was assessed by analysis of serum amylase activity, pancreatic water content and histology. Samples of pancreas were taken for measuring malondialdehyde and myeloperoxidase. Apoptosis in pancreatic acinar cell was determined with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL). Expression of proliferating cell nuclear antigen (PCNA) and nuclear factor kappa B (NF-{kappa}B) were detected with immunohistochemistry. Hydrogen-rich saline treatment significantly attenuated the severity of L-Arg-induced AP by ameliorating the increased serum amylase activity, inhibiting neutrophil infiltration, lipid oxidation and pancreatic tissue edema. Moreover, hydrogen-rich saline treatment could promote acinar cell proliferation, inhibit apoptosis and NF-{kappa}B activation. These results indicate that hydrogen treatment has a protective effect against AP, and the effect is possibly due to its ability to inhibit oxidative stress, apoptosis, NF-{kappa}B activation and to promote acinar cell proliferation.

  11. In vitro and in vivo human herpesvirus 8 infection of placenta.

    Directory of Open Access Journals (Sweden)

    Mariantonietta Di Stefano

    Full Text Available Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8 infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.

  12. Human equilibrative nucleoside transporter 1 and carcinoma of the ampulla of Vater: expression differences in tumour histotypes

    Directory of Open Access Journals (Sweden)

    G. Perrone

    2010-09-01

    Full Text Available The human equilibrative nucleoside transporter 1 (hENT1 is the major means by which gemcitabine enters human cells; recent evidence exists that hENT1 is expressed in carcinoma of the ampulla of Vater and that it should be considered as a molecular prognostic marker for patients with resected ampullary cancer. Aim of the present study is to evaluate the variations of hENT1 expression in ampullary carcinomas and to correlate such variations with histological subtypes and clinicopathological parameters. Forty-one ampullary carcinomas were histologically classified into intestinal, pancreaticobiliary and unusual types. hENT1 and Ki67 expression were evaluated by immunohistochemistry, and apoptotic cells were identified by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL method. hENT1 overexpression was detected in 63.4% ampullary carcinomas. A significant difference in terms of hENT1 and Ki67 expression was found between intestinal vs. pancreaticobiliary types (P=0.03 and P=0.009 respectively. Moreover, a significant statistical positive correlation was found between apoptotic and proliferative Index (P=0.036, while no significant correlation was found between hENT1 and apoptosis. Our results on hENT1 expression suggest that classification of ampullary carcinoma by morphological subtypes may represent an additional tool in prospective clinical trials aimed at examining treatment efficacy; in addition, data obtained from Ki67 and TUNEL suggest a key role of hENT1 in tumour growth of ampullary carcinoma.

  13. Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer.

    LENUS (Irish Health Repository)

    Bennett, M W

    2012-02-03

    BACKGROUND: Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. AIM: To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. SPECIMENS: Thirty paraffin wax embedded human gastric adenocarcinomas. METHODS: FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). RESULTS: Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. CONCLUSIONS: Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.

  14. The Fas counterattack in vivo: apoptotic depletion of tumor-infiltrating lymphocytes associated with Fas ligand expression by human esophageal carcinoma.

    LENUS (Irish Health Repository)

    Bennett, M W

    2012-02-03

    Various cancer cell lines express Fas ligand (FasL) and can kill lymphoid cells by Fas-mediated apoptosis in vitro. FasL expression has been demonstrated in several human malignancies in vivo. We sought to determine whether human esophageal carcinomas express FasL, and whether FasL expression is associated with increased apoptosis of tumor-infiltrating lymphocytes (TIL) in vivo, thereby contributing to the immune privilege of the tumor. Using in situ hybridization and immunohistochemistry, respectively, FasL mRNA and protein were colocalized to neoplastic esophageal epithelial cells in all esophageal carcinomas (squamous, n = 6; adenocarcinoma, n = 2). The Extent of FasL expression was variable, with both FasL-positive and FasL-negative neoplastic regions occurring within tumors. TIL were detected by immunohistochemical staining for the leukocyte common Ag, CD45. FasL expression was associated with a mean fourfold depletion of TIL when compared with FasL-negative areas within the same tumors (range 1.6- to 12-fold, n = 6,p < 0.05). Cell death of TIL was detected by dual staining of CD45 (immunohistochemistry) and DNA strand breaks (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). There was a mean twofold increase in detectable cell death among TIL in FasL-positive areas compared with FasL-negative areas (range 1.6- to 2.4-fold, n = 6, p < 0.05). In conclusion, we demonstrate a statistically significant, quantitative reduction of TIL concomitant with significantly increased TIL apoptosis within FasL-expressing areas of esophageal tumors. Our findings suggest Fas-mediated apoptotic depletion of TIL in response to FasL expression by esophageal cancers, and provide the first direct, quantitative evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in human cancer.

  15. Assessment of Retrograde Coronary Venous Infusion of Mesenchymal Stem Cells Combined with Basic Fibroblast Growth Factor in Canine Myocardial Infarction Using Strain Values Derived from Speckle-Tracking Echocardiography.

    Science.gov (United States)

    Sun, Qi-Wei; Zhen, Lei; Wang, Qin; Sun, Yan; Yang, Jiao; Li, Yi-Jia; Li, Rong-Juan; Ma, Ning; Li, Zhi-An; Wang, Lu-Ya; Nie, Shao-Ping; Yang, Ya

    2016-01-01

    Speckle-tracking echocardiography was used to assess retrograde coronary venous infusion of mesenchymal stem cells (MSCs) combined with basic fibroblast growth factor (bFGF) in a canine model of acute myocardial infarction (AMI). AMI was induced by ligation of the left anterior descending coronary artery. Coronary venous retroperfusion was performed at 1 wk after AMI. Twenty-eight animals were randomized into four groups: saline, bFGF+saline, saline+MSCs and bFGF+MSCs. Echocardiography was performed before AMI, at 7 d post-AMI and 40 d after retroperfusion. Apoptotic cardiomyocytes in the border zone of the ischemic region were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Vascular endothelial growth factor and factor VIII concentrations were measured by western blotting. The left ventricular end-systolic volume increased significantly, whereas the left ventricular ejection fraction and global and segmental strain values decreased significantly after AMI. After retroperfusion, the strain values of the infarct zone, but not conventional echocardiographic parameters, were significantly different between control and bFGF+MSC groups. Cardiomyocyte apoptosis decreased, whereas vascular endothelial growth factor and factor VIII concentrations were higher in the bFGF+MSC, bFGF and MSC groups. Cardiomyocyte apoptosis was well correlated with the strain values. Although retrograde coronary venous infusion of bFGF and MSCs promoted neo-vascularization of the infarcted myocardium and inhibited apoptosis, there was only a slight strain improvement without a substantial increase in global cardiac functions.

  16. Programmed cell death in kiwifruit stigmatic arms and its relationship to the effective pollination period and the progamic phase.

    Science.gov (United States)

    Ferradás, Yolanda; López, Marián; Rey, Manuel; González, Ma Victoria

    2014-07-01

    Kiwifruit is a crop with a highly successful reproductive performance, which is impaired by the short effective pollination period of female flowers. This study investigates whether the degenerative processes observed in both pollinated and non-pollinated flowers after anthesis may be considered to be programmed cell death (PCD). Features of PCD in kiwifruit, Actinidia chinensis var. deliciosa, were studied in both non-pollinated and pollinated stigmatic arms using transmission electron microscopy, DAPI (4',6-diamidino-2-phenylindole) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assays, DNA gel electrophoresis and caspase-like activity assays. In the secretory tissues of the stigmatic arms, cell organelles disintegrated sequentially while progressive vacuolization was detected. At the same time, chromatin condensation, nuclear deformation, and DNA fragmentation and degradation were observed. These features were detected in both non-pollinated and pollinated stigmatic arms; they were evident in the stigmas of pollinated flowers by the second day after anthesis but only by 4 d after anthesis in non-pollinated flowers. In addition, in pollinated stigmatic arms, these features were first initiated in the stigma and gradually progressed through the style, consistent with pollen tube growth. This timing of events was also observed in both non-pollinated and pollinated stigmatic arms for caspase-3-like activity. The data provide evidence to support the hypothesis that PCD processes occurring in the secretory tissue of non-pollinated kiwifruit stigmatic arms could be the origin for the observed short effective pollination period. The results obtained in the secretory tissue of pollinated kiwifruit stigmatic arms upon pollination support the idea that PCD might be accelerated by pollination, pointing to the involvement of PCD during the progamic phase. © The Author 2014. Published by Oxford University Press on behalf of the

  17. Exploring the optimal operation time for patients with hypertensive intracerebral hemorrhage:tracking the expression and progress of cell apoptosis of prehematomal brain tissues

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xin-qing; ZHANG Zhi-min; YIN Xiao-liang; ZHANG Kun; CAI Hui; LING Feng

    2010-01-01

    Background Hypertensive intracerebral hemorrhage (HICH) is a severe disease with high morbidity and mortality.Timely removal of the hematoma through surgical procedures may effectively reduce secondary injuries.However, there has long been a debate over the proper timing of such surgery.In this study, we explored the optimal operation time for HICH patients by observing the pathological changes in perihematomal brain regions during different stages of onset.Methods Twenty-five specimens of brain tissue, obtained from perihematomal region of HICH patients in different phases, were subjected to haematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) staining and Caspase-3, matrix metalloproteinases-9 (MMP-9)immunohistochemical staining.The changing roles of necrosis and apoptosis and the expression of MMP-9 and Caspase-3 positive cells were all observed using image analysis.Results The obvious expression of TUNEL positive cells was recognized within 6 hours of ICH onset, reaching its peak between 6 hours and 24 hours in the early phase.Results were highly consistent with Caspase-3 and MMP-9 positive cell counts.Necrosis was found 6 hours after ICH onset and aggravated after 12 hours.Conclusions In the early phase, apoptosis was seen as a major modality of injury in the brain tissue of the perihematomal region and was strongly correlated with the expression of MMP-9 and Caspase-3.The results of the present study suggest that an operation performed as soon as possible after iCH onset may be optimal for preserving the nervous system function.

  18. Inhibition of human breast cancer xenograft growth by cruciferous vegetable constituent benzyl isothiocyanate.

    Science.gov (United States)

    Warin, Renaud; Xiao, Dong; Arlotti, Julie A; Bommareddy, Ajay; Singh, Shivendra V

    2010-05-01

    Benzyl isothiocyanate (BITC), a constituent of cruciferous vegetables such as garden cress, inhibits growth of human breast cancer cell lines in culture. The present study was undertaken to determine in vivo efficacy of BITC against MDA-MB-231 human breast cancer xenografts. The BITC administration retarded growth of MDA-MB-231 cells subcutaneously implanted in female nude mice without causing weight loss or any other side effects. The BITC-mediated suppression of MDA-MB-231 xenograft growth correlated with reduced cell proliferation as revealed by immunohistochemical analysis for Ki-67 expression. Analysis of the vasculature in the tumors from BITC-treated mice indicated smaller vessel area compared with control tumors based on immunohistochemistry for angiogenesis marker CD31. The BITC-mediated inhibition of angiogenesis in vivo correlated with downregulation of vascular endothelial growth factor (VEGF) receptor 2 protein levels in the tumor. Consistent with these results, BITC treatment suppressed VEGF secretion and VEGF receptor 2 protein levels in cultured MDA-MB-231 cells. Moreover, the BITC-treated MDA-MB-231 cells exhibited reduced capacity for migration compared with vehicle-treated control cells. In contrast to cellular data, BITC administration failed to elicit apoptotic response as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. In conclusion, the present study demonstrates in vivo anti-cancer efficacy of BITC against MDA-MB-231 xenografts in association with reduced cell proliferation and suppression of neovascularization. These preclinical observations merit clinical investigation to determine efficacy of BITC against human breast cancers.

  19. Colitogenic role of tumour necrosis factor (TNF) receptors in trinitrobenzene sulphonic acid colitis: TNF-R1 ablation does not affect systemic inflammatory response.

    Science.gov (United States)

    Yang, Y; Wang, H; Dou, Y; Wang, Y; Han, G; Wang, Renxi; Wang, L; Guo, R; Xiao, H; Li, X; Shen, B; Shi, Y; Chen, G; Li, Y

    2011-09-01

    Tumour necrosis factor (TNF)-α plays a critical role in the pathogenesis of T helper type 1-mediated colitis such as Crohn's disease. However, the roles of its two receptors in mediating pathology remain largely unknown. In this study, trinitrobenzene sulphonic acid (TNBS) was used to induce colitis in TNF-receptor single or double knock-out (DKO) BALB/c mice and in wild-type counterparts. TNF-R1(-/-) mice had significantly less weight loss, reduced mortality, colon shortening and oedema, colon histological damage and lower levels of colon myeloperoxidase compared with wild-type (WT) BALB/c mice. A similar manifestation was also observed in TNF-R2(-/-) and TNF-R1(-/-) TNF-R2(-/-) (TNF-R DKO) mice. Strikingly, systemic inflammatory response (including splenomegaly and monocyte expansion) was found in WT and TNF-R1(-/-) mice after TNBS, instead of TNF-R2(-/-) and TNF-R DKO mice. Attenuated pathology of colitis in TNF-R1(-/-) or TNF-R2(-/-) mice correlated with lower amounts of interleukin (IL)-6, IL-1β, monocyte chemotactic protein (MCP)-1, IL-12p70 and interferon (IFN)-γ production in the colons. Importantly, ablation of TNF-R1 or TNF-R2 reduced the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL)-positive apoptotic epithelial cells in the affected colons compared with WT TNBS-instilled controls, which might be due to the heightened ratio of Bcl-2/Bax and reduced activity of nuclear factor (NF)-κB. These findings suggest that either TNF-R1 or TNF-R2 plays a pathogenic role in the pathology of colitis and TNF signalling via TNF-R1 or TNF-R2 alone is not sufficient for inducing mucosal damage.

  20. Infliximab protects against pulmonary emphysema in smoking rats

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiang-yan; HAN Jing; ZHANG Cheng; SUN Qian-yun; LI Dan; LUO Rong-rong; WAN Zi-fen; YE Xian-wei; LIU Wei-jia; RAO Shan-shan

    2011-01-01

    Background It is widely accepted that tumor necrosis factor-α (TNF-a) plays an important role in the pathogenesis of emphysema. This study aimed at investigating the protective effects of anti-TNF-α antibody, infliximab, in the development of emphysema induced by passive smoking in rats.Methods Thirty-nine rats were randomly divided into a normal control group (group 1), an emphysema group (group 2),and an infliximab-intervention group (group 3). Rat models of emphysema were established by exposure to cigarette smoking daily for 74 days. After 1 month, the infliximab intervention group was treated with infliximab via subcutaneous injection. The levels of TNF-α, iL-8 and vascular endothelial growth factor (VEGF) in bronchoalveolar lavage fluid (BALF)were measured with enzyme linked immunosorbent assay (ELISA). The number and classification of cells in the BALF were measured. Lung tissue sections stained by hematoxylin and eosin (HE) were observed, and mean linear intercept (MLI) and mean alveolar numbers (MAN) were measured. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods were used to examine the percentage of positive cells and distribution of apoptotic cells.Results The levels of TNF-α and IL-8 in BALF were higher in group 2 than in group 1 and group 3. The MLI was greater in group 2 than that in group 1 and group 3 while MAN was decreased. The concentration of VEGF in BALF of group 2 was significantly decreased as compared with group 1. The total cells and neutrophils number was significantly increased in group 2 as compared with group 1 and group 3, so was the percentage of neutrophils. The number of TUNEL positive cells in the alveolar septa was significantly increased in group 2 as compared with group 1 and group 3.Conclusion Infliximab protects against cigarette smoking-induced emphysema by reducing airway inflammation,attenuating alveolar septa cell apoptosis and improving pathological changes.

  1. Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

    Institute of Scientific and Technical Information of China (English)

    Bei ZHOU; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Fumiaki UCHIUMI; Takashi IKEJIMA

    2007-01-01

    Aim: To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.Methods: The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.Results: The protective effects of silibinin were significantly sup-pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously,the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expres-sion of phosphorylated ERK was increased by silibinin, the expression of phos-phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphory-lated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.Conclusion: Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.

  2. Changes in the Expression and Distribution of Claudins, Increased Epithelial Apoptosis, and a Mannan-Binding Lectin-Associated Immune Response Lead to Barrier Dysfunction in Dextran Sodium Sulfate-Induced Rat Colitis

    Science.gov (United States)

    Yuan, Bosi; Zhou, Shuping; Lu, Youke; Liu, Jiong; Jin, Xinxin; Wan, Haijun; Wang, Fangyu

    2015-01-01

    Background/Aims This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. Methods Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. Results The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%±1.2% in controls and was significantly increased to 7.2%±1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. Conclusions There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability. PMID:25717051

  3. Urinary metabolites of di(2-ethylhexyl) phthalate relation to sperm motility, reactive oxygen species generation, and apoptosis in polyvinyl chloride workers.

    Science.gov (United States)

    Huang, Li-Ping; Lee, Ching-Chang; Fan, Jer-Pei; Kuo, Po-Hsiu; Shih, Tung-Sheng; Hsu, Ping-Chi

    2014-08-01

    To measure the concentrations of urinary di(2-ethylhexyl) phthalate (DEHP) metabolites in polyvinyl chloride (PVC) workers and a control group for determining the relationship of DEHP exposure to semen quality, sperm reactive oxygen species (ROS) generation, and sperm apoptosis. We assessed the metabolites of DEHP, namely urinary mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), and semen quality, such as sperm concentration, motility, morphology, ROS generation, and DNA damage by using terminal deoxynucleotidyl transferase-mediated nick end labeling assay obtained from 47 workers employed within two PVC pellet plants and 15 graduate students in Taiwan. Sperm concentration and motility were significantly affected in the high-exposure group. The percentage and intensity of sperm ROS generation were higher in the high-exposure group than those in the control group. After adjustment for age, smoking status, and coffee consumption, the decrease in sperm motility was inversely associated with the concentration of MEHP (β = -0.549, p = 0.0085), MEHHP (β = -0.155, p = 0.0074), and MEOHP (β = -0.201, p = 0.0041). Moreover, sperm apoptosis and ROS generation were positively associated with MEHHP and MEOHP concentration, respectively. This was the first study to explore the associations between levels of DEHP exposure, sperm motility, ROS generation, and apoptosis. The results suggested that urinary MEHHP and MEOHP were sensitive biomarkers for reflecting the relationship between DEHP exposure and semen quality.

  4. A hexane fraction of American ginseng suppresses mouse colitis and associated colon cancer: anti-inflammatory and proapoptotic mechanisms.

    Science.gov (United States)

    Poudyal, Deepak; Le, Phuong Mai; Davis, Tia; Hofseth, Anne B; Chumanevich, Alena; Chumanevich, Alexander A; Wargovich, Michael J; Nagarkatti, Mitzi; Nagarkatti, Prakash S; Windust, Anthony; Hofseth, Lorne J

    2012-04-01

    Ulcerative colitis is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American ginseng extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of American ginseng that suppress colitis and prevent colon cancer. Among five different fractions of American ginseng (butanol, hexane, ethylacetate, dichloromethane, and water), a hexane fraction has particularly potent antioxidant and proapoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex vivo model (CD4(+)/CD25(-) primary effector T cells). A key in vivo finding was that compared with the whole American ginseng extract, the hexane fraction of American ginseng was more potent in treating colitis in a dextran sodium sulfate (DSS) mouse model, as well as suppressing azoxymethane/DSS-induced colon cancer. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling of inflammatory cells within the colonic mesenteric lymph nodes was elevated in mice consuming DSS + the hexane fraction of American ginseng. Results are consistent with our in vitro data and with the hypothesis that the hexane fraction of American ginseng has anti-inflammatory properties and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of American ginseng that suppress colitis and prevent colon cancer associated with colitis.

  5. Effect of L-glutamine on liver Bcl-2 mRNA expression after total hepatic inflow occlusion in rats%谷氨酰胺对大鼠肝门阻断后肝脏Bcl-2 mRNA表达的影响及其保护作用

    Institute of Scientific and Technical Information of China (English)

    刘国平; 朱闻溪; 杨广顺; 周文平; 程广明

    2008-01-01

    目的:探讨谷氨酰胺(L-glutamine,Gln)对肝门阻断后肝脏细胞凋亡及Bcl-2 mRNA表达的影晌.方法:雄性Wistar大鼠,随机分为假手术组(A组)、对照组(B组)和实验组(C组)3组.采用Pringle's法进行肝门阻断,持续35 min,肝门阻断前C组大鼠腹腔注射Gln.分别于肝门阻断前及再灌注后2、4和24 h,每组各选取10只大鼠,测定血清ALT、AST、乳酸脱氢酶(lactic dehydrogenase,LDH)含量;检测肝组织谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)的含量;采用原位末端脱氧核苷酸转移酶法(terminal deoxynucleotidyl transferase-mediated DUTP nick end labeling method,TUNEL)检测肝脏细胞凋亡,并计算凋亡指数(apoptosic index,Al);采用RT-PCR方法检测肝Bcl-2 mRNA的表达.结果:与B组相比,再灌注后C组肝组织中MDA含量下降(P<0.05),而GSH水平增高(P<0.05);血清ALT、AST、LDH含量及Al均明显降低(P<0.05);Bcl-2mRNA表达则显著增强(P<0.05).结论:Gln能够减轻过氧化损伤,上调肝脏Bcl-2 mRNA的表达,抑制肝细胞凋亡,从而在肝门阻断中发挥保护作用.

  6. THE EXPERIMENTAL STUDY ON THE CELL APOPTOSIS AND EXPRESSION OF BCL-2 PROTEIN IN INTRACEREBRAL HEMORRHAGE IN MODEL OF RATS

    Institute of Scientific and Technical Information of China (English)

    Bao Gang; Guo Ning; Zhang Zhonglin; Chen Wei; Bao Dehu

    2006-01-01

    Otjective To study whether there is the apoptosis of neural cells and the expressionof Bcl-2 protein in intracerebral hemorrhage (ICH) in model of rats, for the further understanding the mechanism of the delayed damage of the neural cells around the hematoma after ICH. Methods Fifty SD rats were randomly divided into 5 groups, ten in each. With the Group A as the control, the rest 40 were used to set up intracerebral hemorrhage model. The brains were taken out at 12th, 24th, 48th and 72th hours, respectively. Apoptosis cells were detected with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), and the expression of Bcl-2 protein was detected with immunochemical stainging methed (SP). Results In the control group, no apoptosis cells and Bcl-2protein were detected. In rest groups, the apoptosis cells and Bcl-2 protein were expressed in different degree.Apoptosis rates verified and corresponded with the time after ICH, with the peak at 48th -72th hour after hemorrhage.The peak rate of apoptosis cells was (24. 50± 2.69)% and Bcl-2 protein expression was (20. 76 ± 1.97)% . There was significant difference between the experimental groups and control (P<0.05), and no linear relationship between the apoptosis rate and the expression of Bcl-2 protein. Conclusion Apoptosis may be an important factor in the secondary trauma of ICH. There is a time leg after hemorrhage. All this is instructive to clinical treatment in time. Bcl-2 protein keeps increasing in a certain time after hemorrhage, but not synchronize with the cell apoptosis. This indicates that bcl-2 has the effect to reduce the apoptosis of neural cells.

  7. Pro-apoptotic role of NF-κB pathway inhibition in lipopolysaccharide-stimulated polymorphonuclear neutrophils

    Institute of Scientific and Technical Information of China (English)

    刘艳梅; 张君岚; 赵占胜; 凌亦凌

    2003-01-01

    Objective To investigate the role of nuclear factor kappa B (NF-κB) pathway inhibition in lipopolysaccharide (LPS)-stimulated apoptosis of polymorphonuclear neutrophils (PMNs).Methods Rats with acute lung injury induced by LPS intratracheal instillation and cultured human venous PMNs were studied. Pyrrolidine dithiocarbamate (PDTC) and gliotoxin were used as NF-κB inhibitors. Additionally, to explore the role of extracellularly regulated protein kinase as an upstream signal in NF-κB pathway on regulating LPS-stimulated PMN apoptosis, PD098059, the specific inhibitor of extracellularly regulated protein kinase, was also applied. The lung injury was determined by protein content and PMN numbers in bronchoalveolar lavage fluid. PMN apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) end labeling and DNA fragmentation. IκBα degradation was analyzed by Western blot. NF-κB DNA binding activity was detected by an electrophoretic mobility shift assay.Results (1) The increase of protein content and PMN numbers in bronchoalveolar lavage fluid induced by LPS (100 μg per rat) intratracheal instillation were alleviated by PDTC (50, 100, or 200 mg/kg, I.p.) in a dose-dependent manner. (2) PMNs apoptosis in vivo or in vitro was delayed by LPS, and accelerated by PDTC, gliotoxin or PD098059 pretreatment. (3) IκBα degradation and increased NF-κB DNA binding activity mediated by LPS were inhibited by PDTC, gliotoxin or PD098059 pretreatment.Conclusion Inhibition of either NF-κB itself or the upstream signals in NF-κB pathway such as extracellularly regulated protein kinases has therapeutic effect on LPS-induced acute lung injury, in which the dysregulation of PMN apoptosis plays an important role.

  8. Proliferation, migration, and differentiation of endogenous ependymal region stem/progenitor cells following minimal spinal cord injury in the adult rat.

    Science.gov (United States)

    Mothe, A J; Tator, C H

    2005-01-01

    Ependymal cells of the adult mammalian spinal cord exhibit stem/progenitor cell properties following injury. In the present study, we utilized intraventricular injection of 1,1'-dioctadecyl-6,6'-di(4-sulfophenyl)-3,3,3',3'-tetramethylindocarbocyanine (DiI) to label the ependyma lining the central canal to allow tracking of the migration of endogenous ependymal cells and their progeny after spinal cord injury (SCI). We developed a minimal injury model that preserved the integrity of the central canal and did not interfere with ependymal cell labeling. Three days following SCI, there was an 8.6-fold increase in the proliferative labeling index of the ependymal cells at the level of the needle track based on bromodeoxyuridine labeling, compared with 1 day post-injury. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells were not detected in the ependyma or surrounding gray matter, indicating that ependymal cells do not undergo apoptosis in response to minimal injury. Nestin was rapidly induced in the ependyma by 1 day and expression peaked by 7 days post-injury. We quantitated the number and distance of ependymal cell migration following minimal injury. The number of ependymal cells migrating from the region of the central canal increased by 3 days following minimal injury and DiI-labeled glial fibrillary acidic protein expressing cells were detected 14 days post-SCI, most of which migrated within 70 microm of the region of the central canal. These results show that a minimal SCI adjacent to the ependyma is sufficient to induce an endogenous ependymal cell response where ependymal stem/progenitor cells proliferate and migrate from the region of the central canal, differentiating primarily into astrocytes.

  9. Sialoadenitis progression in nonobese diabetic mice and its correlation with expression of apoptosis-associated proteins in salivary glands and serum IgG levels

    Institute of Scientific and Technical Information of China (English)

    QI Ge; HUA Hong; GAO Yan; LIN Qin; YU Guang-yan

    2007-01-01

    Background Sj(o)gren syndrome (SS) is an autoimmune disorder characterized by chronic lymphocytic infiltration and decreased secretion in salivary glands. Apoptosis is one of the possible mechanisms involved in acinar epithelial destruction in SS. The role of apoptosis in the initiation and effect phase of sialoadenitis is still controversial. The aim of this study was to observe the roles of apoptosis-associated proteins and serum IgG levels in sialoadenitis progression in nonobese diabetic (NOD) mice.Methods 2-, 5-, 10-, 15-, 20-week female NOD and matched BALB/c control mice were selected. Saliva and tear flow rate were measured. Serum IgG level was tested by enzyme-linked immunosorbent assay (ELISA). Number of lymphocyte foci (NLF) in submandibular glands (SMGs) was counted under routine hematoxylin/eosin-stained sections.Expression of Fas, Bcl-2 and procaspase3 proteins as well as apoptotic cells in the SMGs were detected by immunohistochemical staining and by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay respectively.Results Decreased stimulated total flow rate (STFR) and lymphocyte foci in SMGs were first observed in the 10-week NOD group. STFR was negatively correlated with NLF (P<0.05). Serum IgG in NOD mice was significantly higher than that of the control group (P<0.05) and showed a positive correlation with NLF (P<0.05). Fas expression in SMGs acinar cells in NOD mice increased with age and was significantly higher compared with that in the control group. Bcl-2 expression and procaspase3 expression in SMG acinar cells in each NOD group were lower compared with those of the age-matched control mice.Conclusion Abnormal expression of Fas and Bcl-2 in the SMGs and higher level of serum IgG may contribute to the initiation of sialoadenitis and cause the glandular destruction in NOD mice.

  10. Hyperphosphorylation of tau protein in the ipsilateral thalamus after focal cortical infarction in rats.

    Science.gov (United States)

    Dong, Da-Wei; Zhang, Yu-Sheng; Yang, Wan-Yong; Wang-Qin, Run-Qi; Xu, An-Ding; Ruan, Yi-Wen

    2014-01-16

    Hyperphosphorylation of tau has been considered as an important risk factor for neurodegenerative diseases. It has been found also in the cortex after focal cerebral ischemia. The present study is aimed at investigating changes of tau protein expression in the ipsilateral thalamus remote from the primary ischemic lesion site after distal middle cerebral artery occlusion (MCAO). The number of neurons in the ventroposterior thalamic nucleus (VPN) was evaluated using Nissl staining and neuronal nuclei (NeuN) immunostaining. Total tau and phosphorylated tau at threonine 231 (p-T231-tau) and serine 199 (p-S199-tau) levels, respectively, in the thalamus were measured using immunostaining and immunoblotting. Moreover, apoptosis was detected with terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay. It was found that the numbers of intact neurons and NeuN(+) cells within the ipsilateral VPN were reduced significantly compared with the sham-operated group, but the levels of p-T231-tau and p-S199-tau in the ipsilateral thalamus were increased significantly in rats subjected to ischemia for 3 days, 7 days and 28 days. Furthermore, the number of TUNEL-positive cells was increased in the ipsilateral VPN at 7 days and 28 days after MCAO. Thus, hyperphosphorylated tau protein is observed in ipsilateral thalamus after focal cerebral infarction in this study. Our findings suggest that the expression of hyperphosphorylated tau protein induced by ischemia may be associated with the secondary thalamic damage after focal cortical infarction via an apoptotic pathway.

  11. Rebamipide attenuates 5-Fluorouracil-induced small intestinal mucositis in a mouse model.

    Science.gov (United States)

    Kim, Hyun Jin; Kim, Jin Hyun; Moon, Won; Park, Jongha; Park, Seun Ja; Song, Geun Am; Han, Seung Hee; Lee, Jong Hun

    2015-01-01

    5-Fluorouracil (5-FU)-induced intestinal mucositis is one of the most common morbidities in chemotherapy and involves the reactive oxygen species (ROS) system, apoptosis, and inflammatory cytokines. Rebamipide exerts a mucosal-protective effect, mediated through several mechanisms. The aim of this study was to evaluate the effects of rebamipide in 5-FU-induced mouse small-intestinal mucositis. BALB/c mice were assigned randomly to four groups; (1) control group (n=10; receiving saline orally for 6 d), (2) rebamipide group (n=10; 150 mg/kg rebamipide for 6 d orally), (3) 5-FU group (n=10; 30 mg/kg 5-FU for 5 d, intraperitoneally (i.p.)), and (4) rebamipide +5-FU group (n=10; 150 mg/kg rebamipide for 6 d orally and 30 mg/kg 5-FU for 5 d, i.p.). Body weights and diarrhea scales were assessed. At day 5, the mice were sacrificed. Small intestinal tissue was used for: (1) hematoxylin and eosin (HE) staining for determination of small intestinal villi height, (2) terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, (3) immunohistochemistry for inducible nitric oxide synthase (iNOS), F4/80, and transforming growth factor (TGF)-β1, (4) measurement of serum and tissue GSH levels, and (5) measurement of serum tumor necrosis factor (TNF)-α levels. Rebamipide attenuated the severity of mucosal injury reflected by body weight changes, degrees of diarrhea, and heights of villi. Rebamipide reduced the expression of iNOS and TGF-β1, apoptosis, macrophage accumulation, serum TNF-α levels, and prevented reductions in serum and tissue glutathione (GSH) levels by 5-FU administration. These results suggest that rebamipide promotes several mechanisms of mucosal protection and attenuated the 5-FU-induced mucosal injury. In conclusion, administration of rebamipide may have significant protective effects against 5-FU-induced intestinal mucositis.

  12. Apoptosis of motor neurons in the spinal cord after ischemia reperfusion injury delayed paraplegia in rabbits

    Institute of Scientific and Technical Information of China (English)

    Liu Bibo; Liu Miao; Ma Wei; Wang Duoning

    2007-01-01

    Objective To clarify the pathologic change of the motor neuron on spinal cord ischemia reperfusion injury delayed paraplegia. Methods The infrarenal aorta of White New Zealand rabbits (n=24) was occluded for 26 minutes using two bulldog clamps. Rabbits were killed after 8, 24, 72, or 168 hours (n=6 per group), respectively. The clamps was placed but never clamped in sham-operated rabbits (n=24). The lumbar segment of the spinal cord (L5 to L7) was used for morphological studies, including hematoxylin and eosin staining, the expression of bcl-2 and bax proteins in spinal cord was detected with immunohistochemistry. The apoptotic neurons in spinal cord were measured with terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end-labeling of DNA fragments (TUNEL) staining. Results Delayed paraplegia occurred in all rabbits of ischemia reperfusion group at 16-24 hours, but not in sham groups. Motor neurons were selectively lost at 7 days after transient ischemia. After ischemia, the positive expression of bcl-2 protein were in the sham controls but decreased significantly as compared with that of the IR group (P<0.01), especially in 72 hours reperfusion. The positive expression of bax protein were also in the sham controls, but increased in the IR group, especially in 72 hours reperfusion; In addition, TUNEL study demonstrated that no cells were positively labeled until 24 hours after ischemia, but nuclei of some motor neurons were positively labeled at peak after ischemia reperfusion at 72 hours. Conclusion Spinal cord ischemia in rabbits induces morphological and biochemical changes suggestive of apoptosis. These data raise the possibility that apoptosis contributes to neuronal cell death after spinal cord ischemia reperfusion.

  13. Ultrasound-guided direct delivery of 3-bromopyruvate blocks tumor progression in an orthotopic mouse model of human pancreatic cancer.

    Science.gov (United States)

    Ota, Shinichi; Geschwind, Jean-Francois H; Buijs, Manon; Wijlemans, Joost W; Kwak, Byung Kook; Ganapathy-Kanniappan, Shanmugasundaram

    2013-06-01

    Studies in animal models of cancer have demonstrated that targeting tumor metabolism can be an effective anticancer strategy. Previously, we showed that inhibition of glucose metabolism by the pyruvate analog, 3-bromopyruvate (3-BrPA), induces anticancer effects both in vitro and in vivo. We have also documented that intratumoral delivery of 3-BrPA affects tumor growth in a subcutaneous tumor model of human liver cancer. However, the efficacy of such an approach in a clinically relevant orthotopic tumor model has not been reported. Here, we investigated the feasibility of ultrasound (US) image-guided delivery of 3-BrPA in an orthotopic mouse model of human pancreatic cancer and evaluated its therapeutic efficacy. In vitro, treatment of Panc-1 cells with 3-BrPA resulted in a dose-dependent decrease in cell viability. The loss of viability correlated with a dose-dependent decrease in the intracellular ATP level and lactate production confirming that disruption of energy metabolism underlies these 3-BrPA-mediated effects. In vivo, US-guided delivery of 3-BrPA was feasible and effective as demonstrated by a marked decrease in tumor size on imaging. Further, the antitumor effect was confirmed by (1) a decrease in the proliferative potential by Ki-67 immunohistochemical staining and (2) the induction of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling staining. We therefore demonstrate the technical feasibility of US-guided intratumoral injection of 3-BrPA in a mouse model of human pancreatic cancer as well as its therapeutic efficacy. Our data suggest that this new therapeutic approach consisting of a direct intratumoral injection of antiglycolytic agents may represent an exciting opportunity to treat patients with pancreas cancer.

  14. Endocytosed 2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils.

    Directory of Open Access Journals (Sweden)

    Tadakazu Okoshi

    Full Text Available Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid

  15. Neuroprotective effect of exogenous vascular endothelial growth factor on rat spinal cord neurons in vitro hypoxia

    Institute of Scientific and Technical Information of China (English)

    DING Xin-min; MAO Bo-yong; JIANG Shu; LI Sheng-fu; DENG Yi-ling

    2005-01-01

    Background Vascular endothelial growth factor (VEGF) is well known as a hypoxia-induced protein. That it markedly increased expression of VEGF and improvement of rat motor function after spinal cord injury suggested that VEGF could play a neuroprotective role in ischaemic tolerance. This study investigated whether vascular endothelial growth factor has direct neuroprotective effects on rat spinal cord neurons. Methods We employed primary cultures of embryonic rat spinal cord neurons, then administrated different concentrations of VEGF164 in the culture medium before hypoxia when the number of neurons was counted and the cell viability was detected by MTT. The neuronal apoptosis and expression of VEGF and its receptor genes were evaluated by terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and immunohistochemistry. The VEGFR2/FLK-1 inhibitor, SU1498, was used to confirm whether the neuroprotective effect of VEGF was mediated through VEGFR2/Flk-1 receptors. Result In hypoxic conditions,the number and viability of neurons decreased progressively, while the number of TUNEL-positive cells increased along with the prolongation of hypoxic exposure. When the concentration of VEGF in cell culture medium reached 25 ng/ml, the cell viability increased 11% and neuronal apoptosis reduced to half, this effect was dose dependent and led to an approximately 25% increase in cell viability and about threefold decrease in TUNEL-positive cells at a maximally effective concentration of 100 ng/ml. In normal conditions, VEGF/Flk-1 but not VEGF/Flt-1 gene expressed at a low level: after hypoxia, the expression of VEGF/Flk-1, but not VEGF/Flt-1 was significantly increased. The protective effect of VEGF was blocked by the VEGFR2/Flk-1 receptor tyrosine kinase inhibitor, SU1498. Conclusions VEGF has direct neuroprotective effects on rat spinal cord neurons, which may be mediated in vitro through VEGFR2/Flk-1 receptors.

  16. Bone Marrow Stromal Cells Promote Neuronal Restoration in Rats with Traumatic Brain Injury: Involvement of GDNF Regulating BAD and BAX Signaling

    Directory of Open Access Journals (Sweden)

    Qin Shen

    2016-02-01

    Full Text Available Background/Aims: To investigate the effects of bone marrow stromal cells (BMSCs and underlying mechanisms in traumatic brain injury (TBI. Methods: Cultured BMSCs from green fluorescent protein-transgenic mice were isolated and confirmed. Cultured BMSCs were immediately transplanted into the regions surrounding the injured-brain site to test their function in rat models of TBI. Neurological function was evaluated by a modified neurological severity score on the day before, and on days 7 and 14 after transplantation. After 2 weeks of BMSC transplantation, the brain tissue was harvested and analyzed by microarray assay. And the coronal brain sections were determined by immunohistochemistry with mouse anti-growth-associated protein-43 kDa (anti-GAP-43 and anti-synaptophysin to test the effects of transplanted cells on the axonal regeneration in the host brain. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay and Western blot were used to detect the apoptosis and expression of BAX and BAD. Results: Microarray analysis showed that BMSCs expressed growth factors such as glial cell-line derived neurotrophic factor (GDNF. The cells migrated around the injury sites in rats with TBI. BMSC grafts resulted in an increased number of GAP-43-immunopositive fibers and synaptophysin-positive varicosity, with suppressed apoptosis. Furthermore, BMSC transplantation significantly downregulated the expression of BAX and BAD signaling. Moreover, cultured BMSC transplantation significantly improved rat neurological function and survival. Conclusion: Transplanted BMSCs could survive and improve neuronal behavior in rats with TBI. Mechanisms of neuroprotection and regeneration were involved, which could be associated with the GDNF regulating the apoptosis signals through BAX and BAD.

  17. Prosurvival effect of cumulus prostaglandin G/H synthase 2/prostaglandin2 signaling on bovine blastocyst: impact on in vivo posthatching development†.

    Science.gov (United States)

    Nuttinck, Fabienne; Jouneau, Alice; Charpigny, Gilles; Hue, Isabelle; Richard, Christophe; Adenot, Pierre; Ruffini, Sylvie; Laffont, Ludivine; Chebrout, Martine; Duranthon, Véronique; Guienne, Brigitte Marquant-Le

    2017-03-07

    Apoptotic activity is a common physiological process which culminates at the blastocyst stage in the preimplantation embryo of many mammals. The degree of embryonic cell death can be influenced by the oocyte microenvironment. However, the prognostic significance of the incidence of apoptosis remains undefined. Prostaglandin E2 (PGE2) derived from prostaglandin G/H synthase-2 (PTGS2) activity is a well-known prosurvival factor that is mainly studied in oncology. PGE2 is the predominant PTGS2-derived prostaglandin present in the oocyte microenvironment during the periconceptional period. Using an in vitro model of bovine embryo production followed by transfer and collection procedures, we investigated the impact of periconceptional PGE2 on the occurrence of spontaneous apoptosis in embryos and on subsequent in vivo posthatching development. Different periconceptional PGE2 environments were obtained using NS-398, a specific inhibitor of PTGS2 activity, and exogenous PGE2. We assessed the level of embryonic cell death in blastocysts at day 8 postfertilization by counting total cell numbers, by the immunohistochemical staining of active caspase-3, and by quantifying terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling signals and apoptosis regulator (BCL-2/BAX) mRNA expression. Morphometric parameters were used to estimate the developmental stage of the embryonic disk and the extent of trophoblast elongation on day 15 conceptuses. Our findings indicate that periconceptional PGE2 signaling durably impacts oocytes, conferring increased resistance to spontaneous apoptosis in blastocysts and promoting embryonic disk development and the elongation process during preimplantation development.

  18. Intracerebroventricular administration of riluzole prevents morphine-induced apoptosis in the lumbar region of the rat spinal cord.

    Science.gov (United States)

    Hassanzadeh, Kambiz; Habibi-asl, Bohlool; Roshangar, Leila; Nemati, Mahboob; Ansarin, Masood; Farajnia, Safar

    2010-01-01

    Opiates are the most effective drugs for pain relief. However, the repeated use of opiates induces tolerance to their analgesic effects. It has been shown that this morphine-induced tolerance is associated with apoptosis in the central nervous system. The aim of this study is to evaluate the effects of intracerebroventricular (i.c.v.) administration of riluzole, an anti-glutamatergic drug, on morphine-induced apoptosis in the lumbar region of the rat spinal cord. Animals were given daily injections of morphine and vehicle, morphine and riluzole, or riluzole alone. Nociception was assessed using a hot plate apparatus, and apoptosis was assessed using the in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The levels of anti-apoptotic factors Bcl-2 and HSP 70 and the pro-apoptotic agent caspase-3 were evaluated using immunoblotting. The glutamate concentration in the lumbar spinal cord was measured with high performance liquid chromatography (HPLC). The results indicate that the i.c.v. administration of riluzole attenuated morphine tolerance and reduced the number of TUNEL positive cells. Immunoblotting revealed that the levels of the selected anti-apoptotic agents were greater in the treatment groups compared to the controls. Furthermore, the results demonstrated that the administration of riluzole can attenuate the morphine-induced elevation of glutamate in the lumbar spinal cord. In conclusion, i.c.v. administration of riluzole attenuated morphine-induced tolerance to analgesia and apoptosis in addition to preventing the morphine-induced increase of glutamate in the lumbar spinal cord of rats.

  19. The leaves of Diospyros kaki exert beneficial effects on a benzalkonium chloride–induced murine dry eye model

    Science.gov (United States)

    Kim, Kyung-A; Hyun, Lee Chung; Jung, Sang Hoon

    2016-01-01

    Purpose In this study, the beneficial effects of the oral administration of ethanol extract of Diospyros kaki (EEDK) were tested on a mouse dry eye model induced by benzalkonium chloride (BAC). Methods A solution of 0.2% BAC was administered topically to mouse eyes for 14 days, twice daily, to induce dry eye. Various concentrations of EEDK were administrated daily by oral gavage for 14 days after BAC treatment. Preservative-free eye drops were instilled in the positive-control group. The tear secretion volume (Schirmer’s test), tear break-up time (BUT), and fluorescein score were measured on the ocular surface. BAC-induced corneal damage was tested with hematoxylin-eosin staining. Moreover, apoptotic cell death in the corneal epithelial layer was investigated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The protein expression level of interleukin-1alpha (IL-1α), IL-1β, IL-6, tumor necrosis factor- alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1) was determined with western blot analysis. Furthermore, squamous metaplasia in the corneal epithelial layer was detected with immunofluorescent staining for cytokeratine-10. The cellular proliferation in the cornea was examined with immunohistochemical staining for Ki-67. Results EEDK treatment resulted in prolonged BUT, decreased fluorescein score, increased tear volume, and smoother epithelial cells compared with BAC treatment alone in the cornea. Moreover, EEDK treatment inhibited the inflammatory response and corneal epithelial cell death in a BAC-induced murine dry eye model, and changes in squamous cells were inhibited. Proliferative activity in the corneal epithelium cells was improved with EEDK. Conclusions EEDK could be a potential therapeutic agent in the clinical treatment of dry eye. PMID:27110091

  20. Pollination-induced Apoptosis in Tobacco Related to Expression of Calcium/calmodulin-dependent Protein Kinase T1%烟草花粉发育过程中的细胞凋亡及其与钙/钙调素依赖性蛋白激酶T1表达的关系

    Institute of Scientific and Technical Information of China (English)

    王玲; 王盈; 张美; 吕应堂

    2001-01-01

    用原位末端标记及免疫组化显色方法,对烟草花药发育过程中的细胞凋亡进行了检测.结果表明绒毡层、环形细胞、药隔组织细胞在减数分裂后四分体时期开始凋亡(Plate I,C&D),而花药维管束细胞从小孢子母细胞时期就开始凋亡(Plate I,E).这些特定的细胞在花药发育特定时期的凋亡担负着特定的作用.钙/钙调素依赖性蛋白激酶T1在这些凋亡细胞中大量特异表达(PlateII),表明钙信号途径参与了烟草花粉发育的细胞凋亡调控.%Using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and immunohistochemical methods, the pollination-induced cell death in tobacco (Nicotiana tabacum L.) was detected. The results indicated that pollination induced apoptotic death in specific cells. Apoptosis was initiated during the tetrad stage after meiosis in the tapetal cells, cyclic cells, and connective cells (Plate I, C and D), and during pollen mother cell stage in the vascular bundle (Plate I, E). Those apoptotic events,which occurred in the specific cells in the specific stages of antherdevelopment, play some vital roles of specific biological function. The highly expression of calcium/calmodulin dependent protein kinase T1 related closely to these apoptotic processes (Plate II) in these cells, suggesting that calcium-mediated signal pathway is involved in the regulation of apoptosis during anther development in tobacco.

  1. Effects of the Combined Use of Benazepril and Valsartan on Apoptosis in the Kidney of Rats with Adriamycin-induced Nephritic Glomerulosclerosis

    Institute of Scientific and Technical Information of China (English)

    韩子明; 邢燕; 王宏伟; 梁秀玲; 周建华

    2004-01-01

    Summary: The effects of the combined use of angiotensin converting enzyme inhibitor (ACEI)benazepril and angiotensin Ⅱ type 1 receptor antagonist (AT1RA) valsartan on apoptosis and the expression of apoptosis-related proteins Fas and FasL in the kidney of rats with adriamycin-induced nephritic glomerulosclerosis was investigated. Uninephrectomy and the injection of adriamycin induced the rat model of glomerulosclerosis. Benazepril (6 mg/kg), valsantan (20 mg/kg), or benazepril (3 mg/kg) plus valsantan (20 mg/kg) was respectively delivered daily by gavage to the rats in three treatment groups for 12 weeks. Apoptosis was examined by means of terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL). Immunohistochemistry was adopted to detect the expression of Fas and FasL. Software of pathological analysis quantitated the levels of Fas and FasL. The results showed that as compared with those in the control group, the kidneys in the model group had more severe glomerulosclerosis, much more apoptotic cells and higher levels of expression of Fas and FasL. The degree of glomerulosclerosis, the number of apoptotic cells and the levels of expression of Fas and FasL were reduced by benazepril and valsartan. The combined use of benazepril and valsartan had the best therapeutic effect. It was concluded that benazepril and valsartan could suppress the excessive apoptosis of kidney cells by lowering the expression of the apoptosis-related proteins Fas and FasL, so as to postpone the process of glomerulosclerosis. The combined use of benazepril and valsartan has better therapeutic effect.

  2. Hyperlipidemia exacerbates cerebral injury through oxidative stress, inflammation and neuronal apoptosis in MCAO/reperfusion rats.

    Science.gov (United States)

    Cao, Xiao-Lu; Du, Jing; Zhang, Ying; Yan, Jing-Ting; Hu, Xia-Min

    2015-10-01

    Recent studies showed that hyperglycemia enhanced brain damage when subjected to transient cerebral ischemic stroke. However, the etiologic link between them has been less known. In the present study, based on an experimental rat's model of hyperlipidemia combined with cerebral ischemia-reperfusion injury (I/R), we herein showed that hyperlipidemia induced by high-fat diet (HFD) resulted in considerable increase in serum triglycerides, cholesterol and low-density lipoprotein cholesterol, and remarkable decrease in serum high-density lipoprotein cholesterol, which associated with an exacerbation on neurological deficit, cerebral infarct and terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells in the ischemic hemisphere of cerebral I/R rats treated with HFD diet. The data showed that serum superoxide dismutase activity and glutathione peroxides content were significantly decreased, while malondialdehyde level was obviously increased by hyperlipidemia or cerebral I/R alone, especially by coexistence of hyperlipidemia and cerebral I/R; meantime, hyperlipidemia also enhanced cerebral I/R-induced protein expression of cytochrome P450 2E1 (CYP2E1) and the levels of pro-inflammatory factors tumor necrosis factor-α and IL-6 in the ischemic hemispheres. Furthermore, the combined action of hyperlipidemia and cerebral I/R resulted in a protein increase expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 compared to hyperlipidemia or cerebral I/R alone. Meanwhile, this study also showed that hyperlipidemia significantly enhanced cerebral I/R-induced transfer of cytochrome c from mitochondria to cytosolic and the protein expressions of Apaf-1 and caspase-3, but also decreased cerebral I/R-induced bcl-2 protein expression. The results reveal that hyperlipidemia exacerbates cerebral I/R-induced injury through the synergistic effect on CYP2E1 induction, which further induces reactive oxygen species formation, oxidative

  3. Inhibition of RET increases the efficacy of antiestrogen and is a novel treatment strategy for luminal breast cancer.

    Science.gov (United States)

    Spanheimer, Philip M; Park, Jung-Min; Askeland, Ryan W; Kulak, Mikhail V; Woodfield, George W; De Andrade, James P; Cyr, Anthony R; Sugg, Sonia L; Thomas, Alexandra; Weigel, Ronald J

    2014-04-15

    Recent findings suggest that combination treatment with antiestrogen and anti-RET may offer a novel treatment strategy in a subset of patients with breast cancer. We investigated the role of RET in potentiating the effects of antiestrogen response and examined whether RET expression predicted the ability for tyrosine kinase inhibitor (TKI) to affect extracellular signal-regulated kinase 1/2 (ERK1/2) activation in primary breast cancer. Growth response, ERK1/2 activation, Ki-67, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were assessed in breast cancer cell lines in vitro and in xenografts with vandetanib and/or tamoxifen. Thirty tumors with matched normal breast tissue were evaluated for RET expression and response to TKI treatment. Vandetanib potentiated the inhibitory effect of tamoxifen in hormone responsive (P = 0.01) and hormone insensitive (P < 0.001) estrogen receptor α (ERα)-positive breast cancer cells. Vandetanib significantly repressed tumorigenesis of MCF-7 xenografts (P < 0.001), which displayed decreased activation of ERK1/2 and AKT. Vandetanib and tamoxifen reduced the growth of established tumors with a greater effect of dual therapy compared with single agent (P = 0.003), with tamoxifen-reducing proliferative index and vandetanib-inducing apoptosis. In primary breast cancers, RET expression correlated with the ERα-positive subtype. Relative decrease in ERK1/2 phosphorylation with TKI treatment was 42% (P < 0.001) in RET-positive tumors versus 14% (P = ns) in RET-negative tumors. Vandetanib potentiated the antigrowth effects of tamoxifen in breast cancer, which was mediated through RET activation. RET predicted response to TKI therapy with minimal effects on ERK1/2 activation in RET-negative tumors. The preclinical data support evaluation of antiestrogen in combination with TKI as a potential treatment strategy for RET-positive luminal breast cancer. ©2014 AACR.

