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Sample records for cultured pc12 cells

  1. Serum-free culture conditions for serial subculture of undifferentiated PC12 cells.

    Science.gov (United States)

    Ohnuma, Kiyoshi; Hayashi, Yohei; Furue, Miho; Kaneko, Kunihiko; Asashima, Makoto

    2006-03-15

    PC12 cells, a widely used model neuronal cell line, are usually cultured in serum-supplemented medium. This report describes a serum-free medium for the culture of PC12 cells. PC12 cells grown in the two media types had similar growth rates and released dopamine in response to high potassium-induced calcium elevation. However, the levels of dopamine and of dopamine release in cells cultured in the serum-free medium were less than 10% of that in cells cultured in serum-supplemented medium. Dopamine levels recovered within 10 days if cells were returned to serum-supplemented medium, but dopamine release could not be recovered. Nerve growth factor (NGF) induced similar responses in PC12 cells cultured in both media, including phosphorylation of extracellular signal-regulated protein kinases and neurite extension. Transferrin was necessary for survival of neurite-bearing PC12 cells subcultured in serum-free medium and insulin promoted the cells proliferation. Ten days culture with NGF produced a similar increase in neurofilament expression and acetylcholinesterase activity in both media. These results suggest that PC12 in the hormonally defined serum-free media are qualitatively the same as those cultured in serum-supplemented media, and therefore this new culture protocol should enable more precise studies of PC12 cells culture in the absence of confounding unknown factors.

  2. Physiological and morphological studies of rat pheochromocytoma cells (PC12) chemically fused and grown in culture.

    OpenAIRE

    O'Lague, P. H.; Huttner, S L

    1980-01-01

    Cell fusion induced by polyethylene glycol has been used to produce in culture giant multinucleate PC12 cells (up to 300 micron in diameter compared to 10-20 micron for unfused cells). Fused cells, like their unfused counterparts, were found to express various neuronal properties. They contained catecholamines. In the presence of nerve growth factor they extended long processes and expressed Na+, Ca2+, and K+ conductances generally associated with excitable cells. In the absence of nerve grow...

  3. Physiological and morphological studies of rat pheochromocytoma cells (PC12) chemically fused and grown in culture.

    Science.gov (United States)

    O'Lague, P H; Huttner, S L

    1980-03-01

    Cell fusion induced by polyethylene glycol has been used to produce in culture giant multinucleate PC12 cells (up to 300 micron in diameter compared to 10-20 micron for unfused cells). Fused cells, like their unfused counterparts, were found to express various neuronal properties. They contained catecholamines. In the presence of nerve growth factor they extended long processes and expressed Na+, Ca2+, and K+ conductances generally associated with excitable cells. In the absence of nerve growth factor these cells neither grew long processes nor generated Na+-spikes. Other neuronal properties were also observed.

  4. Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

    Science.gov (United States)

    Song, Linyang; Song, Wei; Schipper, Hyman M

    2007-08-01

    The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage. (c) 2007 Wiley-Liss, Inc.

  5. Effects of cultured astrocytes from rat cerebral cortex onthe neurite development of PC12 cells%星形神经胶质细胞对PC12神经元突起生长发育的影响

    Institute of Scientific and Technical Information of China (English)

    莫永炎; 邵紫韫; 陈瑗; 周玫; 张宝

    2004-01-01

    背景:星形细胞对神经元有提供营养、支持及调节突触活性作用,但它对神经元发育的影响还尚不清楚.目的:探讨体外培养的Sprague Dawley大鼠大脑皮质星形细胞对PC12神经元突起生长发育的作用.设计:完全随机设计,对照实验研究.方法:以培养的星形细胞与PC12神经元按不同细胞数目比例(50:1~1:1)共同培养,并用其制备的条件培养液培养PG12细胞.主要观察指标:用快速灵敏的MTT'比色法测定PC12神经元的细胞活力,用光学相差显微镜观察PC12细胞形态学变化.结果:①星形细胞条件培养液可增强PG12细胞活力(MTT测定的A值由0.255±0.012提高到0.510±0.036,P<0.001),却不能促使PC12神经元突起的生出.②当将星形细胞与PC12细胞按30:1~1:1的比例共同培养时,既可提高PC12细胞折光性和光晕又可促使其突起的生长;但按50:1~40:1的比例共同培养时,只观察到提高PC12细胞折光性和光晕,而无促使其突起生长发育的作用.结论:PC12神经元细胞活力的提高与星形细胞分泌到条件培养液中的可溶性因子有关,而PC12神经元突起生长发育可能是和与星形细胞的直接接触以及二者的细胞数目比有关.%BACKGROUND: Although astrocytes are kown to provide structural andtrophic support to neurons and modulate synaptic activity, their role is farfrom being completely understood.OBJECTIVE: To investigate effects of cultured astrocytes fromSprague-Dawley rat cerebral cortex on the neurite development of PC12 cellsderived from rat pheochromocytoma.DESIGN: Completely randomized controlled trial.METHODS: PC12 cells were co-cultured with astrocyte according to dif-ferent astrocytes/neurons ratio(50: 1 -1: 1), or cultured with serum-freeconditioned medium of astrocytes (ACM).MAIN OUTCOME MEASURES: The vitality of PC12 cells was measuredby sensitive MTT method and their morphologic features were observed byOlympus light microscope.RFSULTS: When PC

  6. EFFECTS OF CULTURED ASTROCYTES FROM RAT CEREBRAL CORTEX ON THE DEVELOPMENT OF PC12 CELLS%星形神经胶质细胞对PC12细胞生长发育的影响

    Institute of Scientific and Technical Information of China (English)

    莫永炎; 陈瑗; 周玫; 张宝

    2000-01-01

    在神经系统的生长发育过程中,星形胶质细胞对神经元生长发育的作用是一项重要的研究课题。本文以体外培养的SD大鼠大脑皮质星形胶质细胞与PC12神经元按不同细胞数目比例(50:1~1:1)共同培养,并用其制备的条件培养液培养PC12细胞,用快速灵敏的MTT比色法测定PC12神经元的细胞活力,用光学相差显微镜观察PC12细胞形态学变化。结果显示,星形胶质细胞条件培养液可增强PC12细胞活力(MTT测定的 OD值由0.255±0.012提高到0.510±0.036,P<0.001,且细胞折光性较对照组强),却不能促使PC12神经元突起的生出。将星形胶质细胞与PC12细胞按30:1~1:1的比例共同培养时,既可提高PC12细胞折光性和光晕又可促使其突起的生长;如按50:1~40:1的比例共同培养时,只观察到提高PC12细胞折光性和光晕,而无促使其突起生长发育的作用。本文结果提示,PC12神经元细胞活力的提高与星形胶质细胞分泌到条件培养液中的可溶性因子有关,而PC12神经元突起生长发育可能是和与星形胶质细胞的直接接触以及二者的细胞数且比有关。%To investigate effects of cultured astrocytes from Sprague Dawley rat cerebral cortex on the development of PC12 cellsderived from rat pheochromocytoma, PC12 cells were cocultured with astrocyte according to different astrocytes/neurons ratio(50:1~1:1) , or with serum-free conditioned medium of astrocytes(ACM). The vitality of PC12 cells was measured by sensi-tive MTT method and their morphologic features were observed by Olympus light microscope. The results showed: (1) WhenPC12 cells were cultured with ACM, compared with the control group, the vitality of PC12 cells was increased significantly (0.255+0. 012 vs 0. 510±0. 036, P<0. 001) and the morphological changes were not obvious in the experimental group. (2) WhenPC12 cells were cocultured with astrocyte in the ratio of 30: 1

  7. Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Yue Tang; Yongchun Cui; Fuliang Luo; Xiaopeng Liu; Xiaojuan Wang; Aili Wu; Junwei Zhao; Zhong Tian; Like Wu

    2012-01-01

    In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal α-synuclein accumulation in cells. Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

  8. NGF诱导PC12细胞分化的神经细胞行为观察%Observation on differentiated neuron behaviour induced by NGF in cultured PC12 cells

    Institute of Scientific and Technical Information of China (English)

    丁明杰; 史学义; 张娓; 丁一

    2001-01-01

    Aim:To study the cell division, neurite extension, migration of neurons differentiated by NGF in subcultured PC12 cells.Methods: The Giemsa staining, methyl green-pyronin staining and proliferating cell nuclear antigen immunohistochemical assay were used to study the subcultured PC12 cells.Results: The differentiated neuron appeared different patterns of amitotic figures such as transverse sever, longitudinal split, etc. The proliferating neurons divided in an asymmetric way morphologically and chemically. The differentiated neuron appeared an obvious movement of cell migration and neurite extension.Conclusion: The neurons differentiated by NGF in cultured PC12 cells can divide in amitotic way.The differentiated neuron can form junction by cell migration, neurite extension or cytoplasmic bridge.%目的:观察PC12细胞分化的神经细胞的分裂、突起延伸、迁徙和分化等细胞行为。方法:采用Giemsa、甲绿-派郎宁染色和增殖细胞核抗原(PCNA)免疫组织化学染色方法观察培养的PC12细胞。结果:培养分化的神经细胞呈现横裂、纵裂等不同方式的无丝分裂像;分裂中的神经细胞可呈现形态及化学不对称性;分化的神经细胞有明显的细胞迁徙运动和突起延伸。结论:NGF诱导PC12细胞分化的神经细胞能以无丝分裂方式增殖;分化神经元可以通过细胞迁徙、突起延伸及共同胞质桥等方式相连接。

  9. Evaluation of silicon nitride as a substrate for culture of PC12 cells: an interfacial model for functional studies in neurons.

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    Johan Jaime Medina Benavente

    Full Text Available Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions.

  10. Inhibitory effects of multiwall carbon nanotubes with high iron impurity on viability and neuronal differentiation in cultured PC12 cells.

    Science.gov (United States)

    Meng, Li; Jiang, Aihua; Chen, Rui; Li, Chen-zhong; Wang, Liming; Qu, Ying; Wang, Peng; Zhao, Yuliang; Chen, Chunying

    2013-11-08

    The increasing use of carbon nanotubes (CNTs) in biomedical applications has garnered a great concern on their potential negative effects to human health. CNTs have been reported to potentially disrupt normal neuronal function and they were speculated to accumulate and cause brain damage, although a lot of distinct and exceptional properties and potential wide applications have been associated with this material in neurobiology. Fe impurities strapped inside the CNTs may be partially responsible for neurotoxicity generation. In the present study, we selected rat pheochromocytoma (PC12) cells to investigate and compare the effects of two kinds of multiwall carbon nanotubes (MWCNTs) with different concentrations of Fe impurities which usually come from the massive production of CNTs by chemical vapor deposition. Exposure to Fe-high MWCNTs can reduce cell viability and increase cytoskeletal disruption of undifferentiated PC12 cells, diminish the ability to form mature neurites, and then adversely influence the neuronal dopaminergic phenotype in NGF-treated PC-12 cells. The present results highlight the critical role of iron residue in the adverse response to MWCNTs exposure in neural cells. These findings provide useful information for understanding the toxicity and safe application of carbon nanotubes.

  11. Song Bu Li Decoction, a Traditional Uyghur Medicine, Protects Cell Death by Regulation of Oxidative Stress and Differentiation in Cultured PC12 Cells

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    Maitinuer Maiwulanjiang

    2013-01-01

    Full Text Available Song Bu Li decoction (SBL is a traditional Uyghur medicinal herbal preparation, containing Nardostachyos Radix et Rhizoma. Recently, SBL is being used to treat neurological disorders (insomnia and neurasthenia and heart disorders (arrhythmia and palpitation. Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness. Here, we aimed to evaluate the protective and differentiating activities of SBL in cultured PC12 cells. The pretreatment of SBL protected the cell against tBHP-induced cell death in a dose-dependent manner. In parallel, SBL suppressed intracellular reactive oxygen species (ROS formation. The transcriptional activity of antioxidant response element (ARE, as well as the key antioxidative stress proteins, was induced in dose-dependent manner by SBL in the cultures. In cultured PC12 cells, the expression of neurofilament, a protein marker for neuronal differentiation, was markedly induced by applied herbal extract. Moreover, the nerve growth factor- (NGF- induced neurite outgrowth in cultured PC12 cells was significantly potentiated by the cotreatment of SBL. In accord, the expression of neurofilament was increased in the treatment of SBL. These results therefore suggested a possible role of SBL by its effect on neuron differentiation and protection against oxidative stress.

  12. Clivorine, an otonecine pyrrolizidine alkaloid from Ligularia species, impairs neuronal differentiation via NGF-induced signaling pathway in cultured PC12 cells.

    Science.gov (United States)

    Xiong, Aizhen; Yan, Artemis Lu; Bi, Cathy W C; Lam, Kelly Y C; Chan, Gallant K L; Lau, Kitty K M; Dong, Tina T X; Lin, Huangquan; Yang, Li; Wang, Zhengtao; Tsim, Karl W K

    2016-08-15

    Pyrrolizidine alkaloids (PAs) are commonly found in many plants including those used in medical therapeutics. The hepatotoxicities of PAs have been demonstrated both in vivo and in vitro; however, the neurotoxicities of PAs are rarely mentioned. In this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PC12 cells. PC12 cell line was employed to first elucidate the neurotoxicity and the underlying mechanism of clivorine, including cell viability and morphology change, neuronal differentiation marker and signaling pathway. PC12 cells were challenged with series concentrations of clivorine and/or nerve growth factor (NGF). The cell lysates were collected for MTT assay, trypan blue staining, immunocytofluorescent staining, qRT-PCR and western blotting. Clivorine inhibited cell proliferation and neuronal differentiation evidenced by MTT assay and dose-dependently reducing neurite outgrowth, respectively. In addition, clivorine decreased the level of mRNAs encoding for neuronal differentiation markers, e.g. neurofilaments and TrkA (NGF receptor). Furthermore, clivorine reduced the NGF-induced the phosphorylations of TrkA, protein kinase B and cAMP response element-binding protein in cultured PC12 cells. Taken together, our results suggest that clivorine might possess neurotoxicities in PC12 cells via down-regulating the NGF/TrkA/Akt signaling pathway. PAs not only damage the liver, but also possess neurotoxicities, which could possibly result in brain disorders, such as depression. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor

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    Sherry L. Xu

    2012-01-01

    Full Text Available Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer’s disease and depression.

  14. Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor.

    Science.gov (United States)

    Xu, Sherry L; Choi, Roy C Y; Zhu, Kevin Y; Leung, Ka-Wing; Guo, Ava J Y; Bi, Dan; Xu, Hong; Lau, David T W; Dong, Tina T X; Tsim, Karl W K

    2012-01-01

    Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-)induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.

  15. Study of effect of oxygen/glucose-deprived culture on the brain-pancreas relative protein in PC12 cells and the mechanism

    Institute of Scientific and Technical Information of China (English)

    Yan-huaLIN; LuTIE; Ai-huaLIU; Xue-junLI

    2004-01-01

    AIM: To study the effect of oxygen/glucose-deprived (OGD)culture on the expression of a novel protein, brain-pancreas relative protein (BPRP), and the possible regulating mechanism in vitro. BPRP was a key protein found in our previous study of cerebral ischemia. METHODS: PC12 cells was selected and exposed to the Eagle's solution containing 1 mmol/L Na2S2O4 for

  16. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane

    The brain is the center of the nervous system, where serious neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s are products of functional loss in the neural cells (1). Typical techniques used to investigate these diseases lack precise control of the cellular surroundings...

  17. Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

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    Giada Graziana Genchi

    2015-01-01

    Full Text Available Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g. Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

  18. Characterization of RNA interference in rat PC12 cells

    DEFF Research Database (Denmark)

    Thonberg, Håkan; Schéele, Camilla C; Dahlgren, Cecilia

    2004-01-01

    Double-stranded RNA can initiate post transcriptional gene silencing in mammalian cell cultures via a mechanism known as RNA interference (RNAi). The sequence-specific degradation of homologous mRNA is triggered by 21-nucleotide RNA-duplexes termed short interfering RNA (siRNA). The homologous...... of the rat Golgi-ER protein 95 kDa (GERp95), an Argonaute family protein, by siRNA methodology. After GERp95-ablation, sequential knockdown of NPY by siRNA was shown to be impaired. Thus, we report that the GERp95 protein is functionally required for RNAi targeting NPY in rat PC12 cells....

  19. Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures

    DEFF Research Database (Denmark)

    Tanner, V A; Ploug, Thorkil; Tao-Cheng, J H

    1996-01-01

    We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide...

  20. Damaged PC12 cells in Transwell culture system for promoting the transdifferentiation of bone marrow stromal stem cells into neuron-like cells%Transwell培养体系中受损PC12细胞促进骨髓间质干细胞向神经元样细胞的转分化

    Institute of Scientific and Technical Information of China (English)

    周进; 罗晓光; 张朝东

    2007-01-01

    BACKGROUND: Drug treatment has unsatisfactory effect on Alzheimer disease, while many studies have indicated that the transplantation of bone marrow stromal cells (MSCs) is effective on Parkinson disease, cerebral ischemia, etc., but the mechanism is still unclear.OBJECTIVE: To imitate transplantation environment by co-culture of amyloid β1-40 (Aβ1-40) damaged PC12 cells and MSCs, observe the effect of bi-directional information feedback in the microenvironment on the transdifferentiation of MSCs to nerve cells, and observe its protective effect on the apoptosis of damaged PC12 cells.DESTGN: A comparative observation.SETTING: Department of Neurology, China Medical University.MATERIALS: SD rats of 2-3 weeks after birth either male or female were used. PC12 cell lines were purchased from the Institute of Cell Biology, Chinese Academy of Sciences; neuro-specific enolase (1:50, Boster, Wuhan);Methyl-thiazol-tetrazolium (MTT) 15 μL (terminal concentration of 0.5 g/L).METHODS: The experiment was carried out in the Experimental Center (provincial experimental animal center) of China Medical University from June to July in 2004. Bilateral femurs were aseptically removed from 1 SD rat, and MSCs were identified using CD44 antibody immunofiuorescently. PC12 was used to replace nerve cells. The PC12 cells were stimulated by Aβ1-40 then transferred by Transwell. There were 5 groups: Group A: normally cultured PC12 co-cultured with MSCs; Group B: Aβ1-40 stimulated PC12 co-cultured with MSCs; Group C: normal PC12 supernatant+MSCs; Group D: damaged PC12 supernatant+MSCs; Group E: MSCs cultured with common medium 1640.MAIN OUTCOME MEASURES:Routine immunohistochemical staining was performed. NSE positive cells were observed under inverted fluorescence microscope, 10 visual fields (200×) were randomly selected to count the positive cells. MTT metabolic rate was used to detect the proliferation of MSCs in each group. The differences of measurement data were compared using the

  1. Rho激酶抑制剂诱导PC12PC12Adh细胞突起生长的差异比较%Differences of neurite outgrowth induced by rho kinase inhibitors between in PC12 cell line and PC12 Adh cell line

    Institute of Scientific and Technical Information of China (English)

    却翎; 段为钢; 张陆勇; 江振洲

    2011-01-01

    PC12 cell line is one of the basic tool cell lines used in neural differentiation research.The cell line is able to grow neurite by exposure to rho kinase ( ROCK) inhibitors.Recently, American Type Culture Collection ( ATCC) provided PC12 cell line and PC12 Adh cell line.The main aim of the study was to clarify the differences of the two cell lines in neurite outgrowth induced by ROCK inhibitors.PC12 cell line and PC12 Adh cell line (from ATCC) were treated with nerve growth factor (NGF,1000 ng/mL) or ROCK inhibitors (Y27632 of 33 μmol/L and fasudil of 33 μmol/L) for 1 -4 days.NGF was able to promote neurite outgrowth both in PC12 cell line and PC12 Adh cell line,while ROCK inhibitors was able to do so only in PC12 Adh cell line.These results suggested that PC12 Adh cell line is more suitable for ROCK inhibitors study in neural differentiation.%PC12细胞是研究神经分化最常用的细胞之一.在rho激酶(ROCK)抑制剂的作用下,PC12细胞能够长出神经样突起.最近,美国菌种保存中心(ATCC)同时提供PC12细胞和PC12 Adh细胞.研究的主要目的是观察ROCK抑制剂诱导这2种细胞长突起是否存在差异.PC12细胞和PC12Adh细胞按照ATCc方法进行培养,用神经生长因子(NGF,1 000 ng/mL)或ROCK抑制剂(33 μmol/L Y27632,33 μmol/L法舒地尔)处理细胞1~4 d.结果发现NGF能够诱导这2种细胞生长突起,而ROCK抑制剂只诱导PC12Adh细胞长突起,对PC12细胞不明显.因此,ROCK抑制剂诱导这2种细胞突起生长存在明显差异,PC12Adh细胞更适合用于ROCK抑制剂的神经诱导分化实验.

  2. Serum containing Tongqiaohuoxue decoction suppresses glutamate-induced PC12 cell injury☆

    OpenAIRE

    Wang, Ning; Deng, Yi; Wei, Wei; Song, Lihua; Wang, Yan

    2012-01-01

    Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctorius, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase...

  3. Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen-Glucose Deprivation (OGD).

    Science.gov (United States)

    Liu, Zhao; Huang, Yue-yang; Wang, Yu-xiang; Wang, Hong-gang; Deng, Fei; Heng, Bin; Xie, Lai-hua; Liu, Yan-qiang

    2015-01-01

    To elucidate the role of Zn(2+)-associated glutamate signaling pathway and voltage-dependent outward potassium ion currents in neuronal death induced by hypoxia-ischemia, PC12 cells were exposed to Oxygen-Glucose Deprivation (OGD) solution mimicking the hypoxic-ischemic condition in neuron, and the effect of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn(2+) chelating agent on OGD-induced neuronal death was assessed in the present study. The cell survival rate, apoptosis status, potassium channel currents, intracellular free glutamate concentration and GluR2 expression in PC12 cells exposed to OGD in the absence or presence of TPEN for different time were investigated. The results showed that OGD exposure increased apoptosis, reduced the cell viability (P concentration of intracellular glutamate (P cells. TPEN partially reversed the influence resulted from OGD. These results suggest that OGD-induced cell apoptosis and/or death is mediated by the alteration in glutamate signaling pathway and the voltage-dependent outward potassium ion currents, while TPEN effectively prevent cell apoptosis and/or death under hypoxic-ischemic condition.

  4. Aspartame-induced apoptosis in PC12 cells.

    Science.gov (United States)

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity.

  5. Anti-oxidative effects of the biennial flower of Panax notoginseng against H2O2-induced cytotoxicity in cultured PC12 cells

    Directory of Open Access Journals (Sweden)

    Chen Jijun

    2010-10-01

    Full Text Available Abstract Background Radix notoginseng is used in Chinese medicine to improve blood circulation and clotting; however, the pharmacological activities of other parts of Panax notoginseng have yet to be explored. The present study reports the anti-oxidative effects of various parts of Panax notoginseng. Methods Various parts of Panax notoginseng, including the biennial flower, stem-leaf, root-rhizome, fiber root and sideslip, were used to prepare extracts and analyzed for their anti-oxidation effects, namely suppressing xanthine oxidase activity, H2O2-induced cytotoxicity and H2O2-induced ROS formation. Results Among various parts of the herb (biennial flower, stem-leaf, root-rhizome, fiber root and sideslip, the water extract of the biennial flower showed the strongest effects in (i inhibiting the enzymatic activity of xanthine oxidase and (ii protecting neuronal PC12 cells against H2O2-induced cytotoxicity. Only the water extracts demonstrated such anti-oxidative effects while the ethanol extracts did not exert significant effects in suppressing xanthine oxidase and H2O2-induced neuronal cytotoxicity. Conclusions The present study demonstrates the biennial flower of Panax notoginseng to have neuroprotection effect on cultured neurons and the underlying protection mechanism may involve anti-oxidation.

  6. Induction of cytoprotective autophagy in PC-12 cells by cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Qiwen [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China); Bijie Pilot Area Research Institute of Bijie University, Bijie 551700 (China); Zhu, Jiaqiao; Zhang, Kangbao; Jiang, Chenyang; Wang, Yi; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Gu, Jianhong [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China); Liu, Zongping, E-mail: liuzongping@yzu.edu.cn [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China)

    2013-08-16

    Highlights: •Cadmium can promote early upregulation of autophagy in PC-12 cells. •Autophagy precedes apoptosis in cadmium-treated PC-12 cells. •Cadmium-induced autophagy is cytoprotective in PC-12 cells. •Class III PI3K/beclin-1/Bcl-2 signaling pathway plays a positive role in cadmium-triggered autophagy. -- Abstract: Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.

  7. Investigation of Apoptosis Induction in Differentiated PC-12 Cells after Exposure to Hydrostatic Pressure

    Directory of Open Access Journals (Sweden)

    S. Sadri

    2008-01-01

    Full Text Available Objective: Hydrostatic pressure is crucial component of cell environment andfundamental physical quantity, also it is the main factor of both cell integrity andfunction. Pressure variation disorder, beyond physiological limits, may lead topathological states. In this study, we examined the effect of hydrostatic pressureon apoptosis induction, viability, morphology, adhesion potency to substrate andmigration of differentiated PC-12 cells.Materials and Methods: PC-12 as a neuronal cell line maintained in RPMI1640 culture medium supplemented with 10% fetal bovine serum. Staurosporinewas used for differentiating of mitotic PC-12 cells to post mitotic anddifferentiated neuronal cells. Exclusion Dye was used for viability assay, totalneurite length of each cell as well as morphometry. TUNEL staining was alsoperformed for apoptosis detection, adhesion potency of cells to substrate andevaluation of cell migration.Results: Hydrostatic pressure, over physiological limits, induced apoptosis indifferentiated PC-12 cells. It changed cell viability gradually and reduction happenedsignificantly after 24 hours (p<0.05. In compare to the control group, hydrostaticpressure reduced total neurite length, adhesion potency to substrate and migrationof cells in the examined group (p<0.05.Conclusion: Hydrostatic pressure induced apoptosis in differentiated PC-12 cellsas a result of inappropriate interaction between cells and substrate. We proposethat apoptosis in differentiated PC-12 cells may be an anoikis causing to lose theattachment to the substrate.