  4. Quantitative Analysis of Chemotherapeutic Effects in Tumors Using In Vivo Staining and Correlative Histology

    Directory of Open Access Journals (Sweden)

    Heung Kook Choi

    2005-01-01

    Full Text Available Aims: To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis. Methods: Lewis Lung Carcinomas cells, that are sensitive (CS-LLC and resistant (CR-LLC to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution. Results: We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be ∼2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a ∼1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line. Conclusions: Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.

  5. Stem cell factor-mediated wild-type KIT receptor activation is critical for gastrointestinal stromal tumor cell growth

    Institute of Scientific and Technical Information of China (English)

    Chen-Guang Bai; Xiao-Wei Hou; Feng Wang; Cen Qiu; Yan Zhu; Ling Huang; Jing Zhao

    2012-01-01

    AIM:To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth.METHODS:The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry,and the results were correlated with clinicopathological parameters,including the mitotic count,proliferative index (Ki-67 immunohistochemical staining),mitotic index (phospho-histone H3 immunohistochemical staining)and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling).Using primary cultured GIST cells,the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting,methyl thiazolyl tetrazolium (MTT),and apoptosis assays.RESULTS:We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples,respectively,and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis,including larger tumor size (P =0.0118),higher mitotic count (P =0.0058),higher proliferative index (P =0.0012),higher mitotic index (P =0.0282),lower apoptosis index (P =0.0484),and increased National Institutes of Health risk level (P =0.0012).We also found that the introduction of exogenous SCF potently increased KIT kinase activity,stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation,while a KIT immunoblocking antibody suppressed proliferation (P =0.01) and promoted apoptosis (P < 0.01)in cultured GIST cells.CONCLUSION:SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth.The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.

  6. Environmentally induced programmed cell death in leaf protoplasts of Aponogeton madagascariensis.

    Science.gov (United States)

    Lord, Christina E N; Gunawardena, Arunika H L A N

    2011-02-01

    Within plant systems, two main forms of programmed cell death (PCD) exist: developmentally regulated and environmentally induced. The lace plant (Aponogeton madagascariensis) naturally undergoes developmentally regulated PCD to form perforations between longitudinal and transverse veins over its leaf surface. Developmental PCD in the lace plant has been well characterized; however, environmental PCD has never before been studied in this plant species. The results presented here portray heat shock (HS) treatment at 55 °C for 20 min as a promising inducer of environmental PCD within lace plant protoplasts originally isolated from non-PCD areas of the plant. HS treatment produces cells displaying many characteristics of developmental PCD, including blebbing of the plasma membrane, increased number of hydrolytic vesicles and transvacuolar strands, nuclear condensation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive nuclei, as well as increased Brownian motion within the vacuole. Results presented here for the first time provide evidence of chloroplasts in the vacuole of living protoplasts undergoing environmentally induced PCD. Findings suggest that the mitochondria play a critical role in the cell death process. Changes in mitochondrial dynamics were visualized in HS-treated cells, including loss of mitochondrial mobility, reduction in ΔΨ(m), as well as the proximal association with chloroplasts. The role of the mitochondrial permeability transition pore (PTP) was examined by pre-treatment with the PTP agonist cyclosporine A. Overall, HS is depicted as a reliable method to induce PCD within lace plant protoplasts, and proves to be a reliable technique to enable comparisons between environmentally induced and developmentally regulated PCD within one species of plant.

  7. Silencing livin gene by siRNA leads to apoptosis induction, cell cycle arrest, and proliferation inhibition in malignant melanoma LiBr cells

    Institute of Scientific and Technical Information of China (English)

    Hao WANG; Sheng-shun TAN; Xin-yang WANG; Dong-hua LIU; Chun-shui YU; Zhuan-li BAI; Da-lin HE; Jun ZHAO

    2007-01-01

    Aim: The aim of the present study was to investigate the effects of silencing the livin gene by small interfering RNA (siRNA) on the expression of livin and the effects on apoptosis, cell cycle, and proliferation in human malignant melanoma LiBr cells. Methods: Three chemically-synthetic siRNA duplexes targeting livin were transiently transfected into the LiBr cells, and the effects on livin expression were detected both at the mRNA level by real-time RT-PCR and at the protein level by Western blotting. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, flow cytometric analysis, and the expression of procaspase-3 and activated caspase-3 analysis by Western blotting. Cell cycle was analyzed by flow cytometry. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Results: One of the 3 designed siRNA could effectively knock down the livin expression both at the mRNA and protein levels in dose- and time-dependent manners; 100 nmol/L with maximum downregulation on mRNA at 48 h, and on the protein at 72 h after transfection. Silencing livin could significantly induce apoptosis, arrest cell cycle at the GJG1 phase, and inhibit proliferation in LiBr cells. Meanwhile, caspase-3 was activated. Conclusion: The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma.

  8. Effects of Nitric Oxide on Proliferation and Apoptosis of Cultured Bovine Trabecul ar Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    薛蔚; 杜蜀华; 李勇; 杨业金; 孙京华

    2002-01-01

    The effects of different doses of nitric oxide (NO) on the proliferation and apoptosis of the cultured bovine trabecular meshwork (TM) cells were studied. L-arginine and NG-nitro-L-arginine methyl (L-NAME) were incubated with TM cells for 48 h. In the control group, no medicine was given. In the experimental groups, concentrations of L-arginine and L-NAME were 1 × 10- 7 mol/L,1 × 10-6 mol/L, 1 × 10-5 mol/L, 1 × 10-4 mol/L, 1 × 10-3 mol/L and 1 × 10-2 mol/L, respectively.NO2- in supernate, the proliferation and apoptosis of TM cells and mRNA expression of bcl-2 and bax were measured by Griess reagent, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), MTT assay and in situ hybridization,respectively. The results showed that Larginine with concentration ≥1 × 10-4 mol/L could induce apoptosis of the TM cells and inhibit the proliferation of TM cells through increasing the NO levels, down-regulating bcl-2 mRNA expression and up-regulating bax mRNA expression; L-NAME with concentration ≥1 × 10-5 mol/L could induce the proliferation of the TM cells through suppressing the production of NO. It was concluded that NO in high level could induce apoptosis of the TM cells and suppress the proliferation of the TM cells.

  9. The anti-cancer effects of poi (Colocasia esculenta) on colonic adenocarcinoma cells In vitro.

    Science.gov (United States)

    Brown, Amy C; Reitzenstein, Jonathan E; Liu, Jessie; Jadus, Martin R

    2005-09-01

    Hawaiians tend to have lower incidence rates of colorectal cancer and it was hypothesized that this may be due to ethnic differences in diet, specifically, their consumption of poi, a starchy paste made from the taro (Colocasia esulenta L.) plant corm. Soluble extracts of poi were incubated at 100 mg/mL in vitro for antiproliferative activity against the rat YYT colon cancer cell line. (3)H-thymidine incorporation studies were conducted to demonstrate that the poi inhibited the proliferation of these cancer cells in a dose-dependent manner. The greatest suppression of YYT colon cancer growth occurred when 25% concentration was used. When poi was incubated with the YYT cells after 2 days, the YYT cells underwent apoptotic changes as evidenced by a positive terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) stain. Poi enhanced the proliferation of normal mouse splenocyte control cells, suggesting that poi is not simply toxic to all cells but even has a positive immunostimulatory role. By flow cytometry, T cells (CD4+ and CD8+) were predominantly activated by the poi. Although numerous factors can contribute to the risk of colon cancer, perhaps poi consumption may contribute to the lower colon cancer rates among Hawaiians by two distinct mechanisms. First, by inducing apoptosis within colon cancer cells; second, by non-specifically activating lymphocytes, which in turn can lyse cancerous cells. Our results suggest for the first time that poi may have novel tumor specific anti-cancer activities and future research is suggested with animal studies and human clinical trials.

  10. Effect of nerve growth factor on neuronal apoptosis after spinal cord injury in rats

    Institute of Scientific and Technical Information of China (English)

    曹晓建; 汤长华; 罗永湘

    2002-01-01

    To explore the molecular mechanism of the protective effect of nerve growth factor (NGF) on injured spinal cord. Methods: The posterior T8 (the 8th thoracic segment) spinal cords of 60 Wistar rats were injured by impacts caused by objects (weighing 10 g) falling from a height of 2.5 cm with Allens way. Solution with nerve growth factors (NGF) was given to 30 rats (the NGF group) through a microtubule inserted into the subarachnoid cavity immediately, and at 2, 4, 8, 12 and 24 hours after spinal cord injury (SCI) respectively. Normal saline (NS) with same volume was given to the other 30 rats (the NS group) with the same method. And 5 normal rats were taken as the normal controls. The expression of bcl-2 and bax proteins in spinal cord was detected with immunohistochemistry. The apoptotic neurons in spinal cord were measured with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of DNA fragments (TUNEL) staining. Results: The positive expression of bcl-2 protein was strong in the normal controls, but decreased in the NS group, and increased significantly in the NGF group as compared with that of the NS group (P<0.01). The positive expression of bax protein was also strong in the normal controls, but increased in the NS group, and decreased significantly in the NGF group as compared with that of the NS group (P<0.01). Apoptotic neurons were found in the NS group, and they decreased significantly in the NGF group as compared with that of the NS group (P<0.01). Conclusions: NGF can protect the injured nerve tissues through stimulating the expression of bcl-2 protein, inhibiting the expression of bax protein and inhibiting the neuronal apoptosis after SCI.

  11. Nerve growth factor delivery by ultrasound-mediated nanobubble destruction as a treatment for acute spinal cord injury in rats

    Science.gov (United States)

    Song, Zhaojun; Wang, Zhigang; Shen, Jieliang; Xu, Shengxi; Hu, Zhenming

    2017-01-01

    Background Spinal cord injuries (SCIs) can cause severe disability or death. Treatment options include surgical intervention, drug therapy, and stem cell transplantation. However, the efficacy of these methods for functional recovery remains unsatisfactory. Purpose This study was conducted to explore the effect of ultrasound (US)-mediated destruction of poly(lactic-co-glycolic acid) (PLGA) nanobubbles (NBs) expressing nerve growth factor (NGF) (NGF/PLGA NBs) on nerve regeneration in rats following SCI. Materials and methods Adult male Sprague Dawley rats were randomly divided into four treatment groups after Allen hit models of SCI were established. The groups were normal saline (NS) group, NGF and NBs group, NGF and US group, and NGF/PLGA NBs and US group. Histological changes after SCI were observed by hematoxylin and eosin staining. Neuron viability was determined by Nissl staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining was used to examine cell apoptosis. NGF gene and protein expressions were detected by quantitative reverse transcription polymerase chain reaction and Western blotting. Green fluorescent protein expression in the spinal cord was examined using an inverted fluorescence microscope. The recovery of neural function was determined using the Basso, Beattie, and Bresnahan test. Results NGF therapy using US-mediated NGF/PLGA NBs destruction significantly increased NGF expression, attenuated histological injury, decreased neuron loss, inhibited neuronal apoptosis in injured spinal cords, and increased BBB scores in rats with SCI. Conclusion US-mediated NGF/PLGA NBs destruction effectively transfects the NGF gene into target tissues and has a significant effect on the injured spinal cord. The combination of US irradiation and gene therapy through NGF/PLGA NBs holds great promise for the future of nanomedicine and the development of noninvasive treatment options for SCI and other diseases.

  12. Interleukin-1 beta induction of neuron apoptosis depends on p38 mitogen-activated protein kinase activity after spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Xin-jia WANG; Kang-mei KONG; Wei-li QI; Wei-lian YE; Pei-song SONG

    2005-01-01

    Aim: Interleukin-1 beta (IL-1β) has been implicated as an extracellular signal in the initiation of apoptosis in neurons and oligodendrocytes after spinal cord injury (SCI). To further characterize the apoptotic cascade initiated by IL-1β after SCI, we examined the expression of IL-1 β, p38 mitogen-activated protein kinase (p38 MAPK) and caspase-3 after SCI, and further investigated whether p38 MAPK was involved in neuron apoptosis induced by IL-1 β. Methods: Adult rats were given contusion SCI at the T-10 vertebrae level with a weight-drop impactor (10 g weight dropped 25.0 mm). The expression levels of IL-1β, p38 MAPK and caspase-3after SCI were assessed with Western blots, immunohistochemistry staining, and real time reverse transcription polymerase chain reactions (RT-PCR). Neuron apoptosis was assessed with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method. Results:Increased levels of IL-1β and p38 MAPK were observed soon after injury, with a peak in expression levels within 6 h of injury. By 24 h after injury, caspase-3expression was markedly increased in the injured spinal cord. TUNEL-positive cells were first observed in the lesioned area 6 h after SCI. The largest number of TUNEL-positive cells was observed at 24 h post-SCI. Intrathecal injection of the IL-1 receptor antagonist IL-1Ra significantly reduced expression of p38 MAPK and caspase-3, and reduced the number of TUNEL-positive cells. Moreover,intrathecal injection of an inhibitor of p38 MAPK, SB203580, also significantly reduced the expression of caspase-3, and reduced the number of TUNEL-positive cells in the injured spinal cord. Conclusion: The p38MAPK signaling pathway plays an important role in IL-1β mediated induction of neuron apoptosis following SCI in rats.

  13. Effects of 60 Hz electromagnetic field exposure on testicular germ cell apoptosis in mice

    Institute of Scientific and Technical Information of China (English)

    JinSangLee; SangSeokAhn; KyeongCheonJung; Yoon-WonKim; SangKonLee

    2004-01-01

    Aim:To evaluate the effects of 60 Hz extremely low frequency (ELF) elelctromagnetic field (EMF) exposure on germ cell apoptosis in the testis of mice.Methods:Adult male BALB/c mice (7 weeks of age) were exposed to a 60 Hz EMF of 0.1 mT or 0.5 mT for 24 h/day.A sham-exposed group served as the control.After 8 weeks of exposure,the mice were sacrificed.Germ cell apoptosis in the testis was assessed by histopathological examination,the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and flow cytometric examination of isolated spermatogenic cells stained with 7 aminoactinomycin D (7-AAD).Results:EMF exposure did not significantly affect the body and testis weights,but significantly increased the incidence of germ cell death.The distinguishing morphological feature of EMF exposure was a decrement in the number of well organized seminiferous tubules.Quantitative analysis of TUNEL-positive germ cells showed a significantly higher apoptotic rate in the 0.5 mT exposed mice than that in the sham controls (P<0.05),while the difference between the two exposed groups was insignificant.The TUNEL-positive cells were mainly spermatogonia.In flow cytometryanalysis,the percentage of live cells [forward scatter count (FSC)hgh7-AAD-] was lower in the exposed groups than that in the controls (Figure 5A),but the decrease in viability was not statistically significant.Conclusion:Continuousexposure to ELF EMF may induce testicular germ cell apoptosis in mice.(Asian J Androl 2004 Mar;6:29-34)

  14. Study on TUNEL Technique%TUNEL技术方法的探讨

    Institute of Scientific and Technical Information of China (English)

    陈春莲; 蓝儒竹; 叶章群; 吴翠环; 周晟

    2001-01-01

    探讨末端脱氧核苷酸转移酶介导的dUTP缺口标记技术(TUNEL)的染色方法,以期提高特异性与敏感性。采用以去势(经手术切除睾丸)后天数不同的大白鼠前列腺,用TUNEL法染色,观察不同时期的细胞凋亡水平,对经典的TUNEL法进行改良,将染色结果与TUNEL经典法和HE染色法进行对比。结果表明:采用改良法后,阳性细胞明显增加,而且着色深,凋亡指数明显增高(P<0.01) 。%The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique was studied in order to improve its sensitivity and specificity. The prostates from the castrated rats were stained by TUNEL for the observation of cellular apoptosis,represented as apoptosis indices (AI),in different stages. The improved TUNEL technique was applied and the effects of improved TUNEL,traditional TUNEL and HE method were compared. It was showed that,by using the improved method,the number of positive cells was increased significantly and the staining of cells was much denser,which reflected an increase in AI.

  15. Gap junction proteins in the light-damaged albino rat.

    Science.gov (United States)

    Guo, Cindy X; Tran, Henry; Green, Colin R; Danesh-Meyer, Helen V; Acosta, Monica L

    2014-01-01

    Changes in connexin expression are associated with many pathological conditions seen in animal models and in humans. We hypothesized that gap junctions are important mediators in tissue dysfunction and injury processes in the retina, and therefore, we investigated the pattern of connexin protein expression in the light-damaged albino rat eye. Adult Sprague-Dawley rats were exposed to intense light for 24 h. The animals were euthanized, and ocular tissue was harvested at 0 h, 6 h, 24 h, 48 h, and 7 days after light damage. The tissues were processed for immunohistochemistry and western blotting to analyze the expression of the gap junction proteins in the light-damaged condition compared to the non-light-damaged condition. Cell death was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Intense light exposure caused increased TUNEL labeling of photoreceptor cells. Immunocytochemistry revealed that connexin 36 (Cx36) was significantly increased in the inner plexiform layer and Cx45 was significantly decreased in the light-damaged retina. The pattern of Cx36 and Cx45 labeling returned to normal 7 days after light damage. Cx43 significantly increased in the RPE and the choroid in the light-damaged tissue, and decreased but not significantly in the retina. This elevated Cx43 expression in the choroid colocalized with markers of nitration-related oxidative stress (nitrotyrosine) and inflammation (CD45 and ionized calcium-binding adaptor molecule-1) in the choroid. The results suggest that connexins are regulated differently in the retina than in the choroid in response to photoreceptor damage. Changes in connexins, including Cx36, Cx43, and Cx45, may contribute to the damage process. Specifically, Cx43 was associated with inflammatory damage. Therefore, connexins may be candidate targets for treatment for ameliorating disease progression.

  16. Protection of neurons in the retinal ganglion cell layer against excitotoxicity by the N-acylethanolamine, N-linoleoylethanolamine

    Directory of Open Access Journals (Sweden)

    Duncan RS

    2011-04-01

    Full Text Available R. Scott Duncan1,*, Hua Xin1,*, Daryl L Goad1, Kent D Chapman2,3, Peter Koulen1,31Vision Research Center and Departments of Ophthalmology and Basic Medical Science, School of Medicine, University of Missouri, Kansas City, MO, USA; 2Department of Biological Sciences, University of North Texas, Denton, TX, USA; 3Center for Plant Lipid Research, University of North Texas, Denton, TX, USA *Authors contributed equallyAbstract: Retinal ganglion cell (RGC death is a hallmark of neurodegenerative diseases and disease processes of the eye, including glaucoma. The protection of RGCs has been an important strategy for combating glaucoma, but little clinical success has been reported to date. One pathophysiological consequence of glaucoma is excessive extracellular glutamate subsequently leading to excitotoxicity in the retina. Endocannabinoids, such as the N-acylethanolamine (NAE, arachidonylethanolamine (NAE 20:4, exhibit neuroprotective properties in some models of neurodegenerative disease. The majority of NAEs, however, are not cannabinoids, and their physiological function is not clear. Here, we determined whether the noncannabinoid NAE, linoleoylethanolamine (NAE18:2, protects neurons in the RGC layer against glutamate excitotoxicity in ex-vivo retina cultures. Using a terminal deoxynucleotidyl transferase-mediated dUTP (2´-deoxyuridine 5´-triphosphate nick-end labeling (TUNEL assay, we determined that NAE18:2 reduces the number of apoptotic RGC layer neurons in response to glutamate and conclude that NAE18:2 is a neuroprotective compound with potential for treating glaucomatous retinopathy.Keywords: neuroprotection, glutamate, calcium signaling, immunocytochemistry, eye, vision, glaucoma.

  17. Cytoplasmic phospholipase A2 deletion enhances colon tumorigenesis.

    Science.gov (United States)

    Ilsley, Jillian N M; Nakanishi, Masako; Flynn, Christopher; Belinsky, Glenn S; De Guise, Sylvain; Adib, John N; Dobrowsky, Rick T; Bonventre, Joseph V; Rosenberg, Daniel W

    2005-04-01

    Cellular pools of free arachidonic acid are tightly controlled through enzymatic release of the fatty acid and subsequent utilization by downstream enzymes including the cyclooxygenases. Arachidonic acid cleavage from membrane phospholipids is accomplished by the actions of phospholipase A(2) (PLA(2)). Upon release, free arachidonic acid provides substrate for the synthesis of eicosanoids. However, under certain conditions, arachidonic acid may participate in ceramide-mediated apoptosis. Disruption of arachidonic acid homeostasis can shift the balance of cell turnover in favor of tumorigenesis, via overproduction of tumor-promoting eicosanoids or alternatively by limiting proapoptotic signals. In the following study, we evaluated the influence of genetic deletion of a key intracellular phospholipase, cytoplasmic PLA(2) (cPLA(2)), on azoxymethane-induced colon tumorigenesis. Heterozygous and null mice, upon treatment with the organotropic colon carcinogen, azoxymethane, developed a significant (P < 0.05) increase in colon tumor multiplicity (7.2-fold and 5.5-fold, respectively) relative to their wild-type littermates. This enhanced tumor sensitivity may be explained, in part, by the attenuated levels of apoptosis observed by terminal deoxynucleotidyl transferase-mediated nick end labeling staining within the colonic epithelium of heterozygous and null mice ( approximately 50% of wild type). The lower frequency of apoptotic cells corresponded with reduced ceramide levels (69% and 46% of wild-type littermates, respectively). Remarkably, increased tumorigenesis resulting from cPLA(2) deletion occurred despite a significant reduction in prostaglandin E(2) production, even in cyclooxygenase-2-overexpressing tumors. These data contribute new information that supports a fundamental role of cPLA(2) in the control of arachidonic acid homeostasis and cell turnover. Our findings indicate that the proapoptotic role of cPLA(2) in the colon may supercede its contribution to

  18. Testicular development and modes of apoptosis during spermatogenesis in various castes of the termite Reticulitermes labralis (Isoptera:Rhinotermitidae).

    Science.gov (United States)

    Su, Xiao Hong; Chen, Jiao Ling; Zhang, Xiao Jing; Xue, Wei; Liu, He; Xing, Lian Xi

    2015-11-01

    The separation of reproductive and non-reproductive roles based on caste differentiation is the most prominent characteristic of termites. However, little is known about the mechanism of male reproductive division that underlies caste differentiation. In the present study, testicular development and stage-specific apoptotic patterns were investigated and compared during spermatogenesis in reproductives, workers and soldiers of the termite Reticulitermes labralis. The results showed that male workers were divided into two types, the workers with spermatozoa (WS) and the workers without spermatozoa (WN). Spermatogenesis in WN and soldiers arrested at the spermatocyte stage. Moreover, there were significant differences in testicular size and spermatogenesis among the various castes. The mode of apoptosis in late instar WS was similar to the reproductives, as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) analysis. First, the majority of apoptotic cells were spermatogonia, and the spermatogonia of both late instar WS and reproductives exhibited lower apoptotic rates compared with late instar WN and soldiers. Second, the spermatocytes and spermatids showed very little apoptosis in the late instar WS and reproductives, and no TUNEL signal was detected in any of the examined spermatozoa. Our findings suggest that the male workers undergo a basal developmental schema comprising two undifferentiated larval instars, followed by a bifurcated development into either (i) the sexual lineage, in which the workers are able to provide normal spermatozoa to queens, or (ii) the neuter lineage, in which the male workers lose reproductive options. The level of testicular development may explain the significant discrepancies in reproductive capacity among the reproductives, workers and soldiers and reveal the reproductive division in male workers. These differences are controlled by apoptosis during early spermatogenesis.

  19. Comparison of the effects of erdosteine and N-acetylcysteine on apoptosis regulation in endotoxin-induced acute lung injury.

    Science.gov (United States)

    Demiralay, Rezan; Gürsan, Nesrin; Ozbilim, Gülay; Erdogan, Gülgün; Demirci, Elif

    2006-01-01

    This study was carried out to investigate comparatively the frequency of apoptosis in lung epithelial cells after intratracheal instillation of endotoxin [lipopolysaccharide (LPS)] in rats and the role of tumor necrosis factor alpha (TNF-alpha) on apoptosis, and the effects of erdosteine and N-acetylcysteine on the regulation of apoptosis. Female Wistar rats were given oral erdosteine (10-500 mg kg(-1)) or N-acetylcysteine (10-500 mg kg(-1)) once a day for 3 consecutive days. Then the rats were intratracheally instilled with LPS (5 mg kg(-1)) to induce acute lung injury. The rats were killed at 24 h after LPS administration. Lung tissue samples were stained with hematoxylin-eosin for histopathological assessments. The apoptosis level in the lung bronchial and bronchiolar epithelium was determined using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling) method. Cytoplasmic TNF-alpha was evaluated by immunohistochemistry. Pretreatment with erdosteine and pretreatment with N-acetylcysteine at a dose of 10 mg kg(-1) had no protective effect on LPS-induced lung injury. When the doses of drugs increased, the severity of the lung damage caused by LPS decreased. It was found that as the pretreatment dose of erdosteine was increased, the rate of apoptosis induced by LPS in lung epithelial cells decreased and this decrease was statistically significant in doses of 300 mg kg(-1) and 500 mg kg(-1). Pretreatment with N-acetylcysteine up to a dose of 500 mg kg(-1) did not show any significant effect on apoptosis regulation. It was noticed that both antioxidants had no significant effect on the local production level of TNF-alpha. These findings suggest that erdosteine could be a possible therapeutic agent for acute lethal lung injury and its mortality.

  20. The effects of erdosteine, N-acetylcysteine and vitamin E on nicotine-induced apoptosis of cardiac cells.

    Science.gov (United States)

    Demiralay, Rezan; Gürsan, Nesrin; Erdem, Havva

    2007-01-01

    This study was conducted to investigate the frequency of apoptosis in rat cardiomyocytes after intratraperitoneal nicotine injection, in order to examine the roles of inflammatory markers [myeloperoxidase (MPO) and tumor necrosis factor alpha (TNF-alpha)] in nicotine-induced cardiac damage and to determine the protective effects of three known antioxidant agents (N-acetylcysteine (NAC), erdosteine and vitamin E) on nicotine toxicity in the heart. Female Wistar rats were divided into seven groups, each composed of nine rats: two negative control groups, two positive control groups, one erdosteine-treated group (500 mg kg(-1)), one NAC-treated group (500 mg kg(-1)) and one vitamin E-treated group (500 mg kg(-1)). Nicotine was intraperitoneally injected at a dosage of 0.6 mg kg(-1) for 21 days. Following nicotine injection, the antioxidants were administered orally; treatment was continued until the rats were killed. Heart tissue samples were stained with hematoxylin-eosin for histopathological assessments. Apoptosis level in cardiomyocytes was determined by using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling) method. Staining of cytoplasmic TNF-alpha in cardiomyocytes and heart MPO activity were evaluated by immunohistochemistry. The treatments with erdosteine, NAC and vitamin E significantly reduced the rate of nicotine-induced cardiomyocyte apoptosis. The effect of vitamin E on apoptosis regulation was weaker than the effects of erdosteine and NAC. Erdosteine, NAC and vitamin E significantly reduced the increases in the local production of TNF-alpha and heart MPO activity. This findings suggest that the effects of erdosteine and NAC on apoptosis regulation are stronger than that of vitamin E.

  1. Regulation of sepsis-induced apoptosis of pulmonary cells by posttreatment of erdosteine and N-aceylcysteine.

    Science.gov (United States)

    Demiralay, Rezan; Gürsan, Nesrin; Erdem, Havva

    2006-12-07

    This study investigated the frequency of apoptosis in rat pulmonary epithelial cells after intraperitoneal endotoxin (LPS) injection, the effects of LPS on inflammatory markers [myeloperoxidase (MPO), tumor necrosis factor alpha (TNF-alpha), and vascular endothelial growth factor (VEGF)] in lung damage and the protective effects of two known antioxidant agents, erdosteine and N-acetylcysteine (NAC). Male Wistar rats were divided into six groups, each composed of nine rats: two control groups, two LPS-treated groups, one erdosteine-treated group (150 mg/kg), and one NAC-treated group. LPS was intraperitoneally injected at a dosage of 20mg/kg. Following LPS injection, the antioxidants were administered orally. The rats were killed 24h after LPS administration. Lung tissue samples were stained with hematoxylin-eosin (H&E) for histopathological assessments. Apoptosis level in epithelial cells was determined by using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling) method. Staining of cytoplasmic TNF-alpha in epithelial cells and VEGF in endothelial cells, and epithelial MPO activity were evaluated by immunohistochemistry. Posttreatment with erdosteine and NAC significantly reduced the rate of LPS-induced epithelial cell apoptosis. Posttreatment with erdosteine and NAC significantly reduced the increases in the local production of TNF-alpha and VEGF, and epithelial MPO activity. The effects of NAC on apoptosis, the increases in the local production of TNF-alpha and VEGF, were weaker than the effects of erdosteine. This finding suggests that the effects of erdosteine at the administered dose on apoptosis regulation are stronger than that of NAC.

  2. Treadmill exercise ameliorates symptoms of Alzheimer disease through suppressing microglial activation-induced apoptosis in rats.

    Science.gov (United States)

    Baek, Seung-Soo; Kim, Sang-Hoon

    2016-12-01

    Alzheimer disease (AD) is a most common form of dementia and eventually causes impairments of learning ability and memory function. In the present study, we investigated the effects of treadmill exercise on the symptoms of AD focusing on the microglial activation-induced apoptosis. AD was made by bilateral intracerebroventricular injection of streptozotocin. The rats in the exercise groups were made to run on a treadmill once a day for 30 min during 4 weeks. The distance and latency in the Morris water maze task and the latency in the step-down avoidance task were increased in the AD rats, in contrast, treadmill exercise shortened these parameters. The numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive and caspase-3-positive cells in the hippocampal dentate gyrus were decreased in the AD rats, in contrast, treadmill exercise suppressed these numbers. Expressions of glial fibrillary acidic protein (GFAP) and cluster of differentiation molecule 11B (CD11b) in the hippocampal dentate gyrus were increased in the AD rats, in contrast, treadmill exercise suppressed GFAP and CD11b expressions. Bax expression was increased and Bcl-2 expression was decreased in the hippocampus of AD rats, in contrast, treadmill exercise decreased Bax expression and increased Bcl-2 expression. The present results demonstrated that treadmill exercise ameliorated AD-induced impairments of spatial learning ability and short-term memory through suppressing apoptosis. The antiapoptotic effect of treadmill exercise might be ascribed to the inhibitory effect of treadmill exercise on microglial activation.

  3. Argininosuccinate Synthetase 1 Loss in Invasive Bladder Cancer Regulates Survival through General Control Nonderepressible 2 Kinase-Mediated Eukaryotic Initiation Factor 2α Activity and Is Targetable by Pegylated Arginine Deiminase.

    Science.gov (United States)

    Sahu, Divya; Gupta, Sounak; Hau, Andrew M; Nakashima, Kazufumi; Leivo, Mariah Z; Searles, Stephen C; Elson, Paul; Bomalaski, John S; Casteel, Darren E; Boss, Gerry R; Hansel, Donna E

    2016-12-09

    Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2-dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.

  4. Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis.

    Science.gov (United States)

    Cook, Julia L; Singh, Akannsha; DeHaro, Dawn; Alam, Jawed; Re, Richard N

    2011-11-01

    Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.

  5. Fisetin alleviates early brain injury following experimental subarachnoid hemorrhage in rats possibly by suppressing TLR 4/NF-κB signaling pathway.

    Science.gov (United States)

    Zhou, Chen-hui; Wang, Chun-xi; Xie, Guang-bin; Wu, Ling-yun; Wei, Yong-xiang; Wang, Qiang; Zhang, Hua-sheng; Hang, Chun-hua; Zhou, Meng-liang; Shi, Ji-xin

    2015-12-10

    Early brain injury (EBI) determines the unfavorable outcomes after subarachnoid hemorrhage (SAH). Fisetin, a natural flavonoid, has anti-inflammatory and neuroprotection properties in several brain injury models, but the role of fisetin on EBI following SAH remains unknown. Our study aimed to explore the effects of fisetin on EBI after SAH in rats. Adult male Sprague-Dawley rats were randomly divided into the sham and SAH groups, fisetin (25mg/kg or 50mg/kg) or equal volume of vehicle was given at 30min after SAH. Neurological scores and brain edema were assayed. The protein expression of toll-like receptor 4 (TLR 4), p65, ZO-1 and bcl-2 was examined by Western blot. TLR 4 and p65 were also assessed by immunohistochemistry (IHC). Enzyme-linked immunosorbent assay (ELISA) was performed to detect the production of pro-inflammatory cytokines. Terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) was perform to assess neural cell apoptosis. High-dose (50mg/kg) fisetin significantly improved neurological function and reduced brain edema at both 24h and 72h after SAH. Remarkable reductions of TLR 4 expression and nuclear factor κB (NF-κB) translocation to nucleus were detected after fisetin treatment. In addition, fisetin significantly reduced the productions of pro-inflammatory cytokines, decreased neural cell apoptosis and increased the protein expression of ZO-1 and bcl-2. Our data provides the evidence for the first time that fisetin plays a protective role in EBI following SAH possibly by suppressing TLR 4/NF-κB mediated inflammatory pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. L-carnitine protects against carboplatin-mediated renal injury: AMPK- and PPARα-dependent inactivation of NFAT3.

    Directory of Open Access Journals (Sweden)

    Yuh-Mou Sue

    Full Text Available We have previously shown that carboplatin induces inflammation and apoptosis in renal tubular cells (RTCs through the activation of the nuclear factor of activated T cells-3 (NFAT3 protein by reactive oxygen species (ROS, and that the ROS-mediated activation of NFAT3 is prevented by N-acetyl cysteine and heme oxygenase-1 treatment. In the current study, we investigated the underlying molecular mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Balb/c mice and RTCs were used as model systems. Carboplatin-induced apoptosis in RTCs was examined using terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling. We evaluated the effects of the overexpression of the peroxisome-proliferator-activated receptor alpha (PPARα protein, the knockdown of PPARα gene, and the blockade of AMPK activation and PPARα to investigate the underlying mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Carboplatin reduced the nuclear translocation, phosphorylation, and peroxisome proliferator responsive element transactivational activity of PPARα. These carboplatin-mediated effects were prevented by L-carnitine through a mechanism dependent on AMPK phosphorylation and subsequent PPARα activation. The activation of PPARα induced cyclooxygenase 2 (COX-2 and prostacyclin (PGI2 synthase expression that formed a positive feedback loop to further activate PPARα. The coimmunoprecipitation of the nuclear factor (NF κB proteins increased following the induction of PPARα by L-carnitine, which reduced NFκB transactivational activity and cytokine expression. The in vivo study showed that the inactivation of AMPK suppressed the protective effect of L-carnitine in carboplatin-treated mice, indicating that AMPK phosphorylation is required for PPARα activation in the L-carnitine-mediated protection of RTC apoptosis caused by carboplatin. The results of our study provide molecular evidence

  7. Efficacy of Compound Therapy by Ginseng and Ciprofloxacin on Bacterial Prostatitis

    Directory of Open Access Journals (Sweden)

    Maryam Miri

    2016-04-01

    Full Text Available Objective: Genitourinary tract infections play a significant role in male infertility. Infections of reproductive sex glands, such as the prostate, impair function and indirectly affect male fertility. The general aim of this study is to investigate the protective effect of Korean red ginseng (KRG on prostatitis in male rats treated with ciprofloxacin (CIPX. Materials and Methods: In this experimental study, we randomly divided 72 two male Wistar rats into 9 groups. The groups were treated as follows for 10 days: i. Control (no medication, ii. Sham [(normal saline injection into the vas deferens and oral administration of phosphate-buffered saline (PBS], iii. Ginseng, iv. CPIX, v. CIPX+ginseng, vi. Uropathogenic Escherichia coli (E. coli (UPEC, vii. UPEC+ginseng, viii. UPEC+CIPX, and ix. UPEC+ginseng+CIPX. The rats were killed 14 days after the last injection and the prostate glands were removed. After sample preparation, routine histology was performed using hematoxylin and eosin staining. The terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL method was used to determine the presence of apoptotic cells. Results: The severity score for acinar changes and inflammatory cell infiltration in the UPEC+CIPX group did not significantly different from the UPEC group. However this score significantly decreased in the UPEC+CIPX+ginseng group compared to the UPEC group. Apoptotic index of all ginseng treated groups significantly decreased compared to the UPEC and CPIX groups. Conclusion: These results suggested that ginseng might be an effective adjunct in CIPX treatment of prostatitis. The combined use ginseng and CIPX was more effective than ginseng or CIPX alone.

  8. HSP70 immune reactivity and TUNEL positivity in the liver of toluene-inhaled and melatonin-treated rats.

    Science.gov (United States)

    Tas, Ufuk; Ogeturk, Murat; Kuloglu, Tuncay; Sapmaz, Hilal Irmak; Kocaman, Nevin; Zararsiz, Ismail; Sarsilmaz, Mustafa

    2013-07-01

    Toluene is a clear, colorless and volatile hydrocarbon that is metabolized in liver, produced free oxygen radicals and can mediate cellular damage. Melatonin which is a pineal gland hormone is a very potent antioxidant. It can make the cellular membrane more durable against oxidative attacks and protect nuclear DNA from oxidative damage. This study aimed to investigate heat shock protein (HSP)70 immune reactivity and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positivity (apoptotic activity) in the liver of toluene-inhaled and melatonin-treated rats. A total of 21 adult male Wistar albino rats were divided at random into 3 equal groups. Animals in group I were designated as control. The rats in group II were exposed to toluene (3000 ppm/1 h/day) for 30 days, while the rats in group III were treated with melatonin (10 mg/kg/day, intraperitoneally) plus toluene inhalation. At the end of the 30-day experimental period, all rats were killed by decapitation. Then the liver tissues of rats were removed and tissue specimens were embedded in paraffin blocks. The specimens were stained with periodic acid-schiff (PAS) following routine histological procedures. Sections obtained from paraffin blocks were used for immune detection of TUNEL and HSP70. In light microscopic observations of tissues from toluene-inhaled rats, massive hepatocyte degeneration, ballooning degeneration and decreased PAS positivity were observed. Increased TUNEL positivity and HSP70 immune reactivity were determined in toluene-inhaled group and melatonin treatment decreased all these adverse effects.

  9. Epigenetic Changes are Associated with Programmed Cell Death Induced by Heat Stress in Seedling Leaves of Zea mays.

    Science.gov (United States)

    Wang, Pu; Zhao, Lin; Hou, Haoli; Zhang, Hao; Huang, Yan; Wang, Yapei; Li, Hui; Gao, Fei; Yan, Shihan; Li, Lijia

    2015-05-01

    Histone modification plays a crucial role in regulation of chromatin architecture and function, responding to adverse external stimuli. However, little is known about a possible relationship between epigenetic modification and programmed cell death (PCD) in response to environmental stress. Here, we found that heat stress induced PCD in maize seedling leaves which was characterized by chromatin DNA laddering and DNA strand breaks detected by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) test. The activities of the reactive oxygen species (ROS)-related enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were progressively increased over time in the heat-treated seedlings. However, the concentration of H2O2 remained at relatively lower levels, while the concentration of superoxide anion ([Formula: see text]) was increased, accompanied by the occurrence of higher ion leakage rates after heat treatment. The total acetylation levels of histones H3K9, H4K5 and H3 were significantly increased, whereas the di-methylation level of histone H3K4 was unchanged and the di-methylation level of histone H3K9 was decreased in the seedling leaves exposed to heat stress compared with the control seedlings, accompanied by increased nucleolus size indicative of chromatin decondensation. Furthermore, treatment of seedlings with trichostatin A (TSA), which always results in genomic histone hyperacetylation, caused an increase in the [Formula: see text] level within the cells. The results suggested that heat stress persistently induced [Formula: see text], leading to PCD in association with histone modification changes in the maize leaves.

  10. Quercetin attenuates high fructose feeding-induced atherosclerosis by suppressing inflammation and apoptosis via ROS-regulated PI3K/AKT signaling pathway.

    Science.gov (United States)

    Lu, Xue-Li; Zhao, Cui-Hua; Yao, Xin-Liang; Zhang, Han

    2017-01-01

    Quercetin is a dietary flavonoid compound extracted from various plants, such as apple and onions. Previous studies have revealed its anti-inflammatory, anti-cancer, antioxidant and anti-apoptotic activities. This study investigated the ability of quercetin to inhibit high fructose feeding- or LPS-induced atherosclerosis through regulating oxidative stress, apoptosis and inflammation response in vivo and in vitro experiments. 50 and 100mg/kg quercetin were used in our study, showing significant inhibitory role in high fructose-induced atherosclerosis via reducing reactive oxygen species (ROS) levels, Caspase-3 activation, inflammatory cytokines releasing, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and collagen contents as well as modulating apoptosis- and inflammation-related proteins expression. We also explored the protective effects of quercetin on atherosclerosis by phosphatidylinositide 3-kinases (PI3K)/Protein kinase B (AKT)-associated Bcl-2/Caspase-3 and nuclear factor kappa B (NF-κB) signal pathways activation, promoting AKT and Bcl-2 expression and reducing Caspase-3 and NF-κB activation. Quercetin reduced the atherosclerotic plaque size in vivo in high fructose feeding-induced mice assessed by oil red O. Also, in vitro experiments, quercetin displayed inhibitory role in LPS-induced ROS production, inflammatory response and apoptosis, which were linked with PI3K/AKT-regulated Caspase-3 and NF-κB activation. In conclusion, our results showed that quercetin inhibited atherosclerotic plaque development in high fructose feeding mice via PI3K/AKT activation regulated by ROS.

  11. Quercetin and vitamin E attenuate Bonny Light crude oil-induced alterations in testicular apoptosis, stress proteins and steroidogenic acute regulatory protein in Wistar rats.

    Science.gov (United States)

    Ebokaiwe, Azubuike P; Mathur, Premendu P; Farombi, Ebenezer O

    2016-10-01

    Studies have shown the reproductive effects of Bonny Light crude oil (BLCO) via the mechanism of oxidative stress and testicular apoptosis. We investigated the protective role of quercetin and vitamin E on BLCO-induced testicular apoptosis. Experimental rats were divided into four groups of four each. Animals were orally administered 2 ml/kg corn oil (control: group 1), BLCO-800 mg/kg body weight + 10 mg/kg quercetin (group 2), BLCO-800 mg/kg body weight + 50 mg/kg vitamin E (group 3) and BLCO-800 mg/kg body weight only (group 4) for 7 d. Protein levels of caspase 3, FasL, NF-kB, steroidogenic acute regulatory protein and stress response proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunofluorescence staining was used to quantify the expression of caspase 3, FasL and NF-kB. Apoptosis was quantified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptosis-related proteins by 50% and above after 7 d following BLCO exposure and a concomitant increase in expression of caspase 3, FasL and NF-kB expression by immunofluorescence staining. Apoptosis showed a significant increase in TUNEL positive cells. Co-administration with quercetin or vitamin E reversed BLCO-induced apoptosis and levels of stress protein, relative to control. These findings suggest that quercetin and vitamin E may confer protection against BLCO-induced testicular oxidative stress-related apoptosis.

  12. Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe

    Directory of Open Access Journals (Sweden)

    Guerin Jean F

    2011-06-01

    Full Text Available Abstract Background The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures. Methods Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group and frozen tissue. In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE, DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL in primordial and primary follicles (ApopDETEK Kit system Enzo and morphology in the two groups. 100 Follicles (primordial and primary were counted on both fresh and frozen hemiovary to assess this various tests. Results Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing. After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r = 0.82, p Conclusion We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.

  13. Biochemical and histopathological alterations in TAR DNA-binding protein-43 after acute ischemic stroke in rats.

    Science.gov (United States)

    Kanazawa, Masato; Kakita, Akiyoshi; Igarashi, Hironaka; Takahashi, Tetsuya; Kawamura, Kunio; Takahashi, Hitoshi; Nakada, Tsutomu; Nishizawa, Masatoyo; Shimohata, Takayoshi

    2011-03-01

    Nuclear factor TAR DNA-binding protein-43 (TDP-43) is considered to play roles in pathogenesis of human neurodegenerative diseases, so-called TDP-43 proteinopathy, via its proteolytic cleavage, abnormal phosphorylation, subcellular redistribution, and insolubilization generating TDP-43-positive neuronal intracellular inclusions. The purpose of this study was to elucidate biochemical and histopathological alternations in TDP-43 specific to acute ischemic stroke. Adult male rats were subjected to a 90-min middle cerebral artery occlusion. We examined the proteolytic cleavage, phosphorylation, subcellular localization, and solubility of TDP-43 by immunoblottings and histopathological examinations using the ischemic and sham-operated cortex. The level of full-length TDP-43 (43 kDa) decreased and that of the 25-kDa C-terminal fragment increased after acute ischemic stroke, which can be explained by proteolytic cleavage of TDP-43. Cytoplasmic redistribution and altered nuclear distribution of TDP-43 was observed after acute ischemic stroke, whereas abnormal phosphorylation and insolubilization of TDP-43 as well as formation of intracellular inclusions were not observed. Ischemic neurons with the cytoplasmic redistribution of TDP-43 expressed ubiquitin and activated caspase 3 and were terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling-positive. In conclusion, biochemical and histopathological alterations in TDP-43 were identified in rats after acute ischemic stroke, although there was very less similarity between TDP-43 alterations observed in acute ischemic stroke and those observed in TDP-43 proteinopathy. © 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.

  14. Alteration of oxidative stress and inflammatory cytokines induces apoptosis in diabetic nephropathy.

    Science.gov (United States)

    Sha, Jibin; Sui, Bo; Su, Xiaoqing; Meng, Qingfang; Zhang, Chenggang

    2017-09-19

    Diabetic nephropathy (DN) is one of the most significant long‑term complications in terms of morbidity and mortality for diabetic patients; however, the exact cause remains unknown. To address this, the DN model was established, and oxidative stress indexes, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH‑Px), and inflammatory cytokines, includinginterleukin‑6 (IL‑6), tumor necrosis factor‑alpha (TNF‑α) and transforming growth factor‑beta (TGF‑β), were examined by ELISA. Renal pathological alterations and cell apoptosis was examined by hematoxylin and eosin and terminal deoxynucleotidyl transferase mediated dUTP nick‑end labeling staining, respectively. The expression levels of B‑cell lymphoma‑2 (Bcl‑2), Bcl‑2 associated X (Bax) and caspase‑3 wereexamined by immunohistochemistry and western blotting. The DN model was correctly established, with lower body weight and the higher blood glucose in the diabetes model group. The expression levels of SOD and GSH‑Px were significantly decreased in the diabetes model group; however, MDA, IL‑6, TNF‑α and TGF‑β were significantly increased. The kidney was severely damaged in the diabetes model group, with inflammatory cell invasion, increasing amount of interstitial matrix and hypertrophy with vacuolar degeneration of renal tubular cells. Cell apoptosis levels were significantly increased, and Bcl‑2 was significantly decreased in the diabetes model group in contrast with that of the sham group; however, Bax and caspase‑3 were significantly increased. It suggested that increased oxidative stress and inflammatory cytokines may enhance the apoptosis levels in DN, and may provide a significant diagnostic reference for DN in diabetes patients.

  15. Protective effects of batimastat against hemorrhagic injuries in delayed jellyfish envenomation syndrome models.

    Science.gov (United States)

    Wang, Beilei; Liu, Dan; Liu, Guoyan; Zhang, Xin; Wang, Qianqian; Zheng, Jiemin; Zhou, Yonghong; He, Qian; Zhang, Liming

    2015-12-15

    Previously, we established delayed jellyfish envenomation syndrome (DJES) models and proposed that the hemorrhagic toxins in jellyfish tentacle extracts (TE) play a significant role in the liver and kidney injuries of the experimental model. Further, we also demonstrated that metalloproteinases are the central toxic components of the jellyfish Cyanea capillata (C. capillata), which may be responsible for the hemorrhagic effects. Thus, metalloproteinase inhibitors appear to be a promising therapeutic alternative for the treatment of hemorrhagic injuries in DJES. In this study, we examined the metalloproteinase activity of TE from the jellyfish C. capillata using zymography analyses. Our results confirmed that TE possessed a metalloproteinase activity, which was also sensitive to heat. Then, we tested the effect of metalloproteinase inhibitor batimastat (BB-94) on TE-induced hemorrhagic injuries in DJES models. Firstly, using SR-based X-ray microangiography, we found that BB-94 significantly improved TE-induced hepatic and renal microvasculature alterations in DJES mouse model. Secondly, under synchrotron radiation micro-computed tomography (SR-μCT), we also confirmed that BB-94 reduced TE-induced hepatic and renal microvasculature changes in DJES rat model. In addition, being consistent with the imaging results, histopathological and terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL)-like staining observations also clearly corroborated this hypothesis, as BB-94 was highly effective in neutralizing TE-induced extensive hemorrhage and necrosis in DJES rat model. Although it may require further clinical studies in the near future, the current study opens up the possibilities for the use of the metalloproteinase inhibitor, BB-94, in the treatment of multiple organ hemorrhagic injuries in DJES.

  16. The protective effects of human umbilical cord mesenchymal stem cells on damaged ovarian function: A comparative study.