  8. Melatonin attenuates 1-methyl-4-phenylpyridinium-induced PC12 cell death

    Institute of Scientific and Technical Information of China (English)

    Jin-feng BAO; Ren-gang WU; Xiao-ping ZHANG; Yan SONG; Chang-ling LI

    2005-01-01

    Aim: To explore the effect of melatonin on PC12 cell death induced by 1-methyl-4-phenylpyridinium (MPP+). Methods: MTT assay, lactate dehydrogenase (LDH)efflux assay, and immunohistochemistry methods were used to measure neurotoxicity of PC 12 cells treated acutely with MPP+ in low glucose and high glucose conditions, and to assess the neuroprotective effect of melatonin on PC 12 cell death induced by MPP+. Results: In a low glucose condition, MPP+ significantly induced PC 12 cell death, which showed time and concentration dependence. In a serum-free low glucose condition, the percentages of viability of cells treated with MPP+ for 12, 24, 48, 72, and 96 h were 85.1%, 75.4%, 64.9%, 28.15%, and 9%, respectively. The level of LDH in the culture medium increased and tyrosine hydroxylase positive (TH+) cell count decreased. However, in a serum-free high glucose condition, MPP+ did not significantly induce PC12 cell death compared with control at various concentrations and time regimens. When the cells were preincubated with melatonin 250 μmol/L for 48, 72, and 96 h in a serum-free low glucose condition, cell survival rate significantly increased to 78.1%, 58.8%, and 31.6%, respectively. Melatonin abolished the LDH leakage of cells treated with MPP+ and increased TH+ cells count. Conclusion: MPP+ caused concentrationdependent PC12 cell death. The level of glucose was an important factor to MPP+induced dopaminergic PC12 cell death. Low glucose level could potentiate MPP+toxicity, while high glucose level could reduce the toxicity. In addition, melatonin attenuated PC12 cell death induced by MPP+.

  9. 木豆叶醇提物对皮质酮诱导的PC12细胞损伤的保护作用%Protective Effect of Alcohol Extract of Cajanus cajan on Corticosterone-induced Lesion in Cultured PC12 Cells

    Institute of Scientific and Technical Information of China (English)

    姜保平; 刘亚旻; 李宗阳; 宋波; 潘瑞乐

    2012-01-01

    建立皮质酮诱导的PC12细胞损伤模型并观察木豆叶醇提物及不同组分对皮质酮损伤PC12细胞的保护作用.以100μ mol/L的皮质酮诱导PC12细胞损伤;损伤后的PC12细胞与木豆叶醇提物及不同组分孵育24h,通过形态学观察、MTT检测、LDH测定,研究各组分对皮质酮损伤PC12细胞的保护作用.结果表明,PC12细胞与皮质酮孵育48 h后细胞存活率明显降低,而LDH水平显著升高.而加入木豆叶醇提物及各组分时上述效果明显减轻,且存在明显的剂量依赖关系.从以上结果可知,木豆叶醇提物及不同组分对皮质酮损伤的PC12细胞均有保护作用,且醇提物的效果最好.%The aim of this paper was to establish the corticosterone-induced damage model in PC12 cells and study the protective activity of alcohol extracts and its fractions from Cajanus cajan on corticosterone-induced PC12 cells. PC12 cells were exposed to different concentrations of corticosterone to determine the optimum concentration of establishing the damage model,and then the injured PC12 cells were incubated with the alcohol extract and its fractions for 24 h. After that the neuroprotective effect on injured PC12 cells was investigated by morphological observation, LDH detection and MTT determination. The results showed that the survival rate of PC12 cells and the cells exposed to corticosterone (100 (μmol/L) for 48 h decreased obviously,with an increase in lactate dehydrogenase (LDH) level,but the effect became weak when the alcohol extracts and its fractions from the C. cajan was added to the PC 12 cells,and it was in a dose-dependent manner. The author suggests that the alcohol extract and its fractions from C. cajan can generate a neuroprotective effect on injured PC 12 cells induced by corticosterone,and that of alcohol extract is the most significant.

  10. Mutant alpha-synuclein and autophagy in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Kangyong Liu; Chunfeng Liu; Chuancheng Ren; Yaping Yang; Liwei Shen; Xuezhong Li; Fen Wang; Zhenghong Qin

    2011-01-01

    Several studies have demonstrated that overexpression of mutant α-synuclein in PC12 cells is related to occurrence of autophagy.The present study established mutant a-synuclein (A30P)-transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively.Results demonstrated that mutant o-synuclein resulted in cell death via autophagy and involved α-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity.Mutant α-synuclein (A30P) also mediated toxicity of1-methyl-4-phenylpyridinium ion.Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted o-synuclein aggregation and cell death.To further determine the role of autophagy due to mutant a-synuclein, the present study measured expression of microtubule-associated protein light chain 3.Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3,while rapamycin promoted its expression.These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.

  11. Effects of epigallocatechin gallate on ultra-violet-induced cell death in PC12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hideo; Seki, Sakiko; Sakamoto, Naotaka; Nakagawa, Shigeki [Nihon Univ., Tokyo (Japan). School of Medicine

    2002-04-01

    We examined the effects of catechin on ultra-violet-induced cell death in PC12 cells. PC12 cells were irradiated by ultra-violet C (254 nm) (UVC). We found that the lactate dehydrogenase (LDH) activities in culture media and lipid peroxide in PC12 cells, which indicate cell death and cell membrane damage, respectively, were increased by UVC irradiation in a time-dependent manner. Cell death was gradually stimulated for 9 hours of cultivation after a UVC irradiation period of 10 or 30 min. Epigallocatechin gallate (EGCG), which is one of the main catechins found in green tea, suppressed the increase in LDH activity in culture medium and also inhibited the formation of lipid peroxide. I{kappa}B, a member of the cell death signaling system, was phosphorylated at 1 hour after 10 min of UVC irradiation. Stimulation of phosphorylation of I{kappa}B by UVC was suppressed by the addition of EGCG. We concluded that EGCG protects the PC12 cell from cell damage caused by UVC irradiation. (author)

  12. Mechanisms of rotenone-induced neurotoxicity in PC 12 cells

    Institute of Scientific and Technical Information of China (English)

    Wei Han; Lizhong Sun; Jiafeng Chen; Ming Chang; Hongyan Huo; Linsen Hu

    2008-01-01

    BACKGROUND: Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson's disease remains unclear.OBJECTIVE: To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level.DESIGN, TIME AND SETTING: A controlled proteomics study was performed at the Department of Nearology, First Hospital, Jilin University between March 2006 and March 2007.MATERIALS: PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China.Rotenone was provided by Sigma, USA.METHODS: PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μ mol/L rotenone, or the same amount of Dulbecco's modified eagle's medium (DMEM), was added in the experimental and control conditions, respectively.MAIN OUTCOME MEASURES: Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was used.RESULTS: In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition (P < 0.01 ). Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition (P < 0.01 ), as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein

  13. Serum containing Tongqiaohuoxue decoction suppresses glutamate-induced PC12 cell injury.

    Science.gov (United States)

    Wang, Ning; Deng, Yi; Wei, Wei; Song, Lihua; Wang, Yan

    2012-05-25

    Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctorius, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase and Na(+)-K(+)-ATPase activity, induced by glutamate. It also reduced the concentration of malondialdehyde, enhanced the mitochondrial transmembrane potential, inhibited the elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase II. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells.

  14. Serum containing Tongqiaohuoxue decoction suppresses glutamate-induced PC12 cell injury

    Institute of Scientific and Technical Information of China (English)

    Ning Wang; Yi Deng; Wei Wei; Lihua Song; Yan Wang

    2012-01-01

    Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctorius, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase and Na+-K+-ATPase activity, induced by glutamate. It also reduced the concentration of malondialdehyde, enhanced the mitochondrial transmembrane potential, inhibited the elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase II. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells.

  15. Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Li-li TANG; Rui WANG; Xi-can TANG

    2005-01-01

    Aim: To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). Methods: After being treated with 10 μmol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH)release. AChE activity, mRNA and protein expression were measured by the Ellman's method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Results: Treatment of PC 12 cells with 10 μmol/L HupA for 48 h markedly increased the number of neuritebearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 μmol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 μmol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Conclusion: Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

  16. Differentiation of PC12 cells induced by total saponin of panax ginseng%人参总皂苷对PC12细胞分化的影响

    Institute of Scientific and Technical Information of China (English)

    李晗宇; 王顺和; 郑娟; 姜蓉

    2011-01-01

    目的:研究人参总皂苷(Total saponin ofpanax ginseng,TSPG)诱导PC12细胞神经元性分化的作用.方法:体外培养PC12细胞,观察PC12细胞在TSPG的影响下细胞发生的形态学改变.诱导48 h透射电镜观察突触连接形成情况,72 h利用免疫细胞化学技术,观察TSPG作用后PC12细胞向MAP-2(Microtubule-associatedprotein 2)阳性细胞分化的情况.结果:TSPG作用后的PC12细胞其突起长度和细胞直径较对照组明显增长和增大[长度由(13.95±2.59)μm增长至(30.33±3.82)μm;直径由(8.25±1.82)μm提高到(14.33±2.84)μm,P<0.05].TSPG能促进PC12细胞形成突触连接.TSPG组表达MAP2的阳性细胞率明显高于对照组(由4.55%提高到12.22%,P<0.05).结论:TSPG具有诱导PC12细胞神经元性分化的作用.%Objective: To study the potential ahility of TSPG ( Total saponin of panax ginseng ) to induce the neuronal differentiation of PC12 cells. Methods : PC12 cells were cultured in vitro , and their morphological changes under the TSPG were observed. The synaptic linkage were ohserved by transmitting electronic microscope after the PC12 cells were exposed to TSPG for 48 h and the expression of MAP-2 was detected by immunocytochemistry after 72 h. Results : The length of neurite and max diameter of PC12 cells in TSPG group were obviously longer and bigger than those in control group ((13.95 ± 2.59) μm to ( 30.33 ± 3.82) μ m; ( 8.25 ± 1.82) μm to ( 14.33 ± 2.84) μm , P < 0.05 ) TSPG could induce PC12 cells to form the synaptic linkage.compared with the control group , the numher of MAP-2 positive cells was significantly increased in TSPC group (4.55%to 12.22% ,P < 0.05 ) .Conclusion : TSPG can induce the neuronal differentiation of PC12 cells.

  17. Minocycline protects the apoptosis of PC12 cells induced by 1-methyl-4-phenylpyridinium

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective: To explore the protective effect of minocycline on the apoptosis of cellular parkinsonism models induced by MPP+ . Methods: Using PC12 cells as the apoptotic model of dopaminergic neurons, MC and MPP+ were added into the culture medium of PC12 cells, and using MTT to assay the cell viability and metabolic state; The cells apoptosis was assayed by electrophoresis method and using flow cytometry FACS to assay the apoptosis ratio. Results: Added the MPP+ to get the concentration of 10μmol/L, the cellular parkinsonism model of apoptosis had been prepared. The pre-treatment of MC (100 μmol/L) could significantly increase the PC12 cell viability. The apoptosis ratio of MC + MPP+ group was significantly lower than that of MPP+ group, but was still significantly higher than that of control group. Conclusion: MC may protect the cell apoptosis induced by MPP+ to some extent.

  18. ADENO-ASSOCIATED VIRUS INTRODUCED INTEGRATION AND EXPRESSION OF FOREIGN GENES IN PC12 CELLS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous system. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plasmids were encapsidated as recombinant virions. PC12 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus of AAV vectors by Southern blot and transcript situation of foreign genes by dot blot. Results The hybridization tests showed that AAV introduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PC12cells. Conclusion AAV vectors can serve as high efficiency vectors of target genes in neuronal PC12 cells.

  19. Chorein Sensitive Dopamine Release from Pheochromocytoma (PC12 Cells

    Directory of Open Access Journals (Sweden)

    Sabina Honisch

    2015-12-01

    Full Text Available Background: Chorein, a protein supporting activation of phosphoinositide 3 kinase (PI3K, participates in the regulation of actin polymerization and cell survival. A loss of function mutation of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A leads to chorea-acanthocytosis (ChAc, a neurodegenerative disorder with simultaneous erythrocyte akanthocytosis. In blood platelets chorein deficiency has been shown to compromise expression of vesicle-associated membrane protein 8 (VAMP8 and thus degranulation. The present study explored whether chorein is similarly involved in VAMP8 expression and dopamine release of pheochromocytoma (PC12 cells. Methods: Chorein was down-regulated by silencing in PC12 cells. Transmission electron microscopy was employed to quantify the number of vesicles, RT-PCR to determine transcript levels, Western blotting to quantify protein expression and ELISA to determine dopamine release. Results: Chorein silencing significantly reduced the number of vesicles, VAMP8 transcript levels and VAMP8 protein abundance. Increase of extracellular K+ from 5 mM to 40 mM resulted in marked stimulation of dopamine release, an effect significantly blunted by chorein silencing. Conclusions: Chorein deficiency down-regulates VAMP8 expression, vesicle numbers and dopamine release in pheochromocytoma cells.

  20. Enhanced PC12 cells proliferation with self-assembled S-layer proteins scaffolds.

    Science.gov (United States)

    Babolmorad, Ghazal; Emtiazi, Giti; Ghaedi, Kamran; Jodeiri, Mohamad

    2015-01-01

    Finding 3D biocompatible and biodegradable scaffold is important in tissue engineering which plays a critical role in transplanting methods. Several biomaterials, such as poly-L-lactide, poly(lactic-co-glycolic acid), poly(L-lactic acid)/poly(lactide-co-glycolide), alginate, collagen gel, and so on, have been applied as scaffold to culture cells in 3D environment. The most significant problem of the synthetic materials is lack of biocompatibility and bioactivity. Herein, self assemble S-layer proteins are used as a scaffold for PC12 cells culturing. For this purpose, S-layer protein was extracted from Bacillus coagulans HN68. Then, extracted S-layer was studied by SDS page and AFM. Using MTS test and Immunochemistry staining methods, the effect of self assembled S-layer scaffold on proliferation of PC12 cells was assayed. This study provides that S-layer could be an appropriate scaffold for PC12 cells culturing. Even though poly-L-ornithine is a common scaffold in PC12 cells culturing, the results show that (PLO)/S-layer is more protective.

  1. Codonopsis pilosula (Franch) Nannftotal alkaloids potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    LIUJian-Hui; BAOYong-Ming; SONGJi-Jun; ANLi-Jia

    2003-01-01

    AIM:To explore the effect of Codonopsis pilosula (Franch) Nannf total alkaloids (DSA) on differentiation inducedby nerve growth factor (NGF) in PC12 cells. METHODS: After culturing PC12 cells with DSA in the presence orabsence of NGF, neurite outgrowth in PC12 cells and correlated protein kinases were assayed. RESULTS: DSAalone did not exhibit neuritogenic activity, but caused a significant enhancement of NGF (2 μg/L)-induced neuriteoutgrowth in PC12 cells, and increased the phosphorylation of mitogen-activated protein kinase (MAPK).Furthermore, this enhancing effect was completely blocked by a specific MAPK kinase inhibitor, PD98059.CONCLUSION: DSA enhanced the NGF-induced neurite outgrowth in PC12 cells by amplifying an up-streamstep of the MAPK-dependent signaling pathway.

  2. Enhanced proliferation of PC12 neural cells on untreated, nanotextured glass coverslips

    Science.gov (United States)

    Islam, Muhymin; Atmaramani, Rahul; Mukherjee, Siddhartha; Ghosh, Santaneel; Iqbal, Samir M.

    2016-10-01

    Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro- and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter—towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma.

  3. Susceptibility of naïve and differentiated PC12 cells to Japanese encephalitis virus infection.

    Science.gov (United States)

    Li, Jian-Ri; Wu, Chih-Cheng; Chang, Cheng-Yi; Ou, Yen-Chuan; Lin, Shih-Yi; Wang, Ya-Yu; Chen, Wen-Ying; Raung, Shue-Ling; Liao, Su-Lan; Chen, Chun-Jung

    2017-02-01

    Japanese encephalitis is a mosquito-borne disease caused by Japanese encephalitis virus (JEV) infection. Although JEV infects and replicates in cells with multiple tissue origins, neurons are the preferential cells for JEV infection. Currently, the identities of JEV cell tropism are largely unclear. To gain better insight into the underlying identities of JEV cell tropism, this study was designed to compare the JEV cell tropism with naïve or differentiated PC12 cells. Through nerve growth factor-differentiated PC12 cells, we discovered that JEV efficiently replicated in differentiated PC12 cells rather than naïve cells. Mechanistic studies revealed that viral adsorption/attachment seemed not to be a crucial factor. Supporting data showed that antagonizing postreceptor intracellular signaling of interferons, along with the activation of suppressor of cytokine signaling-3 (SOCS3) expression and protein tyrosine phosphatase activity, were apparent in differentiated PC12 cells after JEV infection. Independent of differentiating inducing agents, the upregulation of SOCS3 expression and protein tyrosine phosphatase activity, as well as preferential JEV tropism, were common in JEV-infected differentiated PC12 cells. Using cultured primary neurons, JEV efficiently replicated in embryonic neurons rather than adult neurons, and the preference was accompanied by higher SOCS3 expression and protein tyrosine phosphatase activity. Given that both SOCS3 and protein tyrosine phosphatases have been implicated in the process of neuronal differentiation, JEV infection seems to not only create an antagonizing strategy to escape host's interferon antiviral response but also takes advantage of cellular machinery to favor its replication. Taken together, current findings imply that dynamic changes within cellular regulators of antiviral machinery could be accompanied by events of neuronal differentiation, thus concurrently playing roles in the control of JEV cell tropism and

  4. KCl stimulation increases norepinephrine transporter function in PC12 cells.

    Science.gov (United States)

    Mandela, Prashant; Ordway, Gregory A

    2006-09-01

    The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

  5. Differentiation of PC12 Cells Results in Enhanced VIP Expression and Prolonged Rhythmic Expression of Clock Genes

    DEFF Research Database (Denmark)

    Pretzmann, C.P.; Fahrenkrug, J.; Georg, B.

    2008-01-01

    PC12 cultures lasted only one 24-h period, while in differentiated cultures, the rhythms continued for at least 3 days. Thus, neuronal differentiation provided PC12 cells the ability to maintain rhythmicity for an extended period. Both vasoactive intestinal polypeptide (VIP) and its receptor VPAC(2......) are expressed in the suprachiasmatic nucleus (SCN), and in agreement with VIP signaling being crucial for maintenance of rhythmicity, we found both VIP and VPAC(2) mRNA increased after differentiation of PC12 cells. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts time- and concentration-dependent......To examine for circadian rhythmicity, the messenger RNA (mRNA) amount of the clock genes Per1 and Per2 was measured in undifferentiated and nerve-growth-factor-differentiated PC12 cells harvested every fourth hour. Serum shock was needed to induce circadian oscillations, which in undifferentiated...

  6. Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway

    Science.gov (United States)

    Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun

    2016-11-01

    In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5 ± 1.2 μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

  7. 人工合成多肽FGL对神经细胞模型PC12细胞增殖与凋亡的影响%Effects of synthetic peptides FG loop on PC12 cells proliferation and apoptosis

    Institute of Scientific and Technical Information of China (English)

    付洪龙; 马学晓; 于腾波; 陈伯华; 李宁

    2011-01-01

    背景:FGL是NCAM的核心活性多肽片段,可直接作用于成纤维细胞生长因子受体1,激活NCAM的信号传导途径.目的:观察FGL人工合成多肽联合培养对PC12细胞增殖和凋亡的作用.方法:将培养的PC12细胞分为对照组和实验组,实验组预先加入1%的FGL多肽溶液.分别于培养1,3,5,7,9 d采用细胞计数试剂8法检测细胞增殖情况.将PC12细胞分为正常组、实验组和损伤组,损伤组加入H2O2刺激16 h.实验组加入H2O2与FGL人工合成多肽刺激16 h,流式细胞仪检测细胞凋亡,荧光定量PCR法检测PC12中的核转录因子κB mRNA表达.结果与结论:FGL人工合成多肽与PC12复合培养细胞生长良好,可明显促进PC12细胞的活性并且减低PC12 细胞凋亡并可明显降低凋亡模型中PC12细胞核转录因子κB基因的表达.说明FGL多肽可以明显促进PC12细胞增殖,并可以抑制PC12细胞凋亡.%BACKGROUND: FG loop (FGL) is a core active peptide fragment of neural cell adhesion molecule (NCAM), which can directly act on fibroblast growth factor receptor 1 (FGFR1) to activate NCAM signal pathway.OBJECTIVE: To observe the effects of synthetic peptides FGL on PC12 cells proliferation and apoptosis.METHODS: ①PC12 cells proliferation and apoptosis: The cultured PC12 cells were divided into control group and experiment group. The experimental group was added with 1% FGL peptide solution. The control group was pre-coated with poly-lysine plates. The cells were cultured 1, 3, 5, 7, 9 d respectively to detect cell proliferation by using Cell Counting Kit-8. ②PC12 apoptosis and nuclear factor kappa B mRNA detection: The PC12 cells were divided into normal group, experimental group and injury group. H2O2 was added into the injury group for 16 hours stimulation. In the experimental group, H2O2 and FGL were used for 16 hours stimulation. The cell apoptosis were detected by flow cytometry; mRNA expression of nuclear factor kappa B was detected by quantitative

  8. Oxidative stress-mediated cytotoxicity of zirconia nanoparticles on PC12 and N2a cells

    Energy Technology Data Exchange (ETDEWEB)

    Asadpour, Elham [Shiraz University of Medical Sciences, Anesthesiology and Critical Care Research Center (Iran, Islamic Republic of); Sadeghnia, Hamid R. [Mashhad University of Medical Sciences, Department of Pharmacology, Faculty of Medicine (Iran, Islamic Republic of); Ghorbani, Ahmad [Mashhad University of Medical Sciences, Pharmacological Research Center of Medicinal Plants (Iran, Islamic Republic of); Sedaghat, Mehran, E-mail: m-sedaghat81@yahoo.com [Mashhad University of Medical Sciences, Department of Neurosurgery (Iran, Islamic Republic of); Boroushaki, Mohammad T., E-mail: boroushakimt@mums.ac.ir [Mashhad University of Medical Sciences, Department of Pharmacology, Faculty of Medicine (Iran, Islamic Republic of)

    2016-01-15

    In recent years, there is a growing interest in the application of nanoparticles like zirconium dioxide (zirconia <100 nm), for many purposes. Since a comprehensive study on the toxic effects of zirconia has not been done, we decided to investigate the effects of zirconia nanoparticles on cultured PC12 and N2a cells. In this study, cytotoxic effect of different concentrations of zirconia nanoparticles at three different time intervals were evaluated using MTT and ROS (reactive oxygen species) assays. Also, Lipid peroxidation, glutathione (GSH) content changes, and DNA damage were measured. Zirconia nanoparticles caused a significant reduction in cell viability and GSH content of the cells, and induce a significant increase in intracellular ROS and MDA content of PC12 and N2a cells. Moreover, it increases the percentage of DNA tail of treated cells as compared with control group. Zirconia nanoparticles have cytotoxic and genotoxic effects in PC12 and N2a cells in a time and concentration-dependent manner in concentration more than 31 µg/mL.

  9. Geniposide inhibits CoCl_2-induced PC12 cells death via the mitochondrial pathway

    Institute of Scientific and Technical Information of China (English)

    GUO Li-xia; LIU Jian-hui; XIA Zhi-ning

    2009-01-01

    Background A number of studies have shown that oxidative stress and mitochondrial involvement are major triggering factors in the development of neurodegenerative diseases. Cobalt chloride (CoCl_2)-induced cell death in PC12 cells may serve a simple and convenient in vitro model of hypoxia-induced neuronal cytotoxicity. To explore the effect of geniposide on CoCl_2 which induced cytotoxicity and mitochondrial function in rat pheochromocytoma PC12 cells, we analyzed the influence of geniposide on the expression of apoptosis-related proteins. Methods PC12 cells and RNAi PC12 cells were treated with 0, 12.5, 25, 50, 100 μmol/L geniposide for 12 hours and then exposure to 400 μmol/L CoCl_2 for 12 hours. Cell viability, cell morphology, and expression of Bcl-2, Bax, P53 and caspase-9 were determined using Western blotting. Results Pretreatment with geniposide markedly improved the cells viability and morphology, decreased the expression of Bax, P53 and caspase-9, and increased the expression of Bcl-2 in PC12 cells challenged by CoCl_2. However, in the RNAi PC12 cells, geniposide had no significant effect on the expression of these proteins. Conclusion Geniposide protects PC12 cells from CoCl_2 involved in mitochondrial mediated apoptosis, and GLP-1 R might play a critical role in the neuroprotection of geniposide in PC12 cells.

  10. Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

    Science.gov (United States)

    Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

    2008-01-01

    Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions. PMID:19081771

  11. In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells

    Directory of Open Access Journals (Sweden)

    Nabiuni Mohammad

    2012-06-01

    Full Text Available Abstract Background Fetal cerebrospinal fluid (CSF contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since they differentiate into sympathetic neuron-like cells in response to growth factors. This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described. Methods In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and β-III tubulin was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis. Results PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive. Conclusions Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important

  12. Internalization of nerve growth factor by pheochromocytoma PC12 cells: absence of transfer to the nucleus.

    Science.gov (United States)

    Rohrer, H; Schäfer, T; Korsching, S; Thoenen, H

    1982-06-01

    The intracellular distribution of 125I-labeled nerve growth factor (NGF) in rat pheochromocytoma PC12 cells was studied by quantitative electron microscopic (EM) autoradiography and by subcellular fractionation. PC12 cells were grown as monolayer cultures in medium supplemented with serum in the presence of 125I-NGF. EM autoradiography showed that 125I-NGF was localized at the plasma membrane and cytoplasmic compartments but did not accumulate in the nuclear chromatin or in the nuclear membrane compartment of cells analyzed after 1 hr and 1, 2, and 8 d of incubation with 125I-NGF. 125I-NGF also was not detected in nuclear subcellular fractions prepared from cells grown in serum-supplemented medium either in suspension for 1 d or in monolayer cultures for 1 to 8 d. In contrast, and in confirmation of the results of Yankner and Shooter (Yankner, B. A., and E. M. Shooter (1979) Pro. Natl. Acad. Sci. U. S. A. 76: 1269-1273), about 60% of the cell-bound 125I-NGF was found in the nuclear pellet after cell fractionation if the cells had been kept previously in suspension for 1 d in phosphate-buffered saline supplemented with 0.2% glucose, 0.1% bovine serum albumin, and 125I-NGF. The ultrastructure of PC12 cells grown under such conditions, however, revealed signs of varying degrees of damage. Autoradiography of the nuclear pellet from these cells showed the grains to be located mainly over damaged nuclei or over cell debris between nuclei. It is concluded that NGF, after binding to specific receptors at the plasma membrane, is transferred to membrane-confined cytoplasmic compartments but does not have to be transferred further to the nuclear membrane or to the nuclear chromatin as a prerequisite for its physiological action.

  13. Effect of insulin on 1-methyl-4-phenylpyridinium ion-induced apoptosis of PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Liping Guo; Jian Wang; Yuping Jiang

    2006-01-01

    BACKGROUND: Insulin receptor (IR) expression in the substantia nigra of patients with Parkinson disease (PD) is not only significantly lower than that in the substantia nigra of normal persons of the same age, but also significantly lower than that in other regions in brain of himself/herself. It suggests that the abnormal effect of insulin receptor-mediated insulin, as a neurotrophic factor, is very possibly related to the loss of dopaminergic neurons in the substantia nigra and striatum in patients with Parkinson disease.OBJECTIVE: To observe the interventional effect of insulin on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis of PC12.DESIGN: Controlled observation.SETTINGS: Department of Neurology, Beijing China-Japan Friendship Hospital; Department of Neurology,Huashan Hospital Affiliated to Fudan University.MATERIALS: PC12 cells were provided by the Cell Bank, Shanghai Institute of Cell Biology, Chinese Academyof Science. MPP+, MTT, HOECHST 33258 (Invitrogen Life Technologies), reverse transcription-polymerase chain reaction (RT-PCR) reagent (Takara Shuzo Co., Ltd.), flow cytometer (Bacton Dickionson, San Jose, CA),enzyme labelling instrument (Bio-Tek, Winooski, VT) and PCR circulation instrument (Takara Shuzo Co., Ltd)were used in this study.METHODS: This study was carried out in the Department of Neurology, Huashan Hospital Affiliated to Fudan University during June 2003 to August 2004. ① Cell culture and experimental grouping: PC12 cells were cultured according to the method from Peng et al, then were randomized into 3 groups; blank control group, MPP+ group and insulin group. ② Detection of relative survival rate of cells: The relative survival rate of cells at different MPP+ final concentrations (0, 50, 100, 200, 300, 1 000 μmol/L) and at different culture time (0, 4, 8, 12, 18, 24hours) in the 300 μmol/L MPP+ group and different concentrations of insulin (0, 15, 50, 100 nmol/L) in the insulin group was detected with MTT method according

  14. Interleukin 1 expression is inducible by nerve growth factor in PC12 pheochromocytoma cells.

    OpenAIRE

    Alheim, K; Andersson, C; Tingsborg, S; Ziolkowska, M; Schultzberg, M. (Marianne); Bartfai, T

    1991-01-01

    Expression of the cytokine interleukin 1 alpha (IL-1 alpha) was demonstrated in the rat PC12 pheochromocytoma cell line by (i) immunohistochemistry using rabbit polyclonal antisera raised against the recombinant murine IL-1 alpha, (ii) an ELISA, and (iii) a specific cell conversion bioassay based on the use of LBRM33-1A5 cells. IL-1 alpha mRNA was demonstrated in the PC12 cells, by PCR amplification. Constitutive expression of IL-1 alpha in PC12 cells was demonstrated in all experiments, alth...

  15. Role of Notch-1 signaling pathway in PC12 cell apoptosis induced by amyloid beta-peptide (25-35)

    Institute of Scientific and Technical Information of China (English)

    Huimin Liang; Yaozhou Zhang; Xiaoyan Shi; Tianxiang Wei; Jiyu Lou

    2014-01-01

    Recent studies have demonstrated that Notch-1 expression is increased in the hippocampus of Alzheimer’s disease patients. We speculate that Notch-1 signaling may be involved in PC12 cell apoptosis induced by amyloid beta-peptide (25-35) (Aβ25-35). In the present study, PC12 cells were cultured with different doses (0, 0.1, 1.0, 10 and 100 nmol/L) of N-[N-(3,5-Dilfuorophen-acetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a Notch-1 signaling pathway inhibitor, for 30 minutes. Then cultured cells were induced with Aβ25-35 for 48 hours. Pretreatment of PC12 cells with high doses of N-[N-(3,5-Dilfuorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (> 10 nmol/L) prolonged the survival of PC12 cells after Aβ25-35 induction, decreased the expression of apoptosis-related proteins caspase-3, -8, -9, increased the activity of oxidative stress-related su-peroxide dismutase and catalase, inhibited the production of active oxygen, and reduced nuclear factor kappa B expression. This study indicates that the Notch-1 signaling pathway plays a pivotal role in Aβ25-35-induced PC12 apoptosis.

  16. Toxicity induced by cumene hydroperoxide in PC12 cells: protective role of thiol donors.

    Science.gov (United States)

    Vimard, F; Saucet, M; Nicole, O; Feuilloley, M; Duval, D

    2011-01-01

    Oxidative shock and production of reactive oxygen species are known to play a major role in situations leading to neuron degeneration, but the precise mechanisms responsible for cell degeneration remain uncertain. In the present article, we have studied in PC 12 cells the effect of cumene hydroxyperoxide on both cell metabolism and morphology. We observed that relatively low concentrations of the drug (100 μM) led to a significant decrease in the cellular content of ATP and reduced glutathione as well as to a decreased mitochondrial potential. These metabolic alterations were followed by an important increase in intracellular free calcium and membrane disruption and death. In parallel, we observed profound changes in cell morphology with a shortening of cell extensions, the formation of ruffles and blebs at the cell surface, and a progressive detachment of the cells from the surface of the culture flasks. We also showed that addition of thiol donors such as N-acetylcysteine or β-mercaptoethanol, which were able to enhance cell glutathione content, almost completely protected PC 12 cells from the toxic action of cumene hydroperoxide whereas pretreatment by buthionine sulfoximine, a selective inhibitor of GSH synthesis, enhanced its action.

  17. The interaction of manganese nanoparticles with PC-12 cells induces dopamine depletion.

    Science.gov (United States)

    Hussain, Saber M; Javorina, Amanda K; Schrand, Amanda M; Duhart, Helen M; Ali, Syed F; Schlager, John J

    2006-08-01

    This investigation was designed to determine whether nano-sized manganese oxide (Mn-40 nm) particles would induce dopamine (DA) depletion in a cultured neuronal phenotype, PC-12 cells, similar to free ionic manganese (Mn(2+)). Cells were exposed to Mn-40 nm, Mn(2+) (acetate), or known cytotoxic silver nanoparticles (Ag-15 nm) for 24 h. Phase-contrast microscopy studies show that Mn-40 nm or Mn(2+) exposure did not greatly change morphology of PC-12 cells. However, Ag-15 nm and AgNO(3) produce cell shrinkage and irregular membrane borders compared to control cells. Further microscopic studies at higher resolution demonstrated that Mn-40 nm nanoparticles and agglomerates were effectively internalized by PC-12 cells. Mitochondrial reduction activity, a sensitive measure of particle and metal cytotoxicity, showed only moderate toxicity for Mn-40 nm compared to similar Ag-15 nm and Mn(2+) doses. Mn-40 nm and Mn(2+) dose dependently depleted DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), while Ag-15 nm only significantly reduced DA and DOPAC at concentrations of 50 mug/ml. Therefore, the DA depletion of Mn-40 nm was most similar to Mn(2+), which is known to induce concentration-dependent DA depletion. There was a significant increase (> 10-fold) in reactive oxygen species (ROS) with Mn-40 nm exposure, suggesting that increased ROS levels may participate in DA depletion. These results clearly demonstrate that nanoscale manganese can deplete DA, DOPAC, and HVA in a dose-dependent manner. Further study is required to evaluate the specific intracellular distribution of Mn-40 nm nanoparticles, metal dissolution rates in cells and cellular matrices, if DA depletion is induced in vivo, and the propensity of Mn nanoparticles to cross the blood-brain barrier or be selectively uptaken by nasal epithelium.

  18. Signal transduction pathway of nitric oxide inducing PC12 cell death

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To study signal transduction pathway of nitric oxideinducing death of PC12 cells.Methods: Cell survival rate was measured with MTT assay, and caspase-3 activity with caspase-3 assay kits after PC12 cells were incubated with sodium nitroprusside (SNP), caspase-3 inhibitor Ⅱ plus SNP or p38 inhibitor-SB203580 plus SNP.Results: SNP induced death of PC12 cells in dose- and time-dependent manner and enhanced caspase-3 activity gradually. Both caspase-3 inhibitor Ⅱ and SB203580 reduced cell death, but SB203580 reduced caspase-3 activity significantly.Conclusions: NO may induce death of PC12 cells through activation of p38 and caspase-3.

  19. Adenovirus-mediated transfection with glucose transporter 3 suppresses PC12 cell apoptosis following ischemic injury

    Institute of Scientific and Technical Information of China (English)

    Junliang Li; Xinke Xu; Shanyi Zhang; Meiguang Zheng; Zhonghua Wu; Yinlun Weng; Leping Ouyang; Jian Yu; Fangcheng Li

    2012-01-01

    In this study, we investigated the effects of adenovirus-mediated transfection of PC12 cells with glucose transporter 3 after ischemic injury. The results of flow cytometry and TUNEL showed that exogenous glucose transporter 3 significantly suppressed PC12 cell apoptosis induced by ischemic injury. The results of isotopic scintiscan and western blot assays showed that, the glucose uptake rate was significantly increased and nuclear factor kappaB expression was significantly decreased after adenovirus-mediated transfection of ischemic PC12 cells with glucose transporter 3. These results suggest that adenovirus-mediated transfection of cells with glucose transporter 3 elevates the energy metabolism of PC12 cells with ischemic injury, and inhibits cell apoptosis.

  20. Influence of Panax quinquefolium saponins on increased intracellular Ca2+ in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Lixin Guan; Xiudong Jin; Yanhui Chu; Yufei Zhang; Yan Wu; Xin Yi; Fengguo Zhai; Mengquan Li

    2009-01-01

    BACKGROUND: Previous studies have demonstrated that intracellular Ca2+ ([Ca2+]i) overload,excitotoxicity, free radical injury, and nitric oxide toxicity are involved in mechanisms of neuronal death in the ischemic brain.OBJECTIVE: To investigate the influence of Panax quinquefolium saponins (PQS) on multiple factors-induced Ca2+ overload in the rat pheochromocytoma (PC12) cell line.DESIGN, TIME AND SETTING: Intergroup comparison, in vitro study. The experiment was performed at the Heilongjiang Key Laboratory of Anti-fibrosis Biotherapy, Mudanjiang Medical University between November 2007 and April 2008.MATERIALS: In vitro cultured PC12 cells in the logarithmic phase were assigned into blank control, model, and drug treatment groups (10 μmol/L nimodipine; 40 μg/L, 100 μg/L, and 250 μg/L PQS). Nimodipine was purchased from Jiangsu Yangtze River Pharmacy Group Co.,China; PQS (purity > 95%, HLPC grade) was provided by School of Basic Medical Sciences, Jilin University. Caffeine, Na2S204, L-glutamic acid (Glu), Fura-2/AM, and calcium ionophore A23187 were purchased from Sigma, USA.METHODS: PC12 cells in the model and drug treatment groups were separately incubated in glucose-free Hank's buffered saline solution + Na2S2O4, (2 mmol/L) for 6 hours, Glu (200 μ mol/L)plus A23187 (0.05 μmol/L) for 6 hours, KCI (50 mmol/L) for 1 hour, and caffeine (5 mmol/L) for 3 hours to establish models of intracellular Ca2+ overload induced by oxygen and glucose deprivation, Glu, A23187, high K+, or caffeine. In addition, control cells were incubated in high-glucose DMEM culture medium.MAIN OUTCOME MEASURES: [Ca2+]i changes in PC12 cells exposed to oxygen-glucose deprivation, Glu, A23187, high K+, or caffeine were detected using spectrofluorometer.RESULTS: PQS blocked the [Ca2+]i increase induced by oxygen-glucose deprivation, Glu,A23187, high K+, or caffeine. In particular, high-dose PQS was most effective (P < 0.01). PQS significantly inhibited Glu- or caffeine-induced [Ca2+]i

  1. Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells.

    Science.gov (United States)

    Chen, Pei-Yi; Ho, Yi-Ru; Wu, Ming-Jiuan; Huang, Shun-Ping; Chen, Po-Kong; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

    2015-01-01

    Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl₂)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl₂-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl₂ exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.

  2. Polysaccharides purified from Cordyceps cicadae protects PC12 cells against glutamate-induced oxidative damage.

    Science.gov (United States)

    Olatunji, Opeyemi J; Feng, Yan; Olatunji, Oyenike O; Tang, Jian; Wei, Yuan; Ouyang, Zhen; Su, Zhaoliang

    2016-11-20

    Two polysaccharides CPA-1 and CPB-2 were isolated purified from Cordyceps cicadae by hot water extraction, ethanol precipitation and purification using anion exchange and gel filtration chromatography. Preliminary structural characterization of CPA-1 and CPB-2 were performed. The protective effect of CPA-1 and CPB-2 against glutamate-induced oxidative toxicity in PC12 cells was analyzed. The results indicated that pretreatment of PC12 cells with CPA-1 and CPB-2 significantly increased cell survival, Ca(2+) overload and ROS generation. CPA-1 and CPB-2 also markedly up-regulated the antioxidant status of pretreated PC12 cells. Our results suggested that Cordyceps cicadae polysaccharides can protect PC12 cells against glutamate excitotoxicity and might serve as therapeutic agents for neuronal disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. DIFFERENTIAL MODULATION OF CATECHOLAMINES BY CHLOROTRIAZINE HERBICIDES IN PHEOCHROMOCYTOMA (PC12) CELLS IN VITRO

    Science.gov (United States)

    Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.Das PC, McElroy WK, Cooper RL.Curriculum in Toxicology, University of North Carolina, Chapel Hill 27599, USA.Epidemiological, wildlife, and lab...

  4. Antioxidative effects of berberine pre-treatment on hydrogen peroxide-induced PC12 cell toxicity

    Institute of Scientific and Technical Information of China (English)

    Daohua Xu; Chenhui Zhou

    2010-01-01

    Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease.Oxidative damage could be prevented by augmenting the endogenous defense capacity against oxidative stress by antioxidant intake.As an effective alkaloid component of Chinese herbal medicine Rhizoma coptidis extract,berberine exhibits antioxidative properties and ameliorates memory impairment in a rat model of Alzheimer's disease.The present study investigated the protective effects of berberine on H2O2-induced PC12 cell toxicity.Results demonstrated that berbedne protects PC12 cells from H2O2-induced apoptosis and increases PC12 cell viability.Lactate dehydrogenase release,reactive oxygen content,and malonyl dialdehyde levels were significantly decreased(P < 0.01).The protective effects of berberine on H2O2-induced PC12 cell toxicity were achieved via the antioxidative effects of berberine.

  5. The protective effect of tetramethylpyrazine (TMP) against PC12 cells damages

    Institute of Scientific and Technical Information of China (English)

    Xin-ruiCHENG; LanSUN; LiZHANG; Juan-juanHU; Guan-HuaDU

    2004-01-01

    AIM: To discover the protective effects of tetramethylpyrazine (TMP) against PC12 cells damages and explore its protective mechanisms. METHODS: We established three in vitro models to investigate the protective effects of TMP against the injuries. In both of glutamate and natrium azide-induced PC12 injuries, the action of TMP on the cell viability was measured by MTT assay. The LDH efflux was measured by the assay kit, production

  6. Enhanced neurite outgrowth of PC-12 cells on graphene-monolayer-coated substrates as biomimetic cues

    Science.gov (United States)

    Lee, Jong Ho; Shin, Yong Cheol; Jin, Oh Seong; Han, Dong-Wook; Kang, Seok Hee; Hong, Suck Won; Kim, Jong Man

    2012-11-01

    Neurons are electrically excitable cells that transmit and process information in the nervous system. Recently, the differentiation of human neural stem cells to neurons has been shown to be enhanced on graphene substrates, and differentiated neurons have been shown to be able to still carry electrical signals when stimulated by graphene electrodes. Graphene films grown by using chemical vapor deposition were transferred onto glass coverslips by using the scooping method and were then coated with fetal bovine serum for a neuronal cell culture. The graphene substrates as biomimetic cues have been shown to enhance the neurite outgrowth of PC-12 cells. Our findings suggest that graphene has a unique surface property that can promote neuronal cells, which should open tremendous opportunities in neuroscience, neural engineering and regenerative medicine.

  7. The protective effect of the insulin against paraquat-induced PC12 cells%胰岛素对百草枯诱导的PC12细胞的保护作用

    Institute of Scientific and Technical Information of China (English)

    邬剑军; 蒋雨平; 张晨

    2009-01-01

    Objective To observe dopamine (DA) D2R protein expression in the cultured PC 12 cells and the effect of insulin on the survival rate,morphology,and DA of paraquat (PQ)-induced PC12 cells. Methods Immunoprecipitate Western blotting method was performed to observe DA D2R protein expression in PC 12 cells,MTT assay was used to analyze the changes in viability and morphology of PC 12 ceils which were exposed to different concentrations of PQ and insulin. Results (1) DA D2R protein was expressed in PC 12 ceils. (2) Normal PCI2 cells bodies showed fusiform shape and the synapses were in-tegrity. The cells which were exposed to the 600 μmol/L PQ became ball-like, vacuolar degeneration oc-urred,and the synapse became shorter or disappeared. But the morphology of PC12 cells had a little difference between the normal PC12 and the insulin groups,except that the cells were fusiform shape or a-nomalism but not round shape,and the synapses grew. (3) With the increase of the concentration of PQ, the viability of the cells was decreased. Insulin increased the viability of the ceils which were exposed to 600 μmol/L PQ. Insulin elevated DA concentration both in the normal PC12 cells and those exposed to 600 μmol/L PQ,but there was no significant difference ( P > 0.05 ). Conclusion Insulin could protect the PC12 dopaminergic neurons from injury induced by PQ.%目的 观察培养的PC12细胞中多巴胺D2受体(DA D2R)蛋白的表达,胰岛素对百草枯(paraquat,PQ)诱导的PC12细胞形态学、生存率和DA含量的影响.方法 应用免疫沉淀Western印迹分析技术对培养的PC12细胞中DA D2R表达的检测;应用二甲基噻唑二苯基四唑溴盐(MTT)法,观察PC12细胞暴露于不同浓度PQ组和胰岛素预先干预后再暴露于PQ组后细胞形态学和生存率的改变;应用酶联免疫吸附试验(ELISA)方法检测空白对照PC12细胞组、PQ干预组、胰岛素组和胰岛素加PQ组PC12细胞上清液DA的浓度.结果 (1)Western法检测到培养的PC

  8. Baicalin inhibits colistin sulfate-induced apoptosis of PC12 cells******

    Institute of Scientific and Technical Information of China (English)

    Hong Jiang; Pengfei Lv; Jichang Li; Hongjun Wang; Tiezhong Zhou; Yingzi Liu; Wei Lin

    2013-01-01

    Baicalin, a type of flavonoid extracted from the dried root of Scutel aria baicalensis georgi, has been shown to effectively inhibit cellapoptosis. Therefore, we assumed that baicalin would suppress colistin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate (62.5-500 μg/mL) for 24 hours resulted in PC12 cellapoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin (25, 50 and 100 μg/mL), and exposed to 125 μg/mL colistin sulfate. cellmorphology markedly changed, and cellviability increased. Moreover, caspase-3 activity, lac-tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhib-ited colistin sulfate-induced PC12 cellapoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.

  9. Neurotrophic Effect of Citrus Auraptene: Neuritogenic Activity in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Mitsunari Nakajima

    2012-04-01

    Full Text Available The activation of extracellular signal-regulated kinases (ERK leads to a number of cellular changes associated with the development of long-term memory. Using cultured cortical neurons, we previously showed that the n-hexane extract prepared from the peels of Citrus grandis (Kawachi bankan induces the activation of ERK1/2 and that one of the compounds with this ability in the extract is 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF, a Citrus polymethoxyflavone. In fact, we found that HMF has the ability to rescue mice from drug-induced learning impairment. This hexane extract contains auraptene (AUR, a coumarin derivative with a monoterpene unit, together with HMF. The present study was designed to investigate the effect of AUR in vitro. Our results show that 1 AUR had the ability to induce the activation of ERK1/2 in not only cortical neurons but also the rat pheochromocytoma cell line (PC12 cells, which is a model system for studies on neuronal proliferation and differentiation; and 2 AUR had the ability to promote neurite outgrowth from PC12 cells.

  10. Release of chromaffin granule glycoproteins and proteoglycans from potassium-stimulated PC12 pheochromocytoma cells.

    Science.gov (United States)

    Salton, S R; Margolis, R U; Margolis, R K

    1983-10-01

    Cultured PC12 pheochromocytoma cells were labeled with [3H]glucosamine, and the glycoproteins and proteoglycans released following potassium-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs-Ringers-HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM). The released complex carbohydrates include chromogranins, dopamine beta-hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(beta 1 leads to 3)N-acetylgalactosamine, as well as several mono- and disialyl O-glycosidically-linked oligosaccharides, and the tetrasaccharide AcNeu(alpha 2 leads to 3)Gal(beta 1 leads to 3)[AcNeu(alpha 2 leads to 6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23-68%), heparan sulfate (16-23%), and glycoprotein oligosaccharides (16-54%), which are of the tri- and tetraantennary and O-glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans.

  11. Arecoline Induces Neurotoxicity to PC12 Cells: Involvement in ER Stress and Disturbance of Endogenous H2S Generation.

    Science.gov (United States)

    Jiang, Jia-Mei; Wang, Li; Gu, Hong-Feng; Wu, Keng; Xiao, Fan; Chen, Ying; Guo, Run-Min; Tang, Xiao-Qing

    2016-08-01

    Arecoline is a major alkaloid of areca nut and has been effect on central nervous system. Although arecoline-induced neurotoxicity has been reported, the possible underlying neurotoxic mechanisms have not yet been elucidated. Increasing evidences have shown that both excessive endoplasmic reticulum (ER) stress and disturbance of hydrogen sulfide (H2S) production are involved in the pathophysiology of numerous neurodegenerative diseases. Here, the purpose of present study was to verify whether ER stress and the disturbance of endogenous H2S generation are also involved in arecoline-caused neurotoxicity. We found that treatment of PC12 cells with arecoline induced the down-regulation of cells viability and up-regulation of apoptosis and the activity of caspase-3, indicating the neurotoxic role of arecoline to PC12 cells. In addition, arecoline also increased the expression of Bax (pro-apoptotic protein) and attenuated the expression of Bcl-2 (anti-apoptotic protein) in PC12 cells. Simultaneously, arecoline caused excessive ER stress in PC12 cells, as evidenced by the up-regulations of Glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), and Cleaved caspase-12 expressions. Notably, the level of H2S in the culture supernatant and the expressions of cystathionine β-synthase and 3-mercaptopyruvate sulfurtransferase (two major enzymes for endogenous H2S generation in PC12 cells) were also reduced by arecoline treatment. These results indicate that arecoline-caused neurotoxicity to PC12 cells is involved in ER stress and disturbance of endogenous H2S generation and suggest that the modulation of ER stress and endogenous H2S generation may be potential therapeutic approach in treatment of arecoline-caused neurotoxicity.

  12. Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells.

    Science.gov (United States)

    Soeda, S; Ochiai, T; Paopong, L; Tanaka, H; Shoyama, Y; Shimeno, H

    2001-11-01

    Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

  13. Intracellular dopamine oxidation mediates rotenone-induced apoptosis in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Hua-qing LIU; Xing-zu ZHU; En-qi WENG

    2005-01-01

    Aim: To study the role of dopamine (DA) in rotenone-induced neurotoxicity in PC12 cells. Methods: Cell viability was assessed by detecting the leakage of lactate dehydrogenase (LDH) into the medium. Apoptosis rate was measured by flow cytometry. Caspase-3-1ike activity was measured by fluorescence assay using the probe Ac-DEVD-AMC. The level of intracellular hydrogen peroxide and other peroxides in PC12 cells were quantified by loading cells with 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) in fluorescence assay. Lactic acid was measured spectrophotometrically. The DA levels in PC12 cells were determined by HPLC-ECD. Results: A 48-h incubation of PC12 cells with rotenone caused an apoptotic cell death and elevated intracellular reactive oxygen species (ROS) and lactic acid accumulation. Intracellular DA depletion with reserpine significantly attenuated rotenone-induced ROS accumulation and apoptotic cell death. No change was found in rotenone-induced ROS accumulation when cells were co-treated with deprenyl. Brief treatment with reserpine at the end of rotenone treatment had no effect on rotenone-induced neurotoxicity. However,when cells were first incubated with deprenyl, a monoamine oxidase-B inhibitor for 30 min then co-incubated with rotenone plus deprenyl, a brief treatment with reserpine enhanced cell injury. Conclusion: Rotenone-induced apoptosis in PC 12 cells was mediated by intracellular dopamine oxidation.