    Science.gov (United States)

    Zhang, Jinjin; Xiong, Jiaqiang; Fang, Li; Lu, Zhiyong; Wu, Meng; Shi, Liangyan; Qin, Xian; Luo, Aiyue; Wang, Shixuan

    2016-09-05

    Numerous studies have reported that human umbilical cord mesenchymal stem cell (hUCMSC) therapy can rescue the structure and function of injured tissues. The aims of this study were to explore the protective role of hUCMSC transplantation in a model of accelerated ovarian aging and to compare 2 methods of transplanting hUCMSCs, i.e. i) via intravenous injection (IV) and ii) in situ ovarian micro injection (MI). Female mice were subjected to superovulation and ozone inhalation to create a model of accelerated ovarian aging with a decline in both the quantity and quality of oocytes. Cells were transplanted via IV or MI, and ovaries were removed after 2 weeks or 1 month of treatment. Ovarian reserve and function were evaluated based on the follicle counts, hormone levels, the estrous cycle, and reproductive performance. Cell tracking, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), real-time polymerase chain reaction (PCR), and Western blot analysis were used to assess the inner mechanisms of injury and repair. Results indicated that ovarian function increased significantly after treatment with hUCMSCs. Immunofluorescence revealed reduced TUNEL staining and a decreased percentage of apoptotic cells. A higher level of expression of anti-apoptotic and antioxidant enzymes was noted in the ovaries of groups treated with hUCMSCs. These parameters were enhanced more when mice were treated with hUCMSCs for 1 month than when they treated with hUCMSCs for 2 weeks. IV was better able to restore ovarian function than MI. These results suggest that both methods of transplantation may improve ovarian function and that IV transplantation of hUCMSCs can significantly improve ovarian function and structural parameters more than MI transplantation of hUCMSCs can.

  17. Sodium salicylate prevents paraquat-induced apoptosis in the rat lung.

    Science.gov (United States)

    Dinis-Oliveira, R J; Sousa, C; Remião, F; Duarte, J A; Ferreira, R; Sánchez Navarro, A; Bastos, M L; Carvalho, F

    2007-07-01

    The nonselective contact herbicide, paraquat (PQ), is a strong pneumotoxicant, especially due to its accumulation in the lung through a polyamine uptake system and to its capacity to induce redox cycling, leading to oxidative stress-related damage. In the present study, we aimed to investigate the occurrence of apoptotic events in the lungs of male Wistar rats, 24, 48, and 96 h after PQ exposure (25 mg/kg ip) as well as the putative healing effects provided by sodium salicylate [(NaSAL), 200 mg/kg ip] when administered 2 h after PQ. PQ exposure resulted in marked lung apoptosis, in a time-dependent manner, characterized by the "ladder-like" pattern of DNA observed through electrophoresis and by the presence of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells (TPC) as revealed by immunohistochemistry. The two main caspase cascades (the extrinsic receptor-mediated and the intrinsic mitochondria-mediated) and the expressions of p53 and activator protein-1 (AP-1) were also evaluated, to obtain an insight into apoptotic cellular signaling. PQ-exposed rats suffered a time-dependent increase of caspase-3 and caspase-8 and a decrease of caspase-1 activities in lungs compared to the control group. A marked mitochondrial dysfunction evidenced by cytochrome c (Cyt c) release was also observed as a consequence of PQ exposure. In addition, fluorescence electrophoretic mobility shift assay (fEMSA) revealed a transcriptional induction of the p53 and AP-1 transcription factors in a time-dependent manner as a consequence of PQ exposure. NaSAL treatment resulted in the remission of the observed apoptotic signaling and consequently of lung apoptosis. Taken together, the present results showed that PQ activates several events involved in the apoptotic pathways, which might contribute to its lung toxicodynamics. NaSAL, a recently implemented antidote for PQ intoxications, proved to protect lungs from PQ-induced apoptosis.

  18. Cell birth and survival following seasonal periods of cell proliferation in the chemosensory epithelia of red-backed salamanders, Plethodon cinereus.

    Science.gov (United States)

    Dawley, Ellen M; Nelsen, Meaghan; Lopata, Adrianne; Schwartz, Jessica; Bierly, Alison

    2006-01-01

    In addition to the continuous low levels of neurogenesis typical of adult vertebrates to replace damaged chemoreceptor cells, red-backed salamanders (Plethodon cinereus) experience an up-regulation of chemoreceptor epithelial cell proliferation on a seasonal basis. Significantly more cell division occurs in late spring than at any other time of the year, and we investigated the fate and life span of these newly generated cells. We used 5-bromo-2'-deoxyuridine (BrdU) immunocytochemical cell birth dating to examine cell proliferation and cell migration in the main olfactory and vomeronasal epithelia of red-backed salamanders collected in late spring who were allowed to survive for one hour or three, four, 25, 28, 42, 49 and 100 days post-injection. We examined new neuron growth in the vomeronasal and olfactory epithelia using antibodies against Growth Associated Protein-43 (GAP-43), a protein whose synthesis is up-regulated during axon growth. We also tracked apoptosis within both types of chemosensory epithelia using terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL). BrdU-immunoreactive cells were located extensively throughout the vomeronasal epithelia, particularly in the area posterior to the entrance of the nasolacrimal duct, not only after three days of survival, but also all of the longer experimental survival periods as well; BrdU-ir cells within the olfactory epithelia were rarely located after longer survival periods. Salamanders collected in late spring displayed extensive GAP-43 labeling in the vomeronasal epithelia posterior to the entrance of the nasolacrimal duct, indicating a large population of young vomeronasal receptor neurons. Finally, apoptotic cells were evident in this same post-nasolacrimal-duct-area of the vomeronasal organ and in the olfactory epithelium. We suggest that vomeronasal receptor neurons born in late spring function throughout the summer and may be associated with the animals' extensive territoriality

  19. Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis.

    Science.gov (United States)

    Srivastava, M; Ahmad, N; Gupta, S; Mukhtar, H

    2001-05-04

    Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.

  20. Ultrasound-microbubble mediated cavitation of plant cells: effects on morphology and viability.

    Science.gov (United States)

    Qin, Peng; Xu, Lin; Zhong, Wenjing; Yu, Alfred C H

    2012-06-01

    The interaction between ultrasound pulses and microbubbles is known to generate acoustic cavitation that may puncture biological cells. This work presents new experimental findings on the bioeffects of ultrasound-microbubble mediated cavitation in plant cells with emphasis on direct observations of morphological impact and analysis of viability trends in tobacco BY-2 cells that are widely studied in higher plant physiology. The tobacco cell suspensions were exposed to 1 MHz ultrasound pulses in the presence of 1% v/v microbubbles (10% duty cycle; 1 kHz pulse repetition frequency; 70 mm between probe and cells; 1-min exposure time). Few bioeffects were observed at low peak negative pressures (cavitation presumably occurred. In contrast, at 0.9 MPa peak negative pressure (with more inertial cavitation activities according to our passive cavitation detection results), random pores were found on tobacco cell wall (observed via scanning electron microscopy) and enhanced exogenous uptake into the cytoplasm was evident (noted in our fluorescein isothiocyanate dextran uptake analysis). Also, instant lysis was observed in 23.4% of cells (found using trypan blue staining) and programmed cell death was seen in 23.3% of population after 12 h (determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]). These bioeffects generally correspond in trend with those for mammalian cells. This raises the possibility of developing ultrasound-microbubble mediated cavitation into a targeted gene transfection paradigm for plant cells and, conversely, adopting plant cells as experimental test-beds for sonoporation-based gene therapy in mammalian cells.

  1. Roles of Treg/Th17 Cell Imbalance and Neuronal Damage in the Visual Dysfunction Observed in Experimental Autoimmune Optic Neuritis Chronologically.

    Science.gov (United States)

    Liu, Yuanyuan; You, Caiyun; Zhang, Zhuhong; Zhang, Jingkai; Yan, Hua

    2015-12-01

    Optic neuritis associated with multiple sclerosis and its animal model, experimental autoimmune optic neuritis (EAON), is characterized by inflammation, T cell activation, demyelination, and neuronal damage, which might induce permanent vision loss. Elucidating the chronological relationship among the features is critical for treatment of demyelinating optic neuritis. EAON was induced in C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein subcutaneously, and visual function was assessed by flash-visual evoked potential (F-VEP) at days 7, 11, 14, 19, 23, 28 post-immunization. Retinal ganglion cell (RGC) apoptosis was measured by terminal-deoxynucleotidyl transferase-mediated nick-end labeling. Demyelination and axonal damage were verified with myelin basic protein (MBP) and β-amyloid precursor protein staining, respectively. Real-time polymerase chain reaction quantified IL-17, IL-1β, TGF-β, FoxP3, IL-6, and IL-10 mRNA expression in the optic nerve, as well as FoxP3 and IL-17 staining. Systemic changes of Th17 and Treg cells were tested by flow cytometry in spleen. F-VEP latency was prolonged at 11 days and peaked at 23 days commensurate with demyelination. However, F-VEP amplitude was reduced at 11 days, preceding axon damage, and was exacerbated at 23 days when a peak in RGC apoptosis was detected. Th17 cells up-regulated as early as 7 days and peaked at 11 days, while Treg cells down-regulated inversely compared to Th17 cells change as verified by IL-17 and FoxP3 expression; spleen cell samples were slightly different, demonstrating marked changed at 14 days. Treg/Th17 cell imbalance in the optic nerve precedes and may initiate neuronal damage of axons and RGCs. These changes are commensurate with the appearances of visual dysfunction reflected in F-VEP and hence may offer a novel therapeutic avenue for vision preservation.

  2. Exenatide Reduces Tumor Necrosis Factor-α-induced Apoptosis in Cardiomyocytes by Alleviating Mitochondrial Dysfunction

    Institute of Scientific and Technical Information of China (English)

    Yuan-Yuan Cao; Zhang-Wei Chen; Yan-Hua Gao; Xing-Xu Wang; Jian-Ying Ma; Shu-Fu Chang; Ju-Ying Qian

    2015-01-01

    Background: Tumor necrosis factor-α (TNF-α) plays an important role in progressive contractile dysfunction in several cardiac diseases.The cytotoxic effects of TNF-α are suggested to be partly mediated by reactive oxygen species (ROS)-and mitochondria-dependent apoptosis.Glucagon-like peptide-1 (GLP-1) or its analogue exhibits protective effects on the cardiovascular system.The objective of the study was to assess the effects of exenatide, a GLP-1 analogue, on oxidative stress, and apoptosis in TNF-c-treated cardiomyocytes in vitro.Methods: Isolated neonatal rat cardiomyocytes were divided into three groups: Control group, with cells cultured in normal conditions without intervention;TNF-α group, with cells incubated with TNF-c (40 ng/ml) for 6, 12, or 24 h without pretreatment with exenatide;and exenatide group, with cells pretreated with exenatide (100 nmol/L) 30 mins before TNF-α (40 ng/ml) stimulation.We evaluated apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry, measured ROS production and mitochondrial membrane potential (MMP) by specific the fluorescent probes, and assessed the levels of proteins by Western blotting for all the groups.Results: Exenatide pretreatment significantly reduced cardiomyocyte apoptosis as measured by flow cytometry and TUNEL assay at 12 h and 24 h.Also, exenatide inhibited excessive ROS production and maintained MMP.Furthermore, declined cytochrome-c release and cleaved caspase-3 expression and increased bcl-2 expression with concomitantly decreased Bax activation were observed in exenatide-pretreated cultures.Conclusion: These results suggested that exenatide exerts a protective effect on cardiomyocytes, preventing TNF-α-induced apoptosis;the anti-apoptotic effects may be associated with protection of mitochondrial function.

  3. Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage.

    Science.gov (United States)

    Hsu, Chuan-Chih; Chen, Sheng-Hsien; Lin, Cheng-Hsien; Yung, Ming-Chi

    2014-10-01

    Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65-270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1β, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage

  4. Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility.

    Science.gov (United States)

    Del Olmo, Enrique; Bisbal, Alfonso; García-Álvarez, Olga; Maroto-Morales, Alejandro; Ramón, Manuel; Jiménez-Rabadán, Pilar; Anel-López, Luis; Soler, Ana J; Garde, J Julián; Fernández-Santos, María R

    2015-11-01

    The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r=-0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

  5. Riluzole improves outcome following ischemia-reperfusion injury to the spinal cord by preventing delayed paraplegia.

    Science.gov (United States)

    Wu, Y; Satkunendrarajah, K; Fehlings, M G

    2014-04-18

    The spinal cord is vulnerable to ischemic injury due to trauma, vascular malformations and correction of thoracic aortic lesions. Riluzole, a sodium channel blocker and anti-glutamate drug has been shown to be neuroprotective in a model of ischemic spinal cord injury, although the effects in clinically relevant ischemia/reperfusion models are unknown. Here, we examine the effect of riluzole following ischemia-reperfusion injury to the spinal cord. Female rats underwent high thoracic aortic balloon occlusion to produce an ischemia/reperfusion injury. Tolerance to ischemia was evaluated by varying the duration of occlusion. Riluzole (8mg/kg) was injected intraperitoneally 4h after injury. Locomotor function (Basso, Beattie and Bresnahan (BBB) scale) was assessed at 4h, 1day, and 5days post-ischemia. Spinal cords were extracted and evaluated for neuronal loss using immunohistology (choline acetyltransferase (ChAT) and neuronal nuclei (NeuN)), inflammation (CD11b), astrogliosis (glial fibrillary acidic protein - GFAP) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Ischemic injury lasting between 5.5 and 6.75min resulted in delayed paraplegia, whereas longer ischemia induced immediate paraplegia. When riluzole was administered to rats that underwent 6min of occlusion, delayed paraplegia was prevented. The BBB score of riluzole-treated rats was 11.14±4.85 compared with 1.86±1.07 in control animals. Riluzole also reduced neuronal loss, infiltration of microglia/macrophages and astrogliosis in the ventral horn and intermediate zone of the gray matter. In addition, riluzole reduced apoptosis of neurons in the dorsal horn of the gray matter. Riluzole has a neuroprotective effect in a rat model of spinal cord injury/reperfusion when administered up to 4h post-injury, a clinically relevant therapeutic time window.

  6. Mitochondrial-derived ROS in edelfosine-induced apoptosis in yeasts and tumor cells

    Institute of Scientific and Technical Information of China (English)

    Hui ZHANG; Consuelo GAJATE; Li-ping YU; Yun-xiang FANG; Faustino MOLLINEDO

    2007-01-01

    Aim: To investigate whether a similar process mediates cytotoxicity of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET- 18-OCH3, edelfosine) in both yeasts and human tumor cells.Methods: A modified version of a previously described assay for the intracellular conversion of nitro blue tetrazolium to formazan by superoxide anion was used to measure the generation of reactive oxygen spe-cies (ROS). Apoptotic yeast cells were detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. DNA fragmenta-tion and the generation of ROS were measured by cytofluorimetric analysis in Jurkat cells.Results: Edelfosine induced apoptosis in Saccharomyces cerevisiae,as assessed by TUNEL assay. Meanwhile, edelfosine induced a time- and con-centration-dependent generation of ROS in yeasts. Rotenone, an inhibitor of the mitochondrial electron transport chain, prevented ROS generation and apoptosis in response to edelfosine in S cerevisiae, α-Tocopherol abrogated the edelfosine-induced generation of intracellular ROS and apoptosis. Edelfosine also induced an increase of ROS in human leukemic cells that preceded apoptosis. The overexpression of Bcl-2 by gene transfer abrogated both ROS generation and apoptosis induced by edelfosine in leukemic cells. Changes in the relative mito-chondrial membrane potential were detected in both yeasts and Jurkat cells.Conclusion: These results indicate that edelfosine induces apoptosis in yeasts in addition to human tumor cells, and this apoptotic process involves mitochondria,likely through mitochondrial-derived ROS. These data also suggest that yeasts can be used as a suitable cell model in elucidating the antitumor mechanism of action of edelfosine.

  7. EFFECT OF STENT ABSORBED c-myc ANTISENSE OLIGODEOXYNUCLEOTIDE ON SMOOTH MUSCLE CELLS APOPTOSIS IN RABBIT CAROTID ARTERY

    Institute of Scientific and Technical Information of China (English)

    张新霞; 崔长琮; 李江; 崔翰斌; 徐仓宝; 朱参战

    2002-01-01

    Objective To investigate the effect of gelatin coated Platinium-Iridium stent absorbed c-myc antisense oligodeoxynucleotide (ASODN) on smooth muscle cells apoptosis in a normal rabbit carotid arteries. Methods Gelatin coated Platinium-Iridium stents were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomly divided into control group and treated group receiving c-myc ASODN (n=16, respectively). On 7, 14, 30 and 90 days following the stenting procedure ,morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical method. Apoptotic smooth muscle cells was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). Results At 7 and 14 days after stenting,there were no detectable apoptotic cells in both groups. The apoptotic cells occurred in the neointima 30 and 90 days after stenting, and the number of apoptotic cells at 30 days were less [4.50±1.29 vs 25.75±1.89 (number/0.1mm2)] than that at 90 days [13.50±1.91 vs 41.50±6.46 (number/0.1mm2)]. Meanwhile c-myc ASODN induced more apoptotic cells than the control group(P<0.0001). c-myc protein expression was weak positive or negative in treated group and positive in control group.Conclusion c-myc ASODN can induce smooth muscle cells apoptosis after stenting in normal rabbit carotid arteries,and it can be used to prevent in-stent restenosis.

  8. Expression changes of Rhodopsin and recoverin in MNU-induced photoreceptor degeneration in rats

    Directory of Open Access Journals (Sweden)

    Wei Jin

    2014-10-01

    Full Text Available AIM: To investigate the time-effect relationship between the expression of rhodopsin and recoverin and photoreceptor damage induced by N-nethl-N-nitrosourea(MNU. METHODS: Thirty-six 7-week old Sprague-Dawley(SDrats were intraperitoneally injected with MNU(60mg/kgand were put to death by dislocation of cervical vertebra 6, 12, 24h; 3, 7d after injection(6 per group, respectively. As a control, six rats were injected with phosphate buffer saline(PBS5mL/kg and sacrificed on d3 after injection. The degree of photoreceptor apoptosis was detected by HE staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNELand transmission electron microscope(TEMin the right eyes. The mRNA expressions of rhodopsin and recoverin were detected different time after injection by Western blot and immunohistochemical method in the left eyes. RESULTS: The dissolution of photoreceptor nucleus and apoptosis body were first perceived at 12h by TEM; most of cells at outer nuclear layer were presented positive reaction. The apoptotic index reached peak(29.7%±2.3%at 24h which was coincided with the observation of TEM. The results of immunohistochemistry displayed that rhodopsin and recoverin were on a declining curve with time extension. Furthermore, the results of Western blot indicated that rhodopsin had dramatic decline at 6h after injection(P0.05, and extremely significant difference comparing to control group after 12h(P0.01; while recoverin dramatic declined at 12h, and extremely significant difference after 24h(P0.01. CONCLUSION: 60mg/kg MNU intraperitoneally injection one-time may specifically induce photoreceptor apoptosis, The mechanism of down-regulation of rhodopsin and recoverin may be related to the selected apoptosis of photoreceptors.

  9. Sperm DNA oxidative damage and DNA adducts

    Science.gov (United States)

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  10. Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc

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    Zhao Ying-Zheng

    2010-11-01

    Full Text Available Abstract Background Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. Methods Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. Results Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4 hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. Conclusion Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment.

  11. Physiological disturbance may contribute to neurodegeneration induced by isoflurane or sevoflurane in 14 day old rats.

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    Binbin Wu

    Full Text Available BACKGROUND: Volatile anesthetics are widely used in pediatric anesthesia but their potential neurotoxicity raise significant concerns regarding sequelae after anesthesia. However, whether physiological disturbance during anesthetic exposure contributes to such side effects remains unknown. The aim of the current study is to compare the neurotoxic effects of isoflurane and sevoflurane in 14 day old rat pups under spontaneous breathing or ventilated conditions. METHODS: Postnatal 14 day rats were assigned to one of five groups: 1 spontaneous breathing (SB + room air (control, n = 17; 2 SB + isoflurane (n = 35; 3 SB + sevoflurane (n = 37; 4 mechanical ventilation (MV + isoflurane (n = 29; 5 MV + sevoflurane (n = 32. Anesthetized animal received either 1.7% isoflurane or 2.4% seveoflurane for 4 hours. Arterial blood gases and blood pressure were monitored in the anesthetized groups. Neurodegeneration in the CA3 region of hippocampus was assessed with terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling immediately after exposure. Spatial learning and memory were evaluated with the Morris water maze in other cohorts 14 days after experiments. RESULTS: Most rats in the SB groups developed physiological disturbance whereas ventilated rats did not but become hyperglycemic. Mortality from anesthesia in the SB groups was significantly higher than that in the MV groups. Cell death in the SB but not MV groups was significantly higher than controls. SB + anesthesia groups performed worse on the Morris water maze behavioral test, but no deficits were found in the MV group compared with the controls. CONCLUSIONS: These findings could suggest that physiological disturbance induced by isoflurane or sevoflurane anesthesia may also contribute to their neurotoxicity.

  12. Effects and Mechanism of Action of Inducible Nitric Oxide Synthase on Apoptosis in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

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    Zheng, Li; Ding, Junli; Wang, Jianwei; Zhou, Changman; Zhang, Weiguang

    2016-02-01

    Inducible nitric oxide synthase (iNOS) is a key enzyme in regulating nitric oxide (NO) synthesis under stress, and NO has varying ability to regulate apoptosis. The aim of this study was to investigate the effects and possible mechanism of action of iNOS on neuronal apoptosis in a rat model of cerebral focal ischemia and reperfusion injury in rats treated with S-methylisothiourea sulfate (SMT), a high-selective inhibitor of iNOS. Seventy-two male Sprague-Dawley (SD) rats were randomly divided into three groups: the sham, middle cerebral artery occlusion (MCAO) + vehicle, and MCAO + SMT groups. Neurobehavioral deficits, infarct zone size, and cortical neuron morphology were evaluated through the modified Garcia scores, 2,3,5-triphenyltetrazolium chloride (TTC), and Nissl staining, respectively. Brain tissues and serum samples were collected at 72 hr post-reperfusion for immunohistochemical analysis, Western blotting, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labeling assay (TUNEL) staining, and enzyme assays. The study found that inhibition of iNOS significantly attenuated the severity of the pathological changes observed as a result of ischemia-reperfusion injury: SMT reduced NO content as well as total nitric oxide synthase (tNOS) and iNOS activities in both ischemic cerebral hemisphere and serum, improved neurobehavioral scores, reduced mortality, reduced the infarct volume ratio, attenuated morphological changes in cortical neurons, decreased the rate of apoptosis (TUNEL and caspase-3-positive), and increased phospho (p)-AKT expression in ischemic penumbra. These results suggested that inhibition of iNOS might reduce the severity of ischemia-reperfusion injury by inhibiting neuronal apoptosis via maintaining p-AKT activity.

  13. Polyphenol-rich sweet potato greens extract inhibits proliferation and induces apoptosis in prostate cancer cells in vitro and in vivo.

    Science.gov (United States)

    Karna, Prasanthi; Gundala, Sushma R; Gupta, Meenakshi V; Shamsi, Shahab A; Pace, Ralphenia D; Yates, Clayton; Narayan, Satya; Aneja, Ritu

    2011-12-01

    Sweet potato (Ipomoea batatas) leaves or greens, extensively consumed as a vegetable in Africa and Asia, are an excellent source of dietary polyphenols such as anthocyanins and phenolic acids. Here, we show that sweet potato greens extract (SPGE) has the maximum polyphenol content compared with several commercial vegetables including spinach. The polyphenol-rich SPGE exerts significant antiproliferative activity in a panel of prostate cancer cell lines while sparing normal prostate epithelial cells. Mechanistically, SPGE perturbed cell cycle progression, reduced clonogenic survival, modulated cell cycle and apoptosis regulatory molecules and induced apoptosis in human prostate cancer PC-3 cells both in vitro and in vivo. SPGE-induced apoptosis has a mitochondrially mediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosis regulatory molecules such as inactivation of Bcl2, upregulation of BAX, cytochrome c release and activation of downstream apoptotic signaling. SPGE caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-nick-end labeling (TUNEL) staining of increased concentration of 3'-DNA ends. Furthermore, apoptotic induction was caspase dependent as shown by cleavage of caspase substrate, poly (adenosine diphosphate-ribose) polymerase. Oral administration of 400 mg/kg SPGE remarkably inhibited growth and progression of prostate tumor xenografts by ∼69% in nude mice, as shown by tumor volume measurements and non-invasive real-time bioluminescent imaging. Most importantly, SPGE did not cause any detectable toxicity to rapidly dividing normal tissues such as gut and bone marrow. This is the first report to demonstrate the in vitro and in vivo anticancer activity of sweet potato greens in prostate cancer.

  14. Nuclear factor-κB p65 (RelA) transcription factor is constitutively activated in human colorectal carcinoma tissue

    Institute of Scientific and Technical Information of China (English)

    Liang-Liang Yu; Hong-Gang Yu; Jie-Ping Yu; He-Sheng Luo; Xi-Ming Xu; Jun-Hua Li

    2004-01-01

    AIM: Activation of transcription factor nuclear factor-κB (NF-κB) has been shown to play a role in cell proliferation,apoptosis, cytokine production, and oncogenesis. The purpose of this study was to determine whether NF-κB was constitutively activated in human colorectal tumor tissues and, if so, to determine the role of NF-κB in colorectal tumorigenesis, and furthermore, to determine the association of RelA expression with tumor cell apoptosis and the expression of Bcl-2 and Bcl-xL.METHODS: Paraffin sections of normal epithelial, adenomatous and adenocarcinoma tissues were analysed immunohistochemically for expression of RelA, Bcl-2 and Bcl-xL proteins.Electrophoretic mobility shift assay (EMSA) was used to confirm the increased nuclear translocation of RelA in colorectal tumor tissues. The mRNA expressions of Bcl-2 and Bcl-xL were determined by reverse transcription polymerase chain reaction (RT-PCR) analysis. Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method.RESULTS: The activity of NF-κB was significantly higher in adenocarcinoma tissue in comparison with that in adenomatous and normal epithelial tissues. The apoptotic index (AI)significantly decreased in the transition from adenoma to adenocarcinoma. Meanwhile, the expressions of Bcl-2 and Bcl-xL protein and their mRNAs were significantly higher in adenocarcinoma tissues than that in adenomatous and normal epithelial tissues.CONCLUSION: NF-κB may inhibit apoptosis via enhancing the expression of the apoptosis genes Bcl-2 and BCl-xL. And the increased expression of RelA/nuclear factor-κB plays an important rote in the pathogenesis of colorectal carcinoma.

  15. Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience.

    Science.gov (United States)

    Toker, M Berk; Alcay, Selim; Gokce, Elif; Ustuner, Burcu

    2016-06-01

    The scope of this study was investigation the affects of various antioxidants on 1% soybean lecithin-based semen extenders for ram semen cryopreservation. Ejaculates, collected via electrically stimulated ejaculation, that have a thick consistency, rapid wave motion (3-5 on a 0-5 scale) and >75% initial motility were pooled. The pooled samples were split into four equal aliquots as 5 mM Methionine, 5 mM Cysteamine, 1 mM Cysteine and a sample of antioxidant-free control group. Each sample group was diluted to a ratio of 1/5 (semen/extender, v/v) as final concentration and two step dilution method was used for cryopreservation. Extender groups were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Semen samples also incubated for 6 h in humidified air with 5% CO2 at 39 °C to evaluate post-thaw incubation resilience of semen characteristics. The results showed that freezing and thawing procedures had negative effects on motility (P < 0.05), plasma membrane integrity (P < 0.05) and acrosomal integrity (P < 0.05). After 6 h of incubation time, the Cysteine supplemented extender group yielded significantly higher results than other extender groups in terms of spermatological parameters. Furthermore MDA levels in the antioxidant groups were lower than control group (P < 0.05). Nevertheless, there were no significant differences among antioxidant groups. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Indigestible material attenuated changes in apoptosis in the fasted rat jejunal mucosa.

    Science.gov (United States)

    Kakimoto, Takashi; Fujise, Takehiro; Shiraishi, Ryosuke; Kuroki, Tsukasa; Park, Jae Myung; Ootani, Akifumi; Sakata, Yasuhisa; Tsunada, Seiji; Iwakiri, Ryuichi; Fujimoto, Kazuma

    2008-03-01

    We have previously demonstrated that fasting induced apoptosis and decreased cell proliferation in the rat intestinal mucosa. The aim was to investigate the effect of expanded polystyrene as indigestible material on apoptosis and cell proliferation in rat small intestinal mucosa during fasting. Male SD rats were divided into 3 groups. The first group was fed with chow and water ad libitum. The second group fasted for 72 hrs. The third group was fasted for 24 hrs and was fed expanded polystyrene. Intestinal apoptosis was evaluated by percent fragmented DNA assay, terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end-labeling (TUNEL) staining, and caspase-3 assay. Cell proliferation was analyzed by 5-bromo-2'-deoxyuridine (5-BrdU) uptake. Truncal vagotomy was performed to evaluate a role of the central nervous system. In the 72-hr fasted rat, mucosal height of the rat jejunum was decreased to 73% of that in rats fed ad libitum, and this decrease was partly restored to 90% in rats fed expanded polystyrene. The fragmented DNA was increased in fasted rats (28.0%) when compared with that in rats fed ad libitum (2.6%). The increase in fragmented DNA in fasted rats was recovered by feeding them expanded polystyrene (8.3%). TUNEL staining confirmed this result. The effect of polystyrene on apoptosis was decreased by truncal vagotomy. Expression of cleaved caspase-3 was increased in fasted rats, which was then decreased by feeding of expanded polystyrene. In contrast to apoptosis, feeding of expanded polystyrene had no reconstructive effect on 5-BrdU uptake in the intestinal epithelium, which was decreased by fasting to 60% of that in rats fed ad libitum. In conclusion, feeding of indigestible material partly restored the decrease in intestinal mucosal length in the fasted rats through the apoptotic pathway without any influence on BrdU uptake. Further exploration focused on the mechanism of this effect of indigestible material is required.

  17. DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

    Science.gov (United States)

    Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

    2014-10-15

    Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Expression of Thyroid Transcription Factor-1 (TTF-1) in Lung Carcinomas and Its Correlations with Apoptosis and Angiogenesis

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Bai; Hong Shen; Chunhui Zhou; Hao Wang

    2009-01-01

    OBJECTIVE To investigate the correlations between the expression of thyroid transcription factor-1 (TTF-1) and apoptosis and angiogenesis in lung carcinomas.METHODS A 829 microarray of the paraffin tissue chips was constructed, which contained 196 lung carcinomas, 10 normal lung tissues, and 1 muscular tissue. Terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and immunohistochemical SP method were used to detect apoptosis and expression of TTF-1 and CD34 in different types of lung carcinomas. A Leica Q500 MC image analysis system was used to measure and calculate TTF-1 positive unit (PU), apoptotic index (AI) and microvessel density (MVD).RESULTS AI of lung small cell carcinoma and large cell carcinoma were smaller than those of lung adenocarcinoma and squamous cell carcinoma (P = 0.000). AI of lung carcinomas with lymph node metastases was smaller than that of those without (P = 0.039). AI of lung carcinomas in TNM stage I-W was smaller than that in stage Ⅰ (P = 0.008). The PU of the TTF-1 was negatively correlated with AI in small cell lung carcinoma (r = -0.752, P = 0.000). MVD of lung carcinomas without lymph node metastases was smaller than that of those with lymph node metastasis (P= 0.031). MVD of lung carcinomas in TNM stage Ⅰ was smaller than that in stage Ⅰ-Ⅳ (P -- 0.040). The PU of TTF-1 was positively correlated with MVD in lung adenocarcinoma (r = 0.708, P = 0.000).CONCLUSION There is a negative correlation between TTF-1 PU and AI in small cell lung carcinoma. TTF-1 PU and AI may be correlated with each other. There is a positive correlation between TTF-1 PU and MVD in lung adenocarcinoma. TTF-1 may induce the development of lung adenocarcinoma by inducing tumor angiogenesis.

  19. Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl

  20. Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

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    Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Wu, Honghai; Gai, Renhua; Wu, Youping [Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); Yang, Bo [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Yang, Xiaochun, E-mail: yangxiaochun@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China)

    2012-06-15

    Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib

  1. Pre-exposure to low-power diode laser irradiation promotes cytoprotection in the rat retina.

    Science.gov (United States)

    Sun, Yue; Zhang, Shisheng; Liao, Huaping; Wang, Jing; Wang, Ling

    2015-01-01

    The aim of this study was to investigate whether pre-exposure to low-power laser irradiation can provoke an effect on cellular protection in the rat retina. The right eyes of 40 rats were exposed to a 3-mm diode laser beam for 1 min in different light intensities and different experimental sets: group A low power of 60 mW (34.27 J/cm(2) on the retina in consideration of the energy losses along the optical pathway) prior to high power of 80 mW (44.88 J/cm(2) on the retina in consideration of the energy losses along the optical pathway), group B high power, group C low power, group D (the left eyes from the counterpart of group A) and group E (untreated rat eyes) as controls. Morphological retinal change retinas were assessed using light microscopy and/or transmission electron microscopy. Heat shock protein (Hsp) 70 and cleaved caspase 3 protein expression were analyzed by immunohistochemical staining and Western blot. Cellular injury was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Hsp 70 expression in the inner plexiform layer and the outer plexiform layer in group A were 73.09 ± 6.49 and 78.03 ± 3.05%, respectively, which was significantly higher (P power laser irradiation stimulates a hyperexpression of Hsp70 together with a hypoexpression of cleaved caspase 3 in rat retina, which may suggest a cellular protective effect.

  2. Novel association between sperm deformity index and oxidative stress-induced DNA damage in infertile male patients

    Institute of Scientific and Technical Information of China (English)

    Tamer M. Said; Nabil Aziz; Rakesh K. Sharma; Iwan Lewis-Jones; Anthony J. Thomas Jr; Ashok Agarwal

    2005-01-01

    Aim: To investigate the impact of abnormal sperm morphology using the sperm deformity index (SDI) on reactive oxygen species (ROS) production and its correlation with sperm DNA damage. Methods: Semen samples were collected from men undergoing infertility screening (n = 7) and healthy donors (n = 6). Mature spermatozoa were isolated and incubated with 5 mmol/L β-nicotinamide adenine dinucleotide phosphate (NADPH) for up to 24 h to induce ROS. Sperm morphology was evaluated using strict Tygerberg's criteria and the SDI. ROS levels and DNA damage were assessed using chemiluminescence and terminal deoxynucleotidyl transferase-mediated fluoresceindUTP nick end labeling (TUNEL) assays, respectively. Results: SDI values (median [interquartiles]) were higher in patients than donors (2 [1.8, 2.1] vs. 1.53 [1.52, 1.58], P = 0.008). Aliquots treated with NADPH showed higher ROS levels (1.22 [0.30, 1.87] vs. 0.39 [0.10, 0.57], P = 0.03) and higher incidence of DNA damage than those not treated (10 [4.69, 24.85] vs. 3.85 [2.58, 5.10], P = 0.008). Higher DNA damage was also seen following 24 h of incubation in patients compared to donors. SDI correlated with the percentage increase in sperm DNA damage following incubation for 24 h in samples treated with NADPH (r = 0.7, P = 0.008) and controls (r = 0.58, P = 0.04).Conclusion: SDI may be a useful tool in identifying potential infertile males with abnormal prevalence of oxidative stress (OS)-induced DNA damage. NADPH plays a role in ROS-mediated sperm DNA damage, which appears to be more evident in infertile patients with semen samples containing a high incidence of morphologically abnormal spermatozoa.

  3. Similar to spironolactone, oxymatrine is protective in aldosterone-induced cardiomyocyte injury via inhibition of calpain and apoptosis-inducing factor signaling.

    Directory of Open Access Journals (Sweden)

    Ting-Ting Xiao

    Full Text Available Accumulating evidence indicates that oxymatrine (OMT possesses variously pharmacological properties, especially on the cardiovascular system. We previously demonstrated that activated calpain/apoptosis-inducing factor (AIF-mediated pathway was the key molecular mechanism in aldosterone (ALD induces cardiomyocytes apoptosis. In the present study, we extended the experimentation by investigating the effect of OMT on cardiomyocytes exposed to ALD, as compared to spironolactone (Spiro, a classical ALD receptor antagonist. Cardiomyocytes were pre-incubated with OMT, Spiro or vehicle for 1 h, and then, cardiomyocytes were exposed to ALD 24 h. The cell injury was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and lactate dehydrogenase (LDH leakage ratio. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, annexin V/PI staining, and relative caspase-3 activity assay. Furthermore, expression of pro-apoptotic proteins including truncated Bid (tBid, calpain and AIF were evaluated by western blot analysis. ALD stimulation increased cardiomyocytes apoptosis, caspase-3 activity and protein expression of calpain, tBid and AIF in the cytosol (p<0.05. Pre-incubated with cardiomyocytes injury and increased caspase-3 activity were significantly attenuated (p<0.05. Furthermore, OMT suppressed ALD-induced high expression of calpain and AIF. And these effects of OMT could be comparable to Spiro. These findings indicated that OMT might be a potential cardioprotective-agent against excessive ALD-induced cardiotoxicity, at least in part, mediated through inhibition of calpain/AIF signaling.

  4. The effect of reactive oxygen species of inducing apoptosis on the hepatocacinoma in the rabbits

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To study the effect of reactive oxygen species of inducing apoptosis on the heptocacinoma tissues following ischemia and reperfusion and perfusion hyperoxia liquid of hepatocarcinoma. Methods: The hepatocarcinoma animal models ware established by implantation of VX2 tumor constitution mass into the left middle lobe of liver of rabbits. The animals were subjected to 60 min clamp-induced ischemia of hepatic artery distributing in the left middle lobe followed by reperfusion atlh, Id, 3d and 7 d, respectively, and perfusion hyperoxia liquid (partial pressure of oxygen, PO2>80 kPa) at the same time with reperfusion beginning. The concentration of MDA and NO ware tested. Apoptotic changes in the hepatocarcinoma and normal hepatic tissues were observed by means of HE staining and terminal de-oxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNED. Results: The concentration of MDA in normal hepatic tissues and hepatocarcinoma tissue increased followed ischemia and reperfusion especially for reperfusion 1 h (4. 61 ±0. 40, 3. 10±0. 23) and restored to normal level at reperfusion 7 d in normal hepatic tissues but still kept high concentration in the hepatocarcinoma tissue. Even though concentration of MDA in normal hepatic tissues is higher than that of before ischemia and reperfusion, no difference have been found after perfusion of hyperoxia liquid, and in the hepatocarcinoma tissue. the increasing of concentration of MDA was obvious after simply ischemia and reperfusion at reperfusion Id (4. 25 ± 0. 45). The concentration of NO in normal hepatic tissues increased for reperfusion 3 d and 7 d(18. 17± 0. 13, 17. 45±0. 23),while that of hepatocarcinoma tissue decreased at reperfusion 3 d(15. 95±043). After perfusion of hyperoxia liquid, the concentration of NO in normal hepatic tissues kept increasing and that decreased in the hepatocarcinoma tissues in all time point and reached the lowest level at reperfusion 1 d(14. 62±O. 45). The result

  5. Hydrogen sulfide induces apoptosis of pulmonary artery smooth muscle cell in rats with pulmonary hypertension induced by high pulmonary blood flow

    Institute of Scientific and Technical Information of China (English)

    LI Wei; JIN Hong-fang; LIU Die; SUN Jing-hui; JIAN Pei-jun; LI Xiao-hui; TANG Chao-shu; DU Jun-bao

    2009-01-01

    Background Abnormal apoptosis of pulmonary artery smooth muscle cells (PASMCs) is an important pathophysiological process in the pulmonary artery structural remodeling and pulmonary hypertension. We investigated possible effect of endogenous hydrogen sulfide (H_2S) on apoptosis of PASMCs during the development of pulmonary hypertension induced by high pulmonary blood flow.Methods Thirty-nine male Sprague-Dawley rats were randomly assigned to 4-week control, 4-week shunt, 4-week shunt+propargylglycine (PPG), 11-week control, 11-week shunt and 11-week shunt+sodium hydrosulfide (NaHS) groups. Rats in 4-week shunt, 4-week shunt+PPG, 11-week shunt and 11-week shunt+NaHS groups underwent an abdominal aorta-inferior vena cava shunt. Rats in 4-week shunt+PPG group were intraperitoneally injected with PPG, an inhibitor of endogenous H_2S production, for 4 weeks. Rats in 11-week shunt+NaHS group were intraperitoneally injected with NaHS, a H_2S donor, for 11 weeks. Lung tissue H_2S was evaluated by sulfide-sensitive electrode. Apoptosis of PASMCs were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Expressions of Fas, bcl-2 and caspase-3 in the PASMCs were analyzed with immunochemical staining.Results Four weeks after the shunting operation, the apoptosis of PASMCs and expression of Fas and caspase-3 were significantly decreased (P<0.01), but expression of bcl-2 increased significantly (P<0.01). PPG administration further inhibited the apoptosis of PASMCs, downregulated the expression of Fas and caspase-3 (P <0.01), but increased the expression of bcl-2 (P<0.01). After 11 weeks of shunting operation, the apoptosis of PASMCs and expression of Fas and caspase-3 were significantly decreased (P <0.01), but expression of bcl-2 increased obviously (P <0.01). NaHS administration significantly increased the apoptosis of PASMCs, upregulated the expression of Fas and caspase-3, but inhibited the expression of bcl-2.Conclusions H_2S induces

  6. Insight into hypoxic preconditioning and ischemic injury through determination of nPKCε-interacting proteins in mouse brain.

    Science.gov (United States)

    Feng, Sujuan; Li, Dongguo; Li, Yun; Yang, Xuan; Han, Song; Li, Junfa

    2013-08-01

    Cerebral hypoxic preconditioning (HPC) provides neuroprotection by intracellular signaling pathways. We previously demonstrated that novel protein kinase Cε (nPKCε) activation participated in cerebral HPC development. In this study, we explore the role of nPKCε in HPC-induced neuroprotection against middle cerebral artery occlusion (MCAO)-induced ischemic injury and identify its possible signaling molecules. A total of 131 adult male BALB/c mice were divided into eight groups: normoxic control (n=9), HPC (n=9), HPC+εV1-2 (n=13), Sham (n=19), HPC+sham (n=6), Ischemia (I, 6h MCAO, n=31), HPC+I (n=25) and HPC+εV1-2+I (n=19). nPKCε specific inhibitor εV1-2 was administered via intracerebroventricular injection. Western blot, 2,3,5-triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were applied to determine nPKCε membrane translocation, infarction volume and programmed cell death (PCD), respectively. Two-dimensional gel electrophoresis (2-De) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify nPKCε-interacting proteins, followed by bioinformatics analysis of genee ontology (GO) to predict nPKCε-specific signaling pathways. Our results showed that HPC attenuates MCAO-induced brain injuries and stabilized nPKCεmembrane translocation in peri-infarct region, which was abolished by nPKCε-speecific inhibitor εV1-2. Proteomics analysis revealed 8 up- and 3 down-regulated nPKCε-interacting proteins both in cytosolic and particulate fractions of HPC mouse brain. GO analysis predicted 25 significant nPKCε-specific signaling pathways among the 16 identified nPKCε-interacting proteins in brain of HPC mice. This study is the first to report multiple nPKCε-interacting proteins and their signaling pathways in HPC mouse brain, suggesting that nPKCε signaling molecules is responsible for HPC-induced neuroprotection against cerebral ischemic

  7. Expression of Inducible Nitric Oxide Synthase, p53 and Bcl-2 in Gastric Precancerous and Cancerous Lesions: Correlation with Clinical Features

    Institute of Scientific and Technical Information of China (English)

    Tao Cui; Zu'an Zhu; Ying Liu; Qingyan Kong; Sujuan Fei

    2006-01-01

    OBJECTIVE To explore the expression of inducible nitric oxide synthase(iNOS), p53 and bcl-2 in gastric precancerous and cancerous lesions and to examine the expression of these proteins in relation to clinical features.METHODS The expressions of iNOS, p53 and bcl-2 proteins in gastric precancerous and cancerous lesions and their correlations with the clinical features were determined using immunohistochemical assays (Power VisionTM two-step method) on 84 gastric carcinomas and 54 gastric atypical hyperplastic tissues. Apoptotic cells were evaluated by terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick-end labeling (TUNEL).RESULTS Expression of iNOS, p53 and bcl-2 was significantly higher in gastric carcinoma (GC) tissues than in gastric atypical hyperplastic tissues. Among the 84 carcinomas, the expression of p53 was observed in 50 (59.52%), bcl-2 in 43 (51.19%), and iNOS in 65 (77.58%). Overexpression of iNOS and bcl-2 in gastrlc carcinoma was related to tumor size and iNOS was related to the presence of lymph node metastasis (P<0.05). The expression of proteins did not correlate with age, sex, stage of disease, or differentiation. Expression of iNOS in gastric carcinoma tissues was positively correlated with bcl-2 expression (χ2=8.926, P=0.003),and also with p53 expression (χ2= 5.2430, P= 0.022). The mean apoptotic indexes (Al) were 1.29%±0.50 in low-grade atypical hyperplasia (LG),0.96%±0.36 in high-grade atypical hyperplasia (HG) and 0.70%±0.43 in GC, with the difference being significant between LG, HG and GC (P<0.05). There was a significant positive correlation between iNOS expression and the Al in GC (t=3.0815, P=0.0028).CONCLUSION iNOS was expressed in the majority of gastric carcinoma tissues and correlated with cellular apoptosis associated with p53 and bcl-2 expression. iNOS overexpression is closely associated with p53 and bcl-2 accumulation status. iNOS may play a synergistic role in the pathogenesis of GC.

  8. Promotion versus suppression of rat colon carcinogenesis by chlorophyllin and chlorophyll: modulation of apoptosis, cell proliferation, and {beta}-catenin/Tcf signaling

    Energy Technology Data Exchange (ETDEWEB)

    Blum, Carmen A.; Xu Meirong; Orner, Gayle A.; Dario Diaz, G.; Li Qingjie; Dashwood, Wan Mohaiza; Bailey, George S.; Dashwood, Roderick H

    2003-03-01

    The carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 1,2-dimethylhydrazine (DMH) induce colon tumors in the rat that contain mutations in {beta}-catenin, but the mutation pattern can be influenced by exposure to dietary phytochemicals, such as the water-soluble derivative of chlorophyll called chlorophyllin. Whereas chlorophyllin is an effective blocking agent during the initiation phase, post-initiation responses depend upon the exposure protocol, and can be influenced by the initiating agent and the concentration of chlorophyllin. Post-initiation treatment with 0.001% chlorophyllin (w/v) in the drinking water promoted colon carcinogenesis in the rat, but much higher concentrations (1.0% chlorophyllin) led to suppression. Bromodeoxyuridine and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) indices revealed that the promotional concentration of 0.001% chlorophyllin increased the ratio of cell proliferation to apoptosis in the colonic crypts, whereas concentrations in the range 0.01-1.0% chlorophyllin modestly reduced this ratio. Molecular studies showed that the spectrum of {beta}-catenin mutations was markedly different in chlorophyllin-promoted colon tumors--many of the mutations led to direct substitutions of critical Ser/Thr residues within the glycogen synthase kinase-3{beta} (GSK-3{beta}) region, whereas in all other groups, including DMH and IQ controls, the mutations typically affected amino acids adjacent to Ser{sup 33}. Substitution of critical Ser/Thr residues caused {beta}-catenin and c-Jun proteins to be markedly over-expressed compared with tumors in which the mutations substituted amino acid residues flanking these critical Ser/Thr sites. In a separate study, rats were exposed to IQ or azoxymethane (AOM), a metabolite of DMH, and they were treated post-initiation with chlorophyllin, chlorophyll, copper, or phytol in the diet. Natural chlorophyll (0.08%) suppressed AOM- and IQ-induced aberrant crypt foci (ACF

  9. Promotion versus suppression of rat colon carcinogenesis by chlorophyllin and chlorophyll: modulation of apoptosis, cell proliferation, and beta-catenin/Tcf signaling.

    Science.gov (United States)

    Blum, Carmen A; Xu, Meirong; Orner, Gayle A; Darío Díaz, G; Li, Qingjie; Dashwood, Wan Mohaiza; Bailey, George S; Dashwood, Roderick H

    2003-01-01

    The carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 1,2-dimethylhydrazine (DMH) induce colon tumors in the rat that contain mutations in beta-catenin, but the mutation pattern can be influenced by exposure to dietary phytochemicals, such as the water-soluble derivative of chlorophyll called chlorophyllin. Whereas chlorophyllin is an effective blocking agent during the initiation phase, post-initiation responses depend upon the exposure protocol, and can be influenced by the initiating agent and the concentration of chlorophyllin. Post-initiation treatment with 0.001% chlorophyllin (w/v) in the drinking water promoted colon carcinogenesis in the rat, but much higher concentrations (1.0% chlorophyllin) led to suppression. Bromodeoxyuridine and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) indices revealed that the promotional concentration of 0.001% chlorophyllin increased the ratio of cell proliferation to apoptosis in the colonic crypts, whereas concentrations in the range 0.0l-1.0% chlorophyllin modestly reduced this ratio. Molecular studies showed that the spectrum of beta-catenin mutations was markedly different in chlorophyllin-promoted colon tumors--many of the mutations led to direct substitutions of critical Ser/Thr residues within the glycogen synthase kinase-3beta (GSK-3beta) region, whereas in all other groups, including DMH and IQ controls, the mutations typically affected amino acids adjacent to Ser(33). Substitution of critical Ser/Thr residues caused beta-catenin and c-Jun proteins to be markedly over-expressed compared with tumors in which the mutations substituted amino acid residues flanking these critical Ser/Thr sites. In a separate study, rats were exposed to IQ or azoxymethane (AOM), a metabolite of DMH, and they were treated post-initiation with chlorophyllin, chlorophyll, copper, or phytol in the diet. Natural chlorophyll (0.08%) suppressed AOM- and IQ-induced aberrant crypt foci (ACF), whereas

  10. p75(NTR) antagonists attenuate photoreceptor cell loss in murine models of retinitis pigmentosa.

    Science.gov (United States)

    Platón-Corchado, María; Barcelona, Pablo F; Jmaeff, Sean; Marchena, Miguel; Hernández-Pinto, Alberto M; Hernández-Sánchez, Catalina; Saragovi, H Uri; de la Rosa, Enrique J

    2017-07-13

    ProNGF signaling through p75(NTR) has been associated with neurodegenerative disorders. Retinitis pigmentosa (RP) comprises a group of inherited retinal dystrophies that causes progressive photoreceptor cell degeneration and death, at a rate dependent on the genetic mutation. There are more than 300 mutations causing RP, and this is a challenge to therapy. Our study was designed to explore a common mechanism for p75(NTR) in the progression of RP, and assess its potential value as a therapeutic target. The proNGF/p75(NTR) system is present in the dystrophic retina of the rd10 RP mouse model. Compared with wild-type (WT) retina, the levels of unprocessed proNGF were increased in the rd10 retina at early degenerative stages, before the peak of photoreceptor cell death. Conversely, processed NGF levels were similar in rd10 and WT retinas. ProNGF remained elevated throughout the period of photoreceptor cell loss, correlating with increased expression of α2-macroglobulin, an inhibitor of proNGF processing. The neuroprotective effect of blocking p75(NTR) was assessed in organotypic retinal cultures from rd10 and RhoP mouse models. Retinal explants treated with p75(NTR) antagonists showed significantly reduced photoreceptor cell death, as determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and by preservation of the thickness of the outer nuclear layer (ONL), where photoreceptor nuclei are located. This effect was accompanied by decreased retinal-reactive gliosis and reduced TNFα secretion. Use of p75(NTR) antagonist THX-B (1,3-diisopropyl-1-[2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-purin-7-yl)-acetyl]-urea) in vivo in the rd10 and RhoP mouse models, by a single intravitreal or subconjunctival injection, afforded neuroprotection to photoreceptor cells, with preservation of the ONL. This study demonstrates a role of the p75(NTR)/proNGF axis in the progression of RP, and validates these proteins as therapeutic targets

  11. Propofol Ameliorates Calpain-induced Collapsin Response Mediator Protein-2 Proteolysis in Traumatic Brain Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    Yun Yu; Min-Yu Jian; Yun-Zhen Wang; Ru-Quan Han

    2015-01-01

    Background:Collapsin response mediator protein-2 (CRMP2),a multifunctional cytosolic protein highly expressed in the brain,is degraded by calpain following traumatic brain injury (TBI),possibly inhibiting posttraumatic neurite regeneration.Lipid peroxidation (LP) is involved in triggering postinjury CRMP2 proteolysis.We examined the hypothesis that propofol could attenuate LP,calpain-induced CRMP2 degradation,and brain injury after TBI.Methods:A unilateral moderate controlled cortical impact injury was induced in adult male Sprague-Dawley rats.The animals were randomly divided into seven groups:Sham control group,TBI group,TBI + propofol groups (including propofol 1 h,2 h,and 4 h groups),TBI + U83836E group and TBI + fat emulsion group.The LP inhibitor U83836E was used as a control to identify that antioxidation partially accounts for the potential neuroprotective effects of propofol.The solvent of propofol,fat emulsion,was used as the vehicle control.Ipsilateral cortex tissues were harvested at 24 h post-TBI.Immunofluorescent staining,Western blot analysis,and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling were used to evaluate LP,calpain activity,CRMP2 proteolysis and programmed cell death.The data were statistically analyzed using one-way analysis of variance and a paired t-test.Results:Propofol and U83836E significantly ameliorated the CRMP2 proteolysis.In addition,both propofol and U83836E significantly decreased the ratio of 145-kDa αⅡ-spectrin breakdown products to intact 270-kDa spectrin,the 4-hydroxynonenal expression and programmed cell death in the pericontusional cortex at 24 h after TBI.There was no difference between the TBI group and the fat emulsion group.Conclusions:These results demonstrate that propofol postconditioning alleviates calpain-mediated CRMP2 proteolysis and provides neuroprotective effects following moderate TBI potentially by counteracting LP and reducing calpain activation.

  12. Gastrodin prevents steroid-induced osteonecrosis of the femoral head in rats by anti-apoptosis

    Institute of Scientific and Technical Information of China (English)

    Zheng Huifeng; Yang Erping; Peng Hao; Li Jianping; Chen Sen; Zhou Jianlin; Fang Hongsong

    2014-01-01

    Background Gastrodin,as one of the major components extracted from the Chinese herb Gastrodia elata BI.,has many biologic effects,one of which is anti-apoptosis.Apoptosis is considered to be one of the pathogenetic mechanisms in steroid-induced osteonecrosis of the femoral head (ONFH).Therefore,we performed this study to investigate whether gastrodin has the potential to prevent steroid-induced ONFH.Methods All 18 male adult Wistar rats were divided equally into three groups:the steroid group,the gastrodin+steroid group,and the control group.Osteonecrosis was induced by low-dose lipopolysaccharide and subsequent high-dose methylprednisolone.Histomorphometric method was used to determine the incidence of osteonecrosis.Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was performed to detect apoptotic index of osteocytes and osteoblasts.Real-time PCR and Western blotting were performed to detect mRNA and protein expression of Bax,Bcl-2,and Caspase-3.Fisher's exact probability test and one-way analysis of variance (ANOVA) with Turkey's post hoc test were used to examine significant differences between groups.Results The incidence of osteonecrosis in the gastrodin+steroid group (16.7%) was significantly lower than that in the steroid group (83.3%).According to TUNEL assay,the apoptotic indices in the steroid group,the gastrodin+steroid group,and the control group were 91.1%,27.1%,and 5.4%,respectively,and the differences were significant between groups.Compared with the control group and the gastrodin+steroid group,the mRNA and protein expression levels of Bax and Caspase-3 were significantly higher in the steroid group,but the Bcl-2 mRNA and protein expression levels were significantly lower.Conclusion Gastrodin could prevent steroid-induced ONFH by anti-apoptosis.

  13. Antitumor activity of orally bioavailable farnesyltransferase inhibitor, ABT-100, is mediated by antiproliferative, proapoptotic, and antiangiogenic effects in xenograft models.

    Science.gov (United States)

    Ferguson, Debra; Rodriguez, Luis E; Palma, Joann P; Refici, Marion; Jarvis, Kenneth; O'Connor, Jacqueline; Sullivan, Gerard M; Frost, David; Marsh, Kennan; Bauch, Joy; Zhang, Haiying; Lin, Nan-Horng; Rosenberg, Saul; Sham, Hing L; Joseph, Ingrid B J K

    2005-04-15

    To evaluate the preclinical pharmacokinetics, antitumor efficacy, and mechanism of action of a novel orally active farnesyltransferase inhibitor, ABT-100. In vitro sensitivity of a panel of human cell lines was determined using proliferation and clonogenic assays. In vivo efficacy of ABT-100 was evaluated in xenograft models (flank or orthotopic) by assessing angiogenesis, proliferation, and apoptosis in correlation with pharmacokinetics. Efficacy of the racemate of ABT-100 (A-367074) was also compared with R115777 (tipifarnib). ABT-100 inhibited proliferation of cells in vitro carrying oncogenic H-Ras (EJ-1 bladder; IC(50) 2.2 nmol/L), Ki-Ras (DLD-1 colon, MDA-MB-231 breast, HCT-116 colon, and MiaPaCa-2 pancreatic; IC(50) range, 3.8-9.2 nmol/L), and wild-type Ras (PC-3 and DU-145; IC(50), 70 and 818 nmol/L, respectively) as well as clonogenic potential. ABT-100 shows 70% to 80% oral bioavailability in mice. ABT-100 regressed EJ-1 tumors (2-12.5 mg/kg/d s.c., every day for 21 days) and showed significant efficacy in DLD-1, LX-1, MiaPaCa-2, or PC-3 tumor-bearing mice (6.25-50 mg/kg/d s.c. once daily or twice daily orally). A-367074 showed equivalent efficacy to R115777 given at approximately one-fourth the total dose of R115777 for a shorter duration (EJ-1 and LX-1). Antitumor activity was associated with decreased cell proliferation (Ki-67), increased apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling), and decreased angiogenesis. A reduction in tumor angiogenic cytokine levels (vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8) correlated with a reduction in tumor vascularity (CD31). Overall, ABT-100 has an acceptable pharmacokinetic profile, is well tolerated, and possesses broad-spectrum antitumor activity against a series of xenograft models similar to farnesyltransferase inhibitors in clinical development; therefore, it is an attractive candidate for clinical evaluation.

  14. Induction of immune response against NY-ESO-1 by CHP-NY-ESO-1 vaccination and immune regulation in a melanoma patient.

    Science.gov (United States)

    Tsuji, Kazuhide; Hamada, Toshitada; Uenaka, Akiko; Wada, Hisashi; Sato, Eiichi; Isobe, Midori; Asagoe, Kenji; Yamasaki, Osamu; Shiku, Hiroshi; Ritter, Gerd; Murphy, Roger; Hoffman, Eric W; Old, Lloyd J; Nakayama, Eiichi; Iwatsuki, Keiji

    2008-10-01

    NY-ESO-1 is a cancer/testis antigen highly immunogenic in cancer patients. Cholesterol-bearing hydrophobized pullulan (CHP) is a nanoparticle-forming antigen-delivery vehicle and CHP complexed with NY-ESO-1 protein (CHP-NY-ESO-1) efficiently activates CD4 and CD8 T cells in vitro. In this study we report on a 50-year-old male melanoma patient with multiple skin and organ metastases (T4N3M1c) who was vaccinated with CHP-NY-ESO-1 at biweekly intervals and who had an unusual disease course. We characterized in this patient humoral and cellular immune responses, immune regulatory cells, and cytokine profiles in the peripheral blood and at local tumor sites. Ten days after the second CHP-NY-ESO-1 vaccination (day 25), blisters appeared on the skin at the metastatic lesions associated with inflammatory changes. A skin biopsy showed the presence of many NY-ESO-1-expressing apoptotic melanoma cells as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test. However, the tumors continued to grow, and the patient died of pulmonary failure due to multiple metastases on day 48. Serum antibody responses were detected after the second CHP-NY-ESO-1 vaccination and antibody titer increased with subsequent vaccinations. Th1 dependent IgG1 was the predominant immunoglobulin subtype. Both, NY-ESO-1-specific CD4 and CD8 T cell responses were detected in PBMC by IFN-gamma secretion assays. After CHP-NY-ESO-1 vaccination a slight decrease in CD4(+)CD25(+)Foxp3(+) Tregs was observed in PBMC but significantly increased numbers of CD4(+)CD25(+)Foxp3(+) Tregs and CD68(+) immunoregulatory macrophages were detected at the local tumor sites. CD4(+)CD25(+)Foxp3(+) Tregs were also increased in the blister fluid. Cytokines in the serum suggested a polarization towards a Th1 pattern in the PBMC and those in the blister fluid suggested a Th2-type response at the tumor site. Our observations indicate induction of specific humoral and

  15. The effect of 7 days of letrozole pretreatment combined with misoprostol on the expression of progesterone receptor and apoptotic factors of placental and decidual tissues from first-trimester abortion: a randomized controlled trial.

    Science.gov (United States)

    Yung, Sofie Shuk Fei; Lee, Vivian Chi Yan; Chiu, Philip Chi Ngong; Li, Hang Wun Raymond; Ng, Ernest Hung Yu; Yeung, William Shu Biu; Ho, Pak Chung

    2016-04-01

    To evaluate if letrozole-induced suppression of estradiol reduces progesterone receptor expression and apoptosis in the first-trimester placenta. We performed a double-blinded, randomized, placebo-controlled trial. We randomized 20 women requesting first-trimester abortion with gestation up to 63 days to receive either letrozole 10 mg daily or placebo pretreatment for 7 days before administrating 400 mcg of vaginal misoprostol followed by suction abortion. We collected the placental and decidual tissues on which we performed immunohistochemical staining for progesterone receptor and apoptotic markers (active caspase 3, caspase 3, Bcl2, CD95, fas ligand) and determined H-scores of each based on the intensities of staining. We performed terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay for apoptosis in the samples of four women to confirm the findings from apoptotic markers. We excluded one woman in the letrozole group from the analysis because she had passage of abortus after taking letrozole, leaving 19 women (9 in the letrozole group, 10 in the placebo group) for analysis. There was no significant difference in the H-scorings of progesterone receptor and apoptotic markers, as well as proportion of apoptotic cells on TUNEL assay between the two groups. The H-scores for the progesterone receptor were 8.17 ± 2.67 (mean ± SD) in the letrozole group and 9.01 ± 2.82 in the placebo group (p=0.36). We did not detect a difference in the expression of progesterone receptor and apoptotic markers in placental and decidual tissues after letrozole pretreatment for 7 days in first-trimester abortion. We did not confirm the hypothesis that letrozole reduces progesterone receptor expression and induces apoptosis in the first-trimester placenta. Further studies are required to allow better understanding of the mechanism by which estrogen suppression following the use of letrozole can lead to improved abortion rate in the first

  16. Anti-apoptotic effect of morphine-induced delayed preconditioning on pulmonary artery endothelial cells with anoxia/reoxygenation injury

    Institute of Scientific and Technical Information of China (English)

    DING Weng-ang; ZHOU Hua-cheng; CUI Xiao-guang; LI Wen-zhi; GUO Yue-ping; ZHANG Bing; LIU Wei

    2008-01-01

    Background Opioid preconditioning (PC) reduces anoxiaJreoxygenation (NR) injury to various cells. However, it remains unclear whether opioid-induced delayed PC would show anti-apoptotic effects on pulmonary artery endothelial cells (PAECs) suffering from A/R injury. The present study was conducted to elucidate this issue and to investigate the potential mechanism of opioid-induced delayed PC.Methods Cultured porcine PAECs underwent 16-hour anoxia followed by 1-hour reoxygenation 24 hours after pretreatment with saline (NaCI; 0.9%) or morphine (1 μmol/L). To determine the underlying mechanism, a non-selective KATP channel inhibitor glibenclamide (Glib; 10 μmol/L), a nitric oxide (NO) synthase blocker NG-nitro-L-arginine methyl ester (L-NAME; 100 μmol/L), and an opioid receptor antagonist naloxone (Nal; 10 pmol/L) were given 30 minutes before the A/R load. The percentage of apoptotic cells was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, eNOS mRNA level was measured by real-time polymerase chain reaction (PCR). NO content of PAECs supernatants was measured with the Griess reagent.Results Compared to the A/R PAECs, morphine-induced delayed PC significantly reduced PAECs apoptosis ((18.1±1.9)% vs (5.5±0.3)%; P <0.05), increased NO release ((11.4±1.3) μmol/L vs (20.5±2.1) μmol/L, P <0.05), and up-regulated eNOS gene expression nearly 9 times (P<0.05). The anti-apoptosis effect of morphine was abolished by pretreatment with Glib, L-NAME and Nal, but the three agent-selves did not aggravate the A/R injury. Furthermore, L-NAME and Nal offset the enhanced release of NO caused by pretreatment with morphine.Conclusions Morphine-induced delayed PC prevents A/R injury of PAECs. This effect may be mediated by activation of KATP channel via opioid receptor and NO signaling pathways.

  17. 灵芝孢子粉对杏仁核点燃模型大鼠神经元凋亡的影响%The effect of Reishi Mushroom Powder on neuronal apoptosis of the amygdala kindling model

    Institute of Scientific and Technical Information of China (English)

    张继国; 王艺; 张静; 邱彦龙

    2012-01-01

    Purpose To observe the effect of Reishi Mushroom Powder on neuronal apoptosis of the a-mygdala kindling model. Methods 24 male amygdala kindling rats were divided into Reishi Mushroom Powder (treatment group) , saline ( negative control) , and phenobarbital gavage ( positive control). After 2 weeks treatment, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was used to collect apoptotic cells of the hippocampus. Results The number of apoptotic cells in the treatment group, negative control group and the positive control group were (21. 25 ± 4. 03 ) , (42.08 ±4.29) and (23. 17 ±3. 73)/mm2 .respectively. Compared with the negative control group,apoptotic cells in the treatment group and the positive control group were significantly lower(P<0.05). No significant difference was seen between treatment group and the positive control group (P>0.05). Conclusion The Reishi Mushroom Powder can significantly reduce epilepsy-induced neuronal apoptosis and has protection function on the nervous system.%目的 观察灵芝孢子粉治疗后杏仁核点燃模型大鼠神经元凋亡状况.方法 雄性大鼠24只,制备杏仁核点燃模型后,分为灵芝孢子粉组、生理盐水(阴性对照)组、苯巴比妥(阳性对照)组,灌胃给药2周后,采用原位杂交末端标记(TUNEL)法检测大鼠脑海马组织细胞凋亡状况.结果 灵芝孢子粉组、阴性对照组及阳性对照组的凋亡细胞数分别为(21.25±4.03),(42.08±4.29)和(23.17±3.73)个/mm2,灵芝孢子粉组和阳性对照组凋亡指教均明显低于阴性对照组(P<0.05),给药组与阳性对照组之问无明显差异(P>0.05).结论 灵芝孢子粉能够明显减少癫痫引起的神经元凋亡,对神经系统具有保护功能.

  18. Activation of caspases-3, -6, and -9 during finasteride treatment of benign prostatic hyperplasia.

    Science.gov (United States)

    Bozec, Aline; Ruffion, Alain; Decaussin, Myriam; Andre, Jean; Devonec, Marian; Benahmed, Mohamed; Mauduit, Claire

    2005-01-01

    Benign prostatic hyperplasia (BPH) results from an increase in both epithelial and stromal compartments of the human prostate. Although inhibitors of 5alpha-reductase such as finasteride have been shown to reduce the size of BPH tissues by inducing apoptosis, their mechanisms of action still remain unknown. The present study supports that such a process triggered by finasteride is caspase dependent with a possible involvement of two effector caspases (caspase-3 and 6) and two initiator caspases (caspase-8 and 9). Indeed, by using tissues from patients affected by BPH and treated by finasteride (5 mg/d) for 2-3, 6-8, or 27-32 d, we observed that the 5alpha-reductase inhibitor induced apoptosis in epithelial cells (evaluated through cell number positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) as early as 2-3 d of treatment, with a maximal activity (250-fold increase, P 8 d of treatment. However, after 27-32 d of treatment, the number of apoptotic cells was reduced and was close to control. Caspases-3, -6, -8, and -9 were immunolocalized to (basal and secretory) epithelial cells and to a lesser extent to stromal cells. Activated caspase-3 immunoexpression was restricted to epithelial secretory cells, and its immunostaining intensity appeared to be higher in BPH tissues from patients treated for 2-3 or 6-8 d. Consistently, in Western blotting analyses, activated caspases-3 and -6 were detected as early as 2-3 d of treatment in BPH tissues, and their levels were increased after 6-8 d of treatment. In real time quantitative PCR experiments, caspase-3 and -6 mRNA levels were found to be unchanged after finasteride treatment. Activated caspase-8 was not detected in the different conditions tested, whereas activated caspase-9 protein levels were maximally enhanced after 2-3 d of finasteride treatment. In conclusion, we report here that finasteride treatment of BPH tissues induced a caspase-dependent apoptotic process

  19. Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.

    Science.gov (United States)

    Yousfi, Malika; Lasmoles, Francoise; El Ghouzzi, Vincent; Marie, Pierre J

    2002-02-15

    Saethre-Chotzen syndrome (SCS) is a human autosomal dominant disorder characterized by premature fusion of cranial sutures caused by mutations of the Twist gene encoding a basic helix-loop-helix (bHLH) transcription factor. We previously showed that Twist haploinsufficiency caused by a Y103X nonsense mutation in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of osteoblast differentiation. Here we show that Twist haploinsufficiency alters osteoblast apoptosis in SCS. Analysis of terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) demonstrated increased osteoblast and osteocyte apoptosis in coronal sutures from two SCS patients with nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins, and one patient with a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding. To assess the mechanisms involved, we studied osteoblast apoptosis in mutant (M-Tw) calvarial cells bearing the Y103X mutation resulting in decreased Twist mRNA and protein levels. M-Tw cells cultured in low serum conditions showed enhanced DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Biochemical analysis showed increased activity of initiator caspases-2 and -8 and downstream effector caspases-3, -6 and -7 in mutant osteoblasts. Caspase-2 was upstream of caspase-8 and effector caspases-3, -6 and -7 because their activities were suppressed by a specific caspase-2 inhibitor. M-Tw osteoblasts also showed increased cytochrome c release from the mitochondria. However, the activity of the downstream effector caspase-9 was not increased due to overexpression of the antagonist protein Hsp70. Detection of differentially expressed genes using cDNA expression array revealed increased Bax and TNFalpha mRNA levels in M-Tw compared to Nl cells, a finding confirmed by RT-PCR and western blot analyses. Neutralization of

  20. Synergistic antitumoral activity and induction of apoptosis by novel pan Bcl-2 proteins inhibitor apogossypolone with adriamycin in human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jin-xia MI; Guang-feng WANG; Heng-bang WANG; Xiao-qing SUN; Xin-yan NI; Xiong-wen ZHANG; Jia-ming TANG; Da-jun YANG

    2008-01-01

    Aim: To investigate the in vitro and in vivo activities and related mechanism of apogossypoione (ApoG2) alone or in combination with adriamycin (ADM) against human hepatocellular carcinoma (HCC). Methods: The IC50 of ApoG2 in vitro was tested by WST assay, and the synergistic effect was analyzed using the CalcuSyn method. Cell apoptosis was determined using 4',6-diamidino-2-phenylindole staining and flow cytometric analysis. Western blotting was used to determine the expression of apoptosis-related proteins. In vivo activity was evaluated in the xenograft model in nude mice, and apoptosis in tumor tissues was determined by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay. Results: The IC50 of ApoG2 in HCC cells was 17.28-30.63 μmol/L. When ApoG2 was combined with ADM, in-creased cytotoxicity and apoptosis were observed in SMMC-7721 cells compared to treatment with ApoG2 alone. The Western blotting results indicated that the ApoG2 induced apoptosis in SMMC-7721 cells by downregulating anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-XL, up-regulating pro-apoptotic protein Noxa, and promoting the activities of caspases-9 and -3. The tumor growth of xenograft SMMC-7721 was inhibited in nude mice when ApoG2 was administered orally without causing damage to the normal tissues. The in vivo study also indicated an increasing anti-tumoral effect when ApoG2 at 100 or 200 mg/kg dosages were used together with ADM at 5.5 mg/kg, with relative tumor proliferation rate (T/C) values of 0.456 and 0.323, respectively. Apoptosis induced in vivo by ApoG2 alone or combined with ADM was confirmed by TUNEL assay in tumor tissues. Conclusion: ApoG2 is a potential non-toxic target agent that induces apoptosis by upregulating Noxa, while inhibiting anti-apoptotic proteins and pro-moting the effect of chemotherapy agent ADM in HCC.

  1. Immunoliposome co-delivery of bufalin and anti-CD40 antibody adjuvant induces synergetic therapeutic efficacy against melanoma

    Directory of Open Access Journals (Sweden)

    Li Y

    2014-12-01

    Full Text Available Ying Li,1,* Jiani Yuan,1,* Qian Yang,1 Wei Cao,1 Xuanxuan Zhou,1 Yanhua Xie,1 Honghai Tu,2 Ya Zhang,1 Siwang Wang1 1Department of Natural Medicine and Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an, People’s Republic of China; 2Institute for Drug and Instrument Control, Xinjiang Military Area Command, Urumqi, Xinjiang, People’s Republic of China *These authors contributed equally to this work Abstract: Liposomes constitute one of the most popular nanocarriers for improving the delivery and efficacy of agents in cancer patients. The purpose of this study was to design and evaluate immunoliposome co-delivery of bufalin and anti-CD40 to induce synergetic therapeutic efficacy while eliminating systemic side effects. Bufalin liposomes (BFL conjugated with anti-CD40 antibody (anti-CD40-BFL showed enhanced cytotoxicity compared with bufalin alone. In a mouse B16 melanoma model, intravenous injection of anti-CD40-BFL achieved smaller tumor volume than did treatment with BFL (average: 117 mm3 versus 270 mm3, respectively; the enhanced therapeutic efficacy through a caspase-dependent pathway induced apoptosis, which was confirmed using terminal deoxynucleotidyl transferase-mediated dUTP-Fluorescein nick end labeling and Western blot assay. Meanwhile, anti-CD40-BFL elicited unapparent body-weight changes and a significant reduction in serum levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, interferon-γ, and hepatic enzyme alanine transaminase, suggesting minimized systemic side effects. This may be attributed to the mechanism by which liposomes are retained within the tumor site for an extended period of time, which is supported by the following biodistribution and flow cytometric analyses. Taken together, the results demonstrated a highly promising strategy for liposomal vehicle transport of anti-CD40 plus bufalin that can be used to enhance antitumor effects via synergetic systemic

  2. Sublethal transient global ischemia stimulates migration of neuroblasts and neurogenesis in mice.

    Science.gov (United States)

    Li, Ying; Yu, Shan Ping; Mohamad, Osama; Genetta, Thomas; Wei, Ling

    2010-09-01

    Increasing evidence has shown the potential of neuronal plasticity in adult brain after injury. Neural proliferation can be triggered by a focal sublethal ischemic preconditioning event; whether mild global ischemia could cause neurogenesis has been not clear. The present study investigated stimulating effects of sublethal transient global ischemia (TGI) on endogenous neurogenesis and neuroblast migration in the subventricular zone (SVZ), dentate gyrus, and peri-infarct areas of the adult cortex. Adult mice of 129S2/Sv strain were subjected to 8-min bilateral common carotid artery ligation followed by 5-bromo-2'-deoxyuridine (BrdU; 50 mg/kg, intraperitoneal) administration every day until being sacrificed at 1-21 days after reperfusion. The mild TGI did not induce neuronal cell death for up to 7 days after TGI, as evidenced by negative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining among NeuN-positive cells in the hippocampus and neocortex. In TGI animals, BrdU staining revealed enhanced proliferation of neuroblasts and their migration track from the SVZ into the striatum and neocortex. In the corpus callosum, there were more BrdU-positive cells in the TGI group in the first 2 days. Increasing numbers of BrdU-positive cells were seen 7-21 days later in the striatum and cortex of TGI mice. The cortex of TGI animals showed increased expression of erythropoietin, erythropoietin receptor, fibroblast growth factor 2, vascular endothelial growth factor, and phosphorylated Jun N-terminal kinase; the expression was peaked 2 to 3 days after reperfusion. BrdU and NeuN double staining in the dentate gyrus, striatum, and cortex implied increased neurogenesis induced by the TGI preconditioning. Doublecortin (DCX)-positive cells increased in the cortex of TGI mice, localized to cortical layers II, III, and V, and many stained positive for the mature neuronal markers NeuN, neurofilament, N-methyl-d-aspartic acid receptor subunit gene NR1, or the

  3. Inhibition of P2X7 receptor ameliorates transient global cerebral ischemia/reperfusion injury via modulating inflammatory responses in the rat hippocampus

    Directory of Open Access Journals (Sweden)

    Chu Ketan

    2012-04-01

    Full Text Available Abstract Background Neuroinflammation plays an important role in cerebral ischemia/reperfusion (I/R injury. The P2X7 receptor (P2X7R has been reported to be involved in the inflammatory response of many central nervous system diseases. However, the role of P2X7Rs in transient global cerebral I/R injury remains unclear. The purpose of this study is to determine the effects of inhibiting the P2X7R in a rat model of transient global cerebral I/R injury, and then to explore the association between the P2X7R and neuroinflammation after transient global cerebral I/R injury. Methods Immediately after infusion with the P2X7R antagonists Brilliant blue G (BBG, adenosine 5′-triphosphate-2′,3′-dialdehyde (OxATP or A-438079, 20 minutes of transient global cerebral I/R was induced using the four-vessel occlusion (4-VO method in rats. Survival rate was calculated, neuronal death in the hippocampal CA1 region was observed using H & E staining, and DNA cleavage was observed by deoxynucleotidyl transferase-mediated UTP nick end labeling TUNEL. In addition, behavioral deficits were measured using the Morris water maze, and RT-PCR and immunohistochemical staining were performed to measure the expression of IL-1β, TNF-α and IL-6, and to identify activated microglia and astrocytes. Results The P2X7R antagonists protected against transient global cerebral I/R injury in a dosage-dependent manner. A high dosage of BBG (10 μg and A-0438079 (3 μg, and a low dosage of OxATP (1 μg significantly increased survival rates, reduced I/R-induced learning memory deficit, and reduced I/R-induced neuronal death, DNA cleavage, and glial activation and inflammatory cytokine overexpression in the hippocampus. Conclusions Our study indicates that inhibiting P2X7Rs protects against transient global cerebral I/R injury by reducing the I/R-induced inflammatory response, which suggests inhibition of P2X7Rs may be a promising therapeutic strategy for clinical treatment of

  4. Effect of Ganoderma lucidum spore on expression of insulin-like growth factor-1, nuclear factor-kappa B, and neuronal apoptosis in the epileptic rat brain

    Institute of Scientific and Technical Information of China (English)

    Shuang Zhao; Shuqiu Wang; Shengchang Zhang; Fafang Li

    2008-01-01

    BACKGROUND: It has been reported that Ganoderma lucidum spore powder, a very well known Chinese traditional medicine, can affect immunoregulation, free radical scavenging, and anti-hypoxia responses. OBJECTIVE: To investigate the effect of Ganoderma lucidum spore powder on expression of insulin-like growth factor-1 (IGF-1), nuclear factor-κB (NF-κB) and neuronal apoptosis in rats with pentylenetetrazol (PTZ)-induced epilepsy.DESIGN, TIME AND SETTING: A cellular and molecular biology experiment with randomized controlled study design was performed at the Central Laboratory of Basic Medical College of Jiamusi University from June to August 2005.MATERIALS: Thirty healthy, adult, male, Wistar rats were selected and randomly divided into 3 groups (10rats per group): control, epilepsy model, and Ganoderma lucidum spore powder. A sub-eclampsia PTZ dose (35 mg/kg) was intraperitoneally injected to induce epilepsy in the latter two groups. Wild Ganoderma lucidum spore powder (30 g/L) was provided by the wild Ganoderma lucidum plant nursery at Jiamusi,China. lmmunohistochemical detection and terminal deoxynucleotidyl transferase-mediate dUTP nick end-labeling (TUNEL) kits were purchased from Wuhan Boster Biological Technology Co., Ltd., China.METHODS: Ganoderma lucidum spore powder was intragastrically administered at a dose of 10.0 mL/kg,once a day for 28 days. In the epilepsy and control groups, an equivalent volume of normal saline was intragastrically administered.MAIN OUTCOME MEASURES: lmmunoreactivity for IGF-1 and NF-κB/P65 were detected by immunohistochemical staining. Neuronal apoptosis was detected using TUNEL methods.RESULTS: The hippocampus and cerebral cortex of rats with PTZ-induced epilepsy exhibited a higher number of apoptotic cells at high magnification (x400), compared with the control group. Expression of IGF-1and NF-κB were higher in the epilepsy group, compared with the control group (P < 0.01). In Ganoderma lucidum spore-treated rats, fewer

  5. The decrease in milk yield during once daily milking is due to regulation of synthetic activity rather than apoptosis of mammary epithelial cells in goats.

    Science.gov (United States)

    Ben Chedly, H; Lacasse, P; Marnet, P-G; Boutinaud, M

    2013-01-01

    Once daily milking (ODM) is a management practice that can improve working conditions and reduce production costs in dairy farming compared with twice daily milking (TDM). However, ODM is associated with a decrease in milk yield. Previous research indicated that disruption of tight junctions in the mammary gland may be one of the regulatory factors involved in the milk yield decrease observed during ODM. The aim of this study was to investigate the involvement of mammary epithelium disruption in the regulation of the activity and dynamics of mammary epithelial cells (MEC) during 5 weeks of ODM in goats. Twelve alpine goats (producing 3.67 ± 0.64 kg/day and 47 ± 1.6 days in milk) were assigned to two groups that were milked once or twice a day during 5 weeks and then switched back to TDM. Mammary biopsies were collected before and on days 2 and 16 of both ODM and TDM switchback periods. Milk purified epithelial cells were collected before and on days 1, 7, 21 and 28 during ODM as well on days 1 and 7 of the TDM switchback period. The mRNA levels of genes involved in the regulation of synthetic activity and apoptosis were analysed by RT-PCR in milk MEC and mammary biopsies. ODM decreased yields of milk (-23%), lactose (-23%) and casein (-16%). Lactose synthesis was regulated at the transcriptional level by downregulation of α-lactalbumin mRNA levels in both biopsy samples (-30%) and milk MEC (-74%). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling) staining of mammary gland biopsies did not show any increase in cell apoptosis after 2 and 16 days of ODM (0.8% and 1%, respectively) despite upregulation of Bax mRNA levels in milk MEC. This suggests that the decrease in milk yield observed during ODM is attributable to a decrease in synthetic activity rather than to induction of MEC cell death. ODM induced the disruption of tight junctions in the mammary gland only on the first day of the treatment as indicated by increased blood

  6. Neuregulin-1 preconditioning protects the heart against ischemia/reperfusion injury through a PI3K/Akt-dependent mechanism

    Institute of Scientific and Technical Information of China (English)

    FANG Shan-juan; WU Xue-si; HAN Zhi-hong; ZHANG Xiao-xia; WANG Chun-mei; LI Xin-yan; LU Ling-qiao; ZHANG Jing-lan

    2010-01-01

    Background Neuregulin-1 (NRG-1), the ligand of the myocardial ErbB receptor, is a protein mediator with regulatory actions in the heart. This study investigated whether NRG-1 preconditioning has protective effects on myocardial ischemia/reperfusion (I/R) injury and its potential mechanism.Methods We worked with an in vivo rat model with induced myocardial ischemia (45 minutes) followed by reperfusion (3 hours). NRG-1 message was detected in the heart using RT-PCR and the protein levels of NRG-1 and ErbB4 were detected by Western blotting analysis. Infarct size was assessed using the staining agent triphenyltetrazolium chloride and cardiac function was continuously monitored. The levels of creatine kinase and lactate dehydrogenase in plasma were analyzed to assess the degree of cardiac injury. The extent of cardiac apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and by Western blotting analysis of cleaved caspase-3. We examined the phosphorylation of Akt in the myocardium and the effect of PI3K/Akt inhibition on NRG-1-induced cardioprotection.Results Transcription and expression of NRG-1 and phosphorylation of its ErbB4 receptor were significantly upregulated in the I/R hearts. NRG-1 pretreatment reduced the infarct size following cardiac I/R in a concentration-dependent manner with an optimal concentration of 4 μg/kg in vivo. NRG-1 pretreatment with 4 μg/kg, i.v.markedly reduced the plasma creatine kinase and lactate dehydrogenase levels. Pretreatment with NRG-1 also significantly reduced the percentage of TUNEL positive myocytes and the level of cleaved caspase-3 in the I/R hearts.Pretreatment with NRG-1 significantly increased phosphorylation of Akt following I/R. Furthermore, the cardioprotective effect limiting the infarct size that was induced by NRG-1 was abolished by co-administration of the PI3K inhibitor LY294002.Conclusions The concentration of NRG-1, a new autacoid, was rapidly upregulated

  7. THE PROTECTIVE EFFECTS OF DRUGS ON APOPTOSIC NEURAL CELLS AFTER SPINAL CORD INJURY%细胞凋亡阻断药物对脊髓损伤后神经细胞凋亡的保护作用观察

    Institute of Scientific and Technical Information of China (English)

    傅强; 侯铁胜; 鲁凯伍; 贺石生; 李明; 赵杰; 石志才

    2001-01-01

    To study the protective effects and the mechanism of drugs on apoptosic neural cells after spinal cord injury and the mechanism of their effects on severe degree compression injury to the lower thoracic spinal cord (T8.9). Rats were divided into four groups randomly . The first group of rats were treated with saline solution(20μl) intrathecally after injury, the second group with cyceoheximide (CH)(30μg/20μl) intrathecally, the third group with monosialicganglioside(GM)(100μg/20μl),and the fourth group methylprednisolone(MP)(30mg/kg). The changes in weight, spinal cord blood flow(SCBF), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL),neurological function and quantitative histological changes were observed.The results indicated that drugs had little effects on the SCBF. But drugs could improve the neurological function and area of spared normal tissue, and obviously reduce the number of TUNEL-positive cells. The results suggested that drugs had neuroprotective effects on the injuried spinal cord. The mechanism of the beneficial effects might be a reduction in neuronal cells apoptosis.%为观察放线菌酮、甲基强的松龙和神经节苷脂对损伤脊髓神经组织中凋亡细胞的保护作用,将60只SD大鼠致以重度脊髓损伤,并随机分为对照组、放线菌酮组、甲基强的松龙组和神经节苷脂组(n=15)。给药后分别观察体重、局部血流量、凋亡细胞原位标记等指标,并进行神经功能评价和定量组织学分析。结果显示,药物对局部血流量无明显作用;治疗组神经功能评分、残余神经组织面积明显高于对照组,而TUNEL阳性细胞数量明显低于对照组,说明3种药物均对损伤脊髓组织有明显保护作用,可能是通过对神经细胞凋亡的阻断来发挥作用的。

  8. Role of granulocyte colony-stimulating factor in paclitaxel-induced intestinal barrier breakdown and bacterial translocation in rats

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chi; XU Yang-guang; DUAN Xue-ning; LIU Yin-hua; ZHAO Jian-xin; XU Ling; YE Jing-ming

    2011-01-01

    Background Chemotherapy causes breakdown of the intestinal barrier, which may lead to bacterial translocation. Paclitaxel, an anti-tubulin agent, has many side effects; however, its effect on the intestinal barrier is unknown. Previous studies show that granulocyte colony-stimulating factor (G-CSF) plays an important role in modulating intestinal barrier function, but these studies are not conclusive. Here, we investigated the effects of paclitaxel on the intestinal barrier, and whether G-CSF could prevent paclitaxel-induced bacterial translocation.Methods Twenty-four male Sprague-Dawley rats were divided into three groups: control group, paclitaxel group and paclitaxel + G-CSF group. Intestinal permeability was measured by the urinary excretion rates of lactulose and mannitol administered by gavage. The mesenteric lymph nodes, spleen and liver were aseptically harvested for bacterial culture. Endotoxin levels and white blood cell (WBC) counts were measured and bacterial quantification performed using relative real-time PCR. Jejunum samples were also obtained for histological observation. Intestinal apoptosis was evaluated using a fragmented DNA assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling staining. One-way analysis of variance and Fisher's exact test were used to compare differences between groups.Results Paclitaxel induced apoptosis in 12.5% of jejunum villus cells, which was reduced to 3.8% by G-CSF treatment. Apoptosis in the control group was 0.6%. Paclitaxel treatment also resulted in villus atrophy, increased intestinal permeability and a reduction in the WBC count. G-CSF treatment resulted in increased villus height and returned WBC counts to normal levels. No bacterial translocation was detected in the control group, whereas 6/8,8/8, and 8/8 rats in the paclitaxel group were culture-positive in the liver, spleen and mesenteric lymph nodes, respectively. Bacterial translocation was

  9. Anti-tumor effects of polybutylcyanoacrylate nanoparticles of diallyl trisulfide on orthotopic transplantation tumor model of hepatocellular carcinoma in BALB/c nude mice

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhi-mian; YANG Xiao-yun; DENG Shu-hai; XU Wei; GAO Hai-qing

    2007-01-01

    Background Hepatocellular carcinoma (HCC) ranked the second among the causes of cancer mortality in China since the 1990s. Up to now, medication still plays an important role in the treatment of HCC. The therapies based on the allicin as a potential chemopreventive analog although is in its infancy at the present time, may have a significant role in the future management of HCC. Diallyl trisulfide (DATS) is a natural compound derived from garlic. In this study, we investigated the inhibitory effects of hepatic targeted polybutylcyanoacrylate nanoparticles of diallyl trisulfide(DATS-PBCA-NP) on orthotopic transplanted HepG2 hepatocellular carcinoma in nude mice.Methods DATS-PBCA-NP were detected by transmission electron microscope (TEM) and high-performance liquid chromatography (HPLC). The orthotopic transplantation HCC models were established by implanting HCC HepG2 xenograft bits under the envelope of the mice liver. Successful models (n=29) were divided into 4 groups: normal saline(NS), empty nanoparticles (EN), DATS and DATS-PBCA-NP were intravenously administered to the mice respectively for 2 weeks. In vivo antitumor efficacy was evaluated by the measurement of tumor volume. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and protein levels of apoptosis and cell proliferation proteins by immunoblotting in tumor tissues were performed to elucidate the possible mechanism.Results DATS-PBCA-NP possessed smooth and round appearance, dispersed well, and released in vitro in accord with double phase kinetics model. DATS-PBCA-NP changed the tissue/organ distribution of DATS in vivo. The successful rate of tumor implantation was 100%. Intravenous administration of DATS-PBCA-NP significantly retarded the growth of orthotopically transplanted hepatoma in BALB/c nude mice (compared with the other three groups, all P<0.05) without causing weight loss (P>0.05). TUNEL staining showed that the tumors from DATS-PBCA-NP treated mice

  10. Effects of D1 and D2 dopamine receptor agonists and antagonists on cerebral ischemia/reperfusion injury%多巴胺D1和D2受体激动剂和拮抗剂对脑缺血/再灌注损伤的影响

    Institute of Scientific and Technical Information of China (English)

    纵雪梅; 曾因明; 许铁; 吕建农

    2003-01-01

    实验应用开阔法、组织病理学方法、原位末端标记(in situterminal deoxynucleotidyl transferase-mediated de-oxy-UTP nick end labeling,TUNEL)法及免疫组织化学等方法,探讨多巴胺D1、D2受体激动剂和拮抗剂对沙土鼠前脑缺血/再灌注损伤海马CA1区神经元凋亡及凋亡相关基因bcl-2、bax表达的影响.结果显示:前脑缺血5 min可引起沙土鼠探索活动增加;再灌注3 d,海马CA1区约95%的锥体细胞凋亡;再灌注7 d,海马CA1区仅残存约2%-7%的存活锥体细胞;前脑缺血5 min可抑制bcl-2的表达并诱导bax表达增高;预先应用D2受体激动剂培高利特可减轻缺血后沙土鼠行为学异常、抑制海马CA1区锥体细胞凋亡、提高锥体细胞存活数、显著诱导bcl-2的表达并抑制bax的表达.预先应用SKF38393、SCH23390及螺哌隆对以上结果无明显影响.实验结果提示,培高利特具有确切的脑保护作用,诱导bcl-2并抑制bax的表达可能是其脑保护作用机制之一.

  11. Netrin-1 rescues neuron loss by attenuating secondary apoptosis in ipsilateral thalamic nucleus following focal cerebral infarction in hypertensive rats.

    Science.gov (United States)

    Liao, S-J; Gong, Q; Chen, X-R; Ye, L-X; Ding, Q; Zeng, J-S; Yu, J

    2013-02-12

    Neurological deficit following cerebral infarction correlates with not only primary injury, but also secondary neuronal apoptosis in remote loci connected to the infarction. Netrin-1 is crucial for axonal guidance by interacting with its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5H (UNC5H). DCC and UNC5H are also dependence receptors inducing cell apoptosis when unbound by netrin-1. The present study is to investigate the role of netrin-1 and its receptors in ipsilateral ventroposterior thalamic nucleus (VPN) injury secondary to stroke in hypertensive rats. Renovascular hypertensive Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO). Continuous intracerebroventricular infusion of netrin-1 (600 ng/d for 7 days) or vehicle (IgG/Fc) was given 24h after MCAO. Neurological function was evaluated by postural reflex 8 and 14 days after MCAO. Then, immunoreactivity was determined in the ipsilateral VPN for NeuN, glial fibrillary acidic protein, netrin-1 and its receptors (DCC and UNC5H2), apoptosis was detected with Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay, and the expressions of caspase-3, netrin-1, DCC, and UNC5H2 were quantified by western blot analysis. MCAO resulted in the impaired postural reflex after 8 and 14 days, with decreased NeuN marked neurons and increased TUNEL-positive cells, as well as an up-regulation in the levels of cleaved caspase-3 and UNC5H2 protein in the ipsilateral VPN, without significant change in DCC or netrin-1 expression. By exogenous netrin-1 infusion, the number of neurons was increased in the ipsilateral VPN, and both TUNEL-positive cell number and caspase-3 protein level were reduced, while UNC5H2 expression remained unaffected, simultaneously, the impairment of postural reflex was improved. Taken together, the present study indicates that exogenous netrin-1 could rescue neuron loss by attenuating secondary apoptosis in the

  12. In vivo prediction of anti-tumor effect of 3-bromopyruvate in hepatocellular carcinoma using Tc-99m labeled annexin-v imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Won; Yoon, Jung Hwan; Kim, Chung Yang [Seoul National University College of Medicine, Seoul (Korea, Republic of); Cheon, Gi Jeoog; Lee, Tae Sup; Woo, Kwang Sun; Chung, Wee Sup [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2005-07-01

    We have recently demonstrated that hypoxia stimulates hepatocellular carcinoma (HCC) cell growth through hexokinase II induction, and its inhibition induces apoptotic cell death through activating mitochondrial apoptotic signaling cascades. In this study, we were apt to evaluate the antitumoral effect of 3-bromopyruvate (3-BP) on in vivo model of HCC by apoptotic imaging using Tc-99m labeled annexin V. In vivo model of HCC was established in C3H mice intradermally implanted with MH134 cells, a mouse HCC cell line, and 3-BP (0, 5, 10 mg/kg) was subsequently administered intraperitoneally. Tc-99m-HYNIC-annexin V (185 KBq) was injected via tail vein at one and three days after the 3-BP treatment, planar scan was acquired at a hour after the injection using gamma camera. The anti-tumor effect was evaluated by measuring tumor volumes and quantification of apoptotic cells using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Tumor volume was significantly reduced in mice treated with 3-BP in a dose-dependent manner (mean tumor volume 1.07 vs. 0.58 vs. 0.39 cm{sup 3} in 3-BP 0, 5, 10 mg/kg, respectively: p=0.047). The percentage of TUNEL staining-positive cells was significantly increased in 3-BP-treated mice (0.53 vs. 1.40 vs. 1.84% in 3-BP 0, 5, 10 mg/kg, respectively; p=0.018). On Tc-99m-HYNIC annexin V imaging, tumor-to-background uptake ratio (UR) was 1.92 at one day and 4.23 at three days after 3-BP treatment of 5 mg/kg (non-treated tumor showed UR of 2.93). Apoptosis-inducing anti-tumor effect of 3-BP was able to be demonstrated in in vivo model of HCC by apoptotic in vivo imaging using Tc-99m-HYNIC annexin V.

  13. Characterization of phosphodiesterase 2A in human malignant melanoma PMP cells.

    Science.gov (United States)

    Morita, Hiroshi; Murata, Taku; Shimizu, Kasumi; Okumura, Kenya; Inui, Madoka; Tagawa, Toshiro

    2013-04-01

    The prognosis for malignant melanoma is poor; therefore, new diagnostic methods and treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of 21 phosphodiesterases, which are divided into 11 families (PDE1-PDE11). PDE2 hydrolyzes cyclic AMP (cAMP) and cyclic GMP (cGMP), and its binding to cGMP enhances the hydrolysis of cAMP. We previously reported the expression of PDE1, PDE3 and PDE5 in human malignant melanoma cells. However, the expression of PDE2 in these cells has not been investigated. Herein, we examined the expression of PDE2A and its role in human oral malignant melanoma PMP cells. Sequencing of RT-PCR products revealed that PDE2A2 was the only variant expressed in PMP cells. Four point mutations were detected; one missense mutation at nucleotide position 734 (from C to T) resulted in the substitution of threonine with isoleucine at amino acid position 214. The other three were silent mutations. An in vitro migration assay and a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay revealed that suppressing PDE2 activity with its specific inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), had no impact on cell motility or apoptosis. Furthermore, the cytotoxicity of EHNA, assessed using a trypan blue exclusion assay, was negligible. On the other hand, assessment of cell proliferation by BrdU incorporation and cell cycle analysis by flow cytometry revealed that EHNA treatment inhibited DNA synthesis and increased the percentage of G2/M-arrested cells. Furthermore, cyclin A mRNA expression was downregulated, while cyclin E mRNA expression was upregulated in EHNA-treated cells. Our results demonstrated that the PDE2A2 variant carrying point mutations is expressed in PMP cells and may affect cell cycle progression by modulating cyclin A expression. Thus, PDE2A2 is a possible new molecular target for the treatment of malignant melanoma.

  14. Transcription factor myocyte enhancer factor 2D regulates interleukin-10 production in microglia to protect neuronal cells from inflammation-induced death.

    Science.gov (United States)

    Yang, Shaosong; Gao, Li; Lu, Fangfang; Wang, Bao; Gao, Fei; Zhu, Gang; Cai, Zhibiao; Lai, Juan; Yang, Qian

    2015-02-20

    Neuroinflammatory responses have been recognized as an important aspect in the pathogenesis of Parkinson's disease (PD). Transcriptional regulation plays a critical role in the process of inflammation. Transcription factor myocyte enhancer factor 2D (MEF2D) is identified as a central factor in transmission of extracellular signals and activation of the genetic programs in response to a wide range of stimuli in several cell types, including neurons. But its presence and function in microglia have not been reported. We therefore investigated the effect of MEF2D in activated microglia on the progress of neuroinflammation and the survival of neurons. BV2 cells and primary cultured glial cells were stimulated with lipopolysaccharide (LPS). Samples from cells were examined for MEF2D expression, interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α) by immunoblotting, quantitative real-time PCR (qPCR) or enzyme-linked immunosorbent assay (ELISA). The activity of MEF2D was examined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Recombinant lentivirus expressing shRNA specific to MEF2D was used to silence MEF2D expression in BV2 cells. The role of IL-10 transcriptionally induced by MEF2D on neuronal survival was assessed by anti-IL-10 neutralizing antibody. The survival of neurons was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Male C57bl/6 mice were used to establish an acute PD model. Brain sections and cell slides were tested by immunofluorescence. We demonstrated that MEF2D was present in microglia. Activation of microglia was associated with an increase in MEF2D level and activity in response to different stimuli in vivo and in vitro. MEF2D bound to a MEF2 consensus site in the promoter region of IL-10 gene and stimulated IL-10 transcription. Silencing MEF2D decreased the level of IL

  15. Anterograde degeneration along the visual pathway after optic nerve injury.

    Directory of Open Access Journals (Sweden)

    Yuyi You

    Full Text Available PURPOSE: To investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy. METHODS: Optic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN and the primary visual cortex (V1. The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP, respectively. RESULTS: An early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm(2, P = 0.042 and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10(2 cells/mm(2, P<0.001 were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10(2 cells/mm(2, P = 0.048, cell size (42.5±4.3 vs 62.1±4.7 µm(2, P = 0.001 and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10(2 cells/mm(2, P<0.001 appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway. CONCLUSIONS: Anterograde degeneration along the visual pathway takes place in target relay (LGN and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic

  16. Anterograde degeneration along the visual pathway after optic nerve injury.

    Science.gov (United States)

    You, Yuyi; Gupta, Vivek K; Graham, Stuart L; Klistorner, Alexander

    2012-01-01

    To investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy. Optic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1). The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL) histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP), respectively. An early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm(2), P = 0.042) and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10(2) cells/mm(2), P<0.001) were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10(2) cells/mm(2), P = 0.048), cell size (42.5±4.3 vs 62.1±4.7 µm(2), P = 0.001) and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10(2) cells/mm(2), P<0.001) appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway. Anterograde degeneration along the visual pathway takes place in target relay (LGN) and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic changes.

  17. M867, a novel selective inhibitor of caspase-3 enhances cell death and extends tumor growth delay in irradiated lung cancer models.