  14. Protective Effect of CBV-D on Glu-induced Injury in Cultured PC12 Cells%环维黄杨星D对PC12谷氨酸损伤的保护作用的实验研究

    Institute of Scientific and Technical Information of China (English)

    袁冬平; 龙军; 方泰惠

    2004-01-01

    目的:探讨环维黄杨星D(CBV-D)对PC12细胞谷氨酸(Glu)损伤模型的影响.方法:用Glu复制PC12细胞兴奋性氨基酸损伤模型,通过MTT染色和乳酸脱氢酶(LDH)活性的测定来研究CBV-D对PC12细胞的保护作用.结果:CBV-D抑制Glu造成的损伤,降低LDH的漏出.结论:CBV-D对兴奋性氨基酸引起PC12细胞损伤具有保护作用.

  15. Edaravone protects PC12 cells from ischemic-like injury via attenuating the damage to mitochondria

    Institute of Scientific and Technical Information of China (English)

    SONG Ying; LI Meng; LI Ji-cheng; WEI Er-qing

    2006-01-01

    Background: Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD (oxygen-glucose deprivation)-reperfusion. Methods: Viability of PC 12cells which were injured at different time of OGD injury, was quantified by measuring MTT (2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) staining. In addition, PC 12 cells' viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by (OGD). Furthermore, apoptotic population of PC12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis,electron microscope and Hoechst/PI staining. Finally, change of Bcl-2/Bax protein expression was detected by Westem blot.Results: (1) The viability of PC 12 cells decreased with time (1~12 h) after OGD. We regarded the model of OGD 2 h, then replacing DMEM (Dulbecco's Modified Eagle's Medium) for another 24 h as an OGD-reperfusion in this research. Furthermore,most PC12 cells were in the state of apoptosis after OGD-reperfusion. (2) The viability of PC12 cells preincubated with edaravone at high concentrations (1,0.1, 0.01 μmol/L) increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. (3) Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion: Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.

  16. Beneficial effect of astragalosides on stroke condition using PC12 cells under oxygen glucose deprivation and reperfusion.

    Science.gov (United States)

    Chiu, Bi-Ying; Chang, Ching-Ping; Lin, Jia-Wei; Yu, Jung-Sheng; Liu, Wen-Pin; Hsu, Yao-Chin; Lin, Mao-Tsun

    2014-08-01

    Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.

  17. Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation.

    Science.gov (United States)

    Shepelev, Mikhail V; Chernoff, Jonathan; Korobko, Igor V

    2011-01-01

    Rho GTPases regulate numerous cellular processes including apoptosis. Chp/RhoV is an atypical Rho GTPase which functions are poorly understood. Here we investigated the role of Chp in regulation of cell viability using PC12 cells with inducible expression of Chp as a model. We found that expression of Chp results in apoptosis in PC12 cells. Chp-induced apoptosis was accompanied by activation of JNK signaling and both death receptor-mediated and mitochondrial apoptotic pathways as justified by caspase-8 and caspase-9 activation, respectively. Moreover, inhibition of JNK by SP600125 rescued PC12 cells from Chp-triggered cell death and attenuated activation of caspases-9 and -3/7 suggesting that activation of JNK mediates pro-apoptotic effect of Chp. Expression of Chp resulted in increased phosphorylation of c-Jun in PC12 cells, and Chp expression in HE K293 cells upregulated AP-1-dependent transcription in a JNK-dependent manner. Together results of our study reveal the role of Chp GTPase as a putative regulator of JNK-dependent apoptotic death in PC12 cells, similarly to previously described pro-apoptotic activity of the related Cdc42 and Rac1 GTPases.

  18. Beneficial effects of BV2 cell on proliferation and neuron-differentiating of mesenchymal stem cells in the circumstance of injured PC12 cell supernatant

    Institute of Scientific and Technical Information of China (English)

    Xiao-Guang LUO; Hong WANG; Jin ZHOU; Rong YAN; Zhe WU; Chao-Dong ZHANG; Qiu-Shuang WANG

    2006-01-01

    Objective The microglias is the representative of immune cells in the brain. It plays dual roles of both repairing and damaging in injured nervous system, and works as an inevitable component of the circumstance of injured neurons. This study was aiming at the effects of the microglias on the biological activities of mesenchymal stem cells (MSCs) inthe circumstance of injured neurons. Methods MSCs were obtained by primary culture. We adopted PC12 cells (PC12) and BV2 cells (BV2) to substitute for neurons and microglias, respectively. PC12 were injured by aged Aβ1-40 and the supernatant of the injured PC12 was used to set up the circumstance of injured neurons. Transwells were used for co-culture of BV2 and MSCs, which allowed the independent detection of cells after co-culture. Immunofluorescence was used to identify MSCs and neuron-differentiating cells with CD44 and neuron specific enolase (NSE) staining, respectively. MTT assay was adopted to measure the proliferation. Results In the circumstance of both BV2 presence and injured PC12 supernatant incubation, either the proliferation or the differentiation of MSCs reached the highest, which seemed to be contradictory, but we gave our explanations. With the BV2 co-culture, the proliferation of MSCs tend to be higher, but the neuron-differentiating MSCs were similar to those incubated without BV2 co-culture either in normal or injured in PC12 supernatant. With the incubation of injured PC12 supernatant, the neuron-differentiating cells were significantly higher than that of control (P < 0.05). Conclusion In the circumstance of injured neurons, microlgias tend to promote the MSCs proliferation. Although not helpful in neuron-differentiating, microglias did not exert any negative effect either.

  19. Protective effects of Gynostemma pentaphyllum polysaccharides on PC12 cells impaired by MPP(+).

    Science.gov (United States)

    Deng, Qi; Yang, Xinling

    2014-08-01

    The aim of the present study was to explore the neuroprotective effects of Gynostemma pentaphyllum polysaccharides (GP) in a 1-methyl-4-phenylpyridiniumion (MPP(+))-induced cellular model of Parkinson's disease (PD) and the underlying mechanisms. Our results indicated that exposure of PC12 cells to 1mM MPP(+) significantly decreased the cell viability when examined by MTT assay, LDH assay, and annexin-V-FITC and propidium iodide (PI) apoptosis detection assays. MPP(+)-induced apoptosis in PC12 cells was accompanied by an increased Bax/Bcl-2 ratio, release of mitochondrial cytochrome c into the cytosol, activation of caspase-3/9 and cleavage of poly (ADP-ribose) polymerase (PARP). However, pretreatment of PC12 cells with 50μg/ml GP prior to MPP(+) exposure effectively attenuated the cytotoxicity and improved cell viability via inhibiting elevated Bax/Bcl-2 ratio, as well as the release of cytosolic cytochrome c. Furthermore, GP was effective in attenuating caspase-3/9 activation and cleavage of PARP in MPP(+)-exposed PC12 cells. These results suggest that the GP has protective effects against MPP(+)-induced neuronal apoptosis in PC12 cells by suppressing apoptosis-related protein, and therefore, might likely be a promising candidate for the treatment of Parkinson's disease (PD). Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Luteolin enhances cholinergic activities in PC12 cells through ERK1/2 and PI3K/Akt pathways.

    Science.gov (United States)

    El Omri, Abdelfatteh; Han, Junkyu; Kawada, Kiyokazu; Ben Abdrabbah, Manef; Isoda, Hiroko

    2012-02-09

    Luteolin, a 3', 4', 5, 7-tetrahydroxyflavone, is an active compound in Rosmarinus officinalis (Lamiacea), and has been reported to exert several benefits in neuronal cells. However cholinergic-induced activities of luteolin still remain unknown. Neuronal differentiation encompasses an elaborate developmental program which plays a key role in the development of the nervous system. The advent of several cell lines, like PC12 cells, able to differentiate in culture proved to be the turning point for gaining and understanding of molecular neuroscience. In this work, we investigated the ability of luteolin to induce PC12 cell differentiation and its effect on cholinergic activities. Our findings showed that luteolin treatment significantly induced neurite outgrowth extension, enhanced acetylcholinesterase (AChE) activity, known as neuronal differentiation marker, and increased the level of total choline and acetylcholine in PC12 cells. In addition, luteolin persistently, activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; while the addition of pharmacological MEK/ERK1/2 inhibitor (U0126) and PI3k/Akt inhibitor (LY294002) attenuated luteolin-induced AChE activity and neurite outgrowth in PC12 cells. The above findings suggest that luteolin induces neurite outgrowth and enhanced cholinergic activities, at least in part, through the activation of ERK1/2 and Akt signaling.

  1. Primary investigation of methamphetamine-induced toxicity in PC12 cells%甲基苯丙胺对PC12细胞的毒性损伤作用

    Institute of Scientific and Technical Information of China (English)

    李丽增; 王慧君; 兰江维; 岳霞; 刘超

    2011-01-01

    Objective To investigate the mechanism of methamphetamine (METH)-induced toxicity in PC12 cells. Methods PC12 cells were treated with METH for 24 h at the doses of 0,05,1.0,15,2.0, or 2.5 mmol/L. The morphological changes of the cells were observed under inverted microscope after the treatment. MTT assay and flow cytometry were used to assess the cell viability and apoptotic rates, respectively, and the level of nitric oxide (NO) was measured by enzyme reduction method. Results The PC12 cells exposed to METH were morphologically featured by cell shrinkage, dendrite disruption and disappearance of cell reticular formation. METH exposure caused a dose-dependent reduction in the cell viability (P<0.01), resulting in also increased cell apoptotic rate and significant elevation of NO in the cell culture supernatant (P<0.05). Conclusion METH exposure induces cytotoxicity and injury of differentiated PC12 cells, leading to decreased cell viability and increased cell apoptosis and NO level. Cell apoptosis and excessive NO production are involved in METH-induced cytotoxicity.%目的 探讨甲基苯丙胺(METH)对PC12细胞的毒性损伤作用,为进一步研究METH神经毒性作用机制提供基础.方法 采用已分化PC12细胞为体外神经元模型,分别用浓度0、0.5、1.0、1.5、2.0、2.5 mmol/L的METH处理PC12细胞24 h,倒置显微镜下观察细胞形态改变;MTT法检测细胞存活率;流式细胞术分析细胞凋亡率;硝酸还原酶法检测细胞培养液NO含量.结果 以0mmol/L为对照组,经0.5~2.5mmol/LMETH处理的PC12细胞,24h倒置显微镜下可见PC12细胞胞体变圆,神经突起变短、断裂至消失,神经网络逐渐消失,细胞边缘不清,可呈毛刺样;MTT法检测PC12细胞活力随METH浓度的升高而逐渐下降,与对照组相比均有显著性差异(P<0.01);细胞凋亡率在0.5~1.5 mmol/L浓度范围内逐渐升高,至2.0mmol/L时凋亡率较前下降,但与对照组相比均升高,且与对照

  2. Daidzin protects PC12 cells from serum deprivation-induced apoptosis.

    Science.gov (United States)

    Ji, Zhao-Ning; Liu, Guo-Qing

    2002-12-01

    This article examines the effect of daidzin on PC12 cell apoptosis induced by serum-free medium. PC12 cell survival was measured by MTT assay. The DNA content and percentage of apoptosis were monitored by flow cytometry and DNA fragmentation was analyzed by agarose gel electrophoresis. The results showed that serum-free (12 h) medium induced apoptosis in PC12 cells. When the cells had been treated with daidzin (0.1, 1 microM) for 12 h, the percentage of PC12 cell apoptosis was significantly decreased to 12.21 and 4.24% from 91.94% in the group with serum deprivation, and DNA fragmentation was prevented. Daidzin (0.01-10 microM) attenuated the cytotoxic effect of sodium cyanide (20 mM), glutamate (0.5 mM) and sodium nitroprusside (0.5 mM) in a manner dependent on concentration. The results suggested that daidzin prevented PC12 cell from serum free-induced apoptosis.

  3. Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Seiji [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Kobayashi, Saori [Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Tsubota, Kazuo [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ozawa, Yoko, E-mail: ozawa@a5.keio.jp [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2014-04-04

    Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.

  4. Neurite outgrowth resistance to rho kinase inhibitors in PC12 Adh cell.

    Science.gov (United States)

    Yin, Hua; Hou, Xiaolin; Tao, Tingrui; Lv, Xiaoman; Zhang, Luyong; Duan, Weigang

    2015-05-01

    Rho kinase (ROCK) inhibitor is a promising agent for neural injury disorders, which mechanism is associated with neurite outgrowth. However, neurite outgrowth resistance occurred when PC12 Adh cell was treated with ROCK inhibitors for a longer time. PC12 Adh cells were treated with ROCK inhibitor Y27632 or NGF for different durations. Neurite outgrowth resistance occurred when PC12 Adh cell exposed to Y27632 (33 µM) for 3 or more days, but not happen when exposed to nerve growth factor (NGF, 100 ng/mL). The gene expression in the PC12 Adh cells treated with Y27632 (33 µM) or NGF (100 ng/mL) for 2 or 4 days was assayed by gene microarray, and the reliability of the results were confirmed by real-time RT-PCR. Cluster analysis proved that the gene expression profile of PC12 Adh cell treated with Y27632 for 4 days was different from that treated with Y27632 for 2 days and those treated with NGF for 2 and 4 days, respectively. Pathway analysis hinted that the neurite outgrowth resistance could be associated with up-regulation of inflammatory pathways, especially rno04610 (complement and coagulation cascades), and down-regulation of cell cycle pathways, especially rno04110.

  5. Modulation of vesicular catecholamine release from rat PC12 cells

    NARCIS (Netherlands)

    Westerink, R.H.S.

    2002-01-01

    Intercellular communication is of vital importance for the nervous system, since the nervous system is the main coordinating system in animals. Nerve cell communication is initiated by the release of chemical messengers, neurotransmitters, from the presynaptic nerve cell. The neurotransmitters, such

  6. PC12细胞转染人GPx-1基因后的抗氧化损伤作用%Anti-oxidative damage effect on PC12 cells transfected with human glutathione peroxidase 1 gene

    Institute of Scientific and Technical Information of China (English)

    马琳; 王辉; 王淑荣; 陈蓉; 郑俩燕

    2013-01-01

    Objective To study the protective effect and molecular mechanism of glutathione peroxidase 1 (GPx-1) high expression on PC12 cell damage mediated by hydrogen peroxide (H2O2). Methods PC12 cells were transfected with plncx plasmid containing human GPx-1 gene and plncx plasmid, and a PC12 cell line was established that could stably express human GPx-1 gene and plncx plasmid through continuous screening. Two means of intervention, H2O2 and sodium selenite + H2O2, were given to PC12, GPx-1-PC12 and plncx-PC12 cells, respectively. GPx activity of cells in each group was detected with GPx activity detection kit; the survival and apoptosis of cells were detected by MTT method and flow cytometer, respectively. The expression of NF-κBp65 was detected by immunohistochemistry technique. Results GPx activity of GPx-PC12 group cells was obviously higher than that of controls. Intervention of H2O2 and sodium selenite + H2O2 did not significantly affect the cell survival rate in PC12 and plncx-PC12 groups (P>0. 05). The cell survival rate was higher in GPx-1-PC12 group than in PC12 and plncx-PC12 groups (P0.05),GPx-1-PC12PC12、plncx-PC12组细胞存活率明显增高(P<0.01);GPx-1-PC12PC12、plncx-PC12细胞早期和晚期凋亡率明显下降,存在统计学差异(P<0.01);GPx-1-PC12PC12细胞NF-κBp65表达明显减少(P<0.01).结论 GPx-1基因高表达对H2O2所致的PC12细胞氧化损伤有很好的保护作用;GPx-1可抑制PC12细胞氧化损伤时凋亡的发生及NF-κB的表达.

  7. Protective effects of Ginkgo biloba extract on 6-hydroxydopamine-induced apoptosis in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Jie Wang; Yanbo Cheng; Jiale Yin; Qian Lu; Xingshun Xu; Xiaoxing Yin

    2011-01-01

    The present study analyzed the protective effects of Ginkgo biloba extract against 6-hydroxydopamine-induced PC12 cell apoptosis in a model of Parkinson's disease. The results showed that Ginkgo biloba extract had a potent cytoprotective action and inhibited apoptosis of PC12 cells induced by 6-hydroxydopamine. Ginkgo biloba extract decreased the ratio of Bax to Bcl-2 and markedly inhibited the activation of p53 and caspase-3. These experimental findings indicate that Ginkgo biloba extract may significantly reduce the effects of oxidative stress induced by 6-hydroxydopamine in PC12 cells and suppress cell apoptosis. The potential effects of Ginkgo biloba extract might be greater than those of levodopa in the treatment of Parkinson's disease.

  8. Plasminogen activator inhibitor-1 aids survival of neurites on neurons derived from pheochromocytoma (PC-12) cells.

    Science.gov (United States)

    Soeda, Shinji; Imatoh, Takuya; Ochiai, Takashi; Koyanagi, Satoru; Shimeno, Hiroshi

    2004-04-09

    Plasminogen activator inhibitor-1 is a serpin that regulates the activities of plasminogen activators. However, its physiological roles in the CNS are incompletely understood. We have found that plasminogen activator inhibitor-1 has a novel biological function in the CNS: the contribution to survival of neurites on neurons. PC-12 cells treated with nerve growth factor differentiated into neurons and formed a network of neurites. In a serum-free culture medium, these neurites disappeared within 24 h. The addition of plasminogen activator inhibitor-1 prevented the disintegration of the neuronal networks, while the addition of the serpin inhibitors aprotinin and antipain did not. The plasminogen activator inhibitor-1 maintained or promoted the phosphorylated state of extracellular signal-regulated kinase (ERK), but not of protein kinase B (Akt). These results are the first evidence that plasminogen activator inhibitor-1 in the CNS acts to maintain the morphology of neurites via activation of the ERK-related pathway in the neurons.

  9. Effect of Inhalational Anesthetics on Cytotoxicity and Intracellular Calcium Differently in Rat Pheochromocytoma Cells (PC12)

    Institute of Scientific and Technical Information of China (English)

    Qiujun WANG; Kezhong LI; Shanglong YAO

    2008-01-01

    Isoflurane, a commonly used inhaled anesthetic, induces apoptosis in rat pheochromo-cytoma cells (PC12) in a concentration- and time-dependent manner with unknown mechanism. We hypothesized that isoflurane induced apoptosis by causing abnormal calcium release from the endo-plasmic reticulum (ER) via activation of inositol 1,4,5-trisphosphate (IP3) receptors. Alzheimer's pre-senilin-1 (PS1) mutation increased activity of IP3 receptors and therefore rendered cells vulnerable to isoflurane-induced cytotoxicity. Sevoflurane and desflurane had less ability to disrupt intraceUular calcium homeostasis and thus being less potent to cause cytotoxicity. This study examined and com- pared the cytotoxic effects of various inhaled anesthetics on PC12 cells transfected with the Alz- heimer's mutated Psi (L286V) and the disruption of intracellular calcium homeostasis. PC12 cells transfected with wild type (WT) and mutated PS1 (L286V) were treated with equivalent of 1 MAC of isoflurane, sevoflurane and desflurane for 12 h. MTT reduction and LDH release assays were per- formed to evaluate cell viability. Changes of calcium concentration in cytosolic space ([Ca2+]c) were determined after exposing different types of cells to various inhalational anesthetics. The effects of IP3 receptor antagonist xestospongin C on isoflurane-induced cytotoxicity and calcium release from the ER in L286V PC12 cells were also determined. The results showed that isoflurane at 1 MAC for 12 h induced cytoxicity in L286V but not WT PC12 cells, which was also associated with greater and faster elevation of peak [Ca2+]c in L286V than in the WT cells. Xestospongin C significantly amelio- rated isoflurane cytotoxicity in L286V cells, as well as inhibited the calcium release from the ER in L286V cells. Sevoflurane and desflurane at equivalent exposure to isoflurane did not induce similar cytotoxicity or elevation of peak [Ca2+]c in L286V PC12 cells. These results suggested that isoflurane induced cytoxicity by

  10. DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

    Science.gov (United States)

    Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong

    2015-01-01

    In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

  11. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

    Science.gov (United States)

    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu

    2011-06-01

    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  12. Cell metabolomics reveals the neurotoxicity mechanism of cadmium in PC12 cells.

    Science.gov (United States)

    Zong, Li; Xing, Junpeng; Liu, Shu; Liu, Zhiqiang; Song, Fengrui

    2017-08-17

    The heavy metals such as cadmium (Cd) can induce neurotoxicity. Extensive studies about the effects of Cd on human health have been reported, however, a systematic investigation on the molecular mechanisms of the effects of Cd on central nervous system is still needed. In this paper, the neuronal PC-12 cells were treated with a series of concentrations of CdCl2 for 48h. Then the cytotoxicity was evaluated by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The IC15 value (15% inhibiting concentration) was selected for further mechanism studies. After PC-12 cells incubated with CdCl2 at a dose of IC15 for 48h, the intracellular and extracellular metabolites were profiled using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS)-based cell metabolomics approach. As found, the effects of the heavy metal Cd produced on the PC-12 cell viability were dose-dependent. The metabolic changes were involved in the glycolysis and gluconeogenesis, biopterin metabolism, tryptophan metabolism, tyrosine metabolism, glycerophospholipid metabolism, and fatty acids beta-oxidation. These could cause the perturbation of cell membrane, redox balance, energy supply, cellular detoxification, further affecting the cellular proliferation and apoptosis and other cellular activities. Copyright © 2017. Published by Elsevier Inc.

  13. Autophagy Alleviates Melamine-Induced Cell Death in PC12 Cells Via Decreasing ROS Level.

    Science.gov (United States)

    Wang, Hui; Gao, Na; Li, Zhigui; Yang, Zhuo; Zhang, Tao

    2016-04-01

    Since melamine was illegally added to raw milk for increasing the apparent protein content, such a scandal has not been quite blown out. Previous studies showed that melamine induced apoptosis and oxidative damage in both in vivo and in vitro experiments. It is well known that autophagy is closely related to oxidative stress. In the present study, we examined whether autophagy played an important role in protecting PC12 cells, which were damaged by melamine. Immunofluorescence assay showed that melamine enhanced the number of punctuate dot, indicating the increase of autophagosomes. Western blot assay presented that melamine significantly elevated the expression level of autophagy markers including LC3-II/LC3-I ratio, beclin-1, and Atg 7. Rapamycin further enhanced the effect, whereas 3-methyadenine (3-MA) inhibited it. MTT assay exhibited that rapamycin significantly enhanced the cell viability (P melamine-treated PC12 cells (P melamine-treated PC12 cells (P melamine-induced cell death via inhibiting the excessive generation of ROS. Regulating autophagy may become a new targeted therapy to relieve the damage induced by melamine.

  14. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E

    1994-01-01

    reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted...

  15. Organic Photovoltaics and Bioelectrodes Providing Electrical Stimulation for PC12 Cell Differentiation and Neurite Outgrowth.

    Science.gov (United States)

    Hsiao, Yu-Sheng; Liao, Yan-Hao; Chen, Huan-Lin; Chen, Peilin; Chen, Fang-Chung

    2016-04-13

    Current bioelectronic medicines for neurological therapies generally involve treatment with a bioelectronic system comprising a power supply unit and a bioelectrode device. Further integration of wireless and self-powered units is of practical importance for implantable bioelectronics. In this study, we developed biocompatible organic photovoltaics (OPVs) for serving as wireless electrical power supply units that can be operated under illumination with near-infrared (NIR) light, and organic bioelectronic interface (OBEI) electrode devices as neural stimulation electrodes. The OPV/OBEI integrated system is capable to provide electrical stimulation (ES) as a means of enhancing neuron-like PC12 cell differentiation and neurite outgrowth. For the OPV design, we prepared devices incorporating two photoactive material systems--β-carotene/N,N'-dioctyl-3,4,9,10-perylenedicarboximide (β-carotene/PTCDI-C8) and poly(3-hexylthiophene)/phenyl-C61-butyric acid methyl ester (P3HT/PCBM)--that exhibited open circuit voltages of 0.11 and 0.49 V, respectively, under NIR light LED (NLED) illumination. Then, we connected OBEI devices with different electrode gaps, incorporating biocompatible poly(hydroxymethylated-3,4-ethylenedioxythiophene), to OPVs to precisely tailor the direct current electric field conditions during the culturing of PC12 cells. This NIR light-driven OPV/OBEI system could be engineered to provide tunable control over the electric field (from 220 to 980 mV mm(-1)) to promote 64% enhancement in the neurite length, direct the neurite orientation on chips, or both. The OPV/OBEI integrated systems under NIR illumination appear to function as effective power delivery platforms that should meet the requirements for wirelessly offering medical ES to a portion of the nervous system; they might also be a key technology for the development of next-generation implantable bioelectronics.

  16. Curcumin Protects β-Lactoglobulin Fibril Formation and Fibril-Induced Neurotoxicity in PC12 Cells.

    Directory of Open Access Journals (Sweden)

    Mansooreh Mazaheri

    Full Text Available In this study the β-lactoglobulin fibrillation, in the presence or absence of lead ions, aflatoxin M1 and curcumin, was evaluated using ThT fluorescence, Circular dichroism spectroscopy and atomic force microscopy. To investigate the toxicity of the different form of β-Lg fibrils, in the presence or absence of above toxins and curcumin, we monitored changes in the level of reactive oxygen species and morphology of the differentiated neuron-like PC12 cells. The cell viability, cell body area, average neurite length, neurite width, number of primary neurites, percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different form of β-Lg fibrils. Incubation of β-Lg with curcumin resulted in a significant decrease in ROS levels even in the presence of lead ions and aflatoxin M1. The β-Lg fibrils formed in the presence of lead ions and aflatoxin M1 attenuated the growth and complexity of PC12 cells compared with other form of β-Lg fibrils. However, the adverse effects of these toxins and protein fibrils were negated in the presence of curcumin. Furthermore, the antioxidant and inhibitory effects of curcumin protected PC12 cells against fibril neurotoxicity and enhanced their survival. Thus, curcumin may provide a protective effect toward β-Lg, and perhaps other protein, fibrils mediated neurotoxicity.