    Directory of Open Access Journals (Sweden)

    Kwang Woon Kim

    Full Text Available BACKGROUND: Lung cancer remains the leading cause of cancer death worldwide. Radioresistance of lung cancer cells results in unacceptable rate of loco-regional failure. Although radiation is known to induce apoptosis, our recent study showed that knockdown of pro-apoptotic proteins Bak and Bax resulted in an increase in autophagic cell death and lung cancer radiosensitivity in vitro. To further explore the potential of apoptosis inhibition as a way to sensitize lung cancer for therapy, we tested M867, a novel chemical and reversible caspase-3 inhibitor, in combination with ionizing radiation in vivo and in vitro. METHODS AND FINDINGS: M867 reduced clonogenic survival in H460 lung cancer cells (DER = 1.27, p = 0.007 compared to the vehicle-treated treated cells. We found that administration of M867 with ionizing radiation in an in vivo mouse hind limb lung cancer model was well tolerated, and produced a significant tumor growth delay compared to radiation alone. A dramatic decrease in tumor vasculature was observed with M867 and radiation using von Willebrand factor staining. In addition, Ki67 index showed >5-fold reduction of tumor proliferation in the combination therapy group, despite the reduced levels of apoptosis observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Radiosensitizing effect of M867 through inhibiting caspases was validated using caspase-3/-7 double-knockout (DKO mouse embryonic fibroblasts (MEF cell model. Consistent with our previous study, autophagy contributed to the mechanism of increased cell death, following inhibition of apoptosis. In addition, matrigel assay showed a decrease in in vitro endothelial tubule formation during the M867/radiation combination treatment. CONCLUSIONS: M867 enhances the cytotoxic effects of radiation on lung cancer and its vasculature both in vitro and in vivo. M867 has the potential to prolong tumor growth delay by inhibiting tumor proliferation

  18. Changes in proliferating and apoptotic markers in the oviductal magnum of chickens during sexual maturation.

    Science.gov (United States)

    Hrabia, Anna; Leśniak-Walentyn, Agnieszka; Ocłoń, Ewa; Sechman, Andrzej

    2016-06-01

    The avian oviduct is characterized by dynamic hormonal, biochemical, and cellular changes during its development. To better understand the molecular mechanisms regulating proper development of this organ in birds, the rate of cell proliferation and apoptosis as well as these processes-related gene expressions in the chicken oviduct during the sexual maturation were examined. The oviducts were isolated from Hy-Line Brown chickens at 2-week intervals from 10 to 16 weeks of age, and at 17 weeks, i.e. just after the onset of egg laying. In the tissue from the middle part of the oviduct (the magnum) the following parameters were tested: (1) proliferating (proliferating cell nuclear antigen [PCNA]-positive) and apoptotic (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive) cells, (2) mRNA expression of bcl-2, caspases 2, 3, 8, and 9, PCNA, survivin-142, and ovalbumin by quantitative real-time polymerase chain reaction, (3) protein expression of Bcl-2, PCNA, and caspases 3 and 9 by Western blot, (4) activity of caspases 2, 3, 8, and 9 by fluorometric method, and (5) localization of Bcl-2 and caspases by immunohistochemistry. It was found that the number of proliferating cells per unit area did not change during the examined period. The number of apoptotic cells in the oviductal wall remained on the same level until 14 weeks of age followed by a gradual decrease, reaching the lowest number at 17 weeks. The mRNA expression of all caspases and Bcl-2 gradually decreased during maturation, and PCNA decreased after 14 weeks of age. Survivin-142 mRNA level increased in 14-week-old chickens and then diminished, whereas ovalbumin expression was dramatically elevated in birds 16 weeks old and older. Patterns of protein expression of Bcl-2, PCNA, and caspases and activity of caspases were similar to mRNA, although not as pronounced. In the wall of the magnum the apoptotic cells and examined proteins were localized predominantly in the mucosa

  19. Phagocytosis (cannibalism) of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas.

    Science.gov (United States)

    Barresi, Valeria; Branca, Giovanni; Ieni, Antonio; Rigoli, Luciana; Tuccari, Giovanni; Caruso, Rosario Alberto

    2015-05-14

    To identify those with a micropapillary pattern, ascertain relative frequency and document clinicopathological characteristics by reviewing gastric carcinomas. One hundred and fifty-one patients diagnosed with gastric cancer who underwent gastrectomy were retrospectively studied and the presence of a regional invasive micropapillary component was evaluated by light microscopy. All available hematoxylin-eosin (HE)-stained slides were histologically reviewed and 5 tumors were selected as putative micropapillary carcinoma when cancer cell clusters without a vascular core within empty lymphatic-like space comprised at least 5% of the tumor. Tumor tissues from these 5 invasive gastric carcinomas were immunostained using an anti-mucin 1 (MUC1) antibody (clone MA695) to detect the characteristic inside-out pattern and with D2-40 antibody to determine the presence of intratumoral lymph vessels. Detection of intraepithelial neutrophil apoptosis was evaluated in consecutive histological tissue sections by three independent methods, namely light microscopy with HE staining, the conventional terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemistry for activated caspase-3 (clone C92-605). Among 151 gastric cancers resected for cure, 5 (3.3%) were adenocarcinomas with a micropapillary component. Four of the patients died of disease from 6 to 23 mo and one patient was alive with metastases at 9 mo. All patients had advanced-stage cancer (≥ pT2) and lymph node metastasis. Positive MUC1 immunostaining on the stroma-facing surface (inside-out pattern) of the carcinomatous cluster cells, together with negative immunostaining for D2-40 in the cells limiting lymphatic-like spaces, confirmed the true micropapillary pattern in these gastric neoplasms. In all five cases, several micropapillae were infiltrated by neutrophils. HE staining, TUNEL assay and immunostaining for caspase-3 demonstrated apoptotic neutrophils within

  20. [Regulation of p14(ARF) expression and induction of cell apoptosis with c-myc in a p53-independent pathway].

    Science.gov (United States)

    Liu, Xiang-juan; Li, Fu-nian; Jiang, Dan-dan; Wang, Xin-gang; Liu, Xiang-ping; Zhang, Dian-liang; Meng, Chun-hui

    2012-08-14

    To explore the regulation of p14(ARF) expression and induction of cell apoptosis with the mutant and wild-type c-myc genes in a p53-independent pathway of signal transduction. The mutant and wild-type c-myc genes were transfected by lentivirus into HCC1937 to form the stable over-expression cell lines. Uninfected cells and lentivirus-infected ones carrying no c-myc gene acted as blank and infection controls respectively. And c-myc and p14(ARF) mRNA and protein, proliferation and apoptosis in HCC1937 with mutant and wild-type c-myc were detected by reverse transcription (RT)-PCR, Western blotting, thiazolyl blue tetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling (TUNEL) respectively. After the lentivirus-mediated gene transfer, c-myc mRNA and protein expression increased in the mutant and wild-type groups. p14(ARF) mRNA and protein increased in the wild-type group and the mutant group and there were significant difference between them with blank and infection controls (mutant groups: 0.560 ± 0.010, 0.154 ± 0.011, wild-type groups: 0.651 ± 0.010, 0.382 ± 0.013, both P c-myc could promote the proliferation of cell growth. And c-myc was more effective to induce apoptosis in the wild-type group as compared with the mutant group (7.1% ± 0.7% vs 3.2% ± 0.4%, P c-myc obviously up-regulates the expression of p14(ARF). And cell apoptosis may be induced through the regulation of p14(ARF)-related gene, keep balance of proliferative promotion and apoptosis induction. When there is a loss-of-function of mutant c-myc, tumorigenicity increases via a disturbed balance of proliferative promotion and apoptosis induction.

  1. Exposure to a solar eclipse causes neuronal death in the retina.

    Science.gov (United States)

    Thanos, S; Heiduschka, P; Romann, I

    2001-10-01

    A solar eclipse was observed in Europe on 11 August 1999. Several individuals suffered from transient or persisting retinal damage, caused by gazing at the eclipse without adequate eye protection. Retinal damage is the most serious hazard of exposure to light. but the mechanisms by which this type of exposure produces retinal damage and its cellular correlates are not yet established. We used an animal model to monitor the mechanisms of retinal damage following excessive light exposure, and in particular to study whether observation of the eclipse induces death of retinal cells. In the geographic area where the experiment was conducted, a partial (90%) solar eclipse was observed. Experimental albino rats were exposed to these eclipse conditions, and control rats were exposed to normal sunlight. Another group of control animals was exposed to the same conditions, but was provided with protective light filters of the type recommended for human use. The DNA fragmentation in retinal sections of the various groups was analysed by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labelling. This analysis revealed that exposure to both normal sunlight and to the eclipse resulted in neuronal apoptosis. Immunohistochemical techniques were used to evaluate possible glial-vascular alterations. Dying cells could first be detected 24 h after exposure, the largest number of which were found 6 days later in the photoreceptor layer. Control levels were attained 14 days after the exposure. Retinal ganglion cells underwent apoptosis in both groups (normal sunlight and eclipse exposure), whereas in the neuroglial cells there was an up-regulation of the intermediate filament content. The number of dying cells in both groups was greater in animals whose pupils had been dilated pharmacologically during exposure. On the other hand, the protective filters were effective in preserving the rat retinal cells from apoptosis. These results show, for the first time, that the cellular

  2. RRR-α-tocopheryl succinate inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

    Institute of Scientific and Technical Information of China (English)

    Kun Wu; Yan Zhao; Bai-He Liu; Yao Li; Fang Liu; Jian Guo; Wei-Ping Yu

    2002-01-01

    AIM: To investigate the effects of growth inhibition ofhuman gastric cancer SGC-7901 cell with RRR-α-tocopherylsuccinate (VES), a derivative of natural Vitamin E, viainducing apoptosis and DNA synthesis arrest.METHODS: Human gastric cancer SGC-7901 cells wereregularly incubated in the presence of VES at 5, 10 and20mg@ L 1(VES was dissolved in absolute ethanol anddiluted in RPMI 1640 complete condition mediacorrespondingly to a final concentration of VES and 1mL@L-1 ethanol), succinic acid and ethanol equivalents asvehicle (VEH) control andcondition media only asuntreated (UT) control. Trypan blue dye exclusionanalysis and MTT assay were applied to detect the cellproliferation. 37kBq of tritiated thymidine was added tocells and [3H] TdR uptake was measured to observe DNAsynthesis. Apoptotic morphology was observed byelectron microscopy and DAPI staining. Flow cytometryand terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) assay were performed to detectVES-triggered apoptosis.RESULTS: VES inhibited SGC-7901 cell growth in a dose-dependent manner. The growth curve showed suppressionby 24.7%, 49.2% and 68.7% following 24h of VEStreatment at 5, 10 and 20 mg@L 1, respectively, similar tothe findings from MTT assay. DNA synthesis wasevidently reduced by 35%, 45% and 98% after 24h VEStreatment at 20 mg@ L-1 and 48h at 10 and 20 mg@ L 1,respectively. VES induced SGC-7901 cells to undergoapoptosis with typically apoptotic characteristics,including morphological changes of chromatincondensation, chromatin crescent formation/margination,nucleus fragmentation and apoptotic body formation,typical apoptotic sub-G1 peak by flow cytometry andincrease of apoptotic cells by TUNEL assay in which 90%of cells underwent apoptosis after 48h of VES treatment at20 mcg@L-1.CONCLUSION: VES can inhibit human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesisarrest. Inhibition of SGC-7901 cell growth by VES is dose-and time

  3. Induction of apoptosis in placentas of pregnant mice exposed to lipopolysaccharides: possible involvement of Fas/Fas ligand system.

    Science.gov (United States)

    Ejima, K; Koji, T; Tsuruta, D; Nanri, H; Kashimura, M; Ikeda, M

    2000-01-01

    To explore the pathogenesis in placental dysfunction and abruptio placentae, we analyzed the occurrence of placental cell apoptosis and the role of Fas and Fas ligand (L) in that process in an inflammatory placental dysfunction model of pregnant mice, using lipopolysaccharides (LPS). In the present study, Day 13 pregnant mice were injected i.p. with LPS (50 microg/kg) or saline as a control, and the placentas were isolated at various time points after the injection. Analysis of the isolated DNA in agarose-gel electrophoresis revealed a typical ladder pattern of bands consisting of 180-200 base pairs (bp), which is regarded as a hallmark of apoptosis. The intensity of the bands increased time-dependently, reaching a maximum level at 12 h after LPS injection. In accord with the biochemical data, histochemical analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) revealed that nuclei positive for double-stranded DNA breaks were found in decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts. The number of positive nuclei was maximized at 12 h after LPS injection. As a next step, we investigated the possible involvement of Fas and Fas L in the induction of apoptosis of the placental cells after LPS injection. Western blot analysis indicated that LPS increased the expression of Fas and Fas L in the placenta by about 4-fold at 12 h and 18 h, respectively, after injection. The cells expressing Fas and Fas L were identified, using immunohistochemistry and nonradioactive in situ hybridization, as decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts. Furthermore, when the expression of 4-hydroxy-2-nonenal (HNE)-modified proteins was assessed to evaluate the relation of oxidative stress elicited by LPS to the induction of apoptosis, once again decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts were positive. Therefore, the placental dysfunction by LPS may be brought about

  4. 合欢皮总皂苷所致小鼠肾毒性及其机制的研究%Study of nephrotoxicity and its mechanism of saponin from Albizia julibrissin durazz in mice

    Institute of Scientific and Technical Information of China (English)

    邱丽颖; 李倩; 许天蟾; 刘玲艳; 冯磊; 金坚; 吴鹏西

    2011-01-01

    Objective To Study the nephrotoxicity and its mechanism of saponin from Albizia julibrissin Durazz in mice. Method Giving mice the toxic dose of saponin for one time,and after 14 days, observe the nephrotoxicity of mice. The morphosis was observed by Hematoxylin Eosin staining. The activities of superoxide dismutase ( SOD) and glutathione peroxidase ( CSH-Px) were measured by biochemical methods. The apoptosis of kidney cell was detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling(TUNEL) method. Immunohistochemical detection of renal Bcl-2, Bax, COX-1 and COX-2 gene expression. Results Toxic dose of total saponin caused pathological changes in kidney, Reduced renal SOD and CSH-Px activities; increase in the number of glomerular endothelial cell apoptosis, Bcl-2 expression decreased, COX-2 expression increased; tubular COX-1 expression increased. Conclusion It shows that total saponin has toxicity to kidney. It perhaps related to increased apoptosis, decreased ability of anti-oxygen, reduced the expression of Bcl-2, and increased the expression of COX.%目的 研究合欢皮总皂苷对小鼠肾毒性及其机制.方法 采用一次性灌胃致毒剂量总皂苷,观察14 d时,合欢皮总皂苷对小鼠肾毒性表现.苏木素-伊红(HE)染色观察病理形态的变化,生化法测定其对肾SOD和GSH-Px的影响,末端标记法检测肾细胞凋亡,免疫组织化学法检测肾组织Bcl-2、Bax、COX-1及COX-2基因蛋白的表达.结果 合欢皮总皂苷口服致毒剂量可引起肾组织形态的变化,降低肾SOD和GSH-Px的活性;肾小球内皮细胞凋亡数目增多,Bcl-2表达下降,COX-2表达增加;肾小管COX-1表达增加.结论 致毒量合欢皮总皂苷对肾有一定毒性,促使细胞凋亡,可能与总皂苷降低肾的抗氧化能力和改变凋亡相关基因的表达、升高COX-1的表达有关.

  5. The effects of erdosteine, N-acetylcysteine, and vitamin E on nicotine-induced apoptosis of pulmonary cells.

    Science.gov (United States)

    Demiralay, Rezan; Gürsan, Nesrin; Erdem, Havva

    2006-02-15

    This study was conducted to investigate the frequency of apoptosis in the pulmonary epithelial cells of rats after intratraperitoneal nicotine injection, in order to examine the role of inflammatory markers [myeloperoxidase (MPO) and tumor necrosis factor-alpha (TNF-alpha)] in nicotine-induced lung damage, and to determine the protective effects of three known antioxidant agents [N-acetylcysteine (NAC), erdosteine, and vitamin E] on the lung toxicity of nicotine in the lungs. Female Wistar rats were divided into seven groups, each composed of nine rats: two negative control groups, two positive control groups, one erdosteine-treated group (500 mg/kg), one NAC-treated group (500 mg/kg), and one vitamin E-treated group (500 mg/kg). Nicotine was injected intraperitoneally at a dosage of 0.6 mg/kg for 21 days. Following nicotine injection, the antioxidants were administered orally, treatment was continued until the rats were killed. Lung tissue samples were stained with hematoxylin-eosin (H&E) for histopathological assessments. The apoptosis level in the lung bronchiolar and alveolar epithelium was determined by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method. Cytoplasmic TNF-alpha in the bronchiolar and alveolar epithelial cells and the lung MPO activity were evaluated immunohistochemically. The protective effect of vitamin E on lung histology was stronger than that of erdosteine or NAC. Treatment with erdosteine, NAC, and vitamin E significantly reduced the rate of nicotine-induced pulmonary epithelial cell apoptosis, and there were no significant differences in apoptosis among the three antioxidants groups. Erdosteine, NAC, and vitamin E significantly reduced the increases in TNF-alpha staining and lung MPO activity. The effects of erdosteine on the increases in the local TNF-alpha level and lung MPO activity were weaker than that of NAC or vitamin E. This findings suggest that erdosteine and NAC can be as effective as

  6. Bile salts inhibit growth and induce apoptosis of culture human normal esophageal mucosal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Ru Zhang; Jun Gong; Hui Wang; Li Wang

    2005-01-01

    AIM: To investigate the effect of six bile salts:glycocholate (GC), glycochenodeoxycholate (GCDC),glycodeoxycholate (GDC), taurocholate (TC),taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC), and their mixture on cultured human normal esophageal mucosal epithelial cells.METHODS: Human normal esophageal mucosal epithelial cells were cultured with serum-free keratinocyte medium. 3-[4,5-Dimethylthiaolyl]-2,5-diphenyl-tetrazolium bromide assay was applied to the detection of cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Sub-G1 DNA fragmentations and early apoptotic cells were assayed by flow cytometry (FCM) with propidium iodide (PI) staining and annexin V-FITC conjugated with PI staining.Apoptotic DNA ladders on agarose gel electrophoresis were observed.RESULTS: Except for GC, GCDC, GDC, TC, TCDC, TDC and their mixture could initiate growth inhibition of esophageal mucosal epithelial cells in a dose- and time-dependent manner. TUNEL and FCM assays demonstrated that the bile salts at 500 μmol/L and their mixture at 1 500 μmol/L induced apoptosis except for GC. The percentage of sub-G1 detected by FCM with PI staining was 83.5% in cells treated with 500μmol/L TC for 2 h, and 19.8%, 20.4%, 25.6%, 13.5%, and 75.8% in cells treated with 500 μmol/L GCDC, TCDC, GDC,TDC, and 1 500 μmol/L mixture for 24 h, respectively,which were higher than that of the control (1.5%). The percentage was 1.4% in cells with 500 μmol/L GC for 24 h.DNA ladders on agarose gel electrophoresis were seen in cells treated with 500 μmol/L TC for 2 h and 1 500 μmol/Lmixture for 24 h.CONCLUSION: All GCDC, GDC, TC, TCDC, TDC and their mixture can inhibit growth and induce apoptosis of cultured human normal esophageal mucosal epithelial cells, but GC is well tolerated by the cells.

  7. Bile salts inhibit growth and induce apoptosis of human esophageal cancer cell line

    Institute of Scientific and Technical Information of China (English)

    Ru Zhang; Jun Gong; Hui Wang; Li Wang

    2005-01-01

    AIM: To explore the effect of six bile salts, including glycocholate (GC), glycochenodeoxycholate (GCDC), glycodeoxycholate (GDC), taurocholate (TC), taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC), and two bile acids including cholic acid (CA) and deoxycholic acid (DCA) on esophageal cancer Eca109 cell line.METHODS: Eca109 cells were exposed to six bile salts, two bile acids and the mixed bile salts at different concentrations for 24-72 h. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)assay. Sub-G1 DNA fragmentations and early apoptosis cells were assayed by flow cytometry (FCM) with propidium iodide (PI) staining and annexin V-FITC conjugated with PI staining. Apoptosis DNA ladders on agarose were observed. Activation of caspase-3 was assayed by FCM with FITC-conjugated monoclonal rabbit anti-active caspase3 antibody and expressions of Bcl-2 and Bax proteins were examined immunocytochemically in 500 μmol/L-TC-induced apoptosis cells.RESULTS: Five bile salts except for GC, and two bile acids and the mixed bile salts could initiate growth inhibition of Eca109 cells in a dose- and time-dependent manner.TUNEL, FCM, and DNA ladder assays all demonstrated apoptosis induced by bile salts and bile acids at 500 μmol/L,except for GC. Early apoptosis cell percentages in Eca109 cells treated with GCDC, GDC, TC, TCDC, TDC,CA at 500 μmol/L for 12 h, DCA at 500 μmol/L for 6 h,and mixed bile salts at 1 000 μmol/L for 12 h were 7.5%,8.7%, 14.8%, 8.9%, 7.8%, 9.3%, 22.6% and 12.5%,respectively, all were significantly higher than that in control (1.9%). About 22% of the cell population treated with TC at 500 μmol/L for 24 h had detectable active caspase-3, and were higher than that in the control (1%). Immunocytochemical assay suggested that TC down-regulated Bcl

  8. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

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    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing, E-mail: allenylq@hotmail.com; Liao, Er-Yuan, E-mail: eyliao@21cn.com

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  9. Overexpression of cyclooxygenase-2 in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and mechanism of cyclooxygenase-2 selective inhibitor celecoxib-induced cell growth inhibition and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ning-Bo Liu; Tao Peng; Chao Pan; Yu-Yu Yao; Bo Shen; Jing Leng

    2005-01-01

    AIM: To investigate the cyclooxygenase-2 (COX-2)expression level in human HepG2, Bel-7402 and SMMC-7721hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis.METHODS: Hepatoma cells were cultured and treated with celecoxib. Cell in situ hybridization (ISH) and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium (MTT) bromide colorimetric assay. Celecoxibinduced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM). The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis.RESULTS: Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTr assays and morphological changes.The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 μmol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402and SMMC-7721 cells was 25.01±3.08%, 26.40±3.05%,and 30.60±2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3and dephosphorylation of Akt (Thr308). Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt (Thr308) after treatment with celecoxib.CONCLUSION: COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.

  10. Significance of AT1 receptor independent activation of mineralocorticoid receptor in murine diabetic cardiomyopathy.

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    Yuji Nagatomo

    Full Text Available BACKGROUND: Diabetes mellitus (DM has deleterious influence on cardiac performance independent of coronary artery disease and hypertension. The objective of the present study was to investigate the role of the renin-angiotensin-aldosterone system, especially angiotensin II type 1a receptor (AT1aR and mineralocorticoid receptor (MR signaling, in left ventricular (LV dysfunction induced by diabetes mellitus (DM. METHODS AND RESULTS: DM was induced by intraperitoneal injection of streptozotocin (200 mg/kg BW in wild-type (WT or AT1aR knockout (KO male mice, and they were bred during 6 or 12 weeks. Some KO mice were administered the MR antagonist eplerenone (100 mg/kg body weight. At 6 weeks, LV diastolic function was impaired in WT-DM, but preserved in KO-DM. At that time point MR mRNA expression was upregulated, NADPH oxidase subunit (p47phox and glutathione peroxidase (GPx1 mRNA expression were upregulated, the staining intensities of LV tissue for 4-hydroxy-2-nonenal was stronger in immunohistochemistry, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL positive cells was increased, Bcl-2 protein expression was significantly downregulated, and the expression of SERCA2a and phosphorylated phospholamban was depressed in WT-DM, while these changes were not seen in KO-DM. At 12 weeks, however, these changes were also noted in KO-DM. Eplerenone arrested those changes. The plasma aldosterone concentration was elevated in WT-DM but not in KO-DM at 6 weeks. It showed 3.7-fold elevation at 12 weeks even in KO-DM, which suggests "aldosterone breakthrough" phenomenon. However, the aldosterone content in LV tissue was unchanged in KO-DM. CONCLUSIONS: DM induced diastolic dysfunction was observed even in KO at 12 weeks, which was ameliorated by minelarocorticoid receptor antagonist, eplerenone. AT1-independent MR activation in the LV might be responsible for the pathogenesis of diabetic cardiomyopathy.

  11. The anti-tumour activity of rLj-RGD4, an RGD toxin protein from Lampetra japonica, on human laryngeal squamous carcinoma Hep-2 cells in nude mice.

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    Shao, Fangyu; Lv, Mei; Zheng, Yuanyuan; Jiang, Junshu; Wang, Yue; Lv, Li; Wang, Jihong

    2015-12-01

    The objective of this study is to investigate the antiproliferative activity and mechanism of integrin-binding rLj-RGD4 in a Hep-2 human laryngeal carcinoma-bearing nude mouse model. Human laryngeal squamous carcinoma cells (Hep-2) were inoculated subcutaneously into the axilla of nude mice to generate a Hep-2 human laryngeal carcinoma-bearing nude mouse model. When the Hep-2 xenograft model was successfully established, the animals were randomly separated into five groups. Three groups were treated with different dosages of rLj-RGD4. Cisplatin was administered to the positive control group, and normal saline (NaCl) was administered to the negative control group for 3 weeks. The body weights and the survival of the nude mice were evaluated, and the volumes and weights of the solid tumours were measured. The mechanism underlying rLj-RGD4 inhibition of tumour growth in transplanted Hep-2 human laryngeal carcinoma-bearing nude mice was evaluated by haematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), measurement of intratumoural microvessel density (MVD), Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The tumour volumes and weights of the treatment groups were reduced compared with the model group, and survival times were improved by rLj-RGD4 treatment in Hep-2 human laryngeal carcinoma-bearing nude mice. The number of apoptotic Hep-2 human cells and intratumoural MVD significantly decreased after the administration of rLj-RGD4. In the xenograft tissue of animals treated with rLj-RGD4, FAK, PI3K, and Akt expression was unaltered, whereas P-FAK, P-PI3K, Bcl-2, P-Akt, and VEGF levels were down-regulated. In addition, activated caspase-3, activated caspase-9, and Bax levels were up-regulated. rLj-RGD4 exhibits potent in vivo activity and inhibits the growth of transplanted Hep-2 human laryngeal carcinoma cells in a nude mouse model. Thus, these results

  12. Protective effects of progesterone against gastric mucosal lesion induced by open abdominal wound and seawater immersion in rats

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    Jiang PU

    2013-01-01

    Full Text Available Objective  To investigate the effects of progesterone on gastric mucosal inflammatory response, changes in mucosa structure, and cell apoptosis in rats with open abdominal wound and seawater immersion. Methods  Thirty male Wistar rats were randomly divided into 3 groups (10 each. The rats in group A were subjected to open abdominal wound + subcutaneous injection of sterile water, and rats in group B to open abdominal wound + seawater immersion + subcutaneous injection of sterile water, and rats in group C to open abdominal wound + seawater immersion + progesterone treatment. The animal model of open abdominal wound and seawater immersion was reproduced, and the rats in groups B and C were given subcutaneous injection of sterile water or progesterone (16mg/kg at 1, 6 and 12h after immersion. All rats were executed at 16h after immersion. The ulcer index (UI of gastric mucosa was calculated in the three groups. The pathological changes in gastric mucosa were observed under light microscope. The concentrations of tumor necrosis factor-alpha (TNF-α and interleukin-6 (IL -6 in gastric mucosa were measured by enzyme-linked immunoassay, the expression of caspase-3 in gastric mucosa was detected by immunohistochemistry, and the cell apoptosis of gastric mucosa was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Results  The UI, concentrations of TNF-α and IL-6, and injury score of gastric mucosa in group B were all significantly higher than those in group A (P<0.01 and group C (P<0.05. The expression of caspase-3 (23.8%±3.5% and apoptosis index (26.4%±2.8% of the gastric mucosa in group B were higher than those in group A (10.2%±1.6% and 8.5%±1.5% respectively, P<0.01 and group C (17.1%±2.3% and 19.8%±2.4% respectively, P<0.05. Conclusion  Progesterone administration could suppress gastric mucosa inflammation, protect gastric mucosal structure, and reduce mucosal cell apoptosis in the rats

  13. Hydrogen sulfide attenuates spatial memory impairment and hippocampal neuroinflammation in beta-amyloid rat model of Alzheimer’s disease

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    Xuan Aiguo

    2012-08-01

    Full Text Available Abstract Background Endogenously produced hydrogen sulfide (H2S may have multiple functions in brain. An increasing number of studies have demonstrated its anti-inflammatory effects. In the present study, we investigated the effect of sodium hydrosulfide (NaHS, a H2S donor on cognitive impairment and neuroinflammatory changes induced by injections of Amyloid-β1-40 (Aβ1-40, and explored possible mechanisms of action. Methods We injected Aβ1-40 into the hippocampus of rats to mimic rat model of Alzheimer’s disease (AD. Morris water maze was used to detect the cognitive function. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay was performed to detect neuronal apoptosis. Immunohistochemistry analyzed the response of glia. The expression of interleukin (IL-1β and tumor necrosis factor (TNF-α was measured by enzyme-linked immunosorbent assay (ELISA and quantitative real-time polymerase chain reaction (qRT-PCR. The expression of Aβ1-40, phospho-p38 mitogen-activated protein kinase (MAPK, phospho-p65 Nuclear factor (NF-κB, and phospho-c-Jun N-terminal Kinase (JNK was analyzed by western blot. Results We demonstrated that pretreatment with NaHS ameliorated learning and memory deficits in an Aβ1-40 rat model of AD. NaHS treatment suppressed Aβ1-40-induced apoptosis in the CA1 subfield of the hippocampus. Moreover, the over-expression in IL-1β and TNF-α as well as the extensive astrogliosis and microgliosis in the hippocampus induced by Aβ1-40 were significantly reduced following administration of NaHS. Concomitantly, treatment with NaHS alleviated the levels of p38 MAPK and p65 NF-κB phosphorylation but not JNK phosphorylation that occurred in the Aβ1-40-injected hippocampus. Conclusions These results indicate that NaHS could significantly ameliorate Aβ1-40-induced spatial learning and memory impairment, apoptosis, and neuroinflammation at least in part via the inhibition of p38 MAPK and p65 NF

  14. Overexpression of cellular repressor of E1A-stimulated genes inhibits TNF-{alpha}-induced apoptosis via NF-{kappa}B in mesenchymal stem cells

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    Peng, Cheng-Fei [Xijing Hospital, Fourth Military Medical University, Xi' an (China); Cardiovascular Research Institute and Department of Cardiology, Shenyang Northern Hospital, Shenyang (China); Han, Ya-Ling, E-mail: hanyaling53@gmail.com [Cardiovascular Research Institute and Department of Cardiology, Shenyang Northern Hospital, Shenyang (China); Jie-Deng,; Yan, Cheng-Hui; Jian-Kang,; Bo-Luan,; Jie-Li [Cardiovascular Research Institute and Department of Cardiology, Shenyang Northern Hospital, Shenyang (China)

    2011-03-25

    Research highlights: {yields} CREG protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis. {yields} CREG inhibits the phosphorylation of I{kappa}B{alpha} and prevents the activation of NF-{kappa}B. {yields} CREG inhibits NF-{kappa}B nuclear translocation and pro-apoptosis protein transcription. {yields} CREG anti-apoptotic effect involves inhibition of the death receptor pathway. {yields} p53 is downregulated by CREG via NF-{kappa}B pathway under TNF-{alpha} stimulation. -- Abstract: Bone marrow-derived mesenchymal stem cells (MSCs) show great potential for therapeutic repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their use. Cellular repressor of E1A-stimulated genes (CREG) has been identified as a potent inhibitor of apoptosis. This study therefore aimed to determine if rat bone marrow MSCs transfected with CREG-were able to effectively resist apoptosis induced by inflammatory mediators, and to demonstrate the mechanism of CREG action. Apoptosis was determined by flow cytometric and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays. The pathways mediating these apoptotic effects were investigated by Western blotting. Overexpression of CREG markedly protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis by 50% after 10 h, through inhibition of the death-receptor-mediated apoptotic pathway, leading to attenuation of caspase-8 and caspase-3. Moreover, CREG resisted the serine phosphorylation of I{kappa}B{alpha} and prevented the nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) under TNF-{alpha} stimulation. Treatment of cells with the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC) significantly increased the transcription of pro-apoptosis proteins (p53 and Fas) by NF-{kappa}B, and attenuated the anti-apoptotic effects of CREG on MSCs. The results of this study

  15. Genome-wide microRNA profiling of rat hippocampus after status epilepticus induced by amygdala stimulation identifies modulators of neuronal apoptosis.

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    Zhen Sun

    Full Text Available MicroRNAs (miRNAs are small and endogenously expressed non-coding RNAs that negatively regulate the expression of protein-coding genes at the translational level. Emerging evidence suggests that miRNAs play critical roles in central nervous system under physiological and pathological conditions. However, their expression and functions in status epilepticus (SE have not been well characterized thus far. Here, by using high-throughput sequencing, we characterized miRNA expression profile in rat hippocampus at 24 hours following SE induced by amygdala stimulation. After confirmation by qRT-PCR, six miRNAs were found to be differentially expressed in brain after SE. Subsequent Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that most of the predicted target genes for these six miRNAs were related to neuronal apoptosis. We then investigated the dynamic changes of these six miRNAs at different time-point (4 hours, 24 hours, 1 week and 3 weeks after SE. Meanwhile, neuronal survival and apoptosis in the hippocampus after SE were evaluated by Nissl staining and terminal deoxynucleotidyl transferase-mediated dUTP end-labeling assay. We found that the expression of miR-874-3p, miR-20a-5p, miR-345-3p, miR-365-5p, and miR-764-3p were significantly increased from 24 hours to 1 week, whereas miR-99b-3p level was markedly decreased from 24 hours to 3 weeks after SE. Further analysis revealed that the levels of miR-365-5p and miR-99b-3p were significantly correlated with neuronal apoptosis after SE. Taken together, our data suggest that miRNAs are important modulators of SE-induced neuronal apoptosis. These findings also open new avenues for future studies aimed at developing strategies against neuronal apoptosis after SE.

  16. Effect of the duration of bladder overdistention on renal function and morphology in rats.

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    Meng, Hong-Zhou; Cao, Min; Xiang, Jian-Jian; Zhou, Xie-Lai; Yin, Hong-Ping; Jin, Bai-Ye; Chen, Zhao-Dian; Jin, Xiao-Dong

    2013-06-01

    The aim of this study was to investigate the influence of the duration of bladder overdistention (DOBO) on kidney structure and function in rats. Bladder overdistention was induced in male Sprague-Dawley rats by an infusion of saline. Forty rats were divided into five groups: DOBO 1, 2, 4 or 8 h groups and the control. Renal function was evaluated using serum creatinine (SCr) and blood urea nitrogen (BUN). Apoptotic indices and morphologic changes of the kidney were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining and transmission electron microscopy (TEM). Compared with the control, rats undergoing 2, 4 or 8 h of overdistention showed significant, time-dependent increases in SCr (12.375 vs. 23.125, 34.375 and 51.500 μmol/l, respectively), BUN (6.980 vs. 18.689, 25.184 and 32.079 mmol/l, respectively), renal size (1.041 vs. 1.472, 1.484 and 1.634 cm(3), respectively) and renal pelvis separation (0.000 vs. 0.223, 0.320, 0.308 and 0.277 cm, respectively; Pcontrol vs. DOBO 2, 4 and 8 h), the interlobar renal artery (IRA; 32.095 vs. 39.16 and 51.745 cm(3)/sec, control vs. DOBO 4 and 8 h) and the segmental renal artery (SRA; 21.171 vs. 24.355 and 25.358 cm(3)/sec, control vs. DOBO 4 and 8 h; Pcontrol and DOBO 1, 2, 4 and 8 h, respectively; P<0.01) and TEM indicated that prolonged overdistention resulted in ultrastructural injuries of increased severity. DOBO plays a significant role in the functional and structural impairment of the rat kidney. With increasing duration, the hemodynamic changes, cell apoptosis and ultrastructural injuries of the kidney are more evident, all of which may contribute to the increasingly serious impairment of renal function and morphology.

  17. Expression of E2F -1 and cyclinD1 in colorectal adenocarcinoma and its significance%大肠癌E2F -1、CyclinD1的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    王蕾; 何妍; 李希芳; 刘岩; 时志民; 刘惠民

    2011-01-01

    Objective To study the expression of E2K - 1 and ryclinDl in human colorectal adenocarcinoma and its significance. Methods Terminal deoxynucleotidyl transferase - mediated nick end labeling TUNEL method and immunohistochemical SP method were used to detect 78 cases of colorectal adenocarcinoma, 20 cases of normal eoloreclal mucosa, 30 cases of colorectal polyps, and 30 cases of coloreotal adenoma cells in situ withered death ( Al) , and E2F - 1 and cyclinDl expression. Results The positive expression rate of E2F - 1 and cyclinDl in esophageal carcinoma was much higher than that in colorectal mucosa, colorectal polyps, and colorectal adenoma (P < 0. 05 ). The expression of E2F - 1 was closely correlated with the differentiation, TNM and lymph node metastasis (P <0. 05). Conclusions E2F - 1 and cyclinDl are closely related to the occurrence of colorectal adenocarcinoma. Both of them are important biological markers in colorectal adenocarcinoma occurrence and development.%目的 探讨E2F -1和cyclinD1在大肠腺癌组织中的表达及其意义 方法 采用脱氧核糖核酸末端转移酶介导的TUNEL法缺口末端标记和免疫组化SP法检测大肠腺癌78例、正常大肠黏膜20例、大肠息肉30例,以及大肠腺瘤细胞30例的原位凋亡(Al)及E2F-1和cyclin D1的表达情况.结果 E2F-1和cyclin D1在大肠腺癌中的阳性率显著高于正常大肠黏膜、大肠息肉以及大肠腺瘤(P<0.05).E2F-1表达与大肠腺癌的病理分化程度、淋巴结转移和临床分期具有相关性(P<0 05).从正常大肠黏膜、大肠息肉、大肠腺瘤到大肠腺癌细胞凋亡呈梯度降低.结论 E2F -1和cyclinD1的表达与大肠腺癌发生关系密切,是大肠腺癌发生、发展的重要生物学标记物

  18. Neuroprotective effects of L-carnitine against oxygenglucose deprivation in rat primary cortical neurons

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    Yu Jin Kim

    2012-07-01

    Full Text Available &lt;b&gt;Purpose:&lt;/b&gt; Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD. &lt;b&gt;Methods:&lt;/b&gt; Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO and propidium iodide (PI were counted, and lactate dehydrogenase (LDH activity and reactive oxygen species (ROS levels were measured. The 3-(4,5-dimethylthiazol-2-yl-2,5- diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 μM, 10 μM, and 100 μM on OGD-induced neurotoxicity. &lt;B&gt;Results:&lt;/b&gt; Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 μM and 100 μM of L-carnitine compared with the untreated OGD group (P&lt;0.05. The application of L-carnitine at 100 μM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P&lt;0.05. &lt;B&gt;Conclusion:&lt;/b&gt; L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

  19. Aconitine-induced Ca{sup 2+} overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats

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    Sun, Gui-bo; Sun, Hong; Meng, Xiang-bao [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 (China); Hu, Jin; Zhang, Qiang; Liu, Bo [Academy of Chinese Medical Sciences of Jilin Province, Changchun, Jilin 130021 (China); Wang, Min [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 (China); Xu, Hui-bo, E-mail: xhb_6505@163.com [Academy of Chinese Medical Sciences of Jilin Province, Changchun, Jilin 130021 (China); Sun, Xiao-bo, E-mail: sun_xiaobo163@163.com [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 (China)

    2014-08-15

    Aconitine is a major bioactive diterpenoid alkaloid with high content derived from herbal aconitum plants. Emerging evidence indicates that voltage-dependent Na{sup +} channels have pivotal roles in the cardiotoxicity of aconitine. However, no reports are available on the role of Ca{sup 2+} in aconitine poisoning. In this study, we explored the importance of pathological Ca{sup 2+} signaling in aconitine poisoning in vitro and in vivo. We found that Ca{sup 2+} overload lead to accelerated beating rhythm in adult rat ventricular myocytes and caused arrhythmia in conscious freely moving rats. To investigate effects of aconitine on myocardial injury, we performed cytotoxicity assay in neonatal rat ventricular myocytes (NRVMs), as well as measured lactate dehydrogenase level in the culture medium of NRVMs and activities of serum cardiac enzymes in rats. The results showed that aconitine resulted in myocardial injury and reduced NRVMs viability dose-dependently. To confirm the pro-apoptotic effects, we performed flow cytometric detection, cardiac histology, transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The results showed that aconitine stimulated apoptosis time-dependently. The expression analysis of Ca{sup 2+} handling proteins demonstrated that aconitine promoted Ca{sup 2+} overload through the expression regulation of Ca{sup 2+} handling proteins. The expression analysis of apoptosis-related proteins revealed that pro-apoptotic protein expression was upregulated, and anti-apoptotic protein BCL-2 expression was downregulated. Furthermore, increased phosphorylation of MAPK family members, especially the P-P38/P38 ratio was found in cardiac tissues. Hence, our results suggest that aconitine significantly aggravates Ca{sup 2+} overload and causes arrhythmia and finally promotes apoptotic development via phosphorylation of P38 mitogen-activated protein kinase. - Highlights: • Aconitine-induced Ca

  20. Mitochondrial outer membrane permeabilization increases reactive oxygen species production and decreases mean sperm velocity but is not associated with DNA fragmentation in human sperm.

    Science.gov (United States)

    Treulen, F; Uribe, P; Boguen, R; Villegas, J V

    2016-02-01

    Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MOMP was associated with ΔΨm dissipation (P DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. The results obtained in this study reveal that the alterations of sperm functions caused by MOMP are sufficiently relevant to justify its future study

  1. Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

    2016-01-01

    Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the

  2. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang [Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Yi, Kyu Yang [Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Daejeon, Chungnam, 305-600 (Korea, Republic of); Chung, Hun-Jong [Industrial Medicine Department, Chungju Hospital, Konkuk Medical School, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Park, Jong Seok [Department of Biomedical Laboratory Science, Taegu Health College, Taegu 702-722 (Korea, Republic of); Kim, Bokyung [Department of Physiology, Konkuk Medical School, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Feng, Zhong-Ping [Department of Physiology, College of Medicine, University of Toronto, Toronto, Ont., Canada M5S 1A8 (Canada); Shin, Hwa-Sup, E-mail: hsshin@kku.ac.kr [Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of)

    2013-04-01

    Poly(adenosine 5′-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. - Highlights: ► 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ► 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ► 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ► 5-AIQ activated Akt and GSK-3

  3. Persistence and accumulation of micronucleated hepatocytes in liver of rats after repeated administration of diethylnitrosamine.

    Science.gov (United States)

    Narumi, Kazunori; Ashizawa, Koji; Fujiishi, Yohei; Tochinai, Ryota; Okada, Emiko; Tsuzuki, Yasuhiro; Tatemoto, Hideki; Hamada, Shuichi; Kaneko, Kimiyuki; Ohyama, Wakako

    2013-08-15

    A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of hepatocarcinogens at relatively low doses, as indicated by dose-dependent micronucleus induction. Because the adult rat liver is known to have a long life-span, this desirable property of the assay will be an advantage in detecting micronucleated hepatocytes (MNHEPs) that have persisted for long periods in the liver following repeated dosing. However, no data directly supporting the underlying mechanisms have been published to date. In the present study, we verified the mechanisms by means of pulse-labeling of micronucleated hepatocytes with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The rodent hepatocarcinogen diethylnitrosamine (DEN) was repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old) for up to 2 weeks, and EdU was injected intraperitoneally on days 1, 7, or 14. Hepatocytes were isolated by use of a non-perfusion technique at 24h, 1 week, or 2 weeks after EdU injection and analyzed for EdU incorporation and micronucleus formation. The results of our study confirmed that MNHEPs labeled with EdU on the first day of DEN administration persisted until 2 weeks post-administration in the rat livers. However, the frequency of MHNEPs among EdU-labeled hepatocytes decreased over time. In addition, the number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive cells in the liver tissue increased, suggesting selective removal of micronucleated cells. Theoretical calculation of the cumulative MNHEP frequency on each of the days on which DEN was administered, taking into account the rate of loss, came out closer to the actual value observed in the liver micronucleus test. Taken together, these results indicate that although micronucleated cells induced in rat livers by administration of the genotoxic hepatocarcinogen DEN undergo selective removal, they

  4. Thioredoxin and impaired spatial learning and memory in the rats exposed to intermittent hypoxia

    Institute of Scientific and Technical Information of China (English)

    YANG Xiu-hong; LIU Hui-guo; LIU Xue; CHEN Jun-nan

    2012-01-01

    Background Obstructive sleep apnea (OSA) can cause cognitive dysfunction and may be a reversible cause of cognitive loss in patients with Alzheimer's disease (AD).Chronic exposure to intermittent hypoxia (IH),such as encountered in OSA,is marked by neurodegenerative changes in rat brain.We investigated the change of thioredoxin (Trx),spatial learning and memory in rats exposed to chronic intermittent hypoxia (CIH).Methods Forty healthy male Sprague-Dawley (SD) rats were randomly divided into four groups of ten each:a CIH+normal saline (CIH+NS group),a N-acetylcystein-treated CIH (CIH+NAC) group,a sham CIH group (sham CIH+NS),and a sham NAC-treated sham CIH (CIH+NAC) group.Spatial learning and memory in each group was assessed with the Morris water maze.Real-time PCR and Western blotting were used to examine mRNA and protein expression of Trx in the hippocampus tissue.The terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) method was used to detect the apoptotic cells of the hippocampus CA1 region.Results ClH-rats showed impaired spatial learning and memory in the Morris water maze,including longer mean latencies for the target platform,reduced numbers of passes over the previous target platform and a smaller percentage of time spent in the target quadrant.Trx mRNA and protein levels were significantly decreased in the CIH-hippocampus,meanwhile,an elevated apoptotic index revealed apoptosis of hippocampal neurons of rats exposed to CIH.The rats,which acted better in the Morris water maze,showed higher levels of the Trx mRNA and protein in the hippocampus;apoptotic index of the neurons in the hippocampus of each group was negatively correlated with the Trx mRNA and protein levels.Conclusion The Trx deficit likely plays an important role in the impaired spatial learning and memory in the rats exposed to CIH and may work through the apoptosis of neurons in the hippocampus.

  5. Effect of chronic intermittent hypoxia on the expression of Nip3, cell apoptosis, β-amyloid protein deposit in mice brain cortex

    Institute of Scientific and Technical Information of China (English)

    ZENG Yi-ming; CAI Kai-jin; CHEN Xiao-yong; WU Minx-ia; LIN Xi

    2009-01-01

    Background Chronic intermittent hypoxia (CIH) is the most important pathophysiologic feature of sleep apnea syndrome (SAS). To explore the relationship between SAS and dementia, the effects of CIH on the expression of Nip3, neuron apoptosis andβ-amyloid protein deposit in the brain cortex of the frontal lobe of mice were evaluated in this study. Methods Thirty male ICR mice were divided into four groups: control group (A, n=-10, sham hypoxia/reoxygenation), 2 weeks CIH group (B, n=-5), 4 weeks CIH group (C, n=-5), and 8 weeks CIH group (D, n=10). The ICR mice were placed in a chamber and exposed to intermittent hypoxia (oxygen concentration changed periodically from (21.72±0.55)% to (6.84±0.47)% every two minutes, eight hours per day). Neuron apoptosis of the cortex of the frontal lobe was detected by means of terminal deoxy-nucleotidyl transferase-mediated in situ end labeling (TUNEL). Immunohistochemical staining was performed for measuring expression of Nip3 and β-amyloid protein. The ultrastructure of neurons was observed under a transmission electron microscope. Results TUNEL positive neurons in each square millimeter in the cortex of the frontal lobe were categorized by median or Ri into group A (1,5.5), group B (133, 13), group C (252, 21), and group D (318, 24). There were significant differences among the above four groups (P=0.000). The significance test was performed between the control group and each CIH group respectively: group A and B (P>0.05); group A and C (P 0.05); groups A and C (P<0.005); and groups A and D (P<0.005). There was no significant difference between groups B and C, groups B and D, and groups C and D. The expression of Nip3 was closely correlated with neuron apoptosis in the brain (P <0.05). The expression ofβ-amyloid protein in the brain of mice was negative in all CIH groups and the control group. Ultrastructure observation showed karyopyknosis of nucleus, swelling of chondriosomes, deposit of lipofuscins and degeneration of

  6. Localisation of Abundant and Organ-Specific Genes Expressed in Rosa hybrida Leaves and Flower Buds by Direct In Situ RT-PCR

    Directory of Open Access Journals (Sweden)

    Agata Jedrzejuk

    2012-01-01

    Full Text Available In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant β-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.

  7. Detection of apoptotic cells and immunohistochemical study of bcl-2 and p53 gene protein in primary gastric mucosa-associated lymphoid tissue (MALT) lymphoma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To identify the apoptotic cells in gastric MALT lymphoma and its relationship between bcl-2 and p53 gene expression. Methods: TdT-mediated dUTP biotin Nick End labeling (TUNEL) and immuno-histochemistry ABC method were used to display apoptotic cells and the gene protein expression of bcl-2 and p53 independently. Results: Apoptotic indices (AI) in high-grade MALT lymphomas were significantly higher than in mixed-grade group and low-grade group (P<0.05). Bcl-2 was expressed in 83% of low-grade tumors, 61.6% of the median-grade tumors and 43.7% of high-grade tumors. An inverse correlation was observed between the expression of bcl-2 and apoptotic indices. Only 27 cases were p53 positive. The frequency of p53 positivity was significantly increased as the histologic grade advanced (P<0.05). There was also an inverse correlation between the expression of bcl-2 and p53. Conclusion: Apoptosis may be important in tumors development and transmission. P53 and bcl-2 were important regulatory genes of apoptosis and may be associated with transformation from low-grade to high-grade lymphomas.

  8. Clinicopathological significance and relations of Caspase-3 expression, cell proliferation and apoptosis in gastric cancer and the precancerous lesions%乳腺癌钼靶X线表现特征(118例分析)

    Institute of Scientific and Technical Information of China (English)

    XiaoBin Hu; Jing Zhao; Lin Yang; Yan Xin

    2009-01-01

    Objective: we investigated the relationship between the expression of Caspase-3. cell proiferation and apoptosis in gastric cancer and their precancerous lesions, to explore the tumorigenesis of the stomach mucosa. Methods: Caspase-3 expression in 13 normal gastric mucosa, 6 chronic atrophic gastritis (CAG), 31 intestinal metaplasia (IM), 114 dysplasia (DYS) and 20 gastric carcinomas were investigated immunohistochemically. Cell proliferation was evaluated with anti-Ki-67immunostaining and apoptosis was evaluated using DNA fragmentation in situ by TdT-mediated dUTP biotin nick end label-ing (TUNEL) method. Results: Caspase-3 mild-moderately positive expression was observed in most of normal superficial epithelia, its positively polar distribution in normal mucosa, CAG, IM, DYS and gastric carcinomas changed as seen in TU-NEL, and so did the positive rate. Caspase-3 protein expression showed significantly positive correlation with the number of apoptotic cells labeled with TUNEL (correlation coefficient r=0.94; P 0.05). Conclusion: Caspase-3 protein expression was up-regulated from CAG to IM and mild-moderate atypical dysplasia, but down-regulated in severe dysplasia and gastric carcinoma, indicating that inactivity or reduced expression of Caspase-3 is closely correlated with carcinogenesis of the stomach mucosa.

  9. Inhibition of tumor necrosis factor-α reduces alveolar septal cell apoptosis in passive smoking rats

    Institute of Scientific and Technical Information of China (English)

    ZHANG Cheng; CAI Shan; CHEN Ping; CHEN Jian-bo; WU Jie; WU Shang-jie; ZHOU Rui

    2008-01-01

    Background Recent studies have revealed that lung cell apoptosis plays an important role in pathogenesis of cigarette-induced chronic obstructive pulmonary disease (COPD).Tumor necrosis factor alpha(TNF-α)is one of the most important cytokines which are involved in COPD.This study aimed at investigating the jnfluence of its inhibitor,recombinant human necrosis factor-alpha receptor Ⅱ:IgG Fc fusion protein(rhTNFR:Fc)on alveolar septal cell apoptosis in passive smoking rats.Methods Forty-eight rats were randomly divided into a normal control group,a passive smoking group,an rhTNFR:Fc intervention group and a sham intervention group.The passive smoking rats were treated by exposure to cigarette smoking daily for 80 days.Afcer smoking for one month the rhTNFR:Fc Intervention group was treated with rhTNFR:Fc by subcutaneous injection,the sham intervention group injected subcutaneousIv with a neutral preparation(normal saline 0.1 ml,manicol 0.8 ml,cane sugar 0.2 mg,Tris 0.024 mg as a control.Lung function was determined and the levels of TNF-α in serum and broncho-alveolar lavage fluid(BALF)were measured with enzyme-linked immunosorbnent assay (ELISA).Lung tissue sections stained by hematoxylin and eosin(HE)were observed for study of morphological alternations.Mean linear intercept(MLI)and mean alveolar numbers(MAN)were measured and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)method was carried out to determine the percentage of positive cells and distribution of apoptotic cells.Results Increased MLI and decreased MAN were found in the passive smoking group compared with both the normal control group and the rhTNFR:Fc intervention group(P<0.05).Forced expiratory volume in 0.3 second(FEV0.3)/forced vital capacity(FVC)and peak expiratory flow(PEF)were lower in the passive smoking group than that in the normal control group(P<0.05).Compared with the sham intervention group,FEV0.3/FVC and PEF increased in the rhTNFR:Fc intervention

  10. 人生长素对雌性大鼠心力衰竭心脏重塑及心肌细胞凋亡的影响%Effect of Human Ghrelin on the Cardiac Remodeling and Cardiomyocytes Apoptosis in Heart Failure Female Rats

    Institute of Scientific and Technical Information of China (English)

    孙宇泽; 李艳丽; 赵勇; 袁寰; 田国忠; 李梅秀

    2015-01-01

    Objective To study the effect of human ghrelin on morphosis and cardiomyocytes apoptosis in heart failure ( HF) rats derived from acute myocardial infarction ( AMI) , so as to explore the cardioprotective activity mechanism of growth hormone releasing-peptides( GHRPs) .Methods Under the monitoring of the electrocardiogram ( ECG) by the multichannel physiological signal collection management system to confirm successfully permanent ligation the left anterior descending coronary artery(LAD) to set up AMI model and then by ultrasound B system to rule out the rats without HF or to make the sham group rats which merely pass through the stylolite beneeth LAD;HF model group rats accept three weeks human ghrelin through tail vein injection, using HE stain technique to analyze the changes of morphosis , the cardiomyocytes apoptosis in HF rats were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling( TUNEL) .Results TUNEL staining results showed that the cardiomyocytes in rats after permanent LAD ligation aligned ex-tremely irregularly, granular degeneration,swelling,much karyopyknosis and severe interstitial fibrosis,inflammatory cell infiltrate,while in human ghrelin intervention rats, cardiaomyocytes aligned irregularly,swelling and granular degeneration also could be found but without typical karyopyknosis or severe interstitial fibrosis.The cardiomyocytes apoptotic index in HF group rats is significantly higher than that in ghrelin intervention group rats(P<0.05).Conclusion Extrinsic human ghrelin exerted the cardioprotective activities by reducing the cardiomyocytes apoptosis and inhabitting the cardiac remodeling.%目的:研究人ghrelin对急性心肌梗死( acute myocardial infarction,AMI)致心力衰竭( heart failure,HF)大鼠心肌形态结构变化和心肌细胞凋亡的影响,探索生长素释放肽( growth hormone releasing-peptides,GHRPs)心脏保护活性机制。方法经心电图监测成功永久结

  11. Antagonism of glutathione to oxidative injury induced by manganese in rats testis%谷胱甘肽拮抗锰致雄性大鼠生殖损伤的研究

    Institute of Scientific and Technical Information of China (English)

    王乾兴; 金华; 张先平

    2011-01-01

    Objective: To explore the antagonism of glutathione to oxidative injury induced by manganese in rats testis. Methods ; Forty - eight healthy male SD rats were randomly divided into 3 groups. The rats of manganese exposure group were treated with 30mg/kg MnCl2· 4H2O for 1 week or4 weeks, followed by 1mmol/Kg glutathione (CSH ) for 2 weeks. The control group were received normal saline. The spermatogenic cell apoptosis was detected by terminal deoxynucleotidyl transferase -mediated Dutp nick end labeling technique. The malondialdehyde ( MDA) concentration and the glutathione peroxidase (CSH - Px) activity were measured by colorimetric analysis. Results: When the rats were exposured to manganese for 1 week, the apoptotic index ( AI) of spermatogenic cells and testicular MDA content of manganese exposure group were significantly higher than those of CSH antagonistic group and the control group ( P <0.01). CSH - Px activity showed no significant difference among the three groups. When manganese exposure for 4 weeks, AI of spermatogenic cells and testicular MDA content in manganese exposure group were significantly higher than those in CSH antagonist group and the control group ( P < 0.01) , meanwhile that in CSH antagonist group were also higher when compared with the control group (P <0.01). The activity of CSH - Px in manganese exposure group was significantly lower than those of CSH antagonistic group and the control group (P<0.01). Conclusion; Timely supplement of glutathione could decrease spermatogenic cell apoptosis through inhibiting lipid peroxidation induced by manganese.%目的:研究谷胱甘肽(GSH)对锰致雄性大鼠睾丸氧化损伤的拮抗作用.方法:将48只健康雄性SD大鼠随机分为3组进行急性和亚急性染毒.染锰组腹腔注射30mg/kg MnCl2·4H2O染毒1周(急性染毒)和4周(亚急性染毒),GSH拮抗组染毒后再给予2周1mmol/kg GSH,对照组染毒后给予生理盐水.TUNEL法检测生精细胞凋亡;化学比色法检

  12. Chronic Intake of Green Propolis Negatively Affecting the Rat Testis

    Science.gov (United States)

    Severi-Aguiar, Grasiela Dias de Campos; Pinto, Suellen Josine; Capucho, Cristina; Oliveira, Camila Andrea; Diamante, Maria Aparecida; Barbieri, Renata; Predes, Fabrícia Souza; Dolder, Heidi

    2017-01-01

    spermatozoa in the lumen of seminiferous tubule was showedThis male reproductive disruption can be linked to phenolic compounds present in Brazilian green propolis. Abbreviation Used: AEC: 3-amino-9-ethylcarbazole; AJ: Adherens junction; AME: Aromadendrin-40-methyl ether; CAPE: Caffeic acid phenethyl ester; Co: Control group; C×43: Connexin 43; DAB: Diaminobenzidine; dNTP: Deoxyribonucleotide phosphate; DSP: Daily sperm production; FA: Ferulic acid; FSH: Follicle-stimulating hormone; GJ: Gap junction; GJIC: Gap junction intercellular communication; HPLC: High-performance liquid chromatography; LC: Leydig cell; LH: Luteinizing hormone; N-cad: N-cadherin; PCNA: Proliferating cell nuclear antigen; PCR: Polymerase chain reaction; RT-PCR: Reverse transcription-polymerase chain reaction; SDM: Standard deviation of mean; T1: Group exposed to 3 mg of propolis/kg/day; T2: Group exposed to 6 mg of propolis/kg/day; T3: Group exposed to 10 mg of propolis/kg/day; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; WB-ras 2 cells: Ras-transformed rat liver epithelial cell line.

  13. Aldosterone induces aortic smooth muscle cell apoptosis%醛固酮诱导大鼠主动脉平滑肌细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    闫永吉; 李炯明; 刘建和; 陈戬; 姜永明; 张劲松

    2012-01-01

    目的 观察醛固酮在体内能否诱导主动脉平滑肌细胞凋亡.方法 将24只SD大鼠随机分为3组,每组8只:(1)空白溶剂设为对照组;(2)醛固酮组;(3)醛固酮+血管扩张剂组.渗透泵内分别注入醛固酮(1 μg/h)或空白溶剂,然后埋于大鼠背部皮下.肼苯哒嗪[25 mg/(kg·d)]溶于引用水,每日灌胃1次.8周后脱氧核苷酸末端转移介导的缺口末端标记法(TUNEL)检测凋亡的主动脉平滑肌细胞.结果 醛固酮组和醛固酮+血管扩张剂组大鼠主动脉TUNEL阳性的平滑肌细胞分别占(18.0±1.5)%和(16.5±2.0)%,与对照组(4.7±1.0)%比较,差异有统计学意义(P<0,05);后两组之间差异无统计学意义(P>0.05).结论 醛固酮在体内可以直接诱导主动脉平滑肌细胞凋亡.%Objective To determine whether aldosterone induces aortic smooth muscle cell apoptosis in vivo.Methods 24 Sprague-Dawley rats were randomly divided into 3 groups:vehicle as control; aldosterone and aldosterone plus hydralazine.All animals were then implanted with an osmotic mini-pump containing either aldosterone ( 1 μg/h) or vehicle.Hydralazine [ 25 mg/(kg·d) ] was resolved in drinking water and gavaged once daily.After 8 weeks,aortic smooth muscle cell (ASMC) apoptosis was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay.Results TUNEL-positive aortic smooth muscle cells in aldosterone-infused and aldosterone plus hydralazine rats were ( 18.0± 1.5 ) % and ( 16.5 ± 2.0) % respectively; compared with control rats (4.7 ± 1.0 ) %,there was signifi-cant difference (P < 0.05 ). In contrast,no significant difference was achieved between the latter twogroups (P > 0.05).Conclusion This study' s findings suggest that aldosterone induces aortic smooth muscle cell apoptosis by its direct effect on the aorta.

  14. An experimental study on the effects of curcumin on posterior capsule opacification in young rabbit eyes

    Institute of Scientific and Technical Information of China (English)

    WANG Wen-ying; ZHANG Zhu-jun; WANG Jun; WANG Hai-wei

    2011-01-01

    Background Posterior capsule opacification (PCO) compromises vision development in infants after cataract surgery and lead to amblyopia.To observe the effects of curcumin on PCO in infant rabbits,curcumin was injected under the capaule and into the anterior chamber during phacoemulsification.Methods Seventy-five 1-month-old healthy New Zealand white rabbits were randomized into 3 groups,one eye of each rabbit was randomly selected to be operated.The operation involved continuous circular capsulorhexis,followed by hydrodissection with 0.6 ml each of balanced salt solution (BSS,group A),hydroxypropyl-β-dodextrin (HP-β-CD,90μg/ml,group B) or CUR-HP-β-CD (123 μg/ml,group C),respectively.After phacoemulsification,0.4 ml of each drug solution was injected into the anterior chamber via an incision.The extent of corneal edema and the inflammatory response within the anterior chamber were considered as measures PCO and observed postoperatively.All eyes were examined 1 and 2 months postoperative by slit lamp microscopy and photography after pupil dilation.On the third day postoperative,6 rabbits from each group were executed.Paraffin-embedded sections were stained with hematoxylin-eosin (HE) or terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL,indicative of apoptosis).Stained sections were observed under light microscopy.Proliferation of lens epithelial cells (LECs) was observed microscopically on day 3,day 7,month 1 and month 2 after the operation with HE staining.Results The remission of cornea edema occurred earlier in group C than in groups A and B (P <0.05); there were no significant differences between groups A and B.The remission of anterior chamber exudation in group C was earlier than those in groups A and B (P <0.05).No significant difference in the times when PCO occurred,was observed among groups.Compared to groups A and B,the extent of PCO was less severe (P <0.05).Three days after the operation,LECs aggregated

  15. Correlation between neuronal injury and Caspase-3 after focal ischemia in human hippocampus

    Institute of Scientific and Technical Information of China (English)

    戚基萍; 吴爱萍; 王德生; 王立峰; 李淑霞; 徐凤琳

    2004-01-01

    Background Cerebral ischemia is a significant clinical problem, and cerebral ischemia usually causes neuron injury such as apoptosis in various brain areas, including hippocampus. Cysteinyl aspartate-specific protease (Caspases) are fundamental factors of apoptotic mechanism. Caspase-3 inhibitors show effect in attenuating brain injury after ischemia. But all the results were from animal models in research laboratories. This study aimed at investigating the correlation between the change of ischemic neuronal injury and Caspase-3 post-ischemia in human hippocampus. Methods We selected and systematized 48 post-mortem specimens from 48 patients, who died of cerebral infarction. Morphological change was firstly analyzed by observing hematoxyline/eosin-staining hippocampal sections. The expression of Caspase-3 was investigated using the methods of in situ hybridization and immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick-end labeling (TUNEL) method was used to clarify the involvement of Caspase-3 in neuron death. The loss of MAP 2 (MAP-2) was applied to judging the damaged area and degree of neuronal injury caused by ischemia.Results In the CA1 sector of hippocampus, Caspase-3 immunostaining modestly increased at 8 hours [8.05/high-power field (hpf)], dramatically increased at 24 hours (24.85/hpf), decreased somewhat after 72 hours. Caspase-3 mRNA was detectable at 4 hours (6.75/hpf), reached a maximum at 16 hours (17.60/hpf), faded at 72 hours. TUNEL-positive cells were detectable at 24 hours (10.76/hpf), markedly increased at 48-72 hours. The loss of MAP-2 was obviously detected at 4 hours, progressed significantly between 24 and 72 hours; MAP-2 immunoreactivity was barely detectable at 72 hours. Before 72 hours, the Caspase-3 evolution was related with the upregulation of TUNEL and the loss of MAP-2. The positive correlation between Caspase-3 mRNA and TUNEL was significant at the 0.05 level (correlation

  16. Effects of Chinese Jianpi herbs on cell apoptosis and related gene expression in human gastric cancer grafted onto nude mice

    Institute of Scientific and Technical Information of China (English)

    Ai-Guang Zhao; Hai-Lei Zhao; Xiao-Jie Jin; Jin-Kun Yang; Lai-Di Tang

    2002-01-01

    AIM: To explore the mechanism of the Sijunzi decoction and another Chinese herbal recipe (SRRS) based mainly on the Sijunzi decoction in treatment of gastric cancer.METHODS: A human gastric adenocarcinoma cell line SGC7901 grafted onto nude mouse was used as the animal model. The mice were divided into 3 groups, one control and the two representative experimental conditions. Ahimals in the two experimental groups received either Sijunzi decoction or SRRS over a 40-day period starting at 1st day after grafting. Control animals received saline on an identical schedule. Animals were killed 41 days after being grafted.The effect of therapy was assessed by two ways: (1)tumor size was periodically measured during the life of the animals; (2) tumor weight was determined by a electron balance immediately after the animals killed. For detection of apoptotic cells, apoptotic indices(AI) were examined by the terminal deoxynucleotidyl transferase-mediated deoxyuddine tdphosphate fluorescence nick end labeling (TUNEL) method.Morphological alterations were observed with electron microscopy. S-P immunohistochemical method was used to detect the expression of Ki-67 in xenografts. Expression of bcl-2 and p53 was semiquantitatively detected using a reverse transcriptase-polymerase chain reaction (RT-PCR)technique.RESULTS: When compared with controls, tumor growth (size and weight) was significantly inhibited by treatment with the Sijunzi decoction (P<0.05) or SRRS (P<0.01). The tumor inhibitory rate in the Sijunzi decoction group was 34.33 % and SRRS group 46.53 %. AI of human gastric cancer xenografts in nude mice was significantly increased to 16.24±3.21% using TUNEL method and 11.38±6.46 % by FACScan in the Sijunzi decoction group compared with the controls (TUNEL: 2.63±1.03 %, P<0.01; FACScan: 7.15± 1.32 %, P<0.05). SRRS group was also found a significantly increased AI by using TUNEL method and flow cytometry analysis compared with the controls (TUNEL: 13.18±3

  17. 外源性促红细胞生成素对大鼠视网膜脱离光感受器细胞的保护作用%Protective effects of recombinant erythropoietin on photoreceptor cells in rat with retinal detachment

    Institute of Scientific and Technical Information of China (English)

    解正高; 宫媛媛; 宋毅; 李彩红; 吴星伟

    2010-01-01

    Objective To investigate the protective effect of recombinant erythropoietin (EPO) on the photoreceptor cells in rat with retinal detachment (RD). Methods One hundred and sixty-two normal male rats were randomly divided into normal control (NC) group, RD model group, RD+phosphate buffer solution (RD+PBS) group, RD+EPO 100 ng group, RD+EPO 200 ng group and RD+EPO 400 ng group. Three days after RD, activated caspase-3 and bcl-XL were detected by Western blot and/or immunofluorescence, and apoptosis were measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick-end labeling(TUNEL). Fourteen and 28 days and two months after RD, the outer nuclear layer (ONL) thickness was measured by histopathologic method. Results Western bolt indicated that the protein level of activated caspase-3 and bcl-XL between six groups were statistically significant(F= 35. 96, 30. 75;P<0. 01). The number of TUNEL positive cells and activated caspase-3 positive cells are consistent with each other in different groups. Fourteen days and two months after RD, the differences of ONL thickness between six groups were statistically significant (F= 21. 52, 96. 25;P<0. 01). Conclusion Supplement of EPO after RD can alleviate apoptosis by inhibiting of the caspase-3 activity and increasing the expression of bcl-XL, thus exerts protective effect on photoreceptor cells.%目的 观察外源性促红细胞生成素(EPO)对大鼠视网膜脱离(RD)状态下光感受器细胞的保护作用.方法 采用计算机产生随机数字的方法将162只正常雄性SD大鼠分为正常对照组(NC组)、RD组、RD+磷酸盐缓冲液(PBS)组、RD+EPO 100、200、400 ng组.RD后3 d,采用蛋白免疫印迹(Western blot)检测半胱氨酸蛋白酶3(caspase-3)活性和B细胞淋巴瘤/白血病-XL(bcl-XL)的表达,免疫荧光检测caspase-3活性,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测光感受器细胞凋亡情况.RD后14、28 d,2个

  18. Effects of Wei Chang An on expression of multiple genes in human gastric cancer grafted onto nude mice

    Institute of Scientific and Technical Information of China (English)

    Ai-Guang Zhao; Ting Li; Sheng-Fu You; Hai-Lei Zhao; Ying Gu; Lai-Di Tang; Jin-Kun Yang

    2008-01-01

    AIM:To investigate the expression of multiple genes in Chinese jianpi herbal recipe Wei Chang An (WCA) in human gastric cancer cell line SGC-7901.METHODS:A human gastric adenocarcinoma cell line SGC-7901 grafted onto nude mice was used as the animal model.The mice were randomly divided into 3 groups,one control and the two representing experimental conditions.Animals in the two experimental groups received either WCA over a 34-d period or 5-fluorouracil (5-FU) over 6-d period starting at 8th d after grafting.Control animals received saline on an identical schedule.Animals were killed 41 d after being grafted.The expression profiles in paired WCA treated gastric cancer samples and the N.S.control samples were studied by using a cDNA array representing 14181 cDNA clusters.The alterations in gene expression levels were confirmed by Real-time Quantitative polymerase chain reaction (qPCR).RESULTS:When compared with controls,the average tumor inhibitory rate in WCA group was 44.32%±5.67% and 5-FU 47.04% 4±11.33% (P<0.01,respectively).The average labeling index (LI) for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group.Apoptotic index (AI) was significantly increased to 9.72%±4.51% using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method in WCA group compared with the controls 2.45%±1.37%.5-FU group was also found to have a significantly increased AI compared with the controls.The expression of cleaved Caspase-3 in WCA group and 5-FU group was significantly increased compared with the control group respectively.There were 45 different expressed sequence tags (ESTs) among the control sample pool and WCA sample pool.There were 24 ESTs up-regulated in WCA samples and 21 ESTs down-regulated.By using qPCR,the expression level of Stat3,rap2 interacting protein x (RIPX),regulator of differentiation 1 (ROD1) and Bcl-2 was lower in WCA group than that in control

  19. 氧化还原因子1对体外培养大鼠耳蜗螺旋神经节氧化损伤的保护作用%Adenovirus-mediated APE/Ref-1 expression protects rat spiral ganglion cells from oxidative damage

    Institute of Scientific and Technical Information of China (English)

    姜振东; 张学渊; 袁伟; 魏运军; 钟诚

    2008-01-01

    Objective To address the question if apurinic/apyrimidimic endonuclase/redox factor 1 (APE/Ref-1) involved in preventing spiral ganglion cells oxidative damage after oxidative stress. Methods Primary cultured rat spiral ganglion cells were infected with the adenovirus containing APE/Ref-1 for 48 h, then treated with H2O2 (0, 10,25,50,100,300 μmol/L)for 1 h, and finally changed back into normal medium. Western blot were used to detect the level of APE/Ref-1 protein in the infected cells to ensure APE/Ref-1 over expression as a result of adenovirus infection. The cell viability was determined by MTF and the apoptosis of spiral ganglion cells was determined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Results Western blot showed that infection of adenovirus resulted in APE/Ref-1 over expression in the spiral ganglion cells. Over expression of APE/Ref-1 significantly improved cell viability in cultures treated with different concentration H2O2 from 50 to 300 μmol/L However, the apoptosis of cells was significantly inhibited. Conclusions Overe xpression of APE/Ref-1 could protect spiral ganglion cells from oxidative damage.%目的 观察表达无嘌呤无嘧啶核酸内切酶/氧化还原因子1(apurinic/apyrimidimicendonuclase/redox factor 1,APE/Ref-1)的重组腺病毒感染对H2O2所致体外培养大鼠耳蜗螺旋神经节细胞氧化损伤的保护作用.方法 体外培养大鼠耳蜗螺旋神经节细胞,APE/Ref-1腺病毒表达载体感染48 h后,加入不同浓度H2O2(0、10、25、50、100及300 μmol/L)干预1 h,更换正常培养液后继续培养24 h,通过蛋白免疫印迹分析、四甲基偶氮唑蓝(MTT)法、原位缺口末端标记法(TUNEL)分别检测感染后螺旋神经节细胞APE/Ref-1蛋白表达、细胞活力以及凋亡情况.结果 通过腺病毒感染实现了APE/Ref-1基因在体外培养耳蜗螺旋神经节细胞的过表达,H2O2浓度为50~300 μmol/L时,APE/Ref-1组同对照组比较,细胞活

  20. Effects of gene transfer of adenovirus-mediated brain-derived neurotrophic factor on apoptosis after traumatic brain injury%腺病毒介导脑源性神经营养因子基因转移对大鼠脑损伤后细胞凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    王国强; 廖维宏; 沈岳; 李芳

    2001-01-01

    Objective To investigate the effects of gene transfer of adenovirus-mediated brain-derived neurotrophic factor (BDNF) on apoptosis after traumatic brain injury. Methods  Adult Wistar rats experienced a weight-drop strike on the right cerebral cortex, and then 4 μl recombinant adenovirus vector (RAV) and 4 μl virus buffer were injected into the hippocampus in the expermental group and in the controls, respectively. Immunohistochemistry and/or in situ hybridization, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and flowcytometry were used to determine the expressions of BDNF and apoptosis-associated signals in the hippocampus and cerebral cortex areas at 3 hour, 1, 3, 7 and 14 days following injury.  Results  Compared with the controls at 3 and 7 days after injury, BDNF positive cells increased significantly, while the apoptotic cells decreased significantly in CA1 and CA3 areas in the RAV group (P<0.01). Meanwhile, the cells expressing BDNF showed less apoptotic signals.  Conclusions  RAV-mediated BDNF gene transfer protects hippocampal neurons through up-regulating BDNF expression and inhibiting programmed cell death.%目的研究腺病毒介导的脑源性神经营养因子(BDNF)基因转移对脑损伤后细胞凋亡的影响。方法将重组腺病毒载体4 μl注入承受单侧大脑皮质重锤打击伤的海马区,对照组注射病毒缓冲液。伤后3 h、1,3,7,14 d利用免疫组化单标和(或)双标染色、原位杂交-免疫组化双标染色、DNA末端原位标记以及流式细胞仪等方法,检测伤侧大脑皮质、海马区BDNF及凋亡相关信号表达的改变。结果与对照组相比,注射病毒载体组动物术后3,7 d海马CA1、CA3区BDNF神经元显著增多,而凋亡细胞显著减少(P<0.01);表达BDNF的神经元较少并同时表达凋亡相关信号。结论腺病毒介导的BDNF基因转移对海马神经元具有保护作用。

  1. Effect of Huangzhu Qingnao Granule on The Brain Tissue Apoptosis in Acute Intracerebral Hemorrhage (Tanre Fushi syndrome)Rats%黄竹清脑颗粒对脑出血急性期(痰热腑实证)大鼠脑组织细胞凋亡影响的实验研究

    Institute of Scientific and Technical Information of China (English)

    刘淑霞; 徐雪; 高军宁; 李晓斌; 任惠峰

    2011-01-01

    Objective:To study the Huangzhu Qingnao granule on acute intracerebral hemorrhage (Tanre Fushi syndrome)in rat brain tissue surrounding hematoma apoptosis.Methods: Collagenase Ⅶ - heparin mixture of stereotactic injection and with the SD rats were fed with autologous method of production of faeces rats(60) with acute intracerebral hemorrhage( Tanre Fushi syndrome)rat model,rats were randomly divided into blank control group, Tanre Fushi syndrome model group, Huangzhu Qingnao granules large, medium,low -dose group and Qingkailing oral group, using TUNEL method (terminal deoxynucleotidyl transferase -mediated dUTP nick end labeling)in the brain cell apoptosis detection.Results: (1)Tanre Fushi syndrome model group compared with the blank control group, the former in rat brain tissue surrounding hematoma positive rate of apoptosis, which was significantly higher than normal brain tissue in rats(P<0.01 ), prompted modeling success;(2)Huangzhu Qingnao granule of large, medium -dose group were superior efficacy of oral liquid group and Qingkai ling Huangzhu Qingnao granule is small -dose group( P <0.05).Conclusion: Huangzhu Qingnao granule particles could significantly reduce the cerebral hemorrhage period ( Tanre Fushi syndrome) Rat droppings dry, irritability easy to move, dry mouth, polydipsia, throat Phlegm - ming, greasy yellow tongue and other symptoms and improve the nervous system in rats after intracerebral hemorrhage in pathological signs, and has significantly reduced apoptosis in rat brain cells,play a role in protecting brain cells.%目的:研究黄竹清脑颗粒对脑出血急性期(痰热腑实证)大鼠血肿周围脑组织细胞凋亡的影响.方法:采用胶原酶Ⅶ-肝素混合液立体定位注射及配合SD大鼠自体粪便灌胃的方法制作60只脑出血急性期(痰热腑实证)大鼠模型,将大鼠随机分为空白对照组、疾热腑实证模型组、黄竹清脑颖粒大、中、小量组及清开灵口服液组,采用TUNEL法(

  2. Effects of soybean isoflavones on calpain expression and neuronal apoptosis in ischemic and reperfused brain tissues of ovariectomized female rats%大豆异黄酮对去势雌大鼠缺血再灌注脑组织细胞凋亡及calpain表达的影响

    Institute of Scientific and Technical Information of China (English)

    么晓轶; 于纪珠; 李颖

    2011-01-01

    目的:研究大豆异黄酮对大鼠脑缺血再灌注后缺血脑组织神经细胞凋亡和calpain表达的影响,探讨大豆异黄酮的神经保护作用.方法:成年雌性大鼠36只,随机分为药物组和对照组,药物组再随机分为小剂量和大剂量组.所有动物切除双侧卵巢,1周后各组每日给予大豆异黄酮和生理盐水,1个月后建立大脑缺血再灌注模型,原位末端标记检测神经细胞凋亡,采用原位杂交技术观察calpain mRNA表达情况,同时比较各组calpain活性变化.结果:大豆异黄酮组凋亡细胞数明显减少(P<0.05),神经细胞凋亡指数降低,calpain mRNA表达明显减少,calpain活性下降.药物大剂量组与小剂量组比较无明显差异.结论:大豆异黄酮使脑缺血再灌注后神经细胞凋亡减少,可能通过下调calpain mRNA表达,降低calpain的活性发挥神经保护作用.%AIM: To study the effects of soybean isoflavones on neuronal apoptosis and mRNA expression of calpain in transient focal cerebral ischemia and reperfusion ( FCIR ) rats. METHODS: Thirty - six adult healthy female SD rats were ovariectomized and randomly divided into soybean isoflavone - treated group and control group. The rats in soybean isoflavone - treated group were subdivided into low and high doses of soybean isoflavone groups. After 1 month of treatment, a FCIR model was established by intraluminal middle cerebral artery occlusion ( MCAO ) with a nylon monofila-ment suture. In situ hybridization was performed to examine the mRNA expression of calpain. Apoptosis was observed with terminal deoxynucleotidyl transferase - mediated dUTP nick - end labeling ( TUNEL ). The activity of calpain was also examined. RESULTS: The mRNA expression of calpain and TUNEL positive cells were observed in the cortex and striatum of ischemic hemisphere. Calpain mRNA was present at lower levels in soybean isoflavone - treated groups than that in control group ( P <0. 05 ). The number of apoptotic cells

  3. Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo

    Directory of Open Access Journals (Sweden)

    Wang Haibo

    2010-09-01

    Full Text Available Abstract Background RhoA and RhoC have been proved to be over-expressed in many solid cancers, including colorectal cancer. The reduction of RhoA and RhoC expression by RNA interference (RNAi resulted growth inhibition of cancer cells. The present study was to evaluate the effect of silencing of RhoA and RhoC expression by RNAi on growth of human colorectal carcinoma (CRC in tumor-bearing nude mice in vivo. Methods To establish HCT116 cell transplantable model, the nude mice were subcutaneously inoculated with 1.0 × 107 HCT116 cells and kept growing till the tumor xenografts reached 5-7 mm in diameter. Then the mice were randomly assigned to three groups(seven mice in each group: (1 normal saline(NS group, (2replication-defective recombinant adenovirus carrying the negative control shRNA (Ad-HK group and (3replication-defective recombinant adenovirus carrying the 4-tandem linked RhoA and RhoC shRNAs (Ad-RhoA-RhoC group. Ad-HK (4 × 108 pfu, 30 ul/mouse, Ad-RhoA-RhoC (4 × 108 pfu, 30 ul/mouse or PBS (30 ul/mouse was injected intratumorally four times once every other day. The weight and volumes of tumor xenografts were recorded. The levels of RhoA and RhoC mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay was used to detect the death of cells. Results The xenografts in mice could be seen at 5th day from the implantation of HCT116 cells and all had reached 5-7 mm in size at 9th day. After injection intratumorally, the growth speed of tumor xenografts in Ad-RhoA-RhoC group was significantly delayed compared with those in NS and Ad-HK group(P RhoA and RhoC reduced more in Ad-RhoA-RhoC group than those in NS and Ad-HK group. The relative RhoA and RhoC mRNA transcripts were decreased to 48% and 43% respectively (P RhoA and Rho

  4. Royal jelly supplemented soybean lecithin-based extenders improve post-thaw quality and incubation resilience of goat spermatozoa.

    Science.gov (United States)

    Alcay, Selim; Toker, M Berk; Onder, N Tekin; Gokce, Elif

    2017-02-01

    The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thaw quality and incubation resilience of goat spermatozoa. Semen samples were collected from five goats. Pooled semen were diluted with soybean lecithin-based extender without RJ (control) or supplemented with different concentrations (0.25, 0.5 and 0.75%) of RJ (RJ0.25, RJ0.5, RJ0.75 respectively), at a final concentration of 150 × 10(6) spermatozoon/mL. Semen samples were assessed for sperm motility, plasma membrane integrity using hypoosmotic swelling test (HOST) damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The addition of RJ (0.5%, 0.75%) led to higher percentages of subjective motilities (55.33 ± 2.29%, 57.67 ± 2.58%) compared to control and RJ0.25 groups (49.00 ± 2,80%, 51.67 ± 3.09%) (P  0.05). In the end of incubation, motility and HOST rates of RJ0.5 (14.00 ± 3.87%, 31.20 ± 3.70%) and RJ0.75 (15.00 ± 3.27%, 29.20 ± 2.59%) groups were higher than control (8.00 ± 2.54%, 18.20 ± 3.11%) and RJ0.25 (9.00 ± 2.07%, 20.60 ± 2.88%) groups (P < 0.05). Also defected acrosome and DNA fragmation rates of RJ0.5 (32.20 ± 1.30%, 5.4 ± 0.55%) and RJ0.75 (29.20 ± 1.30%, 5.80 ± 0.45%) groups were significantly lower than control (38.80 ± 0.84%, 7.40 ± 1.34%) and RJ0.25 (39.80 ± 2.05%, 7.00 ± 1.58) groups. This study shows that RJ supplemented extenders have beneficial effect on goat sperm parameters at 0 h and 6 h of incubation. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Inhalation of hydrogen gas reduces liver injury during major hepatotectomy in swine

    Institute of Scientific and Technical Information of China (English)

    Lei Xiang; Jing-Wang Tan; Li-Jie Huang; Lin Jia; Ya-Qian Liu; Yu-Qiong Zhao; Kai Wang; Jia-Hong Dong

    2012-01-01

    Contrast-group (P =0.0005,P =0.0004).In Hydrogen-group,the HA level was also significantly low to Contrast-group (P =0.0005,P =0.0005) although the two groups all increased at PH 1 h and 3 h.The expression of cluster of differentiation molecule 31 molecules Hydrogen-group was low to Contrast-group.However,PCNA index (%) was not statistically significant between the two groups (P =0.802).Microphotometric evaluation of apoptotic index (AI) in terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-stained tissue after hepatotectomy for 3h,the AI% level in the hydrogen was significantly low to Contrast-group (P =0.012).There were no significant difference between Hydrogen-group and Contrast-group at pre-operation (P =0.653,P =0.423),but after massive hepatotectomy,the TNF-α and IL-6 levels increase,and its in Hydrogen-group was significantly low compared with Contrast-group (P =0.022,P =0.013,vs P =0.016,P =0.012),respectively.Hydrogen-gas inhalation reduce levels of these markers and relieved morphological liver injury and apoptosis.CONCLUSION:H2 gas attenuates markedly ischemia and portal hyperperfusion injury in pigs with massive hepatotectomy,possibly by the reduction of inflammation and oxidative stress,maybe a potential agent for treatment in clinic.

  6. Oxidative stress and apoptosis in gut barrier dysfunction of severe acute pancreatitis%氧化应激及凋亡与重症急性胰腺炎肠屏障功能障碍

    Institute of Scientific and Technical Information of China (English)

    田锐; 俞康龙; 许飞; 王瑞兰; 谢晖; 孟潇潇; 钱永兵; 金卫; 胡家昌; 周志刚

    2012-01-01

    Objective By means of animal study,investigated the gut barrier function in severe acute pancreatitis ( SAP),and role of inflammatory factors releasing,gut mucosa oxidative stress,cell apoptosis in it.Methods The animal experiment was done in the animal center of first people' s hospital,shanghai jiaotong university.Twenty four BALB/c mice were randomized ( random number) divided into two groups with twelve mice each group.The SAP group,mice received six intraperitoneal injections of cerulein at 1-hour intervals, the dose was 50μg/kg, then given one intraperitoneal injection of 10 mg/kg lipopolysaccharide ( LPS from E.Coli) for the induction of severe acute pancreatitis.The control ( sham operation) group,the mice received intraperitoneal injection of 2 ml normal saline for six times at 1-hour intervals.All the animals of each group were averaged to two batches,4 h and 8h after being operated respectively,to be anesthetized and adopted blood and tissue specimen.Then we observed the pathological change of pancreas and gut,scored it.We measured the blood value of diamine oxidase ( DAO),amylase and tumor necrosis factor-α (TNF-α).We detected content of malondialdehyde (MDA),superoxide dismutase (SOD),glutathione (GSH) and activity of xanthine oxidase (XO) in gut mucosa.We detected the casepase-3 activity and cell apopotosis by means of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in gut mucosa,and conculated the apopotosis index (AI).Then using the PASW 18.0 software,we analyzed the data by anova and t-test,to make sure if the values were statistically different between the two groups and the mechanism of gut barrier dysfunction in panreatitis.Results At 4 h and 8 h after operation,the SAP-group-mice had significantly higher pancreas pathological score (P <0.01 ),blood amylase value ( P < 0.05 ),gut pathological score and blood DAO and TNF-α value ( P <0.01 ),compared with the contral-group-mice.The gut mucosa MDA content and

  7. Biotherapic of Trypanosoma cruzi 17d increases apoptosis in experimentally infected mice

    Directory of Open Access Journals (Sweden)

    Silvana Marques de Araújo

    2011-09-01

    UTP-biotin Nick End Labeling (ApopTag® Peroxidade-Chemicon. For statistical analysis software Statistica 8.0 was used. This study was approved by the Ethics Committee for Animal Experimentation of UEM. Results and Discussion: in the samples of liver of animals age 4 and 8 weeks either treated or not with biotherapic 17d it was found cells parasitized by amastigotes of T. cruzi with apoptotic bodies, or phagocytic cells with phagocytic vacuole with apoptotic marked material inside them. The number of cells in apoptosis in animals age 4 weeks was not significantly (p=0.03 larger in treated group BIOT-C4 than in control group CI-4 (Figure 1. In animals age 8 weeks, the number of cells in apoptosis was significantly (p<0.001 larger in the treated group BIOT-8 than in control group CI-8 (Figure 1. Figure 1. Average number of cells in apoptosis observed in slices of liver of male Swiss mice age 4 and 8 weeks infected by 1,400 blood trypomastigotes Y strain treated (biotherapic or not (control with biotherapic of Trypanosoma cruzi 17d. Technique employed: Terminal deoxynucleotidyl transferase-mediated UTP nick end labelling - APOPTAG (Millipore. * p <0,05 regarding the corresponding control. These results point to a mechanism of action for the rise in the peak of parasitemia caused by ultradiluted substances in low dilutions in Swiss mice experimentally infected by T. cruzi, as it was observed in several studies [9-12]. It is known that homeopathic preparations are able to increase communication between cells [13-14]. Affected organisms treated with ultradilutions would resort to apoptosis as mechanism of cure, removing altered cells[15]. Literature confirms that the use of homeopathic medicines increases apoptosis[16]. The idea to be assessed is that, when attempting to eliminate infection by T. cruzi, parasitized cells enter into apoptosis and consequently release parasites to the circulation rising the peak of parasitemia up. This hypothesis was demonstrated here

  8. Antenatal taurine supplementation reduces cerebral cell apoptosis in fetal rats with growth restriction%孕鼠补充牛磺酸减少胎儿生长受限胎鼠脑细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    王晓凤; 滕慧云; 刘敬; 杨娜; 任晓暾

    2013-01-01

    目的 通过建立胎儿生长受限(fetal growth restriction,FGR)大鼠模型,探讨孕鼠补充牛磺酸对FGR胎鼠脑细胞凋亡以及胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)和半胱氨酸蛋白酶-3(caspase 3)表达的影响. 方法 将15只Sprague-Dawley (SD)孕鼠随机分为对照组、FGR模型组(模型组)、FGR+孕鼠补充牛磺酸组(牛磺酸组),每组5只.通过低蛋白饮食法建立FGR模型,牛磺酸组自妊娠第12天开始于饲料中添加牛磺酸300 mg/(kg·d)直至自然分娩.对照组每窝随机取2只适于胎龄胎鼠,牛磺酸组与模型组每窝随机取2只FGR胎鼠,应用原位末端脱氧核苷酸转移酶标记(terminal deoxynucleotidyl transferase mediated nick end labeling,TUNEL)法检测各组胎鼠脑神经细胞凋亡情况.用免疫组织化学方法检测GDNF和caspase-3表达情况.统计学方法采用单因素方差分析、Kruskal-Wallis秩和检验、SNK检验和Tamhane's检验. 结果 (1)对照组、模型组及牛磺酸组胎鼠总数分别为65、60和59只,胎鼠平均体重为(6.36±0.44)、(4.55±0.45)和(5.11±0.67)g.模型组胎鼠均发生FGR,牛磺酸组胎鼠FGR发生率为76.3% (45/59),成功建立FGR模型.(2)对照组胎鼠脑组织内偶见TUNEL阳性细胞;模型组明显增多,在大脑皮质、海马、白质区均有分布;牛磺酸组较模型组则明显减少.3组TUNEL阳性细胞数分别为(0.46±0.11)、(14.76±3.42)和(6.78±1.93)个(H=429.80,P=0.000).(3)对照组胎鼠大脑皮质内仅有少量GDNF阳性细胞分布,模型组明显增多,牛磺酸组则进一步增多.3组GDNF阳性细胞数分别为(93.56±6.73)、(120.36±6.23)和(139.56±5.28)个(H=715.17,P=0.000).(4)对照组胎鼠大脑皮质内仅见少数caspase 3阳性细胞分布,模型组明显增多,牛磺酸组较模型组显著减少,但仍多于对照组.3组caspase 3阳性细胞数分别为(7.50±2.31)、(151.32±24.43)和(37.28±11.21)个(F=132.54,P=0.000).

  9. Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin

    DEFF Research Database (Denmark)

    Liang, Y G; Jorgensen, A G; Kaestel, C G;

    2000-01-01

    . METHODS. Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl...

  10. Expression pattern of apoptosis-related markers in Huntington's disease

    NARCIS (Netherlands)

    Vis, José C; Schipper, Ellis; de Boer-van Huizen, Roelie T; Verbeek, Marcel M; de Waal, Rob M W; Wesseling, Pieter; ten Donkelaar, Hans J; Kremer, Berry

    2005-01-01

    Inappropriate apoptosis has been implicated in the mechanism of neuronal death in Huntington's disease (HD). In this study, we report the expression of apoptotic markers in HD caudate nucleus (grades 1-4) and compare this with controls without neurological disease. Terminal transferase-mediated biot

  11. Role of caspase 12 in apoptosis of cochlea induced by intense noise in guinea pigs%Caspase 12在强噪声诱导下豚鼠耳蜗细胞凋亡中的作用

    Institute of Scientific and Technical Information of China (English)

    薛秋红; 陈佳; 龚树生; 何坚; 谢静; 陈小林

    2009-01-01

    目的 探讨caspase 12在强噪声诱导的豚鼠耳蜗细胞凋亡中的作用.方法 选用健康雄性白色豚鼠32只,随机数字表法分为4组,将实验组动物暴露于声压级120 dB、4 kHz窄带噪声环境4 h.分别对健康对照组及噪声刺激后第1、4、14天组豚鼠测试听性脑干反应(ABR).每组取4只豚鼠耳蜗作石蜡切片,另外4只豚鼠提取耳蜗总蛋白.通过透射电镜和脱氧核糖核甘酸末端转移酶介导的缺口末端标记技术(ternial-deoxynucleotidyl transferase mediated nick end labeing,TUNEL)检测耳蜗凋亡细胞,免疫组化及蛋白质印迹(Western blot)法检测Bip/GRP78、caspa8e 12蛋白的表达.结果 透射电镜观察到实验组第1天耳蜗外毛细胞、螺旋神经节细胞出现凋亡的特征性改变.实验组耳蜗Corti器、侧壁的血管纹和螺旋神经节存在TUNEL阳性细胞,第1天组最多;对照组未见阳性细胞.免疫组化、Western blot检测实验组Bip/GRP78蛋白明显高于对照组,且在第1、4、14天都维持在比较高的水平;caspase 12蛋白含量在噪声刺激后迅速增高,第1、4天达到高峰,第14天表达明显下降,但仍维持较高水平.结论 强噪声可以诱导豚鼠耳蜗细胞凋亡,caspase 12活化引起内质网应激反应性凋亡通道可能是此过程的信号途径之一.%Objective To investigate the relationship between caspase 12 activation and endoplasmic reticulum stress mediated apoptosis of guinea pig cochlea cells induced by intense noise. Methods Thirty-two guinea pigs were randomly divided into 4 groups. The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h. After the noise expose for 1,4, 14 days of the experiment guinea pigs, auditory brainstem response (ABR) of the guinea pigs on experiment and control groups were tested before decapitated. Four guinea pig's cochleae of every group were taken to paraffin section, and the rest was extracted the total protein

  12. 磷脂酰肌醇3激酶/蛋白激酶B通过上调内皮型一氧化氮合酶参与远端缺血后处理的脑保护作用%Phosphoinositide-3 kinase/protein kinase B pathway participates in the neuroprotection of remote ischemic postconditioning by up-regulating endothelial nitric oxide synthase

    Institute of Scientific and Technical Information of China (English)

    彭蓓; 郭曲练; 叶治; 王娜; 郑利民

    2012-01-01

    Objective To investigate the roles of endothelial nitric oxide synthase (eNOS) and phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) pathway in the neuroprotection of remote ischemic postconditioning (RIPoC) on global cerebral ischemia/reperfusion (I/R) injury in rats. Methods One hundred male adult SD rats weighing 200 g-250 g were randomly divided into 5 groups (n=20 each):group sham,group I/R,group I/R+RIPoC,group L-NAME+I/R+RIPoC,group LY+I/R+RIPoC.Global cerebral I/R was induced by four-vessel occlusion.Group I/R+RIPoC,group L-NAME+I/R+RIPoC and group LY+I/R+RIPoC received 3 cycles of 15 min ischemia in bilateral femoral arteries at the beginning of cerebral reperfusion followed by 15 min repeffusion.The group L-NAME+I/R+RIPoC:4-VO with coinjection of N (ω)-nitro-L-arginine methyl ester (L-NAME),5 mg/kg,in normal saline.intraperitoneslly,10 min before 4-VO,then followed by RIPoC and 48 h and 7 d of reperfusion.The group LY+I/R+RIPoC:I/R+RIPoC with injection of LY294002(a highly selective inhibitor of PI3K,LY,10 μL,10 mol/L,in 3% DMSO,intracerebroventricularly.10 min before 4-VO),then followedby RIPoC and 48 h and 7 d of reperfusion.The rats were sacrificed at 48 h of cerebral rep effusion,and brains were removed for determination of neuronal apoptosis [by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method] in hippocampal CA1 region and p-eNOS,eNOS,p-Akt and Akt expression (by Western blot)in hippocampal CA1 region.Morris water maze task was used to test the learning and memory function at 4 d of cerebral reperfusion,and the rats were sacrificed at 7 d of cerebral reperfusion,and brains were removed for determination of neuronal density in hippocampal CA1 region.Results Compared with group sham,the number of apoptotic neurons in CA1 region [(0.8±0.8),(84.7±6.8), (52.8±7.8), (74.3±9.0), (79.5±7.3)/mm] increased (P<0.01),learning and memory function decreased (P<0.01) and neuronal density in CA1 region [ (193±7

  13. Effect of Boric Acid Supplementation on the Expression of BDNF in African Ostrich Chick Brain.

    Science.gov (United States)

    Tang, Juan; Zheng, Xing-ting; Xiao, Ke; Wang, Kun-lun; Wang, Jing; Wang, Yun-xiao; Wang, Ke; Wang, Wei; Lu, Shun; Yang, Ke-li; Sun, Peng-Peng; Khaliq, Haseeb; Zhong, Juming; Peng, Ke-Mei

    2016-03-01

    The degree of brain development can be expressed by the levels of brain brain-derived neurotrophic factor (BDNF). BDNF plays an irreplaceable role in the process of neuronal development, protection, and restoration. The aim of the present study was to evaluate the effects of boric acid supplementation in water on the ostrich chick neuronal development. One-day-old healthy animals were supplemented with boron in drinking water at various concentrations, and the potential effects of boric acid on brain development were tested by a series of experiments. The histological changes in brain were observed by hematoxylin and eosin (HE) staining and Nissl staining. Expression of BDNF was analyzed by immunohistochemistry, quantitative real-time PCR (QRT-PCR), and enzyme linked immunosorbent assay (ELISA). Apoptosis was evaluated with Dutp-biotin nick end labeling (TUNEL) reaction, and caspase-3 was detected with QRT-PCR. The results were as follows: (1) under the light microscope, the neuron structure was well developed with abundance of neurites and intact cell morphology when animals were fed with less than 160 mg/L of boric acid (groups II, III, IV). Adversely, when boric acid doses were higher than 320 mg/L(groups V, VI), the high-dose boric acid neuron structure was damaged with less neurites, particularly at 640 mg/L; (2) the quantity of BDNF expression in groups II, III, and IV was increased while it was decreased in groups V and VI when compared with that in group I; (3) TUNEL reaction and the caspase-3 mRNA level showed that the amount of cell apoptosis in group II, group III, and group IV were decreased, but increased in group V and group VI significantly. These results indicated that appropriate supplementation of boric acid, especially at 160 mg/L, could promote ostrich chicks' brain development by promoting the BDNF expression and reducing cell apoptosis. Conversely, high dose of boric acid particularly in 640 mg/L would damage the neuron structure of

  14. Interplay of neuropilin-1 and semaphorin 3A after partial hepatectomy in rats

    Institute of Scientific and Technical Information of China (English)

    Ling Fu; Tsuneo Kitamura; Kazuhisa Iwabuchi; Syozo Ichinose; Mitsuaki Yanagida; Hideoki Ogawa; Sumio Watanabe

    2012-01-01

    AIM:To elucidate the role of neuropilin-1 (Nrp-1) and semaphorin 3A (Sema3A) in sinusoidal remodeling during liver regeneration in rats.METHODS:Male Wistar/ST rats at 7 wk of age,weighing about 200 g,were used for all animal experiments.In vivo,at 24,48,72,96,144 and 192 h after twothirds partial hepatectomy (PHx),the remnant livers were removed.Liver tissues were immunohistochemically stained for Nrp-1,Sema3A and SE-1,a liver sinusoidal endothelial cell (SEC) marker.Total RNA of the liver tissue was extracted and reversely transcribed into cDNA.The mRNA expression of Sema3A was analyzed by quantitative real-time polymerase chain reaction and normalized to that of ribosomal protein S18.In vitro,SECs were isolated from rat liver and cultured in endothelial growth medium containing 20 ng/mL vascular endothelial cell growth factor.Migration of SECs in primary culture was assessed by cell transwell assay with or without recombinant Sema3A.Apoptotic cells were determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method.RESULTS:In vitro,immunohistochemistry study revealed that Sema3A and Nrp-1 were constitutively expressed in hepatocytes and SECs,respectively,in normal rat liver tissues.Nrp-1 expression in SECs was quantified by the percentage of immunostained area with antiNrp-1 antibody in relation to the area stained with SE-1.Between 24 h and 96 h following resection of liver,Nrp-1 expression in SECs was transiently increased.Compared with the baseline (5.2% ± 0.1%),Nrp-1 expression in SECs significantly increased at 24 h (17.3% ± 0.7%,P < 0.05),48 h (39.1% ± 0.6%,P < 0.01),72 h (46.9% ± 4.5%,P < 0.01) and 96 h (29.9% ± 3.8%,P < 0.01) after PHx,then returned to the basal level at termination of liver regeneration.Interestingly,the expression of Sema3A was inversely associated with that of Nrp-1 in liver after PHx.Sema3A mRNA expression was significantly reduced by about 75% over

  15. 131Ⅰ-酪氨酸-奥曲肽对荷人非小细胞肺癌小鼠的抑瘤效果%The experimental study on anti-tumor effect of 131Ⅰ-Tyr-octreotide in nude mice bearing human non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    苏燕; 王峰; 张乐乐; 郑玉民; 孟庆乐; 蒋娥; 李少华; 王自正

    2009-01-01

    was evaluated. The nude mice models bearing human NSCLC were studied and divided into four groups: group A injected 131Ⅰ-Tyr-octreotide through tail vein, group B injected normal saline, group C injected 131Ⅰ-Tyroctreotide through stroma and group D injected 131Ⅰ through stroma. The radioactivity ratio of tumor to normal tissue (T/NT) was calculated over region of interest (ROI). The tumor cell cycle and cell apoptosis were analyzed by flow cytometry (FCM), terminal deoxynucleotidyl transferase mediated dUTP-biotion nick end labeling (TUNEL) and histopathological analysis. Statistical analysis was performed with SPSS 11.0, and the comparison for difference between groups performed with one-way ANOVA analysis. Results The labeled radiochemical purity was (95.23±1.67)% and specific activity of 3.5×106Bq/ug. The biodistributiou showed high uptake in kidney, and low uptake in liver and spleen. The radioactive uptake in group C was higher than the other groups, and the retention time was longer. The T/NT was 52.74±0.13 after 24 h, which was much higher than that the other groups (group D: 8.90±0.23, group A: 6.42±0.02, q=628.81 and 664.33, all P<0.05). The resuits of tmnor cell cycle determined by FCM showed that the G1 phase was blocked mast remarkably in group C than the other groups [group C: (83.17±6.86)%, group A: (57.02±18.81)%, group D: (49.29±7.80)%, group B: (45.88±5.13)%, q=5.29, 6.86, 7.55, 1.56, 2.26, 0.69, all P<0.05]. Apeptotic cells were observed by TUNEL, and apoptotic body was detected by immuno-histochemical examination. Conclusions 131Ⅰ-Tyr-octreotide was easily labeled by Ch-T. 131Ⅰ-Tyr-octreotide could induce tumor cell apoptosis and inhibit the tumor cell of NSCLC. It might be a potential target-directed agent in NSCLC.