  17. Activation of muscarinic receptors inhibits glutamate-induced GSK-3β overactivation in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Ke MA; Li-min YANG; Hong-zhuan CHEN; Yang LU

    2013-01-01

    Aim:To investigate the actions of the muscarinic agonist carbachol on glutamate-induced neurotoxicity in PC12 cells,and the underlying mechanisms.Methods:PC12 cells were treated with different concentrations of glutamate for 24 or 48 h.The cell viability was measured using MTT assay,and the expression and activation of GSK-3β were detected with Western blot.β-Catenin translocation was detected using immunofluorescence.Luciferase reporter assay and real-time PCR were used to analyze the transcriptional activity of β-catenin.Results:Glutamate (1,3,and 10 mmol/L) induced PC12 cell death in a dose-dependent manner.Moreover,treatment of the cells with glutamate (1 mmol/L) caused significant overactivation of GSK-3β and prevented β-catenin translocation to the nucleus.Pretreatment with carbachol (0.01 μmol/L) blocked glutamate-induced cell death and GSK-3β overactivation,and markedly enhanced β-catenin transcriptional activity.Conclusion:Activation of muscarinic receptors exerts neuroprotection in PC12 cells by attenuating glutamate-induced GSK-3β overactivation,suggesting potential benefits of muscarinic agonists for Alzheimer's disease.

  18. Effects of scutellarin on PKCγ in PC12 cell injury induced by oxygen and glucose deprivation

    Institute of Scientific and Technical Information of China (English)

    Wei XU; Ruo-peng ZHA; Wen-yi WANG; Yi-ping WANG

    2007-01-01

    Aim: To evaluate the neuroprotective effect and mechanisms of scutellarin (Scu)against PC12 cell injury after oxygen and glucose deprivation followed by reperfusion (OGD-Rep). Methods: Undifferentiated rat pheochromocytoma PC12 cells, exposed to oxygen and glucose deprivation followed by reperfusion (OGD-Rep), used as an in vitro model of ischemia/reperfusion. Cell survival was evalu-ated by diphenyltetrazolium bromide (MTT) assay and the amount of LDH release was determined using assay kits. [Ca2+]1 was monitored using a fluorescent Ca2+-sensitive dye Fura-2 acetoxymethyl ester. Cell apoptosis was detected by a DNA ladder and by flow cytometric detection. The expression of protein kinase C (PKC)γ was determined using both RT-PCR and Western blotting. The translocation of PKCγ was assayed by subcellular fractionation and Western blotting.Results: OGD-Rep injury significantly elevated the level of LDH release, [Ca2+]1,mRNA expression and the translocation of PKCγ compared in the PC12 cells with those of the normal group. Scu (10-100 μmol/L) exerted a protective effect against OGD-Rep injury by reducing LDH release, [Ca2+]1, the percent of apoptosis, and the translocation of PKCγ. Conclusion: Scu inhibits the increase of [Ca2+]1 and the activation of PKCγ, exerting protective effects against PC12 cell injury induced by OGD-Rep.

  19. Cytotoxic, Genotoxic, and Neurotoxic Effects of Mg, Pb, and Fe on Pheochromocytoma (PC-12) Cells

    Science.gov (United States)

    Sanders, Talia; Liu, Yi-Ming; Tchounwou, Paul B.

    2014-01-01

    Metals such as lead (Pb), magnesium (Mg), and iron (Fe) are ubiquitous in the environment as a result of natural occurrence and anthropogenic activities. Although Mg, Fe and others are considered essential elements, high level of exposure has been associated with severe adverse health effects including cardiovascular, hematological, nephrotoxic, hepatotoxic, and neurologic abnormalities in humans. In the present study we hypothesized that Mg, Pb, and Fe are cytotoxic, genotoxic and neurotoxic, and their toxicity is mediated through oxidative stress and alteration in protein expression. To test the hypothesis, we used the pheochromocytoma (PC-12) cell line as a neuro cell model and performed the LDH assay for cell viability, Comet assay for DNA damage, Western blot for oxidative stress, and HPLC-MS to assess the concentration levels of neurological biomarkers such as glutamate, dopamine (DA), and 3-methoxytyramine (3-MT). The results of this study clearly show that Mg, Pb, and Fe, respectively in the form of MgSO4, Pb(NO3)2, FeCl2, and FeCl3 induce cytotoxicity, oxidative stress, and genotoxicity in PC-12 cells. In addition, exposure to these metallic compounds caused significant changes in the concentration levels of glutamate, dopamine, and 3-MT in PC-12 cells. Taken together the findings suggest that MgSO4, Pb(NO3)2, FeCl2, and FeCl3 have the potential to induce substantial toxicity to PC-12 cells. PMID:24942330

  20. Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway.

    Science.gov (United States)

    Wang, Xi; Lu, Xiaocheng; Zhu, Ronglan; Zhang, Kaixin; Li, Shuai; Chen, Zhongjun; Li, Lixin

    2017-01-25

    Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

  1. Subcellular localization of WD40 repeat 1 protein in PC12 rat pheochromocytoma cells.

    Science.gov (United States)

    Shin, Dong Hoon; Lee, Eunju; Chung, Yoon Hee; Mun, Ga Hee; Park, Ji yeong; Lomax, Margaret I; Oh, Seung Ha

    2004-09-09

    The dynamics of actin filament protein is crucial for various physiological processes of the cells. Among the proteins correlating with actin dynamics, a novel 67-kDa WD40 repeat protein 1 (WDR1) was the vertebrate homologue of actin-interacting protein 1 (Aip1). Even though previous studies have provided the clues on the function of WDR1 in specific organs under pathological conditions, the exact subcellular localization of WDR1 is not known. Therefore, in the present study, we undertook to determine the distribution of WDR1 within PC12 pheochromocytoma cells (PC12 cells) using light and electron microscopic techniques. Double immunocytochemistry clearly showed that WDR1 immunoreactivities (IRs) were co-localized with anti-actin antibody, suggesting the involvement of WDR1 in actin dynamics. WDR1 immunoreactivities (IRs) in PC12 cells showed different distribution patterns as nerve growth factor (NGF) concentrations varied. During active proliferation, the distribution of WDR1 IRs seemed to be similar to those found in cortical actin patches, whereas WDR1 IR was observed in cytoplasmic actin cables after PC12 cells were induced to differentiate by treating with NGF. Though further studies are necessary to determine the function of WDR1, the current data represents a first step towards the in vitro study of WDR1 protein.

  2. Protective effects of red wine flavonols on 4-hydroxynonenal-induced apoptosis in PC12 cells.

    Science.gov (United States)

    Jang, Young Jin; Kang, Nam Joo; Lee, Ki Won; Lee, Hyong Joo

    2009-08-01

    There is accumulating evidence that a moderate consumption of red wine has health benefits, such as the inhibition of neurodegenerative diseases. Although this is generally attributed to resveratrol, the protective mechanisms and the active substance(s) remain unclear. We examined whether and how red wine extract (RWE) and red wine flavonols quercetin and myricetin inhibited 4-hydroxynonenal (HNE)-induced apoptosis of rat pheochromocytoma PC12 cells. RWE attenuated HNE-induced PC12 cell death in a dose-dependent manner. HNE induced cleavage of poly(ADP-ribose) polymerase, which is involved in DNA repair in the nucleus, and this was inhibited by RWE treatment. Treatment with RWE also inhibited HNE-induced nuclear condensation in PC12 cells. Data of 2',7'-dichlorofluorescin diacetate showed that RWE protected against apoptosis of PC12 cells by attenuating intracellular reactive oxygen species. The cytoprotective effects on HNE-induced cell death were stronger for quercetin and myricetin than for resveratrol. HNE-induced nuclear condensation was attenuated by quercetin and myricetin. These results suggest that the neuroprotective potential of red wine is attributable to flavonols rather than to resveratrol.

  3. In vitro toxicity of nanosized copper particles in PC12 cells induced by oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Xu Pengjuan; Xu Jing; Liu Shichang [Nankai University, College of Medicine (China); Ren, Guogang [University of Hertfordshire, School of Aerospace Engineering (United Kingdom); Yang Zhuo, E-mail: zhuoyang@nankai.edu.cn [Nankai University, College of Medicine (China)

    2012-06-15

    Recent evidence suggests that some nanomaterials, which are widely used in many fields, have health effects. In order to investigate the cytotoxicity induced by nanosized copper particles (nano-Cu), PC12 cells, which were widely used as an in vitro model for the neuron research, were treated with different concentrations (0, 1, 10, 30, and 100 {mu}g/mL) of nano-Cu. The cell viability was determined by measurement of the reduction product of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). The oxidative stress induced by nano-Cu and its possible mechanism were studied in relation to the generation of reactive oxygen species (ROS) and the cellular activity of superoxide dismutase (SOD). Results showed that incubation of PC12 cells with increasing concentrations of nano-Cu induced a decrease of cell viability in a concentration- and time-dependent manner. In addition, flow cytometry assay using Annexin V-FITC/PI staining was used to investigate the mode of nano-Cu-induced cell death and quantified the percentage of apoptotic cells. Results showed that nano-Cu induced the significant apoptosis in PC12 cells. Meanwhile, intracellular accumulation of ROS was increased with the increased concentrations of nano-Cu and it was associated with decreased SOD activity, which was probably due to protect effects against the oxidative stress in PC12 cells. Results suggested that both excessive intracellular ROS and decreased SOD contributed to nano-Cu-induced cytotoxicity. In other words, the increasing of oxidative stress was a key mechanism in PC12 apoptosis induced by nano-Cu.

  4. Involvement of stathmin 1 in the neurotrophic effects of PACAP in PC12 cells.

    Science.gov (United States)

    Dejda, Agnieszka; Chan, Philippe; Seaborn, Tommy; Coquet, Laurent; Jouenne, Thierry; Fournier, Alain; Vaudry, Hubert; Vaudry, David

    2010-09-01

    Rat pheochromocytoma PC12 cells have been widely used to investigate the neurotrophic activities of pituitary adenylate cyclase-activating polypeptide (PACAP). In particular, PACAP has been shown to promote differentiation and to inhibit apoptosis of PC12 cells. In order to identify the mechanisms mediating these effects, we sought for proteins that are phosphorylated upon PACAP treatment. High-performance liquid chromatography and 2D gel electrophoresis analysis, coupled with mass spectrometry, revealed that stathmin 1 is strongly phosphorylated within only 5 min of exposure to PACAP. Western blot experiments confirmed that PACAP induced a robust phosphorylation of stathmin 1 in a time-dependent manner. On the other hand, PACAP decreased stathmin 1 gene expression. Investigations of the signaling mechanisms known to be activated by PACAP revealed that phosphorylation of stathmin 1 was mainly mediated through the protein kinase A and mitogen-activated protein kinase pathways. Blockage of stathmin 1 expression with small interfering RNA did not affect PC12 cell differentiation induced by PACAP but reduced the ability of the peptide to inhibit caspase 3 activity and significantly decreased its neuroprotective action. Taken together, these data demonstrate that stathmin 1 is involved in the neurotrophic effect of PACAP in PC12 cells.

  5. Arachidonic acid release from PC12 pheochromocytoma cells is regulated by I1-imidazoline receptors.

    Science.gov (United States)

    Ernsberger, P

    1998-10-15

    Rat PC 12 pheochromocytoma cells lack alpha2-adrenergic receptors but express plasma membrane I1-imidazoline receptors. In response to the I1-agonist moxonidine, diglycerides are generated via phosphatidylcholine-selective phospholipase C, and prostaglandin E2 is released. This report characterizes I-receptor-mediated release of arachidonic acid, the precursor to the prostaglandins. PC12 cells were incubated with [3H]arachidonic acid for 24 h and superfused with 0.01% bovine serum albumin in Krebs' physiological buffer at 1 ml/min. Calcium ionophore increased arachidonic acid release only marginally, implying that in PC12 cells arachidonic acid release is not driven by calcium. The I1-agonist moxonidine at concentrations between 10 nM and 1.0 microM rapidly elicited up to two-fold increases in [3H]arachidonic acid release. Guanabenz, a potent alpha2-agonist and I2-ligand, had no effect. The selective I1-antagonist efaroxan blocked the action of moxonidine. The phospholipase A2 inhibitor aristolochic acid had no effect, suggesting that arachidonic acid release may be through an indirect pathway, possibly involving diglycerides. Thus, I1-imidazoline receptors in PC12 cells are coupled to arachidonic acid release through an as yet unknown pathway.

  6. Toluene-induced, Ca2+-dependent vesicular catecholamine release in rat PC12 cells

    NARCIS (Netherlands)

    Westerink, R.H.S.; Vijverberg, H.P.M.

    2002-01-01

    Acute effects of toluene on vesicular catecholamine release from intact PC12 phaeochromocytoma cells have been investigated using carbon fiber microelectrode amperometry. The frequency of vesicles released is low under basal conditions and is enhanced by depolarization. Toluene causes an increase in

  7. POTENTIAL MECHANISMS RESPONSIBLE FOR CHLOROTRIAZINE-INDUCED ALTERATIONS IN CATECHOLAMINES IN PHEOCHROMOCYTOMA (PC12) CELLS

    Science.gov (United States)

    ABSTRACTPotential Mechanisms Responsible for Chlorotriazine-induced Changes in Catecholamine Metabolism in Pheochromocytoma (PC12) Cells*PARIKSHIT C. DAS1, WILLIAM K. McELROY2 , AND RALPH L. COOPER2+ 1Curriculum in Toxicology, University of North Carolina, Chape...

  8. Toluene-induced, Ca2+-dependent vesicular catecholamine release in rat PC12 cells

    NARCIS (Netherlands)

    Westerink, R.H.S.|info:eu-repo/dai/nl/239425952; Vijverberg, H.P.M.

    2002-01-01

    Acute effects of toluene on vesicular catecholamine release from intact PC12 phaeochromocytoma cells have been investigated using carbon fiber microelectrode amperometry. The frequency of vesicles released is low under basal conditions and is enhanced by depolarization. Toluene causes an increase in

  9. Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells

    DEFF Research Database (Denmark)

    Silverman, Eran; Frödin, Morten; Gammeltoft, Steen;

    2004-01-01

    We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increas...

  10. Inhibitive Effect of Magnolol Against MPTP-Induced Apoptosis in PC12 Cells%厚朴酚对MPTP诱导PC12细胞凋亡的抑制作用

    Institute of Scientific and Technical Information of China (English)

    鄢印根; 林泉峰; 徐焱; 黄小丽

    2011-01-01

    Objective:To investigate the inhibition of magnolol on the apoptosis of PC 12 cell induced by MPTP and its mechanism. Method: Magnolol (final concentration 0.1, 10 μmol·L-1) and /or MPTP ( final concentration 50, 100, 150, 200 μmol·L-1) were added into the culture of PC12 cells. The cell viability was detected with MTT method, apoptosis was measured by flow cytometry and Bcl-2/Bax ratio was detected by western blot. Result: Cell viability was declined sharply by MPTP with different concentrations, the pre-treatment of magnolol 0. 1-10 μmol·L-1 could significantly increase the PC12 cell viability, apoptosis and the decrease in the Bcl-2/Bax ratio. Conclusion: Magnolol may inhibit the cell damage induced by MPTP and the mechanism is likely related to inhibition to expression of Bcl-2/Bax.%探讨厚朴酚( mangolol,Mag)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导PC12细胞凋亡的抑制作用及可能作用机制.方法 将厚朴酚(终浓度0.1,10 μmol· L-1)或/和MPTP(终浓度50,100,150,200 μmol·L-1)加入到培养的PC12细胞中.四甲基偶氮唑盐法(MTT法)检测细胞增殖活性,用流式细胞仪检测细胞凋亡率,以及Western -blot法检测Bax和Bcl-2的蛋白表达变化.结果 在加入不同浓度的MPTP处理细胞24 h,细胞增殖活性渐次降低,而厚朴酚0.1~10μmol·L-1预处理1h可明显减轻MPTP导致的PC12细胞的损伤,抑制细胞凋亡,以及Bcl-2/Bax比值的改变.结论 厚朴酚对MPTP诱导的PC12细胞凋亡损伤有一定的抑制作用,其作用机制涉及到影响细胞内Bcl-2/Bax蛋白的表达.

  11. Synthesis of cystine C60 derivative and its protective effects on hydrogen peroxide-induced apoptosis in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Zhen Hu; Hai Ping Xing; Zhou Zhu; Wei Wang; Wen Chao Guan

    2007-01-01

    Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the usable ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of some free radical scavenger. Water-soluble amino-fullerene is a novel compound that behaves as a free radical scavenger with excellent biology consistent. In the present study, we have synthesized and characterized a novel cystine C60 derivative for the first time, and investigated the effects on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death as determined by MTT, PI/Hoechst 33342 staining and flow cytometry analysis. These results suggested that cystine C60 derivative has the potential to prevent oxidative stress-induced cell death and has no evident toxicity.

  12. Protective effects of components of the Chinese herb grassleaf sweetlfag rhizome on PC12 cells incubated with amyloid-beta42

    Institute of Scientific and Technical Information of China (English)

    Zi-hao Liang; Xiao-hui Cheng; Zhi-gang Ruan; Han Wang; Shan-shan Li; Jing Liu; Guo-ying Li; Su-min Tian

    2015-01-01

    The major ingredients of grassleaf sweetlfag rhizome areβ-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms ofβ-asarone and eugenol, components of the Chinese herb grassleaf sweetlfag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations (between 1 × 10–10 M and 1 × 10–5 M) ofβ-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions (1 × 10–6 Mβ-asarone and eugenol). The survival rates of PC12 cells significantly increased, while expression levels of the mRNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl mRNA increased. In addition, the combination ofβ-asarone with eugenol achieved better results than either component alone. Our experimental ifndings indicate that bothβ-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.

  13. Protective effects of components of the Chinese herb grassleaf sweetflag rhizome on PC12 cells incubated with amyloid-beta42

    Directory of Open Access Journals (Sweden)

    Zi-hao Liang

    2015-01-01

    Full Text Available The major ingredients of grassleaf sweetflag rhizome are β-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms of β-asarone and eugenol, components of the Chinese herb grassleaf sweetflag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations (between 1 × 10 -10 M and 1 × 10 -5 M of β-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions (1 × 10 -6 M β-asarone and eugenol. The survival rates of PC12 cells significantly increased, while expression levels of the mRNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl mRNA increased. In addition, the combination of β-asarone with eugenol achieved better results than either component alone. Our experimental findings indicate that both β-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.

  14. Effect of formaldehyde on endogenous hydrogen sulfide production in PC12 cells%甲醛对PC12细胞内源性H2S生成的影响

    Institute of Scientific and Technical Information of China (English)

    尹蔚兰; 何剑琴; 方恒荣; 杨丝丝; 胡弼; 唐小卿

    2011-01-01

    目的 观察甲醛对PC12细胞内源性硫化氢(hydrogen sulfide,H2S)生成的影响,以探讨甲醛损伤PC12细胞的新机制.方法 RT-PCR方法检测PC12细胞的胱硫醚-β-合酶 (cystathionine-beta-synthase,CBS) mRNA 的表达;亚甲基蓝分光光度计法检测PC12细胞CBS活性及PC12细胞内源性H2S的含量;MTT法观察PC12细胞的存活率.结果 甲醛可以抑制PC12细胞CBS的表达及其活性,减少内源性H2S的生成;甲醛可以明显地降低PC12细胞的存活率.结论 甲醛能抑制CBS的表达和活性,减少内源性H2S生成,这可能与其损伤PC12细胞有关.%Aim To observe the effect of formaldehyde on the production of endogenous hydrogen sulfide ( H2S ) in PC12 cells and to explore the novel molecular mechanisms of formaldehyde-induced damage in PC12 cells. Methods The expression of cystathionine-beta-synthase ( CBS ) mRNA in PC12 cells was determined by RT-PCR. The production of H2S and the activity of CBS in PC12 cells were detected by methylene blue spectrophotometric method. The survival of PC12 cells was observed by MTT assay. Results Formaldehyde inhibited the expression of CBS mRNA and the activity of CBS, and decreased the production of endogenous H2S in PC12 cells. Formaldehyde significantly reduced the survival rate of PC12 cells. Conclusion Formaldehyde inhibits the expression and activity of CBS, as well as endogenous H2S production in PC12 cells, which may be associated with formaldehyde-induced PC12 cells damage.

  15. High Glucose-Induced PC12 Cell Death by Increasing Glutamate Production and Decreasing Methyl Group Metabolism

    Directory of Open Access Journals (Sweden)

    Minjiang Chen

    2016-01-01

    Full Text Available Objective. High glucose- (HG- induced neuronal cell death is responsible for the development of diabetic neuropathy. However, the effect of HG on metabolism in neuronal cells is still unclear. Materials and Methods. The neural-crest derived PC12 cells were cultured for 72 h in the HG (75 mM or control (25 mM groups. We used NMR-based metabolomics to examine both intracellular and extracellular metabolic changes in HG-treated PC12 cells. Results. We found that the reduction in intracellular lactate may be due to excreting more lactate into the extracellular medium under HG condition. HG also induced the changes of other energy-related metabolites, such as an increased succinate and creatine phosphate. Our results also reveal that the synthesis of glutamate from the branched-chain amino acids (isoleucine and valine may be enhanced under HG. Increased levels of intracellular alanine, phenylalanine, myoinositol, and choline were observed in HG-treated PC12 cells. In addition, HG-induced decreases in intracellular dimethylamine, dimethylglycine, and 3-methylhistidine may indicate a downregulation of methyl group metabolism. Conclusions. Our metabolomic results suggest that HG-induced neuronal cell death may be attributed to a series of metabolic changes, involving energy metabolism, amino acids metabolism, osmoregulation and membrane metabolism, and methyl group metabolism.

  16. Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

    Institute of Scientific and Technical Information of China (English)

    Nan Jiang; Yunliang Guo; Hongbing Chen

    2006-01-01

    BACKGROUND: Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation.OBJECTIVE: To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation.DESIGN: Randomized controlled study.SETTING: Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University.MATERIALS: The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells,were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide (MTT) and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA.METHODS: ① Culture of PC12 cells: PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37 ℃. Number of cells was regulated to 4 × 105 L-1, and cells were inoculated at 96-well culture plate.The final volume was 100 μL. ② Model establishing and grouping: Cultured PC12 cells were randomly divided into three groups: phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L, but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group (the final concentration of 3 g/L) and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non

  17. Aβ寡聚体预处理的BV2细胞对PC12细胞凋亡的影响%Effect of BV2 pre-treated with Aβ1 ~42 oligimers upon PC12 cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    蔺慕会; 徐忠信; 陈晓虹

    2011-01-01

    Objective To study the effect of BV2 upon PC 12 before or after it was pre-treated with Aβ1-42 oligim-ers( ADDLs) ,in order to investigate microglia's injury to neuron. Methods In control group ( Aβ+ PC12) ,PC12 cells were treated with Aβ1-42 oligimers of different concentrations. In experimental group( Aβ + BV2 + PC12) ,BV2 cells were pre-cultured with Aβ1-42 oligimers of the same concentration and then co-cultured with PC12 cells through transwells. PC12 cells' inhibition ratio was tested by MTT,cell apoptosis was tested by flow cytometry assay,and tau( pS396) protein expression was tested by Western blot. Results ADDLs of all concentrations could induce PC12 cells apoptosis and PC12 cells' apoptosis increased with ADDLs' concentrations increased. PC 12 cells' inhibition ratio, cell apoptosis and tau (pS396) protein expression were increased with Aβ concentration increased in all group. Compared with control group, the change was more apparent in experimental group. The difference between two group with same Aβ concentration group was significant(P <0.05). Conclusion BV2 cells can significantly attenuates PC 12 cells' inhibition,tau(pS396)protein expression and apoptosis caused by Aβ1-42 oligimers.%目的 研究Aβ寡聚体(Aβ-derived diffusible ligands,ADDLs)干预的BV2细胞对PC12细胞凋亡的影响,进而探讨小胶质细胞对神经元损伤的作用.方法 分别用不同浓度Aβ1~42寡聚体作用于PC12细胞(大鼠肾上腺嗜铬细胞瘤细胞)作为对照组( Aβ+ PC12);用相应浓度的Aβ1 ~42寡聚体预处理BV2细胞,然后通过转移筛网与PC12细胞共育作为实验组(Aβ+ BV2+ PC12).应用MTT方法检测PC12细胞抑制率,流式细胞仪检测细胞凋亡,Western blot方法观察PC12细胞tau、tau( pS396)蛋白表达的变化.结果 所有浓度的ADDLs均可导致PC12细胞凋亡,且PC12细胞凋亡表现为ADDL浓度依赖性关系,即随着Aβ浓度增加PC12抑制率、细胞凋亡、tau( pS396)表达也明显增加,其中实验组PC

  18. Protective effect of arctigenin on ethanol-induced neurotoxicity in PC12 cells.

    Science.gov (United States)

    Huang, Jia; Xiao, Lan; Wei, Jing-Xiang; Shu, Ya-Hai; Fang, Shi-Qi; Wang, Yong-Tang; Lu, Xiu-Min

    2017-04-01

    As a neurotropic substance, ethanol can damage nerve cells through an increase in the production of free radicals, interference of neurotrophic factor signaling pathways, activation of endogenous apoptotic signals and other molecular mechanisms. Previous studies have revealed that a number of natural drugs extracted from plants offer protection of nerve cells from damage. Among these, arctigenin (ATG) is a lignine extracted from Arctium lappa (L.), which has been found to exert a neuroprotective effect on scopolamine‑induced memory deficits in mice with Alzheimer's disease and glutamate-induced neurotoxicity in primary neurons. As a result, it may offer beneficial effects on ethanol-induced neurotoxicity. However, the effects of ATG on ethanol‑induced nerve damage remain to be elucidated. To address this issue, the present study used rat pheochromocytoma PC12 cells to investigate the neuroprotective effects of ATG on ethanol-induced cell damage by performing an MTT reduction assay, cell cycle analysis, Hoechst33342/propidium iodide fluorescence staining and flow cytometry to examine apoptosis. The results showed that 10 µM ATG effectively promoted the proliferation of damaged cells, and increased the distribution ratio of the cells at the G2/M and S phases (P<0.05). In addition, the apoptosis and necrosis of the PC12 cells were significantly decreased following treatment with ATG. Therefore, it was concluded that 10 µM ATG had a protective effect on ethanol‑induced injury in PC12 cells.