  16. 蛋白激酶B和丝裂原活化蛋白激酶在烟酸保护的HaCaT细胞抵抗中波紫外线损伤中的作用%Roles of protein kinase B and mitogen-activated protein kinase pathways in the protection by nicotinic acid against ultraviolet B-induced damage in keratinocytes

    Institute of Scientific and Technical Information of China (English)

    林福全; 许文; 关翠萍; 周妙妮; 洪为松; 刘东银; 许爱娥

    2012-01-01

    Objective To investigate the intracellular signal transduction pathways involved in the protective effect of nicotinic acid against ultraviolet B(UVB)-induced damage in human skin keratinocytes.Methods Cultured human keratinocyte HaCaT cells were divided into several groups to be treated with nicotinic acid,UVB irradiation,LY294002(an inhibitor of Akt),U0126(an inhibitor of extracellular signal-regulated kinase(ERK)1/2),SB203580(an inhibitor of P38)alone or in combination for different durations.Then,Western blot was performed to quantify the phosphorylation levels of the protein kinase B(Akt)/MAPK pathwayassociated proteins including Akt,P38,JNK and ERK1/2,MTT assay to evaluate the activity of HaCaT cells,enzyme-linked immunosorbent assay to determine the levels of endothelin-1(ET-1)and basic fibroblast growth factor(bFGF)in the culture supernatant of HaCaT cells,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)to evaluate the apoptosis in HaCaT cells.Results As Western blot showed,phosphorylated Akt,P38,JNK and ERK1/2 were markedly activated within 60 minutes after pretreatment with nicotinic acid and irradiation with UVB(all P < 0.01),and the activation was more significant for phosphorylated Akt,P38,and ERK1/2 within 2 hours(all P < 0.01).Nicotinic acid effectively suppressed the UVB-induced cell death and apoptosis in HaCaT cells.The levels of supernatant ET-1 and bFGF were significantly decreased in HaCaT cells treated with the above 3 inhibitors followed by UVB irradiation than in those treated with the inhibitors alone(all P < 0.05),and nicotinic acid pretreatment only reversed the decrease in supernatant bFGF in HaCaT cells treated with SB203580 followed by UVB irradiation.Conclusion The Akt signaling pathway may play a regulatory role in the protection by nicotinic acid against UVB-induced damage in HaCaT cells.%目的 探讨烟酸保护由UVB诱导的角质形成细胞损伤的胞内

  17. Study of Gandouling interves wilson disease cognitive dysfunction in rats and the nerve cells apotosis of hippocampus%肝豆灵干预铜负荷大鼠认知障碍及海马细胞凋亡的研究

    Institute of Scientific and Technical Information of China (English)

    王艳昕; 鲍远程; 孙敏; 杨文明; 韩辉; 方向; 张娟; 许珍晶

    2015-01-01

    Objective To observe Gandouling ( GDL ) interves wilson disease cognitive dysfunction in rats and the nerve cells apotosis of hippo-campus.Methods Wistar rats were randomly divided into 6 groups:normal group, model group, low, medium, high dose GDL groups and control group(penicillamine,PCA).Copper loaded rat model is replica-ted according to research literature.Use low,medium and high dosages of GDL and PCA to make intervention in the treatment of copper loaded rats.Cognitive function of copper loaded rats are detected by Morris wa-ter.The pathological changes in the hippocampus of copper loaded rats was observed and apoptosis of hippocampus was observed by using Termi-nal deoxynucleotidyl transferase -mediated dUTP nick -end labeling ( TUNEL) method.Results PCA group, three doses GDL groups have on average significantly reduced escape latency period ( P<0.01 ) than the model group.In comparison on the frequency of rats crossing the original position, GDL high dose group and the PCA group showed signif-icantly increased frequency ( P<0.01 ) than the model group.Compared with model group, GDL high dose and PCA groups all showed significant-ly shorter total swim distance ( P<0.01 ) .GDL medium, high groups and PCA group much smaller quantity compared with model group, apoptotic index( AI) difference was statistically significant ( P<0.01).Conclusion Copper load model shows decline of cognitive function, and GDL has an effect of improving cognitive function in copper loaded rats.%目的:观察肝豆灵( GDL)干预铜负荷大鼠认知障碍及海马细胞凋亡的研究。方法 Wistar大鼠按照体重随机分为正常组、模型组、对照组和3个剂量实验组,复制铜负荷大鼠模型后,实验组给予 GDL 0.48,0.96,1.92 g・ kg-1・ d-1,对照组给予青霉胺0.09 g・ kg-1・ d-1,每日1次灌胃;正常组及模型组给予等量生理盐水,共6周。 Morris水迷宫测试大鼠空间学习及记忆能力,观

  18. 脊髓损伤后Bcl-2抗神经元凋亡的研究%Study on the role of Bcl-2 in anti- neuronal apoptosis after spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    王瑛; 孙志扬; 张夔鸣; 许国强; 李光

    2010-01-01

    目的 通过研究Bcl-2(B-cell lymphoma/Leukemia-2)高表达转基因小鼠的脊髓损伤模型,探寻Bcl-2抗神经元凋亡,减轻脊髓继发性损害的作用.方法 将Bcl-2质粒转入小鼠的受精卵,培育Bcl-2转基因小鼠.转基因(A组)及同窝非转基因小鼠(B组)各9只随机(随机数字法)分为三组(1d,7 d,14 d),采用垂直打击脊髓(weight dropping,WD)方法,以2.5×3.0 g·cm致伤力致伤小鼠脊髓.另取3只非转基因小鼠设为假手术组(C组),仅打开椎板,暴露脊髓,不做打击.于术后1 d,7 d,14 d分别行:①小鼠后肢运动功能(BBB)评分;②HE染色观察;③原位末端标记法(TUNEL)染色.结果 打击后所有小鼠均出现双侧的后肢瘫痪,两周内BBB评分,A组(5.45±0.15)和B组(5.05±0.35),差异无统计学意义,P=0.67;HE染色提示A组小鼠的脊髓损伤程度较B组鼠要轻;原位末端标记法(TUNEL)染色表明A组小鼠TUNEL阳性的神经元(3.67±2.08/HP)明显小于B组小鼠(12±4/HP),P=0.033;积分光密度(Integration Optic Density,IOD)值(458.33±112.7)A组也明显小于B组小鼠(2436.33±228.01),P=0.000,其差异均具有统计学意义;C组未见明显的TUNEL阳性神经元.结论 Bcl-2蛋白是中枢神经系统内在的,重要的抗损伤因子,过表达Bcl-2能够抑制损伤后神经元的凋亡,从而减轻脊髓的损伤.%Objective To study the variables of behavioral function and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) figure in Bcl-2 transgenic (TG) mice and control mice after spinal cord injury (SCI), thus to find new ideas and ways for diagnosing and treating SCI. Method The genesis of Bcl-2 overexpression transgenic (TG) mice were produced by injection of Bcl-2 plasmid into the fertilized ova of mice.Nine Bcl-2 TG mice and nine control mice were subjected to SCI of moderate severity at T10, with the use of weight dropping (WD) method (impact force 2.5~3.0 g·cm). Up to 1 day , 7 days, and 14 days after SCI

  19. Effect of cholecystokinin-8 on in vitro cultured rat cortical neurons against apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ying Liu; Jiangbao Zhou

    2006-01-01

    BACKGROUND: Cholecystokinin (CCK-8) can regulate the synthesis of NO, release of amino acid substance and suppress Ca2+ inflow. It is unknown about neuroprotection of CCK-8 on neuronal apoptosis and its relationship with nerve growth factor (NGF).OBJECTryE: To investigate the protective effect of CCK-8 on in vitro cultured rat cortical neurons against apoptosis induced by glutamate, and explore its effect on expression of NGF in the neurons during apoptosis.DESIGN: Randomized controlled experiment on the basis of cells.SETTING: Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University.MATERIALS: Eighty SD rats of 1-day old; DMEM/F12 culture medium (Biochrom Company, Germany);Fetal bovine serum (TBD Company, Tianjin); CCK-8 (Sigma Company, USA). Glutamate (Bioengineering Company, Shanghai); TUNEL kit and NGF- in situ hybridization kit (Boster Bioengineering Company,Wuhan); anti-NGF polyclonal antibody (Santa-Cluz Company); NGF immunocytochemistry kit (Zhongshan Company, Beijing).METHODS: The experiments were carried out in Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University from December 2004 to September 2005. Primary cultured cortical neurons from SD rats of 1-day oldwere incubated for 7 days. The cultured cells were divided randomly into 3 groups:experimental group, model group and control group. Neurons in experimental groups were added CCK-8 of 1 ×10-6, 1 ×10-7, 1 ×10-8 μ mol/L respectively, and then added 50 μmol/L glutamate solution a hour later. Neurons in model groups were treated with 50 μ mol/L glutamate solution. In the control group, cells were treated with normal medium. Apoptosis of cultured cortical neurons were observed by fluorescent microscope, the expression of NGF protein and mRNA were determined respectively by immunocytochemistry and in situ hybridization, and apoptosis of cortical neurons was detected with terminal deoxynucleotidyl transferase-mediated nick

  20. Effect of valsartan and carnitine on cardiomyocyte Calpain-1 and Bcl-xl expressions of dogs with chronic alcohol intake-induced cardiomyopathy%钙激活中性蛋白酶Calpain-1在犬酒精性心肌病心肌细胞凋亡中的表达及不同药物对其的干预作用

    Institute of Scientific and Technical Information of China (English)

    桑颖; 周立君; 井玲; 原琳; 鲁立新; 张庆辉

    2011-01-01

    dogs with chronic alcohol intake-induced cardiomyopathy.Methods Dogs were randomly assigned into 4 groups(n=7 each):(1)alcohol fed(free access to 5%,1st week;10%2nd week;500 ml 25%bolus plus free access to 5%from 3 to 24 weeks,A);(2)alcohol+valsartan (5 mg·kg-1·d-1,B);(3)alcohol+carnitine(300 mg·kg-1·d-1,C);(4)Control(D).After six months,all animals were assessed for left ventricular(LV)function by echocardiography.The Bad and Bcl-xl protein expressions were evaluated by immunohistochemistry.The expression of Calpain-1 protein was determined with Western blot.Myocardial morphology was quantified on HE stained slices and under electron microscopy.The terminal deoxynucleotidyl transferase-mediated iotin-dUTP nick end-labeling(TUNEL)was performed for apoptosis analysis.Results Compared with group D.LVEDD and LVESD were significantly increased while EF and FS significantly decreased in group A.In alcohol fed group,expressions of Bad and Calpain-1 protein were significantly increased while Bcl-xl protein expression was downregulated,all changes could be significantly attenuated by intervention with valsartan and carnitine(all P<0.05).Conclusion These data suggest that alcohol could promote cardiac myocyte apoptosis,reduce cardiac function and aggravate myocardial remodeling which valsartan and carnitine could reduce alcoholic cardiomyopathy by downregulating Calpain-1 and Bad protein expression and upregulating expression of Bcl-xl protein.

  1. Enhancement of apoptosis by aspirin combined with sorafenib in a highly metastatic murine model of human hepatocellular carcinoma%阿司匹林抑制核因子-κB增强索拉非尼对肝癌的促凋亡作用

    Institute of Scientific and Technical Information of China (English)

    张伟; 孙惠川; 熊宇泉; 庄鹏远; 朱小东; 汤钊猷

    2011-01-01

    Objective To investigate the effect of combination of sorafenib and aspirin on tumor apoptosis,tumor growth,lung metastasis and survival of tumor-beating nude mice in a highly metastatic xenograft murine model of human hepatocellular carcinoma (HCC) and explore the mechanism.Methods The Xenograft of a highly metastatic human HCC tumor (LCI-D20) was used to evaluate survival,primary tumor growth and lung metastasis after treatment with aspirin alone or in combination with sorafenib.Apoptosis of tumor cells in vivo was determined by the terminal deoxynucleotidyl-transferase mediated dUTP nick-end labeling assay (TUNEL).Results Tumor volumes were (4.76 ±0.51 ),( 1.41 ±0.08),(0.66±0.12) and (4.58 ±0.47) cm3 in control,serafenib,serafenib plus aspirin,and aspirin groups,respectively.Lung metastases were 189 ±21,96 ± 15,30 ± 17 and 92 ± 18 in four groups,and median survival was 72,98,112 and 80 days,respectively.Apoptosis rate was (2.6 ± 1.1 )%,(8.6 ±3.7)%,(24.3±6.9)% and (6.8 ± 1.5)% respectively in four groups.Combination of aspirin and sorafenib significantly decreased tumor volume ( P < 0.05 ),inhibited number of lung metastases ( P < 0.01 ),and prolonged survival (P < 0.05 ) as compared with sorafenib alone group.Increased apoptosis of tumor cells (P <0.05 ) was observed by combination of aspirin and sorafenib.Nuclear factor-κB (NF-κB) was upregulated and IκBα was downregulated by sorafenib,which was reversed by combination of aspirin with sorafenib.Conclusion Aspirin significantly promoted apoptosis of tumor cells in combination with sorafenib through inhibiting sorafenib-induced NF-κB activation.%目的 观察合用阿司匹林(Aspirin)与索拉非尼(Sorafenib)对肝癌生长转移的抑制作用和促凋亡作用并探讨其机制.方法 采用人肝癌裸鼠原位模型LCI-D20,分为对照组、Sorafenib 单用组(30 mg/kg灌胃,1d1次)、Sorafenib+ Aspirin合用组、Aspirin单用组(30 mg/kg灌胃,1 d 1 次),治疗4周后观

  2. 右美托咪定对晶状体摘出术后小鼠认知功能的作用%Effect of Dexmedetomidine on cognitive function after lens extraction in mice

    Institute of Scientific and Technical Information of China (English)

    杨丽华; 郝尚臣; 徐玉灿; 李志松; 张卫

    2015-01-01

    Objective To investigate the effect of Dexmedetomidine (Dex) on long-term cognitive function after extracapsular lens extraction (ECLE) in mice.Methods Forty adult male C57BL/6J mice were randomly divided into control group (C group),ECLE group,normal saline group (NS group) and Dex group with 10 mice in each group.Three days after ECLE model was made,the mouse in C group and ECLE group underwent Morris water maze test,then they were euthanized and hippocampus tissue was immediately excised for determination of wet weight to dry weight (W/D) and total water content (TWC).The transcription levels of cycteinyl aspirate-specific protease-12 (caspase-12) mRNA and its protein in hippocampus tissue were detected respectively by reverse transcription polymerase chain reaction (PCR) and Western Blot.Apoptosis index (AI) of hippocampus tissue was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay.After the mice regained consciousness post the operation,animals received intraperitoneally Dex at a dose of 25 μg · kg-1 · d-1 for 30 consecutive days in Dex group.Animals of NS group were accepted intraperitoneally injection with the same volume of normal saline.The evaluation indexes mentioned above were programmed at 30 dyas post intraperitonedlly injection.Results Compared with C group,the escape latency and swimming distance were prolonged transcription in ECLE group (both P < 0.05),the value of W/D,TWC and AI were increased (all P < 0.05),and the expression of caspase-12 and mRNA transcription in hippocampus tissue were significantly higher (both P < 0.05) in ECLE group.Compared with NS group,the escape latency and swimming distance were shortened in Dex group(both P < 0.05),the value of W/D,TWC and AI were decreased (all P < 0.05),and the expression of caspase-12 and mRNA protein transcription in hippocampus tissue were significantly lower (both P < 0.05) in Dex group.Conclusion The treatment of Dex could improve the

  3. Changes in neuronal apoptosis and apoptosis modulatory factors in cerebral ischemia/reperfusion

    Institute of Scientific and Technical Information of China (English)

    Liying Qiu; Ying Li; Hongbin Fan; Bin Du; Zhiyong Yang; Jianqing Cheng

    2007-01-01

    terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling method. The expressions of Bcl-2 protein were determined with immunohistochemical staining. The activity of calcineurin was determined by the inorganic phosphorus method. The content of excitatory amino acid was detected by high performance liquid chromatography.MAIN OUTCOME MEASURES:①Glutanate content in brain tissue;②Conditions of apoptosis;③ Calcineurin activity in brain tissue;④Bcl-2 expression in brain tissue.RESULTS: Totally 30 SD rats were used, 5 died and the other 25 were involved in the analysis of results.①Changes of apoptosis: There were 0 - 3 apoptotic cells in the sham-operated group. In the model group, the numbers of apoptotic cells were obviously increased at 24 and 72 hours of reperfusion (P<0.01), and it was markedly reduced at 72 hours as compared with 24 hours (P<0.01).② Changes of glutanate content: The glutamate contents at 24 and 72 hours of reperfusion in the model group were obviously higher than those in the sham-operated group (P<0.01); In the model group, it was obviously increased at 24 hours as compared with 72 hours (P<0.01).③Changes of Bcl-2 protein: In the model group, the Bcl-2 protein expression had no obvious changes at 24 hours of reperfusion, and it was obviously enhanced at 72 hours, which was obviously different from that in the sham-operated group and that at 24 hours (P<0.01).④Changes of calcinerin activity: In the model group, the activity of calcineurin in brain tissue had no obvious changes at 24 hours of reperfusion; The activity of calcineurin at 72 hours was obviously higher than that in the sham-operated group and that at 24 hours (P<0.01).CONCLUSION: The brain cyto-apoptosis action at different time points following reperfusion incompletely depends on the glutamate levels, while it depends on the interaction of some apoptosis related factors, such as amino acid, calcineurin, and Bcl-2, etc.

  4. Expression of MicroRNA-1,21 in Ischemic Preconditioning,Ischemic Postconditioning and Remote Ischemic Preconditioning in an Isolated Rat Heart Model%MicroRNA-1和microRNA-21在缺血预处理、后处理及远端预处理中的表达变化

    Institute of Scientific and Technical Information of China (English)

    段欣; 王小华; 吉冰洋; 刘晋萍; 龙村

    2012-01-01

    hearts were randomly assigned into one of the four groups, control group (CON group, n=\\2), ischemia preconditioning group (IPC group, n=12), ischemia post-conditioning group (IPO group, n= 12) and remote ischemia preconditioning group (RIPC group, n= 12). Cardiac function was digitalized and analyzed. The expression of HSP70, PDCD4, B-cell lymphoma/leukemia-2 (Bcl-2) and Bax was detected by Western blotting. The expression of miRNA-1 and miRNA-21 was detected by real-time reverse transcriotion-polymerase chain reaction (RT-PCR). Assessment of cardiac infarct size and myocardial apoptosis was determined using triphenyltetrazolium chloride (TTC) assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) assay respectively. Results The expressions of miRNA-1 and miRNA-21 were up-regulated in IPC group, but the expression of miRNA-1 was down-regulated in RIPC group and IPO group (P < 0. 05). The expres-sionsof PDCD4, HSP70 and Bax were down-regulated in 'conditioning' groups compared with CON group (P < 0. 05). The expression of BcI-2 was not statistically different among the four groups. The infarct size and the myocardial apoptosis in 'conditioning' hearts were significantly decreased compared with CON group (P < 0. 05). Conclusion The expressions of the miRNA-1 and miRNA-21 are different in IPC, RIPC and IPO groups, and their target proteins are not inversely correlated with the miRNAs in all the 'conditioning' groups.

  5. Effects on survival of shRNA mediated APE/Ref1 gene silencing in rat spiral ganglion cells in oxidative stress%shRNA沉默APE/Ref1对体外培养大鼠耳蜗螺旋神经节细胞过氧化氢损伤的影响

    Institute of Scientific and Technical Information of China (English)

    姜振东; 钟诚; 李太军; 向召兰; 张学渊

    2014-01-01

    目的 通过转染pGenesil-APE/Ref1-shRNA抑制体外培养大鼠耳蜗螺旋神经节细胞(spiral ganglion cell,SGC)中无嘌呤无嘧啶核酸内切酶/氧化还原因子1(apurinic/apyrimidimic endonuclase/redox factor 1,APE/Ref1)的表达,观察SGC在过氧化氢(H2O2)所致氧化应激环境中的活性及凋亡情况,探讨APE/Ref1在SGC抗氧化损伤过程中的作用.方法 体外培养大鼠SGC,加入pGenesil-APE/Ref1-shRNA转染72 h,更换培养基后加入不同浓度的H2O2 (0、10、25、50、100、300μmol/L)干预1h,更换正常培养基后继续培养24h.通过免疫印迹、分光光度检测法、原位缺口末端标记法(TUNEL)、四甲基偶氮唑蓝法(MTT)分别检测SGC APE/Refl、磷酸化组蛋白H2AX的表达、caspase3活性、细胞活性以及凋亡情况.结果 pGenesil-APE/Ref1-shRNA能明显抑制APE/Ref1的表达,H2O2损伤浓度在50 ~ 300 μmol/L时,与对照组相比,pGenesil-APE/Ref1-shRNA组磷酸化组蛋白H2AX表达增加,caspase 3活性增加,细胞凋亡率增高,细胞活性降低,差异具有统计学意义(P值均<0.05).结论 pGenesil-APE/Ref1-shRNA抑制APE/Refl表达后,大鼠体外培养SGC抗氧化损伤能力降低;APE/Ref1可能通过DNA修复功能,实现对SGC的保护作用.%Objective To investigate the effects of reducing APE/Refl expression in the cultures of rat spiral ganglion cells with oxidative damage induced by H2O2.Methods Primary cultured rat spiral ganglion cells were infected with small interfering RNA to APE/Refl (Apel siRNA) for 72 h,followed by treating with H2O2(0,10,25,50,100 and 300 μmol/L) for 1 h,and then cultured in normal medium for 24 h.Western blot were used to detect the level of APE/Refl protein and phosphorylation of histone protein H2AX in the infected cells.The caspase3 activation was tested by spectrophotometric method.The cell viability was determined by MTT and the apoptosis of spiral ganglion cells was determined by terminal-deoxynucleotidyl transferase mediated nick

  6. 纳秒脉冲治疗裸鼠皮下移植瘤抗癌机制研究%Anti-cancer mechanism of nanosecond Pulsed Electric Fields on xenografts in nude mice

    Institute of Scientific and Technical Information of China (English)

    彭巧; 赵雪; 姚陈果; 唐均英

    2013-01-01

    To investigate the anticancer effect of nanosecond Pulsed Electric Fields (nsPEF) on human melanoma A375 cell xenograft in nude mice. Tumor size was detected by vernier caliper. The cell apoptosis was detected by DNA agarose gel eleetrophoresis and termina I deoxynucleotidyl transferase- mediated dUTP nick end - labeling (TUNEL) assay;Tumor morphology was observed with HE staining; The expression of microvessel density (MVD) 、 vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) were detected by SP immunohistochemistry. Results showed that tumor growth inhibition rate was 42.6% at 4d after nsPEF treatment. DNA ladder were observed more typically in 4d group than other two groups; The apoptosis rate of tumor cells in 4d group was the most highest than other group detected by TUNEL. Tumor turned grey immediately after treatment; A few of necrosis cells were found in 2h group, while numerous necrosis were found in 4d group; The slate of blocking of capillary vessel was found significantly in 4d group. The expressions of MVD, VEGF and PCNA were significantly lower in 4d group than the other two groups, while no significant difference were found between 2h group and control group. The result indicated that the inhibitory effects of nsPEF may be related to inducing cell apoptosis, reducing blood flow in a short time, decreasing the angiogenesis and discouraging tumor revascularization and tumor cell' s proliferative activity.%本研究探讨了纳秒级陡脉冲(nsPEF)对人恶性黑色素瘤A375细胞裸鼠皮下移植瘤模型可能的抗癌机制.通过用游标卡尺测量裸鼠皮下瘤大小,估算抑瘤率.用琼脂糖凝胶电泳、TUNEL法检测肿瘤细胞在纳秒脉冲电场下的细胞凋亡情况,并用HE染色观察细胞形态变化.通过免疫组化法检测瘤体内微血管密度MVD、微血管形态、血管生长因子VEGF和增殖细胞核抗原PCNA的表达.结果发现nsPEF治疗后4d肿瘤生长抑制率达42

  7. Preliminary study of skeletal muscle apoptosis after severe thermal injury in rats%严重烧伤大鼠骨骼肌细胞凋亡的初步研究

    Institute of Scientific and Technical Information of China (English)

    段红杰; 柴家科; 姚咏明; 尹会男; 申传安; 吴焱秋; 蔺静; 梁黎明

    2009-01-01

    目的 探讨严重烧伤后大鼠骨骼肌细胞凋亡状况,以了解其对骨骼肌萎缩的可能作用机制.方法 100只雄性Wistar大鼠被随机分为假伤组和烫伤组;各组又分为伤后1、4、7、10、14 d 5个时间点,每个时间点10只.制备大鼠40%总体表面积m度烫伤模型,于伤后各时间点称体重和胫骨前肌重量.取后肢胫骨前肌,用透射电镜观察骨骼肌细胞凋亡的超微结构变化,用原位末端缺刻标记法(TUNEL)及免疫组化双标检测骨骼肌组织凋亡细胞核.结果 与假伤组比较,严重烫伤后大鼠体重及胫骨前肌重量从伤后1 d起即开始下降,4 d时达到最低(P<0.05和P<0.01),7 d后体重及胫骨前肌莺量开始缓慢上升,但差异仍有统计学意义(P均<0.01).电镜下观察烫伤组骨骼肌可见少部分细胞核核膜皱缩,染色质边集、断裂,核周区域肌丝降解,部分近核胞膜呈泡状突起,而同一骨骼肌细胞的其他核可显示为正常结构.TUNEL检测也发现烫伤组骨骼肌有散在的阳性细胞核,而假伤组骨骼肌细胞核则未见凋亡改变.结论 严重烧伤大鼠骨骼肌细胞存在凋亡现象,这可能是骨骼肌萎缩的原因之一.%Objective To investigate changes in skeletal muscle apoptosis after a severe thermal injury in rats.Methods One hundred Wistar rats were randomly divided into two groups:sham thermal injury group and severe thermal injury group.They were subdivided into 1,4,7,10,14 days post-injury with 10 rats in each subgroup.Rats in severe thermal injury group were subjected to a 40%total body surface area full-thickness scald injury.Both weight and tibialis anterior(TA)mass of rats were weighed on 1,4,7,10, 14 days post-injury.Electron microscope was used for observing ultrastructural changes in skeletal muscle, including apoptosis.Tissues of tibialis anterior from burn and sham burn animals were then examined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

  8. 吗啡成瘾时脑细胞凋亡数目增加%Neuron apoptosis increases in morphine-dependent rats

    Institute of Scientific and Technical Information of China (English)

    刘立伟; 王新华; 傅强; 吴青华; 傅舒昆

    2012-01-01

    deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNED method. Immunohistochemistry was used to detect the expression of Caspase-3 protein. Results The score of abstinent symptom in morphine-dependent group is not statistically different from control group (P>0. 05), while it was significantly lower than that in morphine-abstinent group (P0.05). Conclusion Chronic morphine can promote abnormal neuronal apoptosis in rat brain, which might be related to opiate-induced nerve damage.

  9. The effects of hydrogen-rich saline on renal ischemia/reperfusion injury in mice%静脉注射含饱和氢气生理盐水减轻小鼠肾脏缺血再灌注损伤

    Institute of Scientific and Technical Information of China (English)

    宋少华; 沈筱芸; 傅志仁; 刘芳; 郭闻渊; 傅宏; 王正昕; 丁国善

    2010-01-01

    Objective To evaluate the protective effect of hydrogen-rich saline on renal ischemia/reperfusion (I/R) in mice. Methods Thirty C57BL/6 mice were randomly divided into 3 groups: sham-operated (SO) group, I/R group (mice were injected with 5 ml/kg saline by tail vein just before ischemia induction) and hydrogen-rich saline group (mice were injected with 5 ml/kg hydrogen-rich saline). At the 6th h after reperfusion, the sera and renal samples subject to IR injury were collected. The Scr and BUN levels in serum were determined and renal histological changes were also examined. The apoptosis of renal tubular epithelial cells was measured by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay. Malondialdehyde (MDA) contents in renal samples were measured using specific kits. The infiltration of F4/80 positive macrophages and neutrophils was assayed by using immunohistochemistry. The mRNA expression of TNF-α, IL-6, IL-1β and IL-17 was detected by using real time reverse transcription PCR. Results As compared with LR group, at the 6th h following reperfusion the levels of Scr and BUN were significantly reduced (P<0.05), histological changes obviously alleviated (P<0.01), apoptosis of renal tubular epithelial cells and MDA contents was decreased (P<0.05) in hydrogen-rich saline group. Moreover, the infiltration of macrophages and neutrophils, and the mRNA expression of TNF-α, IL-6, IL-1β and IL-17 in renal tissue in hydrogen-rich saline group were also declined as compared with IR group (P<0.05). Conclusion Hydrogen-rich saline can ameliorate renal IR injury to some extent, which is associated with inhibition of inflammatory response induced by reperfusion.%目的 研究静脉注射含饱和氢气生理盐水对小鼠肾脏缺血再灌注(IR)损伤的保护作用及其机制.方法 健康、雄性的C57BL/6小鼠随机分为3组,每组10只.假手术组(SO组)小鼠仅接受中线开腹、双侧肾蒂游离及关腹操作;缺血再灌注组(IR

  10. 青藤碱减轻小鼠肾脏缺血再灌注损伤%Protective effects of sinomenine on renal ischemia/reperfusion injury in mice

    Institute of Scientific and Technical Information of China (English)

    王博; 徐达; 王锡智; 王祥慧; 周佩军; 邵琨; 舒心雨; 罗斐埜

    2011-01-01

    equal volume of saline). Six and 24 hs later, the serum was collected and the contents of alanine aminotransferase (ALT) and creatinine (SCr) were determined. In the experiment two, C57BL/6 mice were randomly divided into 3 groups: sham-operated (SO) group, SIN group (mice were injected with 200 mg/kg sinomenine just before ischemia induction) and saline group (mice were injected with equal volume of saline at the same time). At the 6th h after reperfusion, the sera and renal samples subject to IR injury were collected. The SCr and BUN levels in serum were determined and renal histological changes were also examined. The apoptosis of renal tubular epithelial cells was measured by using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay. The infiltration of F4/80 positive macrophages was measured by using immunohistochemistry and that of neutrophils with myeloperoxidase (MPO) kits. The mRNA expression of tumor necrosis factor (TNF)-α, chemokine CXC ligand (CXCL)-10, intercellular adhesion molecule (ICAM)-1 and IL-17 was detected by using real-time reverse transcription PCR. The activation of transcription factor NF-κB was measured by using Western blotting. Results In the experiment one, there was no significant difference in ALT and SCr between the two groups at 6 or 24 h. In the experiment two,levels of SCr and BUN were lower in SIN group (P<0. 05 or P<0. 01 ), histological damage was milder (P<0. 01 ), and apoptosis rate of renal tubular epithelial cells apoptosis was lower than in saline group (P<0. 05). The infiltration of macrophages, neutrophils and the mRNA expression of TNF-α, CXCL-10, ICAM-1 and IL-17 in the renal tissue in SIN group were reduced as compared with saline group (P<0. 05 or P<0. 01 ). The activation of NF-κB in SIN group was significantly downregulated as compared with saline group. Conclusion SIN can ameliorate the renal IR injury without hepatic or renal toxicity, which is associated with inhibition of

  11. The intervention effect of Schisandrin B on cisplatin induced acute kidney injury in mice%五味子乙素对顺铂所致小鼠急性肾损伤的干预作用

    Institute of Scientific and Technical Information of China (English)

    吴银娜; 谭小月; 张勉之

    2015-01-01

    Objective To observe the intervention effect of Schisandrin B (Sch B) on cisplatin induced acute kidney injury (AKI) in mice and its possible mechanism.Methods Twenty-five BALB/c mice were randomly divided into blank control group, model group, low and high dose of Sch B intervention groups and Sch B control group. Olive oil with Sch B was administered by gavage at the dose of 20 mg/kg or 100 mg/kg for low and high dose of Sch B intervention groups respectively; olive oil with Sch B 100 mg/kg was applied by gavage to the Sch B control group; the same volume of olive oil was perfused into the gastric cavity in the blank control group and model group; the above measures in various groups were consecutively used for 5 days. On the 3rd day of the experiment, AKI mice model was established by intraperitoneal injection of cisplatin (20 mg/kg) once and the same measure was given to the low and high dose of Sch B intervention groups; 1 mL/kg normal saline was injected into the peritoneal cavity in the bland control group and Sch B control group. At the end of the experiment, the serum creatinine (SCr) level was determined; apoptosis of renal tubular epithelial cells were detected by using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay; the morphological changes of renal tubular epithelial cells were observed by hematoxylin eosin (HE) staining, and renal tubular injury score was evaluated; p53 protein content in the kidney tissue was measured by immunohistochemical analysis; furthermore, expressional level of p53 protein in renal tissue was tested by Western Blot.Results Compared with the blank control group, the level of SCr (μmol/L: 86.77±10.97 vs. 14.37±0.81), renal tubular injury score (9.67±1.20 vs. 1.00±0.45), the count of apoptotic renal tubular epithelial cells (cells/200 power field: 20.00±2.13 vs. 2.30±0.40) in the model group were all increased (P < 0.05 orP < 0.01), and p53 protein content (cells/400 power field: 13

  12. 吡格列酮对高脂血症大鼠主动脉内皮细胞凋亡的作用%Effect of pioglitazone on aortic endothelial cell apoptosis in hyperlipidemic rats

    Institute of Scientific and Technical Information of China (English)

    李蓉; 董晓蕾; 赵凌杰; 蔡辉; 袁爱红

    2012-01-01

    目的 观察吡格列酮对高脂血症大鼠主动脉内皮细胞凋亡的影响,并探讨其可能的作用机制.方法 清洁型SD大鼠26只,随机分为健康对照组(9只)、高脂饮食组(17只),高脂饮食组喂养12周后再随机分为模型组(8只)和吡格列酮组(9只),4周后,检测各组血脂水平,通过免疫组织化学法检测主动脉Bcl-2、Bax的表达,TUNEL染色法观察主动脉内皮细胞凋亡情况,计算细胞凋亡指数.结果 (1)高脂饮食组喂养12周后,血脂明显升高;给药4周后,与模型组比较,吡格列酮组TG、TC水平明显降低(P =0.000);(2)与健康对照组相比,模型组主动脉Bax蛋白表达明显增高(P=0.003),Bcl-2蛋白表达和Bcl-2/Bax比值明显降低(P=0.000);与模型组相比,吡格列酮组主动脉Bax蛋白表达明显降低(P=0.000),Bcl-2蛋白表达(P=0.001)和Bcl-2/Bax比值明显升高(P=0.000),且呲格列酮组主动脉内皮细胞凋亡指数较模型组明显降低,差异有统计学意义(17.5633±7.0584比6.0475±2.2370,P=0.000).结论 吡格列酮可改善高脂血症大鼠血脂水平,调节凋亡蛋白表达,减少主动脉内皮细胞凋亡.%Objective To investigate the influence of pioglitazone on aortic endothelial cell apoptosis in hyperlipidemia rats and its possible mechanism. Methods A total of 26 male SD rats were randomly divided into control group (n = 9) and high-fat diet group ( n = 17). Rats at high-fat diet group raised for 12 weeks were randomly devided into model group (rc=8) and pioglitazone treatment group (n = 9). After4 weeks,serum lipid level of all rats was measured and apoptosis protein expression of Bax and Bcl-2 on the aorta was analyzed by immunohistochemical method. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to observe apoptotic cells in aortic intimal area and to calculate apoptosis index Results (1) Serum levels of TC and TC were significantly higher in model group and pioglitazone treatment group

  13. L-carnitine prevents gentamicin-induced impairment of hair cell in newborn guinea pigs%L-肉碱对庆大霉素致子代豚鼠毛细胞损伤干预作用

    Institute of Scientific and Technical Information of China (English)

    郝帅; 田颖; 姜菲菲; 姜学钧

    2012-01-01

    Objective To evaluate the role of L-camitine(LCAR) supplementation in hearing threshold and cochlear damage in newborn guinea pigs exposed to gentamicin in utero. Methods Pregnant albino guinea pigs were intraperitoneally injected with 100 mg/kg body weight gentamicin once a day for 7 consecutive days,or gentamicin and LCAR at gestational day 51 to 57. At postnatal day 14,the pups were examined in the distortion product otoacoustic emission( DPOAE) task. The temporal bones were immunohistochemically stained for caspase-3, using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) method. Results Gentamicin used during pregnancy decreased the DPOAE threshold shifts at different frequencies of 1,2,4,5,and 8 kHz(8. 38 ±2. 28,12. 56 ±3.22,10.46 ± 1. 98,12. 22 ± 1.72,and 17.73 ±2. 16 dB,respectively). In contrast,LCAR supplementation,coincidentally with gentamicin exposure,ameliorated these changes( 14. 26 ±1.47,20. 98 ±2. 39,19. 23 ±2. 15,22.56 ± 1. 41,and 29. 13 ±3. 14 dB,respectively). In addition, gentamicin used during pregnancy increased the percentage of apoptotic cells in hair cells( 34.6% ), but LCAR supplementation decreased the percentage of apoptotic cells( 15.1% ,P <0. 05). Conclusion Our data offer a conceptual framework for designing clinical trials using a safe nutrient,LCAR,as a simple preventive strategy for gentamicin-induced ototoxicity.%目的 观察围产期L-肉碱对庆大霉素暴露致子代豚鼠听力损伤及耳蜗毛细胞凋亡的影响.方法 将18只孕中期豚鼠按体重随机分为3组,从孕51 ~57 d,对照组腹膜内注射生理盐水100 mg/kg、庆大霉素组腹膜内注射庆大霉素100 mg/kg,L-肉碱组腹膜内注射庆大霉素100 mg/kg同时以饮水方式给予L-肉碱100 mg/mL;出生后14 d,检测子代豚鼠畸变产物耳声发射(distortion product otoacoustic emission,DPOAE)改变,应用原位末端转移酶标记技术(TUNEL)染色和免疫组化检测

  14. Effects of cobalt chloride on the expressions of stromal cell derived factor 1 and chemokine receptor 4 in cerebral ischemia rats%氯化钴对大鼠脑缺血模型基质细胞衍生因子-1及趋化因子受体4表达的影响

    Institute of Scientific and Technical Information of China (English)

    李佳; 邹常林; 张志明; 张春阳

    2016-01-01

    目的:研究氯化钴预处理对脑缺血大鼠基质细胞衍生因子( SDF-1)、CXC家族趋化因子受体4(CXCR4)蛋白表达的影响。方法45只SD大鼠,随机分为假手术组、模型组、实验组,每组均15只。模型组及实验组,用线栓法制作脑缺血模型;在造模前2天与前1天,实验组腹腔注射氯化钴30 mg · kg-1。用蛋白质印迹法检测各组大鼠脑组织SDF-1、CXCR4蛋白表达,原位末端标记法( TUNEL)检测各组大鼠脑细胞凋亡率,用氯化三苯基四氮唑( TTC)染色并计算2d后脑组织缺血坏死面积。结果在各时间点,与假手术组(脑组织SDF-1、CXCR4蛋白表达极低)比较,实验组与模型组SDF-1、CXCR4蛋白表达均明显增高( P<0.05),提示氯化钴可上调SDF-1、CXCR4的表达;2 d后,实验组的脑细胞凋亡率明显低于模型组[(9.31±0.79)% vs (18.99±1.13)%, P<0.05];实验组脑组织梗死灶面积也明显低于模型组( P<0.05)。结论氯化钴可上调SDF-1、CXCR4蛋白表达,减少脑细胞凋亡率,缩小脑细胞缺血坏死灶面积,对缺血脑组织具有保护作用。%Objective To observe the effect of cobalt chloride precondi-tioning on the expression of stromal cell derived factor ( SDF -1 ) and CXC family chemokine receptor 4 ( CXCR4) protein in rats with cerebral ischemia .Methods The 45 SD rats were randomly divided into sham group with 15 rats, model group with 15 rats, and test group with 15 rats.For model group and test group , cerebral ischemia model was estab-lished with suture emboli method.The rats in test group before modeling 2 d, 1 d were given intraperitoneal injection of cobalt chloride ( 30 mg· kg -1 ).Western blotting was used to detect the expression of SDF -1, CXCR4 protein.Apoptosis rate of rat brain cells in each group was tested by Terminal deoxynucleotidyl transferase -mediated dUTP-biotin nick end labeling assay ( TUNEL

  15. 围手术期营养支持对结直肠癌患者细胞周期蛋白D1表达及复发转移的影响%Effects of perioperative total parenteral nutrition support on cyclin D1 expression, recurrence and metastasis of colorectal cancer cells

    Institute of Scientific and Technical Information of China (English)

    刘彦; 陶凯雄; 王国斌

    2010-01-01

    Objective To evaluate the effects of perioperative total parenteral nutrition on cyclin D1, recurrence and metastasis of colorectal cancer cells. Methods A total of 120 patients with colorectal carcinoma were randomly divided into two groups, namely group A (total parenteral nutrition,TPN,60 cases) and group B (non total parenteral nutrition, NTPN, 60 cases). In group A, the patients were given with TPN (including glucose, intralipid, amino acid, and vitamins, etc.) for 10 days perioperation (7 days preoperatively and 3 days postoperatively). In group B, the patients did not receive any nutrition support perioperative nutrition support. The samples were obtained by colonoscopy preoperatively or during operation. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique,expression of proliferating cell nuclear antigen (PCNA) by immunohistochemical staining, and the expression of cyclin D1 by in situ hybridization. The apoptotic index (AI), the proliferating index (PI), and the expression of cyclin D1 were calculated perioperatively and postoperatively. Results After perioperative nutrition support, the expression rates of cyclin D1, PI and AI in group A and group B were (35.23±5.12)% and (37.53±5.31)%, (7.21±2.56)% and (8.75±3.84)%, (53.45±7.74)% and (56.74±8.02)% respectively. There were no significant difference of PI, AI and the expression of cyclin D1 (all P>0.05) between two groups. The 3-year recurrent rates in two groups were 16.7% and 15.0%(P>0.05). Conclusion Perioperative TPN can not promote proliferation and apoptosis of carcinoma cells, and has no significant impact on the expression of cyclin D1, recurrence or metastasis of colorectal cancer.%目的 探讨围手术期营养支持对结直肠癌患者肿瘤细胞cyclin D1表达及复发转移的影响.方法 将120例结直肠癌患者按其是否行营养支持分为营养支持组(60例)和对照组(60例).围手术期营养支持方案:葡

  16. Cell apoptosis in perihematomal brain regions and expression of Caspase-3 protein in patients with hypertensive intracerebral hemorrhage

    Institute of Scientific and Technical Information of China (English)

    Xinqing Zhang; Xiaoliang Yin; Kun Zhang; Zhimin Zhang; Hui Cai; Honglan Xu

    2006-01-01

    hours) and. 5 cases in delayed phase(onset > 24 hours). The brain tissue obtained from distant regions of hematoma was preserved for control group (5 cases). ②All the specimens were subjected to terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) staining and Caspase-3 immunohistochemical staining. The change rule of positive cells was observed with image analyzer, and the relationship between apoptotic cells and Caspase-3 positive cells was analyzed. ③ Analysis of variance and t test were used for comprising the difference of measurement data, and liner correlation analysis was performed. MAIN OUTCOME MEASURES: ①Changes of cell apoptosis and Caspase-3 expression in brain tissue of perihematomal region of HICH patents in different phases. ② Correlation of cell apoptosis and Caspase-3 expression.RESULTS: Cell apoptosis: TUNEL positive cells were hardly or occasionally found in each visual field in the control group, while the number of apoptotic cells in 3 different phases in the patient group was all higher than that of control group, and considerable TUNEL positive cells were found in the super-early stage; The number of TUNEL positive cells reached peak at 8 to 24 hours in early phase, and then declined ndelayed phase, but was still higher than that in the super-early phase(F=92.34, P < 0.01 ). ②Caspase-3 protein expression: Caspase-3 positive cells were hardly seen in each visual field in the control group; Considerable Caspase-3 positive cells were found in the super-early group; The Caspase-3 positive cells were significantly increased and reached the peak in the early-phase, and then declined almost to the same level in delayed phase compared with the super-early phase (P > 0.05). There was no significant difference in the number of Caspase-3 positive cells between super-early phase group and delayed phase group, but significant difference among the other groups (F =143.23, P < 0.01).

  17. 七氟烷对急性肺损伤大鼠肺组织细胞凋亡的影响及其机制%The mechanism of sevoflurane on the apoptosis of pulmonary cells in rats with acute lung injury induced by lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    王乐; 赵建华; 栾恒飞; 方志源; 曾因明

    2011-01-01

    after instillation of LPS, histological examination by lung water contant (wet/dry ,W/D) calculation and light microcopy was performed. Apoptosis was determined by flow cytometry (FCM),TUNEL test (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and electron microscope. At the same time, Fas, Bcl-2 protein expression were studied by using immunocytochemistry and western blot techniques in all groups mentioned above. Results The change of the levels of W/D(6.74±0.26), AI(48.1±1.9,46.6±1.2), Fas(0.223±0.034), Bcl-2(28.5±0.5) of the LPS group was significant compared with the control group (11.2±2.3,8.6±0.5,4.35±0.37,0.091±0.013,75.6±2.7), and lung injury was more severe. The levels of W/D(5.37±0.23), AI(27.2±0.9,26.3±2.4), Fas of the sevofluran group(0.131±0.21 ) were decreased, while,Bcl-2 expression (81.2±5.2) were increased compared with the LPS group (0.223±0.034,28.5±0.5)(P<0.01), and lung injury was attenuated. Conclusion Sevoflurane inhalation protects lung from injury by inhibiting excessive cell apoptosis, Fas expression and up-regulating Bcl-2 expression, which may play an important role in the pathogenesis of LPS-induced ALI.

  18. Induction of injury to endothelium of pulmonary artery due to entero-superantigen by up-regulation of lymphocyte chemokine receptor 5%T淋巴细胞趋化因子受体5表达上调介导了肠毒素超抗原对肺动脉内皮的损伤

    Institute of Scientific and Technical Information of China (English)

    吴礼襄; 萧正伦; 孔天翰

    2011-01-01

    pulmonary artery endothelial cell (HPAEC) and explore its possible mechanism. Methods HPAEC was cocultured with SEB-activated T cells supernatant, and the secretion of chemotactic factors from HPAEC was examined. The Transwell inserts was used in chemoattraction assays. After HPAECs were cocultured with T cells and 10 ng/ml SEB for 3 days, HPAEC damage was monitored by microscopy and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Results Three kinds of tested chemokines showed a time-dependent increase in all supernatant of HPAEC incubated with different concentrations of T cells. After 72 hours, the monocyte chemoattractant protein-1 (MCP-1,ng/ml) in 1 × 10-2, 1 × 10-1 , 1× 100 T cell supernatant groups was 1. 240± 0. 103, 4. 200± 0. 305, 6. 500±0. 500, respectively, macrophage inflammatory protein-1a (MIP-1α, ng/ml) was 0. 210 ± 0. 015, 0. 287 ±0. 012, 0. 531 ± 0. 037, respectively, and Rantes (ng/ml) was 1. 420 ± 0. 074, 7. 634 ± 0. 630, 15. 700 ±1. 300, respectively. Rantes presented a two-phase secretion mode : in early 6 hours it increased swiftly, but relatively slow at 12, 24, 48, 72 hours. T cell adherent to polycarbonate membrane increased after SEB stimulation in superantigen group compared with control group without SEB stimulation (86. 38± 14. 50 vs.16. 50± 2. 50, P< 0. 01 ). When 10 ng/ml SEB activated T cell was cocultured with HPAEC, more of originally suspended cultured T cells adhered to HPAEC monolayer [(15. 50±1.08)% vs. (1.60±0. 22)%,P<0.01], whereas the cell adhesion ratio decreased markedly in 1 μg/ml Met-Rantes group [(4. 39±0. 66)%, P < 0. 01 ). FACs test of HPAEC-adherent T cell showed lymphocyte chemokine receptor 5 (CCR5)/CD4 and CCR5/CD8 increased over 2. 5 folds and 2.8 folds compared with 100 ng/ml SEB activated T cell. Cell death rate of HPAEC was increased when cocultured with SEB-activated T cell in superantigen group compared with HPAEC normal incubation group [(32. 50±4

  19. 大鼠小肠移植排斥反应期移植肠基因表达、ICAM-1和IL-2R分子表达及上皮细胞凋亡的变化%Up-regulated intragraft gene expression,ICAM-1 and IL-2R molecules,and apoptotic epithelial cells during rejection of rat small intestine allografts

    Institute of Scientific and Technical Information of China (English)

    李元新; 李宁; 李幼生; 吴波; 黎介寿

    2001-01-01

    adhesion molecule-1 ( ICAM-1 ) during acute rejection episodes, and to analyze the changes in apoptosis in small intestinal allograft rejection. Methods Heterotopic small intestine transplantation was performed with inbred rats F344/N (RT11) and Wistar/A (RT1-Ak, RT1-Ed). All recipients were divided into four groups: group 1 : Wistar, native control; group 2: Wistar→Wistar; group 3: F344→Wistar and group 4: F344→Wistar + cyclosporine A (6 mg·kg-1 ·d-1 I.M. ). The grafts were harvested on postoperative days (PODs) 3, 5 and 7. All samples were examined pathologically. Intragraft mRNA expression of IL-2, IFN-γ, perforin and granzyme B were detected with reverse transcriptase polymerase chain reaction (RT-PCR) and intragraft expression of IL-2R and ICAM-1 were stained using immunohistochemistry. We also analyzed the change in apoptosis rejection with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Results Mild acute rejection occurred on POD 3 in the ailograft group, moderate acute rejection on POD 5, and severe acute rejection on POD 7, while none of the isografts had histological evidence of acute rejection. Cyclosporine A could effectively control rejection. Gene expression was virtually negative in the native control. Only on POD 5 was IL-2 mRNA expression of ailografts significantly higher than that of isografts ( P < 0.05). IFN-γ mRNA expression was significantly higher than that of the control groups ( P < 0.01 ) on PODs 3, 5 and 7, and the level of perforin and granzyme B mRNA expression reached significantly higher levels than in the other two control groups on POD 5 and POD 7. Intragraft IL-2R expression of the allograft was significantly higher than that of the other three control groups. Only on POD 3 was intragraft ICAM-1 expression of allografts significantly higher than isografts. The number of apoptotic cells per crypt of allografts was significantly higher than that of the other three control groups on POD 3 and POD

  20. 氯雷他定对哮喘豚鼠白细胞介素5及嗜酸性粒细胞凋亡的影响%Effect of Loratadine on Expression of Interleukin -5 and Eosinophil Apoptosis in Asthmatic Guinea Pigs

    Institute of Scientific and Technical Information of China (English)

    宋晓丹; 欧维琳; 莫碧文; 李雄; 秦静廷; 郑伟华; 张世杰

    2012-01-01

    guinea pigs were peritoneal injected with 20 g · L-1 ovaibumin(OVA) 3 times for allergization,inhaled 10 g · L-1 OVA inhalation about 40 min to induce asthma attacks. Guinea pigs in low - dose loratadine group and high — dose loratadine group were drenched with 2 mg · kg-1· d-1 ,10 mg · kg-1 · d-1 of loratadine about 14 days,once a day. The normal control group,the asthmatic group inhaled physiological saline. Enzyme linked immunosorbent assay was used to measure the contents of IL-5 in BALK and serum,meanwhile,eosinophil apoptosis was also detected by terminal deoxynucleotidyl transferase -mediated dUTP nick end labeling(TUNEL). Combined with the hematoxylin - eosin staining slices,the apoptosis cells in every 200 EOS in one slice were counted randomly. Results 1. The serum IL - 5 in the normal control group, asthmatic group, low -dose loratadine group and high -dose loratadine group were(0. 27 ±0.04) ng · L-1 ,(0.41 ±0.03) ng · L-1, (0.38 ±0.02) ng · L-1,(0.31 ±0.03) ng · L-1 ;IL -5 in BALF were respectively(0. 10 ±0.01) ng · L-1, (0. 38 ±0. 04) ng · L-1, (0. 33 ±0.05) ng · L-1 ,(0.23 ±0.09) ng · L-1.comparison between every 2 groups, the differences were significant( P, <0. 05). 2. The apoptosis cells in the 4 groups were 33,95 ,129,182,respectively,the differences between them were statistically significant (x2 =114.5, P< 0.05) ,but the difference between the asthmatic group and the low -dose loratadine group was not significant(x2 = 10.0,P >0.008 3). Conclusions Loraladine can reverse the IL-5 afforded delayed apoptosis,ameliorate the inflamatory state in lung tissue,and is of active therapeutic value potentially in asthma management.

  1. Impacts of chronic sleep deprivation on learning and memory, autophagy and neuronal apoptosis in mice%慢性睡眠剥夺对小鼠学习记忆及神经元自噬和凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    邱红艳; 李崧; 乐卫东

    2015-01-01

    Objective To establish chronic sleep deprivation mouse model,evaluate the learning and memory ability of mice and observe autophagy and apoptosis levels in mouse brain.Methods C57BL/6 mice (n =20) were randomly separated into sleep deprivation group and control group.After 2-month sleep deprivation by using an adapted multiple platform method,the behavioral performance of mice was measured by IntelliCage system.The expression of microtubule associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) and Beclin-1 was detected by Western blotting.Confocol microscopy was used to observe autophagosome.In addition,terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed to detect neuronal apoptosis level in mouse brain.Results The results of behavioral test showed that the incorrect visit ratio was much higher in sleep deprivation group than that in control group.Moreover,the expression of LC3-Ⅱ (sleep deprivation group 1.681 ± 0.186,control group 1.125 ±0.048,t =2.892,P =0.027 6) and Beclin-1 (sleep deprivation group 1.144 ±0.048,control group 1.006 ± 0.017,t =2.721,P =0.018 6) in mouse hippocampus and cortex was significantly elevated in sleep deprivation group than those in control group.Accordingly,the confocal microscopy observation also revealed an increased nuclear LC3-positive puncta in hippocampus and cortex of sleep deprived mice (hippocampus in sleep deprivation group 1.665 ± 0.153,in control group 0.819 ± 0.072,t =5.024,P =0.002 4;cortex in sleep deprivation group 1.925 ± 0.175,in control group 1.195 ± 0.111,t =3.521,P =0.012 5).In addition,TUNEL staining showed a much higher percentage of TUNEL-positive nuclei in these brain regions (hippocampus in sleep deprivation group 47.24 ± 4.15,in control group 19.26 ± 3.72,t =5.025,P =0.007 4;cortex in sleep deprivation group 42.25 ± 1.25,in control group 27.50 ± 3.23,t =4.262,P =0.005 3).Conclusions Chronic sleep deprivation can impair the learning and memory

  2. 癫(痫)持续状态模型中海马神经元损伤及其可能机制%Hippocampal neuronal damage and its possible mechanism in status epilepticus

    Institute of Scientific and Technical Information of China (English)

    丁秀芳; 王纪文; 姚国; 代方方; 张冰

    2015-01-01

    by using LiC1-pilocarpine.The 62 rats were divided into 1 h (6 cases),6 h (12 cases), 24 h (12 cases),72 h (12 cases)and 7 d (20 cases)after status epilepticus.At the same time, the healthy control group (12 cases) was established.Morphologic changes of hippocampus and the amount of apoptotic cells in healthy control and SE model groups at different time points (6 h, 24 h, 72 h, 7 d) (6 cases each group) after status epilepticus were quantified by adopting Nissl staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining respectively.Expressions of GluR2 in healthy control and SE model groups at 1 h,6 h,24 h,72 h and 7 d (6 cases each group) after status epilepticus were detected by using Western blot.Co-precipitation and Western blot techniques were used to investigate whether the GluR2/GAPDH interaction in the hippocampus was increased.Results Compared with the healthy control group, the number of nerve cells in the hippocampal CA1 and CA3 regions was significantly reduced at all studied time points(F =30.866,24.043, all P <0.05).Apoptotic cells in hippocampal CA1 and CA3 regions were significantly increased at 24 h,72 h and 7 d after status epilepticus (F =84.762,52.574, all P < 0.01).GluR2 at 1 h,6 h after status epilepticus was equal to that of the control group (P > 0.05), but it was shown to be significantly down-regulated at other studied time points (F =76.506,P < 0.01);when compared with the healthy control group,the GluR2/GAPDH interaction was significantly up-regulated in the hippocampus at 72 h after status epilepticus (t =7.029, P < 0.05).Conclusions Status epilepticus can lead to neuronal damage in the hippocampus.Down-regulation of GluR2 and increase of the GluR2/GAPDH complex formation might be one of the mechanisms involved in hippocampal neuronal damage.

  3. Effects of Prunella vulgaris L on the growth of hemangioma and the expression of Fas/FasL in nude mice%夏枯草对裸鼠血管瘤生长及Fas/FasL表达的影响

    Institute of Scientific and Technical Information of China (English)

    陈伟; 彭强; 刘铭; 杨春; 肖骁

    2014-01-01

    .At 30 days post-implantation,among 65 survivors,5 nude mice were randomly sacrificed for measuring the volume of hemangioma.The hemangioma tissues were extracted and prepared into paraffin sections as primary control.And another 60 nude mice were randomly divided into 3 groups of vaseline,PVL and triamcinolone acetonide.The growth and status of tumor were observed.At Days 7,14,21 and 28,5 mice in each group were randomly sacrificed for volume measuring and histological examinationt.Fas,FasL and cysteinyl aspartate specific proteinase 8 (caspase-8) antibody were irnmunohistochemically stained in all sections and their expressions analyzed.Tumor cell apoptosis was tested by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL).The experimental data were analyzed by statistical software SPSS 20.0.P<0.05 denoted significant difference.Results After intervention,as compared with vaseline group,hemangioma volumes in PVL and triamcinolone acetonide groups significantly decreased (P<0.05).At 21 days post-intervention,in PVL group,the positive rate of Fas expression was (49.71 ± 3.67) %,the positive expression rate of FasL (32.59 ± 3.01)% and the positive expression rate of Caspase-8 (39.69±3.47)%.And the above three positive rates were (29.01 ± 2.53)%,(19.01 ± 2.13)% and (17.11 ± 1.69)% in vaseline group versus (26.83± 2.37)%,(16.97 ± 1.81)% and (15.86 ± 1.91)% respectively in triamcinolone acetonide group.For comparisons between groups,the positive rates in PVL group were significantly higher than those in two other groups (P<0.05).At Days 7,14,21 and 28 post-intervention,the cell apoptosis index in PVL group were 14.75 ± 2.29,26.69 ± 3.12,38.71 ±3.81 and 53.65 ± 4.63 versus 24.53 ± 2.61,37.35 ± 3.58,56.37 ± 4.25 and 72.92 ± 5.21 respectively in triamcinolone acetonide group.All of them were significantly higher than that pre-intervention (6.79± 1.33) and that in vaseline group during the same period (7.12 ± 1

  4. 1,25 (OH)2D3 influences endothelial cell proliferation, apoptosis and endothelial nitric oxide synthase expression of aorta in apolipoprotein E-deficient mice%1,25(OH)2D3对载脂蛋白E缺乏鼠主动脉内皮细胞增生与凋亡及一氧化氮合酶表达的影响

    Institute of Scientific and Technical Information of China (English)

    向伟; 符生苗; 马建林; 王洁; 郑维; 康红; 何小解; 马燕琳; 易著文; 曹艳; 赵水平; 杨进福; 马志超; 吴明

    2011-01-01

    on endothelial cell proliferation were observed by MTT,apoptosis of cells were quantitated by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling,Bcl-2 mRNA,fas mRNA and eNOS mRNA was detected by reverse transcription-polymerase chain reaction.Result Endothelial cell proliferation rate of aorta did not significantly change in the two control groups (0.162 ±0.031 vs.0.158 ±0.006,P >0.05 ).Compared with control groups,1,25 (OH) 2 D3 stimulated endothelial cell proliferation of aorta ( P <0.05),but endothelial cell proliferation rate did not significantly change in different 1,25 (OH)2D3concentration groups [ 1,25( OH)2D3 concentration:10-4moL/L,10-5 moL/L,10-6 moL/L,10 -7 mol/L,10-8 mol/L,endothelial cell proliferation rate:0.189 ± 0.013 vs.0.285 ± 0.011 vs.0.296 ± 0.026 vs.0.284 ±0.017 vs.0.233 ± 0.010,P > 0.05 ].1,25 ( OH )2D3 research concentration as chosen as 10 -6mol/L.In 1,25 (OH)2D3 10-6 mol/L group,the expression of Bcl-2,eNOS mRNA was significantly increased (0.78 t:0.16 vs.0.46 ± 0.21 vs.0.42 ± 0.17,0.56 ± 0.16 vs.0.39 ± 0.13 vs.0.35 ± 0.11,0.46 ± 0.2 vs.10.42 ± 0.17 vs.0.78 ± 0.16,0.79 ± 0.21 vs.0.81 ± 0.20 vs.0.43 ± 0.12 ),apoptotic index,Fas mRNA was significantly decreased ( 15.14 ±3.19 vs.18.94 ±4.22 vs.19.27 ±4.58,0.43 ±0.12 vs.0.79 ±0.21 vs.0.81 ± 0.20) ( P < 0.05 ).The quantity of eNOS gene expression was inversely associated with apoptosis index and Fas mRNA,was positively associated with Bcl-2 mRNA (r =-0.676,-0.758,0.762,P <0.01).Conclusion 1,25(OH)2D3 stimulated endothelial cell proliferation,inhibited apoptosis and increased eNOS expression of aorta in apoE-/- mice.These results may deepen understanding of the pathogenesis of atherosclerosis.

  5. Protective effect of sodium valproate on motor neurons in the spinal cord following sciatic nerve injury in rats

    Institute of Scientific and Technical Information of China (English)

    Fei Wu; Danmou Xing; Zhengren Peng; Wusheng Kan

    2006-01-01

    BACKGROUND: Sodium valproate (VPA) is used to be an effective anti-epileptic drug. VPA possesses the characteristics of penetrating rapidly through the blood-brain barrier (BBB) and increasing levels of Bcl-2 and growth cone-associated protein (GAP) 43 in spinal cord.OBJECTIVE: To observe the effect of VPA on Bcl-2 expression and motor neuronal apoptosis in spinal cord of rats following sciatic nerve transection.DESIGN: Randomized controlled experiment.SETTING: Department of Hand Surgery and Microsurgery, Wuhan Puai Hospital.MATERIALS: A total of 30 male healthy SD rats of olean grade and with the body mass of 180-220 g were provided by Experimental Animal Center of Medical College of Wuhan University. Sodium Valproate Tablets were purchases from Hengrui Pharmaceutical Factory, Jiangsu.METHODS: The experiment was performed in the Central Laboratory of Wuhan Puai Hospital and Medical College of Wuhan University from February to May 2006. Totally 30 rats were randomly divided into two groups:treatment group (n =15) and model group (n =15). Longitudinal incision along backside of right hind limbs of rats was made to expose sciatic nerves, which were sharply transected 1 cm distal to the inferior margin of piriform muscle after nerve liberation under operation microscope to establish sciatic nerve injury rat models.Sodium Valproate Tablets were pulverized and diluted into 50 g/L suspension with saline. On the day of operation, the rats in the treatment group received 6 Ml/kg VPA suspension by gastric perfusion, once a day,whereas model group received 10 Ml/kg saline by gastric perfusion, once a day. L4-6 spinal cords were obtained at days 1, 4, 7, 14 and 28 after operation, respectively. Terminal deoxyribonucleotidyl transferase (TdT)-mediated Dutp-biotin nick end labeling (TUNEL) technique and immunohistochemical method (SP method) were used to detect absorbance (A) of neurons with positive Bcl-2 expression. Apoptotic rate of cells (number of apoptotic cells

  6. Detection of apoptosis in kidney biopsies of patients with D+ hemolytic uremic syndrome.

    NARCIS (Netherlands)

    Loo, D.M.W.M. te; Monnens, L.A.H.; Heuvel, L.P.W.J. van den; Gubler, M.C.; Kockx, M.M.

    2001-01-01

    In this study we have investigated the presence of apoptotic cells in renal biopsy material of seven patients with hemolytic uremic syndrome (HUS) by using an improved and stringent terminal deoxynucleotidyl nick-end labeling (TUNEL) technique. Renal biopsy material was taken in the second or third

  7. Relation between serum xenobiotic induced receptor activities and sperm DNA damage and sperm apoptotic markers in European and Inuit populations

    DEFF Research Database (Denmark)

    Long, Manhai; Stronati, Alessanda; Bizzaro, Davide;

    2007-01-01

    -mediated luciferase reporter gene expression. Sperm DNA damage was measured using terminal deoxynucleotidyl transferase-driven dUTP nick labeling assay (TUNEL) and pro- (Fas) and anti-apoptotic (Bcl-xL) markers were determined by immune methods. Different features of xenobiotic-induced receptor activity in serum...

  8. Coordinate reduction in cell proliferation and cell death in mouse olfactory epithelium from birth to maturity

    NARCIS (Netherlands)

    Fung, KM; Peringa, J; Venkatachalam, S; Lee, VMY; Trojanowski, JQ

    1997-01-01

    We investigated cell proliferation and cell death in the olfactory epithelium (OE) of mice from birth to maturity using bromodeoxyuridine and terminal deoxynucleotidyl transferase nick end labeling. We show that cell death events and proliferative activity diminish concomitantly with age in the OE.

  9. Coordinate reduction in cell proliferation and cell death in mouse olfactory epithelium from birth to maturity

    NARCIS (Netherlands)

    Fung, KM; Peringa, J; Venkatachalam, S; Lee, VMY; Trojanowski, JQ

    1997-01-01

    We investigated cell proliferation and cell death in the olfactory epithelium (OE) of mice from birth to maturity using bromodeoxyuridine and terminal deoxynucleotidyl transferase nick end labeling. We show that cell death events and proliferative activity diminish concomitantly with age in the OE.

  10. Additional use of immunostaining for active caspase 3 and cleaved actin and PARP fragments to detect apoptosis in patients with chronic heart failure

    NARCIS (Netherlands)

    De Boer, RA; Van Veldhuisen, DJ; Van der Wijk, J; Brouwer, RMHJ; Cole, GM; Suurmeijer, AJH; de Jonge, N.

    2000-01-01

    Background: Apoptosis might play a role in the pathophysiology of chronic heart failure (CHE). Detection of apoptosis in tissue mostly relies on the terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) method. Recent studies showed, however, that TUNEL-positive cardiomyocytes were not in

  11. Programmed cell death in developing human fetal CNS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The spatial and temporal distributions of programmed cell death (PCD) in developing central nervous system (CNS) of human fetuses ranging from 12 to 39 weeks of gestation were investigated using techniques of flow cytometry and terminal transferase-mediated nick end labeling (TUNEL). The results showed that PCD did occur in every representative brain region of all fetuses examined in different stages. It was found that there were two peaks of PCD appearing at the 12th and 39th weeks respectively, which suggested that the first peak of apoptosis may be involved in the selective elimination of neurons overproduced during the early development and the second may play an important role in establishing the correct neuronal circuitry.

  12. Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

    Institute of Scientific and Technical Information of China (English)

    Serafín Pérez-Cerezales; Alberto Miranda; Alfonso Gutiérrez-Adán

    2012-01-01

    It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin,which acquires fully condensed status at the caput epididymidis.The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay,the comet assay,the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation.Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay.

  13. Extract of ginkgo biloba EGb761 inhibits cell apoptosis following spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Jiejun Jiao; Jiannong Jiang; Bin Du; Yuan Mo; Ming Zhou; Hongquan Zhang

    2010-01-01

    The neuroprotective effects of ginkgo biloba extract have been shown in rats following spinal cord injury (SCI). However, the precise protective mechanisms remain unclear. In the present study, low-acid water-soluble extract of ginkgo biloba EGb761 was used to treat rats with SCI. Xanthin oxidase, thiobarbituric acid, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling assay, and immunohistochemistry were utilized to detect lipid peroxidation, neural cell apoptosis, and inducible nitric oxide synthase activity in rats with SCI. Results revealed significantly increased superoxide dismutase activity, decreased malondialdehyde content, apoptotic index, and inducible nitric oxide synthase expression in SCI rats following EGb761 treatment. Therefore, EGb761 suppressed lipid peroxidation following SCI, relieved neural cell apoptosis, inhibited inducible nitric oxide synthase expression, and ultimately exerted protective effects on SCI.

  14. Uterosacral ligament smooth muscle cell apoptosis is increased in women with uterine prolapse.

    Science.gov (United States)

    Takacs, Peter; Nassiri, Mehdi; Gualtieri, Marc; Candiotti, Keith; Medina, Carlos A

    2009-05-01

    The purpose of this study was to compare the smooth muscle content and apoptosis of the uterosacral ligament in women with and without uterine prolapse. Uterosacral ligaments were sampled in women with (n = 9) or without (n = 9) uterine prolapse undergoing hysterectomy. Smooth muscle of the uterosacral ligament was identified by immunohistochemistry. Digital image analysis was used to determine the fractional area of smooth muscle in the histologic cross sections. Apoptosis was assessed by terminal deoxynucelotidyl-transferase-mediated dUTP nick-end-labeling method. The fractional area of nonvascular smooth muscle in the uterosacral ligament of women with uterine prolapse was significantly decreased compared to women without prolapse (0.32 +/- 0.12 vs. 0.42 +/- 0.03, P = .02) and the apoptotic index was significantly higher compared to women without prolapse (0.20 +/- 0.06 vs. 0.08 +/- 0.04, P uterine prolapse compared to women without prolapse.

  15. Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time.

    Science.gov (United States)

    Rougier, Natalia; Uriondo, Heydy; Papier, Sergio; Checa, Miguel Angel; Sueldo, Carlos; Alvarez Sedó, Cristian

    2013-07-01

    To compare the DNA fragmentation of semen samples established by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) after incubation in polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) for different time periods. Comparative prospective study. Center for reproductive medicine. Twenty-seven semen samples from infertile patients. None. Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: A) normal TUNEL (DNA fragmentation significantly decreased after centrifugation gradient, regardless of the initial levels of the sample. Samples with TUNEL ≥ 20% were more susceptible to a significant increase in DNA fragmentation over time, with similar increases being observed over time for samples that were incubated in HA or PVP. These data may be relevant for sperm preparation for intracytoplasmic sperm injection. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. Persephin基因转染对缺氧诱导神经干细胞凋亡的影响%Effect of Persephin gene transfer on hypoxia induced neural stem cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    沈庆煜; 肖颂华; 黎祥喷; 叶剑虹; 王艺东

    2007-01-01

    Interventions: C17.2 neural stem cells cultured in vitro were infected by recombinant adenovirus containing persephin gene, and they were divided into four groups: blank control group (Group A, in which the C17.2 neural stem cells were not treated with hypoxia), hypoxic group [Group B, in which the cells were cultured at 37 ℃ in anaerobic incubation containing N2 (0.95 in volume fraction) and CO2 (0.05 in volume fraction)], hypoxia + pAdCMV persephin infection group [Group C, where the cells were cultured under the conditions as in group B after pAdCMV persephin infection for 48 hours], and hypoxia + pAdCMV persephin infection + anti-sense persephin ODN group (Group D, where the cells were infected by pAdCMV persephin and anti-sense persephin ODN. ② Evaluation: The expression of Persephin protein was analyzed using Western blotting; Apoptotic index was detected with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay; The changes of apoptotic rate was determined with flow cytometry.MAIN OUTCOME MEASURES: Expression of Persephin protein; Apoptotic index; Apoptotic rate.RESULTS: ① Expression of Persephin protein: A specific band (relative molecular mass of 24 000) was detected by Western blotting in pAdCMV persephin infected cells, suggesting the successful expression of persephin gene.Interestingly, the cells infected with both pAdCMV persephin and anti-sense persephin ODN also showed the specific band of about 24 000, but with much less density, indicating that anti-sense persephin ODN could effectively inhibit the expression of pAdCMV persephin. However, this band was not presented in the blank control groups. ② Apoptotic index:The apoptotic index in group C was significantly lower than those in groups B and D (P<0.01), but still higher than that of group A (P<0.01), suggesting that persephin gene transfer could attenuate apoptosis to some extent. ③ Apoptotic rate: The apoptotic rate in groups B and D were

  17. Monitoring patterned enzymatic polymerization on DNA origami at single-molecule level

    Science.gov (United States)

    Okholm, A. H.; Aslan, H.; Besenbacher, F.; Dong, M.; Kjems, J.

    2015-06-01

    DNA origami has been used to orchestrate reactions with nano-precision using a variety of biomolecules. Here, the dynamics of albumin-assisted, localized single-molecule DNA polymerization by terminal deoxynucleotidyl transferase on a 2D DNA origami are monitored using AFM in liquid. Direct visualization of the surface activity revealed the mechanics of growth.DNA origami has been used to orchestrate reactions with nano-precision using a variety of biomolecules. Here, the dynamics of albumin-assisted, localized single-molecule DNA polymerization by terminal deoxynucleotidyl transferase on a 2D DNA origami are monitored using AFM in liquid. Direct visualization of the surface activity revealed the mechanics of growth. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01945a

  18. Spatial and temporal regulation of collagenases—3,—4,and stromelysin —3 implicates distinct functions in apoptosis and tissue remodeling during frog metamorphosis

    Institute of Scientific and Technical Information of China (English)

    DAMJANOVSKISASHKO; ATSUKOISHIZUYAOKA; 等

    1999-01-01

    Matrix metalloproteinases (MMPs) are a family of extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix(ECM).They have been implicated to play important roles in a number of developmental and pathological processes,such as tumor metastasis and inflammation.Relatively few studies have been carried out to investigate the function of MMPs during postembryonic organ-development.Using Xenopus laevis development as a model system,we demonstrate here that three MMPs,stromelysin-3(ST3),collagenases-3(Col3),and Col4,have distinct spatial and temporal expression profiles during metamorphosis as the tadpole transforms into a frog.In situ hybridizations reveal a tight,but distinct,association of individual MMPs with tissue remodeling in the tail and intestine during metamorphosis.In particular,ST3 expression is strongly correlated with apoptosis in both organs as demonstrated by analyses of serial sections with in situ hybridization for ST3 mRNA and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling)for apoptosis,respectively.On the other hand,Col3 and Col4 are present in regions where extensive connective tissue remodeling take place.These results indicate that ST3 is likely to play a role in ECM-remodeling that facilitate apoptotic tissue remodeling or resorption while Col3 and Col4 appear to participate in connective tissue degradation during development.

  19. Increased apoptosis and decreased density of medial smooth muscle cells in human abdominal aortic aneurysms

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian张健; Jan Schmidt; Eduard Ryschich; Hardy Schumacher; Jens R Allenberg

    2003-01-01

    Objective To determine the increase of apoptosis and the decrease of smooth muscle cells (SMCs) density in human abdominal aortic aneurysms (AAA). Methods In situ terminal transferase-mediated dUTP nick end labeling (TUNEL) was employed to detect apoptosis of SMCs in patients with AAA (n=25) and normal abdominal aortae (n=10). Positive cells were identified by specific cell marker in combination with immunohistochemistry. Meanwhile SMC counting was performed by anti-α-actin immunohistostaining to compare the SMC density. Results TUNEL staining revealed that there was significantly increased apoptosis in AAAs (average 8.6%) compared with normal abdominal aortae (average 0.95%, P<0.01). Double staining showed that most of these cells were SMCs. Counting of α-actin positive SMCs revealed that medial SMC density of AAAs (37.5±7.6 SMCs /HPF) was reduced by 79.1% in comparison with that of normal abdominal aortae (179.2±16.1 SMCs /HPF, P<0.01). Conclusions Significantly increased SMCs of AAA bear apoptotic markers initiating cell death. Elevated apoptosis may result in a decreased density of SMCs in AAA, which may profoundly influence the development of AAA.

  20. Epidermal cell death in frogs with chytridiomycosis

    Science.gov (United States)

    Roberts, Alexandra A.; Skerratt, Lee F.; Berger, Lee

    2017-01-01

    Background Amphibians are declining at an alarming rate, and one of the major causes of decline is the infectious disease chytridiomycosis. Parasitic fungal sporangia occur within epidermal cells causing epidermal disruption, but these changes have not been well characterised. Apoptosis (planned cell death) can be a damaging response to the host but may alternatively be a mechanism of pathogen removal for some intracellular infections. Methods In this study we experimentally infected two endangered amphibian species Pseudophryne corroboree and Litoria verreauxii alpina with the causal agent of chytridiomycosis. We quantified cell death in the epidermis through two assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL) and caspase 3/7. Results Cell death was positively associated with infection load and morbidity of clinically infected animals. In infected amphibians, TUNEL positive cells were concentrated in epidermal layers, correlating to the localisation of infection within the skin. Caspase activity was stable and low in early infection, where pathogen loads were light but increasing. In animals that recovered from infection, caspase activity gradually returned to normal as the infection cleared. Whereas, in amphibians that did not recover, caspase activity increased dramatically when infection loads peaked. Discussion Increased cell death may be a pathology of the fungal parasite, likely contributing to loss of skin homeostatic functions, but it is also possible that apoptosis suppression may be used initially by the pathogen to help establish infection. Further research should explore the specific mechanisms of cell death and more specifically apoptosis regulation during fungal infection. PMID:28168107

  1. Inhibition of p38 activity reverses claudin-6 induced cell apoptosis,invasion, and migration

    Institute of Scientific and Technical Information of China (English)

    WU Qiong; LIU Xing; LIU Ya-fang; LU Yan; WANG Li-ping; ZHANG Xiao-wei; LI Yu-lin

    2013-01-01

    Background Claudin-6 is a protein component of tight junctions and its expression could downregulate the malignant phenotype of breast carcinoma.Here we investigated the mechanisms of claudin-6 induced human MCF-7 breast cancer cells apoptosis,invasion,and migration.Methods Terminal deoxyribonucleotide transferase-mediated nick-end labeling assay and Annexin-V/PI double stain assay were carried out to evaluate apoptosis.Inhibitors of each pathway were used to inactivate the signaling pathways.The expression of claudin-6 and phosphate p38,Erk 1/2 and Akt protein levels was confirmed by Western blotting analysis.Invasive and migratory traits of claudin-6 expressing cells were determined by Boyden chamber invasion assay and monolayer wound-healing assay.Results Cells with high-level expression of claudin-6 had a higher rate of apoptosis than control cells.Western blotting assay showed that by contrast to control groups,p38 pathways were more activated in claudin-6 expressing cells.However,after inhibitor SB203580 treatment,the activation status could be significantly counteracted.Furthermore,by applying inhibitors to the apoptotic rate,invasive and migratory traits were also recovered in cells with claudin-6 expression.Conclusion Claudin-6 may function through p38 mitogen-activated protein kinase pathway,of which inhibition may reverse claudin-6-induced cell apoptosis,invasion,and migration.

  2. Fas/Fas ligand-mediated apoptosis in different cell lineages and functional compartments of human lymph nodes.

    Science.gov (United States)

    Kokkonen, Tuomo S; Karttunen, Tuomo J

    2010-02-01

    We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL-mediated apoptosis in lymph node homeostasis.

  3. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2016-01-01

    Full Text Available Genistein is effective against amyloid-β toxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer's disease. A rat model of Alzheimer's disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-β peptide (25–35. In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg was given prior to establishing Alzheimer's disease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genist-ein in Alzheimer's disease rats. These findings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c.

  4. Levistolide A overcomes P-glycoprotein-mediated drug resistance in human breast carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Fei CHEN; Tao WANG; Jia WANG; Zi-qiang WANG; Ming QIAN

    2008-01-01

    Aim:The aim of the present study was to investigate the reversing effect of levistolide A (LA) on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in human breast carcinoma Bcap37/MDR1 cells. Methods:After chemotherapeu-tic drugs (adriamycin or vincristine) used alone or in combination with LA, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-lium bromide assay and cell cycle distribution by flow cytometry. RT-PCR was used to detect MDR1 gene transcription and the Western blot assay was used to assess P-gp expression and the cleavages of poly(ADP-ribose) polymerase and caspase-3. Apoptosis was detected by terminal transferase-mediated dUTP nick end-labeling assay. Moreover, the P-gp function was evaluated by the intracellu-lar accumulation of the P-gp substrate detected by flow cytometry. Results:We found the subcytotoxic doses of LA significantly enhanced adriamycin- or vinc-ristine-induced G2/M arrest and apoptosis. These effects were consistent with the ability of LA to inhibit P-gp function. Moreover, LA dramatically enhanced the verapamil (VER) ability to reverse drug resistance. Conclusion:LA has the poten-tial to be developed as a novel P-gp modulator. Furthermore, the combination of LA and VER might represent a more sufficient but less toxic anti-MDR regimen.

  5. Inhibitory and Apoptosis-Inducing Effects of Newcastle Disease Virus Strain AF2240 on Mammary Carcinoma Cell Line

    Directory of Open Access Journals (Sweden)

    Umar Ahmad

    2015-01-01

    Full Text Available Breast cancer is the malignant tumour that developed from cells of the breast and is the first leading cause of cancer death among women worldwide. Surgery, radiotherapy, and chemotherapy are the available treatments for breast cancer, but these were reported to have side effects. Newcastle disease virus (NDV known as Avian paramyxovirus type-1 (APMV1 belongs to the genus Avulavirus in a family Paramyxoviridae. NDV is shown to be a promising anticancer agent, killing tumour cells while sparing normal cells unharmed. In this study, the oncolytic and cytotoxic activities of NDV AF2240 strain were evaluated on MDA-MB-231, human mammary carcinoma cell line, using MTT assay, and its inhibitory effects were further studied using proliferation and migration assays. Morphological and apoptotic-inducing effects of NDV on MD-MB-231 cells were observed using phase contrast and fluorescence microscopes. Detection of DNA fragmentation was done following terminal deoxyribonucleotide transferase-mediated Br-dUTP nick end labeling staining (TUNEL assay, which confirmed that the mode of death was through apoptosis and was quantified by flow cytometry. Furthermore, analysis of cellular DNA content demonstrated that the virus caused an increase in the sub-G1 phase (apoptotic peak of the cell cycle. It appears that NDV AF2240 strain is a potent anticancer agent that induced apoptosis in time-dependent manner.

  6. Correlation of cell apoptosis with brain edema and elevated intracranial pressure in traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    YANG Xiao-feng; LIU Wei-guo; SHEN Hong; GONG Jiang-biao; YU Jun; HU Wei-wei; L(U) Shi-ting; ZHENG Xiu-jue; FU Wei-ming

    2005-01-01

    Objective: To study the correlation between brain edema, elevated intracranial pressure (ICP) and cell apoptosis in traumatic brain injury (TBI). Methods: In this study, totally 42 rabbits in 7 groups were studied. Six of the animals were identified as a control group, and the remaining 36 animals were equally divided into 6 TBI groups. TBI models were produced by the modified method of Feeney. After the impact, ICP of each subject was recorded continuously by an ICP monitor until the animal was sacrificed at scheduled time. The apoptotic brain cells were detected by an terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. Cerebral water content (CWC) was measured with a drying method and calculated according to the Elliott formula. Then, an analysis was conducted to determine the correlation between the count of apoptotic cells and the clinical pathological changes of the brain. Results: Apoptotic cell count began to increase 2 h after the impact, and reached its maximum about 3 days after the impact. The peak value of CWC and ICP appeared 1 day and 3 days after the impact, respectively. Apoptotic cell count had a positive correlation with CWC and ICP. Conclusions: In TBI, occurrence of brain edema and ICP increase might lead to apoptosis of brain cells. Any therapy which can relieve brain edema and/or decrease ICP would be able to reduce neuron apoptosis, thereby to attenuate the secondary brain damage.

  7. Knockdown of Stat3 expression using RNAi inhibits growth of laryngeal tumors in vivo

    Institute of Scientific and Technical Information of China (English)

    Li-fang GAO; Lian-ji WEN; Hao YU; Ling ZHANG; Yan MENG; Yue-ting SHAO; De-qi XU; Xue-jian ZHAO

    2006-01-01

    Aim:To study the effect of pSilencer1.0-U6-siRNA-stat3 on the growth of human laryngeal tumors in nude mice.Methods:Hep2 cells were transplanted into nude mice,then at the time of tumor fornaation,growth rates were observed.After the tumor formed,pSilencer1.0-U6-siRNA-stat3 was injected.Tumor volumes were calculated,and growth curves were plotted.Representative histological sections were taken from mice beating transplantation tumors in both treated and control groups,and stat3,Ptyr-star3,Bcl-2,cyclin D1,and survivin expression were detected by Western blotting.survivin Mrna levels were detected by Northern blotting,hematoxylin and cosin staining and terminal deoxvribonucleotidvl transferase-mediated Dutp-digoxigenin nick end-1abeling (TUNEL)assay to confirm the apoptosis of tumors.Results:In nude mice,pSilencer1.0-U6-siRNA-stat3 significantly suppressed the growth of tumors compared with controls (P<0.001). It suppressed stat3 expression,and downregulated BcL2,cyclin D1,and survivin expression within the tumor.This significantly induced apoptosis of the tumors.Conclusion:pSilencer1.0-U6-siRNA-stat3 was able to inhibit the growth of transplanted human laryngeal tumors in nude mice and induce apoptosis.

  8. Usage of whey protein may cause liver damage via inflammatory and apoptotic responses.

    Science.gov (United States)

    Gürgen, S G; Yücel, A T; Karakuş, A Ç; Çeçen, D; Özen, G; Koçtürk, S

    2015-07-01

    The purpose of this study was to investigate the long- and short-term inflammatory and apoptotic effects of whey protein on the livers of non-exercising rats. Thirty rats were divided into three groups namely (1) control group, (2) short-term whey (WS) protein diet (252 g/kg for 5 days), and (3) long-term whey (WL) protein diet (252 g/kg for 4 weeks). Interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and cytokeratin 18 (CK-18-M30) were assessed using enzyme-linked immunosorbent assay and immunohistochemical methods. Apoptosis was evaluated using the terminal transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method. Hepatotoxicity was evaluated by quantitation of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Based on the biochemical levels and immunohistochemical results, the highest level of IL-1β was identified in the WL group (p whey protein is used in an uninformed manner and without exercising, adverse effects on the liver may occur by increasing the apoptotic signal in the short term and increasing inflammatory markers and hepatotoxicity in the long term.

  9. Sestrin2 protects against acetaminophen-induced liver injury.

    Science.gov (United States)

    Kim, Seung Jung; Kim, Kyu Min; Yang, Ji Hye; Cho, Sam Seok; Kim, Ji Young; Park, Su Jung; Lee, Sang Kyu; Ku, Sae Kwang; Cho, Il Je; Ki, Sung Hwan

    2017-05-01

    Acetaminophen (APAP) overdose accounts for half of the cases of acute liver failure worldwide. We previously reported that Sestrin2 (Sesn2) protects against d-galactosamine/lipopolysaccharide-induced acute fulminant liver failure. In this study, we demonstrated that Sesn2 protects APAP-induced liver injury in mice, using a recombinant adenovirus encoding Sesn2 (Ad-Sesn2). First, we found that treatment of mice with toxic levels of APAP significantly reduced Sesn2 expression. Tail-vein injection with Ad-Sesn2 inhibited APAP-induced serum alanine aminotransferase and aspartate aminotransferase levels and markedly reduced hepatocyte degeneration and inflammatory cell infiltration. Additionally, APAP-induced glutathione depletion and reactive oxygen species generation were inhibited by Ad-Sesn2 treatment. Consistently, hepatic inflammatory gene expression and proinflammatory cytokine levels were also inhibited in Sesn2-infected mice, and we observed reduced APAP-mediated apoptotic signaling by terminal transferase-mediated dUTP nick-end labeling staining of the hepatic tissue. At a high dose of APAP, the mortality rate of Ad-Sesn2-infected mice was significantly lower than that of control mice. Furthermore, Sesn2 prevented APAP-induced damage through suppression of downstream mitogen-activated protein kinase pathway activation. Therefore, Sesn2 exerted a protective effect against APAP-induced acute liver damage by inhibiting oxidative stress and proinflammatory signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Expression of 8-hydroxy-2'-deoxyguanosine in gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Xiaoyu Chen; Jun Du; Luo Gu

    2007-01-01

    Objective: Reactive oxygen species may be involved in the progression of gastric carcinomas. To clarify whether the pathology of gastric carcinoma are related to oxidative DNA damage, the expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was examined in 30 patients with gastric carcinomas. Methods: The expression of 8-OHdG and apoptosis in the gastric carcinoma were measured using the methods of immunocytochemistry and deoxynucleartididyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), respectively. Results: Of the 30 cases, 25 (83%) showed stronger immunoreactivity than normal control. The patients with poorly differentiated gastric carcinoma had a larger tumor size and higher labeling indices of TUNEL- and 8-OHdG-positive cells than those with well and moderately differentiated gastric carcinoma. Conclusion: Our findings suggest that oxidative DNA damage is increased in association with necroinflammation in chronic gastric injuries and determination of 8-OHdG is useful in assessing high-grade malignancy in gastric carcinomas.

  11. Induction of apoptosis in HeLa cells by chloroform fraction of seed extracts of Nigella sativa

    Directory of Open Access Journals (Sweden)

    Alshatwi Ali A

    2009-11-01

    Full Text Available Abstract Background Cancer remains one of the most dreaded diseases causing an astonishingly high death rate, second only to cardiac arrest. The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy and radiotherapy have not succeeded in reverting the outcome of the disease to any drastic extent, has made researchers investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. This study progresses in the direction of identifying component(s from Nigella sativa with anti cancer acitivity. In the present study we investigated the efficacy of Organic extracts of Nigella sativa seed powder for its clonogenic inhibition and induction of apoptosis in HeLa cancer cell. Results Methanolic, n-Hexane and chloroform extracts of Nigella sativa seedz effectively killed HeLa cells. The IC50 values of methanolic, n-hexane, and chloroform extracts of Nigella sativa were 2.28 μg/ml, 2.20 μg/ml and 0.41 ng/ml, respectively. All three extracts induced apoptosis in HeLa cells. Apoptosis was confirmed by DNA fragmentation, western blot and terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL assay. Conclusion Western Blot and TUNEL results suggested that Nigella sativa seed extracts regulated the expression of pro- and anti- apoptotic genes, indicating its possible development as a potential therapeutic agent for cervical cancer upon further investigation.

  12. p53 and telomerase control rat myocardial tissue response to hypoxia and ageing

    Directory of Open Access Journals (Sweden)

    A. Cataldi

    2009-12-01

    Full Text Available Cellular senescence implies loss of proliferative and tissue regenerative capability. Also hypoxia, producing Reactive Oxygen Species (ROS, can damage cellular components through the oxidation of DNA, proteins and lipids, thus influencing the shortening of telomeres. Since ribonucleoprotein Telomerase (TERT, catalyzing the replication of the ends of eukaryotic chromosomes, promotes cardiac muscle cell proliferation, hypertrophy and survival, here we investigated its role in the events regulating apoptosis occurrence and life span in hearts deriving from young and old rats exposed to hypoxia. TUNEL (terminal-deoxinucleotidyl -transferase- mediated dUTP nick end-labeling analysis reveals an increased apoptotic cell number in both samples after hypoxia exposure, mainly in the young with respect to the old. TERT expression lowers either in the hypoxic young, either in the old in both experimental conditions, with respect to the normoxic young. These events are paralleled by p53 and HIF-1 ? expression dramatic increase and by p53/ HIF-1 ? co-immunoprecipitation in the hypoxic young, evidencing the young subject as the most stressed by such challenge. These effects could be explained by induction of damage to genomic DNA by ROS that accelerates cell senescence through p53 activation. Moreover, by preventing TERT enzyme down-regulation, cell cycle exit and apoptosis occurrence could be delayed and new possibilities for intervention against cell ageing and hypoxia could be opened.

  13. Effect of Tindurin on Immunopathogenesis Mechanism of Collagen-Induced Arthritis

    Directory of Open Access Journals (Sweden)

    F Saadat

    2005-08-01

    Full Text Available Rheumatoid arthritis is a chronic inflammatory disease characterized by the sequestration of various leukocyte subpopulations within both the developing pannus and synovial space. This study was undertaken to examine the therapeutic potency of tindurin in experimental rheumatoid arthritis. Collagen-induced arthritis (CIA was induced by intradermally immunization of Lewis rats at the base of the tail. The paws and knees were then removed for histopathology and radiography analysis. Using fibrosarcoma cell line the apoptosis process was measured by Terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL method. Our data showed that the i.p. injection of tindurin to arthritic rats induced a significant reduction in paw edema. Histopathological assessment showed reduced inflammatory cells infiltrate, tissue edema and bone erosion in joints of treated rats. Moreover, our results in radiography were in line with histological findings as well as tindurin was found to induce apoptosis of treated cells in comparison with positive, negative and non-treated ones. Our findings revealed the therapeutic effect of tindurin in experimental model of rheumatoid arthritis in comparison with methotrexate as a choice drug.

  14. Association of HIF- expression and cell apoptosis after traumatic brain injury in the rat

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To explore the expression of hypoxia inducible factor-1α (HIF-1~) and the correlation between HIF-1α and apoptosis after traumatic brain injury.Methods: Using experimental traumatic brain injury in the rats, the expression of HIF-1α was studied by immunohisto-chemistry in cerebral tissue, apoptotic cell death was evaluated with TUNEL (transferase-mediated XdUTP nick end labeling ), and double-labeled immunohistochemistry and TUNEL methods were used to investigate the relationship between HIF-1α and apoptosis.Results: There was remarkable difference in the expression of HIF-1α between the experimental groups and the control groups (P < 0.01), in the experimental groups,the expression of HIF-1α at 48 hours was highest; the evidence of apoptotic cell death after experimental traumatic brain injury was found by TUNEL; the apoptotic percentage increased or decreased according to the changes of the positive expression of HIF-1α (r = 0.99).Conclusions: The results suggest that secondary brain ischemia plays a crucial role in apoptotic cell death after traumatic brain injury; HIF-1α can prompt apoptotic cell death after experimental traumatic brain injury.e expres

  15. Developmental changes in cell proliferation and apoptosis in the normal duck thymus.

    Science.gov (United States)

    Fang, J; Cui, H; Peng, X; Chen, Z; He, M; Tang, L

    2011-12-01

    Cell proliferation and apoptosis in the normal duck thymus during embryonic and post-embryonic development were studied. The flow cytometry assay shows that the level of G(0)/G(1) thymic cell population and the proportion of apoptotic cells increased with age, while the levels of S phase, G(2) + M phase and the proliferating index decreased with age. Proliferation cell nuclear antigen (PCNA) was mainly detected in the nuclei of lymphocytes. The number of PCNA-positive cells in the cortex and medulla significantly decreased with age. Transferase-mediated dUTP nick-end labelling (TUNEL) reaction stained apoptotic bodies in the cytoplasm of macrophages and free apoptotic bodies or nuclei with condensed chromatin in lymphocytes. The number of TUNEL-positive cells in the cortex and medulla markedly increased with age. The amount of proliferation and apoptotic cells in the thymic cortex was higher than that in the medulla. The balance between proliferation and apoptosis in the duck thymus may account for the process of thymic development and involution.

  16. Profile of hepatocyte apoptosis and bile lakes before and after bile duct decompression in severe obstructive jaundice patients

    Institute of Scientific and Technical Information of China (English)

    ToarJMLalisang; RadenSjamsuhidajat; NurjatiCSiregar; AkmalTaher

    2010-01-01

    BACKGROUND: Excessive hepatocyte apoptosis and bile lakes in severe obstructive jaundice might impair liver functions. Although decompression of the bile duct has been reported to improve liver functions in animal studies, the mechanism of obstruction differs from that in humans. This study aimed to determine the profiles of hepatocyte apoptosis and bile lakes following bile duct decompression in patients with severe obstructive jaundice in the clinical setting. METHODS: We conducted a "before and after study" on severe obstructive jaundice patients as a model of inhibition of the excessive process by bile duct decompression. Specimens of liver biopsies were taken before and after decompression of the bile duct and then stained by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) to identify hepatocyte apoptosis and by hematoxilin-eosin (HE) to identify bile lakes. All measurements were independently done by 2 observers. RESULTS: Twenty-one severe obstructive jaundice patients were included. In all patients, excessive hepatocyte apoptosis and bile lakes were apparent. After decompression, the hepatocyte apoptosis index decreased from 53.1 (SD 105) to 11.7 (SD 13.6) (P CONCLUSION: Bile duct decompression improves hepatocyte apoptosis and bile lakes in cases of severe obstructive jaundice, similar to the findings in animal studies.

  17. Salt Stress-induced Programmed Cell Death in Rice Root Tip Cells

    Institute of Scientific and Technical Information of China (English)

    Jian-You Li; Ai-Liang Jiang; Wei Zhang

    2007-01-01

    Salt stressed rice root tips were used to investigate the changes of reactive oxygen species (ROS) and antioxidant enzymes at the early stages of programmed cell death (PCD). The results indicated that 500 mmol/L NaCl treatment could lead to specific features of PCD in root tips, such as DNA ladder, nuclear condense and deformation, and transferase mediated dUTP nick end labeling positive reaction, which were initiated at 4 h of treatment and progressed thereafter. Cytochrome c release from mitochondria into cytoplasm was also observed, which occurred at 2 h and was earlier than the above nuclear events. In the very early phase of PCD, an immediate burst in hydrogen peroxide and superoxide anion production rate was accompanied by two-phase changes of superoxide dismutases and ascorbate peroxidase. A short period of increase in the activity was followed by prolonged impairment. Thus,we conclude that salt can induce PCD in rice root tip cells, and propose that in the early phase of rice root tip cell PCD, salt stress-induced oxidative burst increased the antioxidant enzyme activity, which, In turn, scavenged the ROS and abrogated PCD. Also, when the stress is prolonged, the antioxidant system is damaged and accumulated ROS induces the PCD process, which leads to cytochrome c release and nuclear change.

  18. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer’s disease

    Institute of Scientific and Technical Information of China (English)

    Yan Wang; Biao Cai; Jing Shao; Ting-ting Wang; Run-ze Cai; Chang-ju Ma; Tao Han; Jun Du

    2016-01-01

    Genistein is effective against amyloid-βtoxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer’s disease. A rat model of Alzheimer’s disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-βpeptide (25–35). In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg) was given prior to establishing Alzheimer’s dis-ease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genist-ein in Alzheimer’s disease rats. These ifndings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c.

  19. Spermatocyte apoptosis, which involves both intrinsic and extrinsic pathways, explains the sterility of Graomys griseoflavus x Graomys centralis male hybrids.

    Science.gov (United States)

    Rodriguez, Valeria; Diaz de Barboza, Gabriela; Ponce, Ruben; Merico, Valeria; Garagna, Silvia; Tolosa de Talamoni, Nori

    2010-01-01

    Spermatogenic impairment and the apoptotic pathways involved in establishing sterility of male hybrids obtained from crossing Graomys griseoflavus females with Graomys centralis males were studied. Testes from G. centralis, G. griseoflavus and hybrids were compared at different ages. Terminal transferase-mediated dUTP nick-end labelling assay (TUNEL), Fas, Bax and cytochrome c labelling were used for apoptosis evaluation, and calbindin D(28k) staining as an anti-apoptotic molecule. In 1-month-old animals, spermatocytes were positive for all apoptotic markers, but moderate TUNEL (+) spermatocyte frequency was only found in G. centralis. At subsequent ages, the apoptotic markers were downregulated in testes from parental cytotypes, but not in hybrid testes. TUNEL (+) spermatocytes were present at 78% and 44% per tubule cross-section in 2- and 3-month-