  19. VAMP-2 promotes neurite elongation and SNAP-25A increases neurite sprouting in PC12 cells.

    Science.gov (United States)

    Shirasu, M; Kimura, K; Kataoka, M; Takahashi, M; Okajima, S; Kawaguchi, S; Hirasawa, Y; Ide, C; Mizoguchi, A

    2000-08-01

    Recent studies suggest that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion in the plasma membrane that allows expansion for neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. In this study, we took the opposite approach by overexpressing green fluorescent protein (GFP)-fusion SNAREs, including VAMP-2, SNAP-25A, and syntaxin1A, in PC12 cells to investigate the role of SNAREs in the neurite outgrowth of PC12 cells. Neurite outgrowth analysis demonstrated that: (1) GFP-VAMP-2 increased the length of individual neurites, without changing the number of neurites per cell; (2) GFP-SNAP-25A increased the number of neurites per cell, with no change in the length of the individual neurites. In both cases, the total length of neurites per cell was increased; (3) GFP-syntaxin1A resulted in no significant change, either in neurite length, or in the number of neurites per cell. These findings suggest that when overexpressed in PC12 cells, VAMP-2 can promote neurite elongation, while SNAP-25A can stimulate neurite sprouting. On the other hand, overexpression of syntaxin1A neither promotes nor inhibits neurite outgrowth. Thus VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.

  20. Activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells and isolated hepatocytes by organophosphates.

    Science.gov (United States)

    Kauffman, F C; Davis, L H; Whittaker, M

    1990-01-15

    Several organophosphates including diisopropylfluorophosphonate (DPF) and a variety of compounds used as chemical warfare agents produced dose- and time-dependent increases in phosphorylase-a, the phosphorylated form of glycogen phosphorylase in rat pheochromocytoma cells, PC12, and isolated hepatocytes. Increases in phosphorylase-a did not occur in cells exposed to the carbamates, physostigmine or pyridostigmine, or to O-ethyl S-2-diisopropylaminoethylmethyl-phosphonathiolate (VX), an organophosphate which is protonated at physiological pH. When extracellular pH was increased to pH 8, VX acted like the other organophosphates and increased phosphorylase-a activity. The possibility that organophosphates increase phosphorylase-a in intact cells by releasing Ca2+ from intracellular binding sites is supported by the following findings: organophosphate-induced increases in phosphorylase-a did not correlate with changes in cyclic AMP in the two cell types studied; in PC12 cells, increases in this activity occurred in the absence of extracellular calcium and were not inhibited by the calcium channel blocker, verapamil; fluorescence of the calcium sensitive dye, Quin-2, in PC12 cells preloaded with the acetoxymethyl ester of the dye was increased by soman; finally, addition of the calcium ionophore, A23187, to PC12 cells maintained in calcium-free medium caused sarin-stimulated phosphorylase-a activity to return rapidly to basal levels. Collectively, these data argue strongly that organophosphates increase phosphorylase-a activity in intact cells via a novel mechanism involving release of calcium from intracellular binding sites.

  1. Nitric oxide promotes nicotine-triggered ERK signaling via redox reactions in PC12 cells.

    Science.gov (United States)

    Miyamoto, Yoshiaki; Sakai, Ryosuke; Maeda, Chiharu; Takata, Tsuyoshi; Ihara, Hideshi; Tsuchiya, Yukihiro; Watanabe, Yasuo

    2011-10-30

    Nitric oxide (NO), produced by neuronal NO synthase (nNOS), serves as a signaling molecule with diverse biological responses in the central nervous system (CNS). In the present study, we demonstrated that nNOS expression enhances the nicotine-triggered activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in nNOS-transfected PC12 (NPC12) cells. Treatment with nicotine increased the phosphorylation level of ERK1/2 in the NPC12 cells as compared with that in control PC12 cells. However, nicotine treatment failed to enhance ERK1/2 phosphorylation when NPC12 cells were pretreated with several selective inhibitors of NOS, the nicotinic acetylcholine receptors, L-type voltage-dependent Ca(2+) channels, protein kinase C, Src, epidermal growth factor receptor, and MEK. The nicotine-induced ERK1/2 phosphorylation in PC12 cells was observed by their pretreatment with a NO donor. Moreover, the enhancement of nicotine-induced ERK1/2 phosphorylation in the NPC12 cells was regulated by intracellular glutathione levels, but not by the soluble guanylate cyclase-cGMP-protein kinase G signaling. Meanwhile, depolarization stimulated ERK1/2 phosphorylation in both PC12 and NPC12 cells. Taken together, these findings suggest that nicotine modulates NO-dependent redox condition; the resulting calcium influx, would increase ERK1/2 phosphorylation in nNOS expressing cells. Blockade of NO pathway may be selective target to reduce ERK1/2 phosphorylation via attenuation of the nicotine responses in the CNS.

  2. The role of ribosylated-BSA in regulating PC12 cell viability.

    Science.gov (United States)

    Kuo, Tsun-Yung; Huang, Chuen-Lin; Yang, Jung-Mou; Huang, Wei-Jung; Huang, Nai-Kuei; Chen, Yue-Wen; Lin, Ren-Jye; Yang, Ying-Chen

    2012-08-01

    Glycation, one of the post-translational modifications, is known to influence protein structure and biological function. Advanced glycation end products (AGEs) have been shown to cause pathologies of diabetes. Glycation levels in patients with Alzheimer's disease (AD) are higher than in normal people. However, whether the glycation of susceptible proteins is a triggering event for cell damage or simply a result remains to be elucidated. In this study, we demonstrated that ribose-conjugated BSA (Rib-BSA) directly induces PC12 cell death in a dose- and time-dependent manner. The IC(50) is 4.6 μM. Unlike glucose-incubated BSA, Rib-BSA rapidly forms cytotoxic AGEs. PC12 is vulnerable to Rib-BSA. However, fructose can induce AGE formation, although no effect on cell survival was observed. This effect of Rib-BSA is reversed by pretreatment of pioglitazone and rosiglitazone, which belongs to thiazolidinediones (TZDs) and are peroxisome proliferator-activated receptor (PPAR-γ) ligands. Moreover, Rib-BSA upregulates inducible nitric oxide synthase (iNOS), cycloxygenase 2 (COX-2) expression, and p-38 phosphorylation and leaves extracellular regulated protein1/2 (ERK1/2) phosphorylation unchanged. The Rib-BSA-induced signaling changes are blocked by rosiglitazone and confirmed by PPAR-γ small-interfering RNA transfection. The reduction of cell survival by Rib-BSA is blocked by the iNOS inhibitor and p38 inhibitor. No effect on cell survival was observed using the COX-2 inhibitor. Consequently, these results show that Rib-BSA directly inducing PC12 cell death is a triggering event and TZDs protect PC12 cell from Rib-BSA damage. Signaling molecules, such as PPAR-γ, P38, and iNOS, are involved in Rib-BSA-mediated cytotoxicity.

  3. Activin A prevents neuron-like PC12 cell apoptosis after oxygen-glucose deprivation

    Institute of Scientific and Technical Information of China (English)

    Guihua Xu; Zhongxin Xu; Jinting He; Hongliang Guo; Chunli Mei; Jiaoqi Wang; Zhongshu Li; Han Chen; Jing Mang; Hong Yang

    2013-01-01

    In this study, PC12 cells were induced to differentiate into neuron-like cells using nerve growth factor, and were subjected to oxygen-glucose deprivation. Cells were treated with 0, 10, 20, 30, 50, 100 ng/mL exogenous Activin A. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and Hoechst 33324 staining showed that the survival percentage of PC12 cells significantly decreased and the rate of apoptosis significantly increased after oxygen-glucose deprivation. Exogenous Activin A significantly increased the survival percentage of PC12 cells in a dose-dependent manner. Reverse transcription-PCR results revealed a significant increase in Activin receptor IIA, Smad3 and Smad4 mRNA levels, which are key sites in the Activin A/Smads signaling pathway, in neuron-like cells subjected to oxygen-glucose deprivation, while mRNA expression of the apoptosis-regulation gene caspase-3 decreased. Our experimental findings indicate that exogenous Activin A plays an anti-apoptotic role and protects neurons by means of activating the Activin A/Smads signaling pathway.

  4. Selective alterations of transcription factors in MPP+-induced neurotoxicity in PC12 cells.

    Science.gov (United States)

    Xu, Z; Cawthon, D; McCastlain, K A; Duhart, H M; Newport, G D; Fang, H; Patterson, T A; Slikker, W; Ali, S F

    2005-08-01

    MPP(+) (1-methyl-4-phenylpyridinium; the active metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine)) depletes dopamine (DA) content and elicits cell death in PC12 cells. However, the mechanism of MPP(+)-induced neurotoxicity is still unclear. In this study, the dose response and time-course of MPP(+)-induced DA depletion and decreased cell viability were determined in nerve growth factor (NGF)-differentiated PC12 cells. The alteration of transcription factors (TFs) induced by MPP(+) from a selected dose level and time point was then evaluated using protein/DNA-binding arrays. K-means clustering analysis identified four patterns of protein/DNA-binding changes. Three of the 28 TFs identified in PC12 cells increased by 100% (p53, PRE, Smad SBE) and 2 decreased by 50% (HSE, RXR(DR1)) of control with MPP(+) treatment. In addition, three TFs decreased within the range of 33-50% (TFIID, E2F1, CREB) and two TFs increased within the range of 50-100% (PAX-5, Stat4). An electrophoretic mobility shift assay (EMSA) was used to confirm the changes of p53 and HSE. The observed changes in TFs correlated with the alterations of DA and cell viability. The data indicates that selective transcription factors are involved in MPP(+)-induced neurotoxicity and it provides mechanistic information that may be applicable to animal studies with MPTP and clinical studies of Parkinson's disease.

  5. Protective effect of telmisartan against oxidative damage induced by high glucose in neuronal PC12 cell.

    Science.gov (United States)

    Eslami, Habib; Sharifi, Ali M; Rahimi, Hamzeh; Rahati, Maryam

    2014-01-13

    Telmisartan is an angiotensin II type 1 receptor blocker and partial agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ). Here, we investigated the protective capacity of telmisartan against high glucose (HG)-elicited oxidative damage in PC12 cells. The activity of lactate dehydrogenase (LDH), NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT) as well as the levels of malondialdehyde (MDA), glutathione (GSH), intracellular reactive oxygen species (ROS), cell viability and DNA fragmentation were measured in HG-treated PC12 cells with and without telmisartan co-treatment. Moreover, the direct antioxidant effect of telmisartan was determined by 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay and protein expression of Bax, Bcl-2, cleaved caspase-3 and NOX subunit p47phox by western blotting. Telmisartan exhibited antioxidant activity in the ABTS assay with the IC50 value of 37.5 μM. Pretreatment of PC12 cells with telmisartan, prior to HG exposure, was associated with a marked diminution in cleaved caspase-3 expression, DNA fragmentation, Bax/Bcl-2 ratio, intracellular ROS and MDA levels. Additionally, the cell viability, GSH level, SOD and CAT activity were notably elevated by telmisartan, whereas the activity and the protein expression of NADPH oxidase subunit p47phox were attenuated. Interestingly, co-treatment with GW9662, a PPAR-γ antagonist, partially inhibited the beneficial effects of telmisartan. These findings suggest that telmisartan has protective effects on HG-induced neurotoxicity in PC12 cells, which may be related to its antioxidant action and inhibition of NADPH oxidase. Furthermore, the results show that PPAR-γ activation is involved in the neuroprotective effects of telmisartan. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  6. Resveratrol Protects PC12 Cell against 6-OHDA Damage via CXCR4 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    2015-01-01

    Full Text Available Resveratrol, herbal nonflavonoid polyphenolic compound naturally derived from grapes, has long been acknowledged to possess extensive biological and pharmacological properties including antioxidant and anti-inflammatory ones and may exert a neuroprotective effect on neuronal damage in neurodegenerative diseases. However, the underlying molecular mechanisms remain undefined. In the present study, we intended to investigate the neuroprotective effects of resveratrol against 6-OHDA-induced neurotoxicity of PC12 cells and further explore the possible mechanisms involved. For this purpose, PC12 cells were exposed to 6-OHDA in the presence of resveratrol (0, 12.5, 25, and 50 μM. The results showed that resveratrol increased cell viability, alleviated the MMP reduction, and reduced the number of apoptotic cells as measured by MTT assay, JC-1 staining, and Hoechst/PI double staining (all p<0.01. Immunofluorescent staining and Western blotting revealed that resveratrol averts 6-OHDA induced CXCR4 upregulation (p<0.01. Our results demonstrated that resveratrol could effectively protect PC12 cells from 6-OHDA-induced oxidative stress and apoptosis via CXCR4 signaling pathway.

  7. Characterization of docking and fusion of synaptic-like microvesicles in PC12 cells using TIRFM

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neurotransmitters are released by the fusion of synaptic vesicles with presynaptic membrane, which has been extensively studied. The analysis of single vesicle fusion kinetics reveals that there exist fusion modes of "kiss and run" and "kiss and stay" which may be favored by neurons especially during strong firing beside full fusion. Pre-fusion steps of translocation, docking and priming along the exocytotic pathway play important roles in neurotransmitter release and its regulation. In the present report, we used dual-color imaging of VAMP2-pHluorin and VAChT-TDimer2 under total internal reflection fluorescence microscope (TIRFM) to monitor the docking and fusion of synaptic-like microvesicles (SLMVs) in PC12 cells stimulated by high K+. Our results show that "kiss and run" is a dominative fusion mode in PC12 cells under high K+-challenge, and the dwell time of SLMVs is prolonged by the high K+ stimulation that suggests an enhancement of vesicle priming.

  8. Relationship between apoptosis and pErk in manganese-treated PC12 cell line%锰致PC12细胞凋亡作用及与p- Erk关系

    Institute of Scientific and Technical Information of China (English)

    徐文; 刘红; 闫文; 杨银书; 卢娟; 徐强; 董大海; 缪珊; 李金翠; 高琨; 高丽莉; 侯顺利; 左晶

    2012-01-01

    目的 以鼠嗜铬神经瘤细胞(PC12)为模型,筛选锰对神经细胞增殖抑制作用的时间及剂量,观察锰作用下PC12细胞的细胞形态学、生化指标改变和磷酸化的胞外信号调节激酶(p - Erk)的表达.方法 用200、400、600、800 μmol/L MnCl2的培养液,分别作用对数生长期PC12细胞1、2、3、4d后,四甲基偶氮塞唑蓝(MTT)筛选锰的细胞毒性剂量;透射电镜观察细胞形态学变化;琼脂糖凝胶电泳检测MnCl2对PCl2细胞基因组DNA的影响.免疫印迹法( western blot)检测p- Erk.结果 MTT显示200~800 μmol/L MnCl2作用1、2、3、4d对PC12有显著的抑制作用,呈剂量和时间依赖趋势,600 μmol/L MnCl2作用4d对PC12的抑制率50%;600 μrnol/L MnCl2作用4d电镜可见细胞凋亡,同样条件下细胞DNA碎片化;Western blot显示600μml/oL MnCl2作用1、2、3、4d可见p- Erk2逐渐降低,其中作用2d时较对照降低75%(n=3,P<0.05),200、400、600 μmol/L MnCl2分别作用4d时,p- Erk亦逐渐降低,当400 μmol/L MnCl2作用4d时较对照明降低78%(n=3,P<0.01);使用Erk通路MEK1/2特异性阻制剂PD98059实验结果表明:锰可能通过MEK1/2磷酸化下游的Erk,下调p- Erk.结论 锰对PC12细胞的毒性作用可能是通过关闭胞外信号调节激酶ErK通路诱导细胞凋亡.%Objective To observe apoptosis related cell morphology, biochemical changes and phosphrylations of phos-phoralated extracellularc signal-regulated kinase(p-Erk)in pheochromocytoma cells(PC12) exposed to manganese at different concentration and exposure time. Methods PC12 cells in logarithm growth period were incubated in culture media with 200,400,600,and 800μmol/L manganese(MnCl2)for 1,2,3 and 4 days,respectively. The cell viability was examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide (MTT) and morphological changes of PC12 cells were investigated with transmission electron microscope. Agarose gel electrophoresis was adopted to

  9. 甲基苯丙胺致PC12细胞的差异蛋白表达%Differentially expressed proteins in PC12 cells induced by methylamphetamine

    Institute of Scientific and Technical Information of China (English)

    李丽增; 王慧君; 兰江维; 刘超

    2012-01-01

    目的 探讨甲基苯丙胺( METH)作用于PC12细胞后的蛋白质表达变化.方法 METH2.5 mmol· L-1作用PC12细胞24 h后,提取细胞总蛋白质,丙酮沉淀法纯化蛋白,Braford法对蛋白质进行定量,并对蛋白质进行双向凝胶电泳,Image scannerⅢ透射扫描仪获取凝胶电泳图谱.应用Image Master 7.0软件对获得的双向凝胶电泳图谱进行差异性蛋白质点分析,并对相应差异蛋白点用高端基质辅助激光解析-飞行时间( MALDI-TOF)串联质谱仪进行差异蛋白质鉴定.结果 应用双向凝胶电泳结合质谱分析技术,METH作用PC12细胞24h后,共鉴定出18个差异表达蛋白质点,其中8个差异蛋白点在METH作用后表达增强,10个蛋白点表达减弱.这些蛋白质主要包括细胞骨架相关蛋白、分子伴侣、氧化应激和凋亡相关的蛋白以及与能量代谢相关的酶类.结论 METH诱导PC12细胞18个蛋白差异表达.%OBJECTIVE To investigate differentially expressed proteins of PC 12 cells exposed to methylamphetamine ( METH ). METHODS PC 12 cells were treated with METH 2.5 mmol·L-1 for 24 h. Total proteins were extracted for two dimensional gel electrophoresis (2-DE). Impurities were removed by acetone precipitation and the quantity of proteins was fixed by Braford method. Proteins were separated by two-dimensional gel electrophoresis (2-DE) , and the 2-DE patterns were obtained by transmission scan with Image scanner HI. The scanned patterns were then analyzed by ImageMaster 7. 0 software to search the differentially expressed proteins. Those protein spots were cutted and then identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) tandem mass spectrum. RESULTS After PC 12 cells were cultured with METH for 24 h, eighteen differentially expressed proteins were identified in PC 12 cells, eight of which were up-regulated and 10 down-regulated. According to functions of proteins, these proteins were divided into 4 categories

  10. Luteolin induces microRNA-132 expression and modulates neurite outgrowth in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Lian-Fang Lin

    Full Text Available Luteolin (3',4',5,7-tetrahydroxyflavone, a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132 in PC12 cells. The correlation between miR-132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB, which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA and MAPK/ERK kinase 1/2 (MEK1/2 inhibitors but not by protein kinase C (PKC or calcium/calmodulin-dependent protein kinase II (CaMK II inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.

  11. Effect of nerve regeneration factor on differentiation of PC12 cells and its signaling pathway

    Institute of Scientific and Technical Information of China (English)

    DING Fei; QIANG Liang; LIU Mei; GU Xingxing; GU Xiaosong

    2004-01-01

    The effects of nerve regeneration factor (NRF) on neuronal differentiation of PC12 cells and its signaling pathway are investigated by morphological observation and immunofluorescent cytochemical method, and the activity of ERK1/2 in NRF-treated PC12 cells in absence of serum is also studied by immuno-coprecipitation and Western blot analysis. The MEK1/2-specific inhibitor U0126, the broad-spectrum protein kinase C (PKC) inhibitor G6983 and tyrosine protein kinase (TPK) inhibitor genistein were used to determine the roles of the activation of ERK1/2 by NRF and the involvement of certain kinds of PKC or TPK receptor in this activation process. The results show that U0126 and G6983 inhibit the activation of ERK1/2 by NRF to different extents, while genistein has no effect on it, demonstrating that NRF remarkably induces neuronal differentiation of PC12 cells through activating ERK1/2 in a dose-dependent and time-dependent manner.

  12. Neuronal differentiation of PC12 cells induced by sciatic nerve and optic nerve conditioned medium

    Institute of Scientific and Technical Information of China (English)

    DU Chan; YANG De-mei; ZHANG Pei-xun; DENG Lei; JIANG Bao-guo

    2010-01-01

    Background Previous work has shown that optic nerve and sciatic nerve conditional medium had neurotrophic activity on neurons. In order to find if the optic nerve conditioned media (CM) had a similar activity to make PC12 cells differentiate as sciatic nerve CM did, we explored the neurotrophic activity in optic nerve CM in the same in vitro system and compared the neurotrophin expression levels in optic and sciatic nerves under both conditions.Methods PC12 cells were used to examine the effects of neurotrophins secreted by the sciatic nerve and optic nerve. RT-PCR and real-time QPCR showed that the sciatic nerve and optic nerve produced a range of neurotrophins including nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3).Results The effects of sciatic nerve and optic nerve CM on neurite outgrowth were tested against a range of neurotrophins, and they had different neuritogenic activities. Only NGF and sciatic nerve CM had obvious neuritogenic activities, although the concentration of NGF in the sciatic nerve CM was very low.Conclusions Our experiment showed that sciatic nerve CM had a higher neurotrophic activity on PC12 cells than optic nerve CM. These results suggested that peripheral nervous system (PNS) and central nervous system (CNS) had different expression levels of neurotrophin, which may in part explain the lack of ability to regenerate the CNS.

  13. Cyclophilin A affects Bcl-2 and Bax expression following beta-amyloid fragment 25-35-induced injury to PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Li Cheng; Chaodong Zhang

    2008-01-01

    BACKGROUND: Cyclophilin A can protect neurons against oxidative stress.OBJECTIVE: To investigate the effect of cyclophilin A on Bcl-2 and Bax protein expression in pheochromocytoma (PCI2) cells treated with beta-amyloid fragment 25-35 (A β25-35), and to verify the protection pathway ofcyclophilin A.DESIGN, TIME AND SETTING: The initial experiment was performed at the Laboratory of Department of Neurology, First Clinical College, China Medical University from November 2006 to July 2007.MATERIALS: PCI2 cells were cultured at the Cell Center of Peking Union Medical College. A β25-35 (Sigma, USA), antibodies of Bcl-2 and Bax (Wuhan Boster, China), and recombinant human cyclophilin A (Biomol, USA) were used in this study.METHODS: PC12 cells were divided into three groups. Cells in the control group were incubated in culture medium. Cells in the Aβ25-35 injury group were incubated in medium containing a final concentration of 10 μ mol/L of Aβ25-35. Cells in the cyclophilin A group were incubated in medium containing a final concentration of 10 nmol/L of cyclophilin A for 30 minutes, and then treated with 10 μmol/L Aβ25-35. MAIN OUTCOME MEASURES: After 24 hours of culture, immunohistochemistry was used to detect Bcl-2 and Bax expression in PC12 cells. Annexin-V flow cytometry was employed to measure the apoptosis rate of PC12 cells. The MTT method was applied to examine the survival rate of PC12 cells.RESULTS: Bcl-2 expression decreased, whereas Bax expression increased in PCI2 cells treated with Aβ25-35 (t = 2.277, 5.957, P<0.05). However, in PC12 cells treated with Aβ25-35 and cyclophilin A, Bcl-2 expression increased and Bax expression decreased (t = 4.497, 2.531, P < 0.05). The survival rate of PC12 cells significantly decreased and the apoptosis rate increased (t=8.509, 22.886, P < 0.05) following Aβ25-35 treatment. Cyclophilin A enhanced the survival rate of PC12 cells to Aβ25-35-induced apoptosis (t = 4.895, 10.042, P< 0.05).CONCLUSION: Cyclophilin A can

  14. Acrylamide decreased dopamine levels and increased 3-nitrotyrosine (3-NT) levels in PC 12 cells.

    Science.gov (United States)

    Tareke, Eden; Lyn-Cook, Beverly D; Duhart, Helen; Newport, Glenn; Ali, Syed

    2009-07-17

    Acrylamide is a chemical known to produce neurotoxicity in animals, as well as in humans. The mechanism of acrylamide-induced neurotoxicity is not fully known. However, recent studies have revealed that acrylamide affects the dopaminergic system. Therefore, the aim of this study was to investigate the effect of acrylamide on dopamine (DA) and the metabolites, 3,4-dihydroxy phenylacetic acid (DOPAC) and homovanillicacid (HVA), levels in Pheochromocytoma (PC 12) cells. In addition, the generation of peroxynitrite (ONOO(-)), measured by 3-nitrotyrosine (3-NT), was investigated as a possible mechanism in acrylamide-induced neurotoxicity. HPLC-coupled to electrochemical detection (ECD) was used to determine DA, DOPAC, HVA and 3-NT levels. Acrylamide (0.01-5mM) exposure produced a dose- and time (1-42h)-dependent decrease in DA levels. The decrease (P<0.05) in DA levels was noted at 24h after exposure to acrylamide. The study also revealed that 3-NT levels in PC 12 increased as a result of treatment with acrylamide. Thus, these data suggest that acrylamide-induced decrease in DA levels in PC 12 cells may be associated with peroxynitrite formation, measured as 3-NT levels.

  15. Protective Effect of Eecdysterone on the PC12 Cell CytotoxicityInduced by beta-amyloid 25-35

    Institute of Scientific and Technical Information of China (English)

    YANGSu-Fen; WUZhong-Jun; YANGZheng-Qin; LIYu; WuQin; ZHOUQi-Xin; SHIJing-Shan

    2004-01-01

    Objective. To study the effect of ecdysterone (ECR) on beta - amyloid peptide fragment 25-35 ( Aβ25-35 )-induced PC12 cell cytotoxicity, and further to expore its mechanism. Methods: PC12 survial was monitored by LDH release and 3-(4, 5-dimethylthiazol-yl-2, 5-diphenyhetrazolium bromide (MTT) assays. The content of malondi-

  16. Protective effects of ginkgo biloba leaves extract on peroxide-induced oxidative stress damage in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Weiqiang Chen; Taiping Hu; Ying Liu

    2007-01-01

    BACKGROUND: Extracts of ginkgo biloba leaves (EGB) and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative stress.OBJECTIVE: To investigate protective effects of EGB on peroxide (H2O2)-induced oxidative stress damage in PC12 cells.DESIGN: Observational contrast study.SETTING: Department of Pathophysiology, Guangdong Pharmacological College.MATERIALS: EGB was provided by Xi'an Fujie Biotechnological Development Company; 1640 culture medium, methylthiazolyl tetrazolium (MTT), trypsin and dimathyl sulfoxide (DMSO) by Sigma Company;PC 12 cell strain by Cell Center of Medical College of Zhongshan University; calf serum by Hangzhou Sijiqing Bioengineering Company; lactate dehydrogenase (LDH) kit by Nanjing Jiancheng Bioengineering Research Institute.METHODS: The experiment was carried out in Department of Cell Biology of Guangdong Pharmacological College from June to December 2005.①Cell culture: PC 12 cells were cultured in 1640 medium containing 200 g/L fetal calf serum. The cells were diluted to 1×107 L-1 and washed every two days. Those cells were used to experiment until they grew in logarithm on solid wall.②Grouping and intervention: PC 12 cells(1×108L-1) were plated in 96-well plates with the density of 200 μL/hole and divided into three groups:normal control group (routinely adding media), H2O2 group (treating with media and H2O2 for 20 hours) and EGB group (adding media, 100 μ mol/L EGB and 100 μmol/L H2O2).③MTT assay: PC12 cells (1×108L-1) were plated in 96-well plates and divided into three groups with 8 holes for each group. Under sterile condition, cells were added with 5 g/L MTT (100 μ L) and cultured for 4 hours. And then, 200 μL DMSO fluid was added and shaken for 30 minutes until blue crystal products formed were dissolved soundly.④Experimental evaluation: Absorbance (A) at 630 nm was measured and LDH activity was measured at the same

  17. Apoptotic effect of cobalt chloride on PC12 cells%氯化钴对PC12细胞的凋亡作用

    Institute of Scientific and Technical Information of China (English)

    曾季平; 王立祥; 胡晓燕; 于清水; 秦文; 崔行

    2005-01-01

    目的确定氯化钴(CoCl2)对PC12细胞的影响.方法构建CoCl2诱导的PC12细胞模型,检测CoCl2对PC12细胞的毒性作用;将Caspases的抑制基因p35转染PC12细胞得到可稳定表达p35基因的细胞株PC12/p35,检测p35对CoCl2诱导的PC12细胞的作用;检测Caspases特异性多肽抑制剂Z-VAD-FMK对CoCl2诱导的PC12细胞的作用.结果分别以100、300、500、700和1 000 μmol/L CoCl2诱导PC12细胞24 h或以500μmol/L CoCl2分别诱导PC12细胞12、24、36、48和60 h后,PC12细胞存活率均明显下降(P<0.01),并与CoCl2诱导的时间和浓度呈正相关;细胞亚显微结构检测,流式细胞分析和DNA片段化结果也表明500 μmol/L CoCl2诱导PC12细胞24 h可使PC12细胞出现明显凋亡特征.构建可稳定表达Caspase蛋白酶抑制基因p35的PC12细胞株,或分别加入50和100 μmol/L Caspases多肽抑制剂Z-VAD-FMK预处理PC12细胞1 h后,以500μmol/L CoCl2诱导PC12细胞24 h,形态学观察结果,细胞存活率检测和流式细胞分析均表明p35基因和Z-VAD-FMK均可有效地抑制CoCl2诱导的PC12细胞凋亡(P<0.01).结论CoCl2可诱导PC12细胞凋亡.

  18. Distinctive effects of rat fibroblast growth factor-2 isoforms on PC12 and Schwann cells.

    Science.gov (United States)

    Müller-Ostermeyer, F; Claus, P; Grothe, C

    2001-01-01

    Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18 kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during post-natal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC12 cells.

  19. 醒脑启智胶囊药物血清对PC12细胞缺氧损伤的保护作用%Protective role of xingnao qizhi capsule in hypoxia injury of PC12 cell

    Institute of Scientific and Technical Information of China (English)

    杨牧祥; 于文涛; 田元祥; 王少贤

    2005-01-01

    .42,12.71,6.35,3.18 g/kg组分别为82.9%,75.6%,65.9%,53.7%.结论:醒脑启智药物血清能明显减轻PC12细胞的缺氧损伤,可增强细胞活力,降低乳酸脱氢酶活性,并呈现一定的剂量依赖关系.%BACKGROUND: The onset of vascular dementia (VD) is closely related to brain ischemic-hypoxia. Previous research has proved that behavioral disturbance can be obviously attenuated by xingnao qizhi capsule in VD rats; however, it remains unclear whether the improvement of VD is due to the protection on ischemic cells.OBJECTIVE: Pheochromocytoma PC12 cells were cultured in vitro and exposed to Na2S2O4 to induce hypoxia injury. Then serum pharmacological technique was used to investigate the protection of xingnao qizhi drug serum on hypoxia-injured PC12 cells.DESIGN: Randomized controlled study.SETTING: School of Traditional Chinese Medicine of Hebei Medical College.PARTICIPANTS: This study was conduced at the Experimental Animal Laboratory and Cell Culture Laboratory, School of Traditional Chinese Medicine of Hebei Medical College, between June and December 2003.Forty SD rats were randomized into 5 groups, namely serum control group (n=12) and xingnao qizhi serum 25.42, 12.71, 6.35 and 3.18 g/kg groups (n=7). PC12 cell line was purchased form the Cell Center of Chinese Academy of Medical Sciences.serum 25.42, 12.71, 6.35 and 3.18 g/kg groups were exposed through gastric lavage to xingnao qizhi of corresponding dosages (composed of wolfberwhile normal saline was given by gastric lavage to rats in control group in dosage of 10 mL/kg, twice a day at an interval of 12 hours for 3 consecutive days. One hour after the last administration, femoral blood samples were collected when rats were deeply anaesthetized and serum was sepainto 6 groups. Cells in control group were cultured with control serum while in experimental model groups cells were exposed to Na2S2O4 to induce hypoxia injury 1 hour later. Cells in Xingnao Qizhi 25.42, 12.71,6.35 and 3.18 g/kg groups were cultured in 5

  20. Bcl-xS and Bax induce different apoptotic pathways in PC12 cells.

    Science.gov (United States)

    Lindenboim, L; Yuan, J; Stein, R

    2000-03-30

    Apoptosis is regulated by the action of the Bcl-2 family of proteins, which includes anti- and pro-apoptotic members such as Bcl-xS and Bax. These proteins may differ from each other in structure, mechanism of action and interactions with anti-apoptotic signaling. The mechanism whereby Bax induces cell death has been studied in some cellular systems, but the mechanism of Bcl-xS-induced apoptosis is largely unknown. In this study we investigated and compared the apoptotic effects of Bcl-xS and Bax in the pheochromocytoma cell line, PC12 (a useful model system for studying neuronal apoptosis), and the extent to which they are protected by the survival factor, nerve growth factor (NGF). PC12 cells express endogenous Bcl-xS, Bax and Bcl-xL proteins. Subcellular fractionation revealed that Bax is presented mainly in the cytosolic and the heavy membrane fractions, Bcl-xS is present only in the cytosol, and the anti-apoptotic protein Bcl-xL is located mainly in the heavy membrane fraction. In contrast to the cytosolic localization of endogenous Bcl-xS, the exogenously overexpressed Bcl-xS is localized to the mitochondria. Overexpression of Bcl-xS or Bax induces cell death in the transfected cells. The cell death induced by overexpression of Bcl-xS was inhibited by coexpression of Bcl-xS with Bcl-2 or Bcl-xL, or by treatment with the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (Z-VAD-FMK) or with NGF. The Bcl-2 mutants deltaC22, which lacks the transmembrane domain, and G145A (mI-3) were able to inhibit the death-inducing effect of Bcl-xS. These results therefore suggest that the apoptotic pathway induced by overexpression of Bcl-xS in PC12 cells can be controlled by Bcl-2 and Bcl-xL, is mediated by caspases, and can be inhibited by the NGF signaling pathway. The Bax-induced cell death was inhibited by co-expression of Bax with Bcl-2 or Bcl-xL, but was not inhibited by Z-VAD-FMK, NGF, or the Bcl-2 ml-3 or deltaC22 mutants. These

  1. Effect of Calmodulin on the Differrentiation and Migration of PC12 Cells%钙调蛋白在PC12细胞分化和迁移中的作用

    Institute of Scientific and Technical Information of China (English)

    袁俊; 李朝军

    2011-01-01

    To investigate the roles of calmodulin during neuronal differentiation and migration,we checked PC12 cells by immunofluorescence staining and single cell tracking assay after NGF treatment. We found that calmodulin showed a dense distribution pattern in top of PC12 cells. Only a small percentage of the cells grown in W7 treatment cells. A single cell tracking experiment showed that calmodulin in PC12 cells could increase cell motility. The data suggested that calmodulin may play an important role in differentiation and migration of PC12 cells.%PC12 细胞经神经生长因子 (NGF)作用后,利用免疫荧光染色、单个细胞迁移率检测等方法,研究了钙调蛋白在PC12 细胞中的分布以及钙调蛋白对PC12 细胞分化和迁移的影响.免疫荧光染色结果表明,钙调蛋白在PC12细胞突起的顶端处呈密集分布.加入钙调蛋白抑制剂W7的细胞仅有少量长出突起.单个细胞迁移率检测表明钙调蛋白可能促进PC12 细胞迁移.提示钙调蛋白可能在PC12细胞的分化和迁移过程中发挥作用.

  2. Reversible effects of vitamins C and E combination on oxidative stress-induced apoptosis in melamine-treated PC12 cells.

    Science.gov (United States)

    An, L; Li, Z; Zhang, T

    2014-02-01

    Due to its high nitrogen content, melamine was deliberately added to raw milk for increasing the apparent protein content. Previous studies showed that melamine-induced apoptosis and oxidative damage on PC12 cells and rats' hippocampus. Several evidences suggested that vitamin antioxidant reduced oxidative stress and improved organic function. Whether treatments with antioxidant vitamins C or E, otherwise combination of them can attenuate oxidative stress after melamine administration remains to be elucidated. In this study, the reversible effects of vitamin antioxidants was investigated on melamine-induced neurotoxicity in cultured PC12 cells, an in vitro model of neuronal cells. When comparing vitamin C and E, the combination of both statistically increased PC12 cells viability. The results further showed that vitamin complex has effectively reduced the formation of reaction oxygen species, decreased the level of malondialdehyde, and elevated the activities of antioxidative enzymes. Hoechst 33342 staining and flow cytometric analysis of apoptosis showed that vitamin combination treatment effectively prevented PC12 cells from this melamine-induced apoptosis. It revealed the apoptotic nuclear features of the melamine-induced cell death. Additionally, a combination treatment of vitamins effectively inhibited apoptosis via blocking the increased activation of caspase-3. In summary, the vitamin E and C combination treatment could rescue PC12 cells from the injury induced by melamine through the downregulation of oxidative stress and prevention of melamine-induced apoptosis.

  3. Recycling of intact dense core vesicles in neurites of NGF-treated PC12 cells.

    Science.gov (United States)

    Bauer, Roslyn A; Khera, Rebecca S; Lieber, Janet L; Angleson, Joseph K

    2004-07-30

    Exocytic fusion in neuroendocrine cells does not always result in complete release of the peptide contents from dense core vesicles (DCVs). In this study, we use fluorescence imaging and immunoelectron microscopy to examine the retention, endocytosis and recycling of chromogranin B in DCVs of NGF-treated PC12 cells. Our results indicate that DCVs retained and retrieved an intact core that was available for subsequent exocytic release. The endocytic process was inhibited by cyclosporine A or by substitution of extracellular Ca(2+) with Ba(2+) and the total recycling time was less than 5 min.

  4. The endothelin-2/vasoactive intestinal contractor gene: expression and promoter activity in PC12 rat pheochromocytoma cells.

    Science.gov (United States)

    Saida, K; Uchide, T; Usui, A; Gao, X D; Tomizuka, N; Oka, S; Masuda, H

    2000-11-01

    In order to understand the physiological roles of vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2), we examined the expression of this peptide by specific reverse transcriptase polymerase chain reaction (RT-PCR) analysis and found that PC12 rat pheochromocytoma cells express the VIC gene. The 5'-flanking 1.0 kilo base pair (kb) region of the mouse VIC gene is sufficient to express a secreted alkaline phosphatase (SEAP) reporter gene in transiently transfected PC12 cells. The 1.0 kb promoter region may contain cis-acting elements that determine the rate of the VIC gene transcription in PC12 cells.

  5. Set up Alzheimer's Disease Cell Apoptosis Model with PC-12 Cell Induced by Aβ25-35

    Institute of Scientific and Technical Information of China (English)

    王亚利; 王中卫; 宋潍; 杨林

    2003-01-01

    Objective: To prepare an apoptosis cell model of Alzheimer Disease ( AD ) by PC-12 cells treated with β-amyloid protein (Aβ). Methods: PC-12 cells were incubated with differ-ent concentrations of Aβ25-35 for different duration in vitro. The cell viability was detected by MTT as-say. Morphological features of apoptosis were analyzed with Hoechst 33258/Prorlidium iodide dual staining, The level of intracellular free calcium ([Ca2+]i) was calculated by Fura-2 / AM fluorescence ratio imaging. Results: ① The viability of PC-12 cells was significantly decreased in prolrrrtion to concentration of Aβ25-35 and duration of exposure to Aβ25-35. ②The apoplotic cells appeared in a time and concentration-dependent manner, and the maximal apoptosis happened at 48 h after execute to 20μmol/ L of Aβ25-35 and 36 h to 30μmol/ L. Cell death reached the peak at 12-24 h later than the apoptotic peak. ③([Ca2+]i) of PC-12 cells was increased in prolrrrtion to duration of exposure to the same concentration of Aβ25-35. The time of the highest increase rate of [Ca2+]i was about 12 h earlier than that of apoptosis. Conclusion : An AD cell model using the PC-12 cells induced with Aβ25-35 displays aseries of chanqes related to apoptosis, which may be related to elevation of [Ca2+]i.

  6. EGb-761 Attenuates the Anti-proliferative Activity of Fluoride via DDK1 in PC-12 Cells.

    Science.gov (United States)

    Zhang, Cai-Yi; Chen, Rui; Wang, Fen; Ren, Chao; Zhang, Peng; Li, Qian; Li, Hui-Hua; Guo, Ke-Tai; Geng, De-Qin; Liu, Chun-Feng

    2017-02-01

    EGb-761 is commonly used as a treatment for ischemic brain injury, neurodegenerative diseases and some types of tumors (Christen and Maixent, in Cell Mol Biol 48(6):601-611, 2002). However, it is unclear whether EGb-761 affects the proliferation of cells exposed to fluoride. In this study, the proliferation and apoptosis of PC-12 cells exposed to fluoride were investigated and EGb-761 was used to protect PC-12 cells against the effects of fluoride. We found that the canonical Wnt signaling pathway was involved in the anti-proliferation of PC-12 cells exposed to fluoride. Furthermore, the results also showed that EGb-761 could attenuate the anti-proliferative activity of fluoride via DDK1 in PC-12 cells. This study may provide a new method for protecting against the inhibition of cell proliferation induced by fluoride.

  7. 神经干细胞对去血清诱导PC12细胞凋亡的作用%Effect of Neural Stem Cells on Apoptosis of PC12 Cells Induced by Serum Deprivation

    Institute of Scientific and Technical Information of China (English)

    李香琴; 刘天庆; 朱蕾蕾; 葛丹; 崔占峰

    2007-01-01

    肾上腺嗜铬细胞瘤细胞(PC12 cells)去血清后,其凋亡与多巴胺能神经元的凋亡有许多共同之处,今通过研究神经干细胞(NSCs)对去血清诱导的PC12细胞凋亡的作用,进一步为NSCs移植治疗神经系统疾病提供相应的实验和理论依据.将正常培养的PC12细胞与NSCs以不同的方式进行去血清共培养,观察PC12细胞的形态,检测PC12细胞的活性,计算PC12细胞的存活率,同时检测培养基中胶质细胞源神经营养因子(GDNF)的浓度,分析不同培养方式下NSCs对去血清诱导凋亡的PC12细胞的作用以及NSCs与去血清诱导凋亡的PC12细胞共培养后,其分泌GDNF的能力.结果表明:①去血清诱导PC12细胞凋亡呈时间依赖性,去血清72 h后,PC12细胞存活的比率为44.25%;②NSCs培养基对去血清诱导凋亡的PC12细胞没有明显的保护作用;③NSCs培养上清及NSCs对去血清诱导凋亡的PC12细胞具有保护作用;④NSCs与去血清诱导凋亡的PC12细胞共培养后,分泌GDNF的能力增强.

  8. Activated RET/PTC oncogene elicits immediate early and delayed response genes in PC12 cells.

    Science.gov (United States)

    Califano, D; Monaco, C; de Vita, G; D'Alessio, A; Dathan, N A; Possenti, R; Vecchio, G; Fusco, A; Santoro, M; de Franciscis, V

    1995-07-06

    The expression of the receptor-like tyrosine kinase RET is associated with tumors, tissues or cell lines of neural crest origin. In addition RET products (Ret) are involved in determining cell fate during the differentiation of the enteric nervous system and during renal organogenesis. However, as yet, no direct evidence exists to indicate that the Ret kinase activity might interfere in a specific way with cellular differentiation, or proliferation, of a neural crest derived cell line. By using two constitutively activated forms of RET (RET/PTC1 and RET/PTC3) in transient transfection experiments, we have obtained evidence that active RET could reprogramme the gene expression pattern in the rat pheochromocytoma PC12 cell line. Transcription driven by gene promoters, such as NGFI-A and vgf, which belong, respectively, to primary and delayed response genes to nerve growth factor (NGF), and by the neuron-specific enolase (NSE) promoter, is rapidly induced by the expression of activated RET oncogenes. This induction is not elicited in other non neural derived cell types tested. We also demonstrate that endogenous ras activity is required for RET induction of these neural markers. Finally, in the RET/PTC transfected PC12 cells, NGF is unable to induce further their transcription. This suggests that RET/PTC could share an intracellular signalling pathway with the NGF-receptor.

  9. Antibody-mediated inhibition of Nogo-A signaling promotes neurite growth in PC-12 cells

    Directory of Open Access Journals (Sweden)

    Iman K Yazdi

    2016-01-01

    Full Text Available The use of a monoclonal antibody to block the neurite outgrowth inhibitor Nogo-A has been of great interest for promoting axonal recovery as a treatment for spinal cord injury. While several cellular and non-cellular assays have been developed to quantify the bioactive effects of Nogo-A signaling, demand still exists for the development of a reliable approach to characterize the effectiveness of the anti-Nogo-A antibody. In this study, we developed and validated a novel cell-based approach to facilitate the biological quantification of a Nogo-A antibody using PC-12 cells as an in vitro neuronal cell model. Changes in the mRNA levels of the neuronal differentiation markers, growth-associated protein 43 and neurofilament light-polypeptide, suggest that activation of the Nogo-A pathway suppresses axonal growth and dendrite formation in the tested cell line. We found that application of anti-Nogo-A monoclonal antibody can significantly enhance the neuronal maturity of PC-12 cells by blocking the Nogo-A inhibitory effects, providing enhanced effects on neural maturity at the molecular level. No adverse effects were observed on cell viability.

  10. Cholecystokinin-2 receptor mediated gene expression in neuronal PC12 cells

    DEFF Research Database (Denmark)

    Hansen, Thomas v O; Borup, Rehannah; Marstrand, Troels;

    2007-01-01

    Cholecystokinin (CCK) is abundantly expressed in the CNS, in which it regulates feeding behavior and long-term memory. Moreover, CCK has been implicated in mental disorders, such as anxiety and schizophrenia. Despite its manifest physiological and pathophysiological role, the molecular targets...... could be identified. Comparison with forskolin- and nerve growth factor (NGF)-treated PC12 cells showed that CCK induced a separate set of target genes. Taken together, we propose that neuronal CCK may have a role in the regulation of the circadian rhythm, the metabolism of cerebral cholesterol...

  11. Histone deacetylase inhibitor attenuates neurotoxicity of clioquinol in PC12 cells.

    Science.gov (United States)

    Fukui, Takao; Asakura, Kunihiko; Hikichi, Chika; Ishikawa, Tomomasa; Murai, Rie; Hirota, Seiko; Murate, Ken-Ichiro; Kizawa, Madoko; Ueda, Akihiro; Ito, Shinji; Mutoh, Tatsuro

    2015-05-01

    Clioquinol is considered to be a causative agent of subacute myelo-optico neuropathy (SMON), although the pathogenesis of SMON is yet to be elucidated. We have previously shown that clioquinol inhibits nerve growth factor (NGF)-induced Trk autophosphorylation in PC12 cells transformed with human Trk cDNA. To explore the further mechanism of neuronal damage by clioquinol, we evaluated the acetylation status of histones in PC12 cells. Clioquinol reduced the level of histone acetylation, and the histone deacetylase (HDAC) inhibitor Trichostatin A upregulated acetylated histones and prevented the neuronal cell damage caused by clioquinol. In addition, treatment with HDAC inhibitor decreased neurite retraction and restored the inhibition of NGF-induced Trk autophosphorylation by clioquinol. Thus, clioquinol induced neuronal cell death via deacetylation of histones, and HDAC inhibitor alleviates the neurotoxicity of clioquinol. Clioquinol is now used as a potential medicine for malignancies and neurodegenerative diseases. Therefore, HDAC inhibitors can be used as a candidate medicine for the prevention of its side effects on neuronal cells.

  12. JWA protein binds to α-tubulin in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Hairong; LI Aiqun; LI Aiping; ZHOU Jianwei

    2004-01-01

    Our previous study elucidated that JWA protein was a newly identified microtubule-associated protein (MAP), which combined to and co-localized with β-tubulin.In the present study, we designed a series of experiments to explore if any interactions between JWA protein and α-tubulin existed and how JWA protein would functionally link to α-tubulin, especially in cell mitosis. Results of coimmunoprecipitation, gene transfection and immunofluorescence microscopy from PC12 and HEK293 cells provided strong evidence for a linkage between JWA protein and α-tubulin. Our data showed that JWA protein bound to α-tubulin stably no matter whether α-tubulin was polymerized or not. In addition, by using antisense oligonucleotides, cell cycle blocking agents and hypothermia disposal techniques,we also found the interaction between JWA protein and α-tubulin. The further analysis using flow cytometry and confocal microscopy showed that both proteins co-existed in PC12 cells and were independent on the cell cycle. In conclusion, JWA protein is a newly identified microtubuleassociated protein, binds to α-tubulin, and probably plays an important role in regulation of microtubular stability.

  13. Nerve growth factor activates calcium-insensitive protein kinase C-epsilon in PC-12 rat pheochromocytoma cells.

    OpenAIRE

    Ohmichi, M; Zhu, G.; Saltiel, A R

    1993-01-01

    Protein kinase C (PKC) family members were examined in PC-12 rat pheochromocytoma cells to evaluate their role in the action of nerve growth factor (NGF). Immunoblot analysis of whole cell lysates using antibodies against various PKC isoforms revealed that PC-12 cells contained PKC-alpha, -delta, -epsilon and zeta. Assay of the protein kinase activity in these different anti-PKC immunoprecipitates demonstrated that NGF stimulated the kinase activity of PKC-epsilon, but not PKC-alpha, -delta a...

  14. Roles of notch signaling in rotenone-induced apoptosis of PC12 cells%Notch信号通路在鱼藤酮诱导的PC12细胞凋亡中的作用

    Institute of Scientific and Technical Information of China (English)

    张媚; 何亮; 李俊; 张临洪

    2012-01-01

    Objective To elucidate the roles of Notch Signaling in rotenone-induced apoptosis of PC12 cells.Methods PC12 ceils were treated with rotenone (5 μmol/L).Apoptosis of PC12 cells and activation of Notch signaling were observed.PC12 cells were pretreated with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butvlester ( DAPT,10 μmol/L) before incubation with rotenone.The effect of Notch signaling on apoptosis of PC12 cells in response to rotenone was evaluated and the relationship between Notch signaling and rotenone-induced apoptosis of PC12 cells was analyzed.Results Rotenone significantly induced apoptosis of PC12 cells [( 35.6 ± 5.4 ) %,P < 0.05] and increased Caspase-3 activity (0.52 ±0.15,P <0.05 ).At the same time,rotenone activated Notch/Jagged1 signaling in PC12 cells.However,inhibition of Notch signaling by DAPT not only decreased apoptosis of PC12 cells [(9.8 ±3.1 ) %,P < 0.05],but also diminished Caspase-3 activity (0.16 ± 0.06,P < 0.05 ).Conclusion Activation of Notch signaling could be one of the main mechanisms for rotenone-induced apoptosis of PC12 cells.%目的 探讨Notch信号通路在鱼藤酮诱导PC12细胞凋亡中的作用机制.方法 用鱼藤酮(5 μmol/L)处理PC12细胞,检测PC12细胞的凋亡和Notch信号通路的活化状态.同时以Notch信号抑制剂(2S)-N-N-(3,5-二氟苯乙酰基)-L-丙氨酰-2-苯基甘氨酸叔丁酯(DAPT,10 μmol/L)处理上述PC12细胞,观察Notch信号通路抑制后PC12细胞对鱼藤酮处理的凋亡反应,分析Notch信号通路与鱼藤酮诱导PC12细胞凋亡之间的关系.结果 鱼藤酮显著诱导PC12细胞凋亡[(35.6±5.4)%,P<0.05]和半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)活性升高(0.52±0.15,P<0.05),并活化其Notch/Jagged1信号通路.DAPT显著抑制鱼藤酮诱导的Notch/Jagged1信号通路活化并降低PC12细胞凋亡率[(9.8±3.1)%,P<0.05]和Caspase-3活性(0.16 ±0.06,P<0.05).结论 Notch信号通路活化是鱼藤酮诱导PC

  15. Protective Effects of Costunolide against Hydrogen Peroxide-Induced Injury in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Chong-Un Cheong

    2016-07-01

    Full Text Available Oxidative stress-mediated cellular injury has been considered as a major cause of neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. The scavenging of reactive oxygen species (ROS mediated by antioxidants may be a potential strategy for retarding the diseases’ progression. Costunolide (CS is a well-known sesquiterpene lactone, used as a popular herbal remedy, which possesses anti-inflammatory and antioxidant activity. This study aimed to investigate the protective role of CS against the cytotoxicity induced by hydrogen peroxide (H2O2 and to elucidate potential protective mechanisms in PC12 cells. The results showed that the treatment of PC12 cells with CS prior to H2O2 exposure effectively increased the cell viability. Furthermore, it decreased the intracellular ROS, stabilized the mitochondria membrane potential (MMP, and reduced apoptosis-related protein such as caspase 3. In addition, CS treatment attenuated the cell injury by H2O2 through the inhibition of phosphorylation of p38 and the extracellular signal-regulated kinase (ERK. These results demonstrated that CS is promising as a potential therapeutic candidate for neurodegenerative diseases resulting from oxidative damage and further research on this topic should be encouraged.

  16. Identification of coffee components that stimulate dopamine release from pheochromocytoma cells (PC-12).

    Science.gov (United States)

    Walker, J; Rohm, B; Lang, R; Pariza, M W; Hofmann, T; Somoza, V

    2012-02-01

    Coffee and caffeine are known to affect the limbic system, but data on the influence of coffee and coffee constituents on neurotransmitter release is limited. We investigated dopamine release and Ca(2+)-mobilization in pheochromocytoma cells (PC-12 cells) after stimulation with two lyophilized coffee beverages prepared from either Coffea arabica (AR) or Coffea canephora var. robusta (RB) beans and constituents thereof. Both coffee lyophilizates showed effects in dilutions between 1:100 and 1:10,000. To identify the active coffee compound, coffee constituents were tested in beverage and plasma representative concentrations. Caffeine, trigonelline, N-methylpyridinium, chlorogenic acid, catechol, pyrogallol and 5-hydroxytryptamides increased calcium signaling and dopamine release, although with different efficacies. While N-methylpyridinium stimulated the Ca(2+)-mobilization most potently (EC(200): 0.14±0.29μM), treatment of the cells with pyrogallol (EC(200): 48±14nM) or 5-hydroxytryptamides (EC(200): 10±3nM) lead to the most pronounced effect on dopamine release. In contrast, no effect was seen for the reconstituted biomimetic mixture. We therefore conclude that each of the coffee constituents tested stimulated the dopamine release in PC-12 cells. Since no effect was found for their biomimetic mixture, we hypothesize other coffee constituents being responsible for the dopamine release demonstrated for AR and RB coffee brews. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Inhibition of ROCK2 expression protects against methamphetamine-induced neurotoxicity in PC12 cells.

    Science.gov (United States)

    Yang, Xingyi; Liu, Yunyun; Liu, Chao; Xie, Weibing; Huang, Enping; Huang, Weiye; Wang, Jiawen; Chen, Ling; Wang, Huipin; Qiu, Pingming; Xu, Jingtao; Zhang, Fu; Wang, Huijun

    2013-10-02

    Methamphetamine is a type of psychoactive drug. It is well known that neurotoxicity caused by Methamphetamine(METH) can damage the nervous system and lead to apoptosis and cell loss of dopaminergic neurons. ROCK2 is a prominent target for gene therapy because its inhibition has proved to have a protective effect in various cell lines and pathophysiological conditions. Although several of the negative effects of METH on the dopaminergic system have been studied, the protective molecular mechanisms and the effective treatment of METH-induced apoptosis remain to be clarified. We hypothesized that ROCK2 is involved in METH-induced apoptosis. We tested our hypothesis using RT-PCR and western blotting to analyze whether silencing of ROCK2 with small interfering RNA (siROCK2) could reduce damage and apoptosis in PC12 cells after METH exposure. Increases in viability and cytomorphological changes were detected by MTT assay and bright field microscopy after pretreatment of METH-treated PC12 cells with 100 nM siROCK2. Apoptosis decreased significantly after ROCK2 silencing, as shown by Annexin V and TUNEL staining. The results show that ROCK2 is a possible gene target for therapeutics in METH-induced neurotoxicity in vitro, providing a foundation for future in vivo research.

  18. The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells

    DEFF Research Database (Denmark)

    Jessen, U; Novitskaya, V; Pedersen, N;

    2001-01-01

    roles in this process by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in co-culture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding wild-type or dominant negative forms of CREB and c-Fos or an activated...... thus present evidence that NCAM-mediated neurite outgrowth involves a series of signal transduction pathways, including the cAMP/PKA pathway, targeting c-Fos and CREB....

  19. Neurite outgrowth of mature retinal ganglion cells and PC12 cells requires activity of CK1δ and CK1ε.

    Directory of Open Access Journals (Sweden)

    Joachim Bischof

    Full Text Available Mature retinal ganglion cells (RGCs do not normally regenerate severed axons after optic nerve injury and show only little neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1δ and CK1ε activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF stimulated PC12 cells, respectively. In both cell types CK1δ and ε were localized in granular particles aligned at microtubules in neurites and growth cones. Although LI treatment did not measurably affect the expression of CK1δ and ε, it significantly elevated the specific kinase activity in the retina. Similarly, CK1δ/ε specific kinase activity was also elevated in NGF treated PC12 cells compared with untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1δ C-terminal targeting kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of CK1δ and ε markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth.

  20. A subcellular distribution of estrogen receptor-alpha is changed during artificially induced senescence of PC12 pheochromocytoma cells.

    Science.gov (United States)

    Lee, Eunju; Mun, Ga Hee; Oh, Chang Seok; Chung, Yoon Hee; Cha, Choong Lk; Lee, Young Soo; Shin, Dong Hoon

    2004-11-30

    Although estrogen has been considered as a sex hormone for decades, recent reports suggest that estrogen might modulate the development and physiological function of the brain. In addition, the subcellular localization of estrogen receptors (ERs) has shown their presence within both the perinuclear cytoplasm and nuclei, suggesting that these ERs may differ functionally. We, therefore, assayed changes in the subcellular localization of ER-alpha immunoreactivity (IR) in rat pheochromocytoma PC12 cells during the artificial senescence induced by the telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT). After 2 months of culture with AZT, PC12 cells showed morphological and biochemical characteristics of cellular senescence. In the cells showing artificial senescence, the ER-alpha IR was mainly localized within the cytoplasm, whereas in control cells, ER-alpha IR was found only in the nuclei. Since senescence was induced by AZT, which inhibits the action of telomerase whenever the cells divide, the change in subcellular distribution of ER-alpha IR may be correlated with the length of the telomere.

  1. Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells.

    Science.gov (United States)

    Han, Jung Min; Kim, Jae Ho; Lee, Byoung Dae; Lee, Sang Do; Kim, Yong; Jung, Yon Woo; Lee, Sukmook; Cho, Wonhwa; Ohba, Motoi; Kuroki, Toshio; Suh, Pann-Ghill; Ryu, Sung Ho

    2002-03-08

    Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.

  2. New physiological function of secoiridoids: neuritogenic activity in PC12h cells.

    Science.gov (United States)

    Chiba, Kenzo; Yamazaki, Matsumi; Kikuchi, Masafumi; Kakuda, Rie; Kikuchi, Masao

    2011-01-01

    Previously, we have reported that geniposide isolated from an extract of Gardenia fructus has neuritogenic activity in PC12h cells, a subclone of rat pheochromocytoma cells. Furthermore, we have indicated that several geniposide-related iridoid compounds also had similar potent neuritogenic activity. In this study, we have examined the effects of various secoiridoid compounds [K-1, sweroside; K-2, swertiamarin; K-3, gentiopicroside; K-4, 6'-O-β-D: -glucopyranosylsweroside; K-5, 6'-O-β-D: -glucopyranosylgentiopicroside; K-6, 6'-O-β-D: -glucopyranosylswertiamarin; K-7, 5'-O-β-D: -glucopyranosylamarogentin; K-8, 5'-O-β-D: -glucopyranosylamaroswertin; H-1, n-butyl vogeloside; H-2, n-butyl epivogeloside; H-3, (7S)-secologanin butyl methyl acetal; H-4, (7R)-secologanin butyl methyl acetal; H-5, secologanin dimethyl acetal] isolated from various medicinal herbs. The secoiridoids H-1, H-2, H-3, H-4, and H-5 induced significant neurite outgrowth. Among these H-series compounds, H-2 was the most potent neuritogenic compound. Among the K-series compounds, K-1, K-2, K-3, and K-8 showed the most potent activity. These results suggest that secoiridoids have neuritogenic activity in PC12h cells and that these secoiridoid compounds are promising starting compounds for the development of neurotrophic factor-like and iridoid compounds.

  3. Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms.

    Science.gov (United States)

    Wang, Ze-Jian; Nie, Bao-Ming; Chen, Hong-Zhuan; Lu, Yang

    2006-01-05

    Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.

  4. Neuroprotective Effects of Exogenous Activin A on Oxygen-Glucose Deprivation in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Zhong-Xin Xu

    2011-12-01

    Full Text Available Ischemic cerebrovascular disease is one of the most common causes of death in the World. Exogenous activin A (ActA protects neurons against toxicity and plays a central role in regulating the brain’s response to injury. In the present study, we investigated the mechanisms involved in the neuroprotective effects of ActA in a model of hypoxic-ischemic brain disease. We found that ActA could effectively increase the survival rate of PC12 cells and relieve oxygen-glucose deprivation (OGD damage. To clarify the neuroprotective mechanisms of ActA, the effects of ActA on the ActA/Smad pathway and on the up-regulation of inducible nitric oxide synthase (NOS and superoxide dismutase (SOD were investigated using OGD in PC12 cells. The results showed that ActA could increase the expression of activin receptor IIA (ActRIIA, Smad3 and Smad4 and that 50 ng/mL and 100 ng/mL of ActA could reduce NO levels and increase SOD activity by 78.9% and 79.9%, respectively. These results suggested that the neuroprotective effects of ActA in ischemia could be related to the activation of the ActA/Smad signaling pathway and to its anti-oxidant activities.

  5. Protective Effects of Periploca forrestii Schltr. Root Extract Against Cytotoxicity Induced by Hydrogen Peroxide in PC12 Cells%黑骨藤对PC12细胞氧化损伤的保护研究

    Institute of Scientific and Technical Information of China (English)

    张贵源; 龚莉莉; 余跃生; 王恒; 陆玉炯; 杨再昌

    2012-01-01

    [Objective] The aim was to study the protective effects of Periploca forrestii Schltr.root extract against cytotoxicity induced by hydrogen peroxide in PC12 cells.[Method] The model of PC12 cells toxicity induced by hydrogen peroxide was established and the protective effects of Periploca forrestii Schltr.root extract on PC12 cells were detected.[Result] Compared to negative control,Periploca forrestii Schltr.root extract could protect PC12 cells at concentration ranges of 16-128 ug/mL at a dose-response manner.[Conclusion] Periploca forrestii Schltr.root extract showed anti-oxidative effect on PC12 cells.%[目的]研究黑骨藤提取物对H2O2诱导的PC12氧化损伤的保护作用。[方法]使用H2O2氧化压力诱导PC12出现氧化损伤,检测黑骨藤乙醇提取物对PC12细胞的保护作用。[结果]与模型对照组相比,黑骨藤乙醇提取物浓度在16~128μg/mL时,能显著提高PC12细胞的存活率,呈典型的量效关系。[结论]黑骨藤乙醇提取物具有抗氧化损伤作用。

  6. Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells

    Directory of Open Access Journals (Sweden)

    Dongdong Zhang

    2015-01-01

    Full Text Available Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer’s disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25–35 (Aβ25–35 induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased Aβ25–35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in Aβ25–35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer’s disease.

  7. The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells

    Science.gov (United States)

    Fan, W.; Ding, J.; Duan, W.; Zhu, Y. M.

    2004-11-01

    The aim of present work was to investigate the influence of magnetic fields exposure on neurite outgrowth in PC12 cells. The neurite number per cell, length of neurites and directions of neurite growth with respect to the direction of the magnetic field were analyzed after exposure to 50 Hz electromagnetic field for 96 h. A promotion was observed under a weak field (0.23 mT), as the average number of neurites per cell increased to 2.38±0.06 compared to 1.91±0.07 neurites/cell of the control dishes, while inhibition and directional outgrowth was evident under a relatively stronger field (1.32 mT). Our work shows that biological systems can be very sensitive to the strength of electromagnetic field.

  8. The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, W.; Ding, J. E-mail: masdingj@nus.edu.sg; Duan, W.; Zhu, Y.M

    2004-11-01

    The aim of present work was to investigate the influence of magnetic fields exposure on neurite outgrowth in PC12 cells. The neurite number per cell, length of neurites and directions of neurite growth with respect to the direction of the magnetic field were analyzed after exposure to 50 Hz electromagnetic field for 96 h. A promotion was observed under a weak field (0.23 mT), as the average number of neurites per cell increased to 2.38{+-}0.06 compared to 1.91{+-}0.07 neurites/cell of the control dishes, while inhibition and directional outgrowth was evident under a relatively stronger field (1.32 mT). Our work shows that biological systems can be very sensitive to the strength of electromagnetic field.

  9. Rectification of Acetylcholine-Elicited Currents in PC12 Pheochromocytoma Cells

    Science.gov (United States)

    Ifune, C. K.; Steinbach, J. H.

    1990-06-01

    The current-voltage (I-V) relationship for acetylcholine-elicited currents in the rat pheochromocytoma cell line PC12 is nonlinear. Two voltage-dependent processes that could account for the whole-cell current rectification were examined, receptor channel gating and single receptor channel permeation. We found that both factors are involved in the rectification of the whole-cell currents. The voltage dependence of channel gating determines the shape of the I-V curve at negative potentials. The single-channel I-V relationship is inwardly rectifying and largely responsible for the characteristic shape of the whole-cell I-V curve at positive potentials. The rectification of the single-channel currents is produced by the voltage-dependent block of outward currents by intracellular Mg2+ ions.

  10. Inhibition by anandamide of 6-hydroxydopamine-induced cell death in PC12 cells.

    LENUS (Irish Health Repository)

    Mnich, Katarzyna

    2010-01-01

    6-hydroxydopamine (6-OHDA) is a selective neurotoxin that is widely used to investigate cell death and protective strategies in models of Parkinson\\'s disease. Here, we investigated the effects of the endogenous cannabinoid, anandamide, on 6-OHDA-induced toxicity in rat adrenal phaeochromocytoma PC12 cells. Morphological analysis and caspase-3 activity assay revealed that anandamide inhibited 6-OHDA-induced apoptosis. The protection was not affected by antagonists of either cannabinoid receptors (CB(1) or CB(2)) or the vanilloid receptor TRPV1. Anandamide-dependent protection was reduced by pretreatment with LY294002 (inhibitor of phosphatidylinositol 3-kinase, PI3K) and unaffected by U0126 (inhibitor of extracellularly-regulated kinase). Interestingly, phosphorylation of c-Jun-NH2-terminal kinase (JNK) in cells exposed to 6-OHDA was strongly reduced by anandamide pre-treatment. Furthermore, 6-OHDA induced c-Jun activation and increased Bim expression, both of which were inhibited by anandamide. Together, these data demonstrate antiapoptotic effects of anandamide and also suggest a role for activation of PI3K and inhibition of JNK signalling in anandamide-mediated protection against 6-OHDA.

  11. Protective effect of curcumin on dopamine-induced apoptosis in PC12 cells%姜黄素对多巴胺诱导PC12细胞凋亡的拮抗作用

    Institute of Scientific and Technical Information of China (English)

    王翔宇; 梅元武; 杨红卫

    2011-01-01

    目的 探讨姜黄素(Cur)对多巴胺氧化应激损伤PC12细胞的保护作用.方法 以多巴胺(DA)损伤PC12细胞为氧化应激损伤的模型,采用MTT法检测细胞增殖状况,Hoechst染色检测细胞核形态,碘化丙啶(PI)染色流式细胞术(FCM)检测细胞凋亡,罗丹明123(Rh123)染色FCM检测细胞线粒体膜电位(MMP).结果 多巴胺(50~800 μmol/L,24 h)呈浓度依赖性地损伤PCI2细胞,姜黄素(10~40μmol/L)对PC12细胞无毒性作用,但呈浓度依赖性地减轻DA对PC12细胞的毒性作用,抑制DA诱导PC12细胞凋亡,减轻DA对PC12 MMP的降低作用.结论 姜黄素可抑制DA诱导PC12细胞凋亡,对DA氧化应激损伤神经元具有保护作用,其机制可能与姜黄素抑制MMP的降低有关.%Objective To explore the protective effect of curcumin (Cur) on apoptosis of PC12 cells induced by dopamine (DA) oxidative stress. Methods Using DA to induce PC12 cell damage, the model of oxidative stress-induced neuron damage was established. Proliferation of PC12 cells was observed by 3-[4 ,5-dimethylthiazolyl ]-2 ,5-diphenyltetrazolium bromide (MTT) assay. Morphology of the nucleus was observed by Hoechst staining. Apoptosis of PC12 cells was detected by propidium iodide(PI) staining flow cytometry (FCM). The mitochondrial membrane potential (△Ψm) was determined by Rh123 staining flow cytometry. Results ① DA damaged PC12 cells in a concentrationdependent manner after application of DA (50-800 μmol/L) for 24 h, and MTT assay showed that Cur ( 10-40 μmol/L)had no toxic effect on PC12 cells. ② Cur reduced the toxic effect induced by DA on PC12 cells in a concentration-dependent manner. ③ Cur reducing the DA-induced toxic effect on PC12 cells was confirmed by cellular morphologic experiments. ④ PI staining FCM showed that Cur inhibited cellular apoptosis induced by DA and this effect was also confirmed by Hoechst staining. ⑤ The level of △Ψm was significantly decreased in PC12 cells after 100 and 200

  12. Preparation of a hydrophobic polythiophene film to improve protein adsorption and proliferation of PC 12 cells.

    Science.gov (United States)

    Li, Da-Feng; Wang, Hua-Jie; Fu, Jian-Xi; Wang, Wei; Jia, Xue-Shun; Wang, Jin-Ye

    2008-12-25

    High quality films of polythiophene with different alkyl side chains were successfully synthesized by a novel method in the presence of sodium dodecylbenzenesulonate (SDBS) under N2 atmosphere on the PTFE (polytetrafluorethylene) substrate. The as-prepared films were characterized by scanning electron microscopy (SEM), conductivity measurement, and water contact angle measurement. The morphologies of the films were homogeneous with micro-/nanostructures, and their conductivities were high enough for biomedical applications. Hydrophobicity of the films could be adjusted easily by inducing alkyl side chains with different length, which could control protein adsorption in succession. Hydrophobic polythiophene film with a long alkyl side chain had a higher ability of protein adsorption and PC 12 cell proliferation. The biocompatibility study of the synthesized films in vitro proved that the synthesized films were not cytotoxic to two cell lines used and could support cell attachment and proliferation well. Polythiophenes films prepared by in-situ deposition will be good candidates for biomedical applications.

  13. Rab3D is critical for secretory granule maturation in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Tanja Kögel

    Full Text Available Neuropeptide- and hormone-containing secretory granules (SGs are synthesized at the trans-Golgi network (TGN as immature secretory granules (ISGs and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs. Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

  14. Satureja khuzestanica attenuates apoptosis in hyperglycemic PC12 cells and spinal cord of diabetic rats.

    Science.gov (United States)

    Kaeidi, Ayat; Esmaeili-Mahani, Saeed; Abbasnejad, Mehdi; Sheibani, Vahid; Rasoulian, Bahram; Hajializadeh, Zahra; Pasban-Aliabadi, Hamzeh

    2013-01-01

    Several studies have indicated the involvement of oxidative stress and high glucose-induced cell death in the development of diabetic neuropathy. Satureja khuzestanica has been recommended in the literature as a remedy for the treatment of diabetes, and also contains antioxidant agents. Here, we investigated the possible neuroprotective effects of Satureja khuzestanica extract (SKE) on in vitro and in vivo models of diabetic neuropathy pain. High-glucose-induced damage to pheochromocytoma (PC12) cells and in streptozotocin-induced diabetic rats was studied. Tail-flick and rotarod treadmill tests were used to access nociceptive threshold and motor coordination, respectively. Cell viability was determined by MTT assay. Activated caspase 3 and Bax/Bcl-2 ratio-biochemical markers of apoptosis-were evaluated using immunoblotting. We found that elevating the glucose in the medium (to 4× normal) increased cell toxicity and caspase-3 activation in PC12 cells. Incubation with SKE (200 and 250 μg/ml) decreased cell damage. Furthermore, the diabetic rats developed neuropathy, which was evident from thermal hyperalgesia and motor deficit. Administering SKE at a daily dose of between 50 and 200 mg/kg to the diabetic animals for 3 weeks ameliorated hyperglycemia, weight loss, hyperalgesia, and motor deficit, inhibited caspase 3 activation, and decreased the Bax/Bcl-2 ratio. The results suggest that SKE exerts neuroprotective effects against hyperglycemia-induced cellular damage. The mechanisms of these effects may be related to (at least in part) the prevention of neural apoptosis, and the results suggest that Satureja has the therapeutic potential to attenuate side effects of diabetes, such as neuropathy.

  15. Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation.

    Science.gov (United States)

    Offermann, Barbara; Knauer, Steffen; Singh, Amit; Fernández-Cachón, María L; Klose, Martin; Kowar, Silke; Busch, Hauke; Boerries, Melanie

    2016-01-01

    The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation.

  16. A rapidly inactivating Ca2(+)-dependent K+ current in pheochromocytoma cells (PC12) of the rat.

    Science.gov (United States)

    Pun, R Y; Behbehani, M M

    1990-01-01

    The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current- and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2-3 microM), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (less than 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd2+ (0.5 mM), Mn2+ (4 mM), and 0 mM Ca2+/4 mM Mg2+ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about -30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd2+, 0.5 mM; Mn2+, 4 mM; Ba2+, 3 mM), and removal of Ca2+ (0 mM Ca2+/4 mM Mg2+) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K+ current IA in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of -40 mV. The reversal potential of this current was -90 mV, and shifted positively when extracellular K+ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca2(+)-dependent K+ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca2+ buffering (uptake or extrusion) system.

  17. 灵芝孢子提取物对Lactacystin致PC12细胞损伤的保护作用%PROTECTIVE EFFECTS OF GANODERMA LUCIDUM SPORE EXTRACT ON LACTACYSTIN INDUCED PC12 CELLS DAMAGE

    Institute of Scientific and Technical Information of China (English)

    张红; 王芳; 刘湧; 王建东

    2012-01-01

    [目的]探讨灵芝孢子提取物对Lactacystin诱导PC12细胞损伤的保护作用.[方法]体外培养PC12细胞,建立Lactacystin诱导PC12细胞损伤的模型,观察细胞形态,用CCK-8法检测细胞活力,annexinV-FITC/PI流式细胞法检测细胞调亡.[结果]用不同浓度的灵芝孢子提取物处理PC12细胞时,细胞存活率与对照几乎一致,并未表现出细胞毒性.经20 μmol/L的Lactacystin处理24h后,PC12细胞活力比对照组降低,仅为对照组的63.2%.模型组经不同浓度的是芝孢子提取物预处理后,细胞活力明显提高,其保护作用随浓度升高而升高.Lactacystin诱导PC12细胞凋亡,处理24h后细胞凋亡率为48.86%,经灵芝孢子提取物处理后,细胞凋亡率明显降低.[结论]Lactacystin 能诱PC12细胞凋亡,灵芝孢子提取物能对Lactacystin致PC12细胞损伤有一定的保护作用.%[Objective] To investigate the effect of Ganodenna lucidum spore extract (GLS extract) on PC12 cells damage iduced by Lactacystin. [Methods] The toxicity model was established by treating PC12 cells with Lactacystin in vitro. Observed the morphological changes of cells by microscope. Cell viability was assayed with CCk-8 method. The cell apoptotic rate was measured by flow cytometry (FCM) method. [Results] After being treated with different concentrations of GLS extract, the viability of PC12 cells as same as the cell control group tested by CCK-8 assay. After being treated with Lactacystin (20? Mol/L) for 24 h, the viability of PC12 cells depressed and the viable cells was only 63.2% when compared with cell control group. Pretreated with GLS extract at different concentrations, the cell viabilities were all increased significantly. The protective effect on cells increased with its concentration increased. Lactacystin induced apoptosis in PC12 cells, and the apoptotic rate was 48.86% after 24 h of treatment Pretreated with GLS extract, the cell apoptotic rates reduced. [Conclusion] Lactacystin

  18. Hypoxia pretreatment and EPO-modification enhance the protective effects of MSC on neuron-like PC12 cells in a similar way.

    Science.gov (United States)

    Feng, Jinli; Wang, Wei

    2017-01-08

    Mesenchymal stem cells (MSC) based cell transplantation therapy is proved to be an attractive strategy with great potential for improvement of hypoxia induced neural damage. In the present study, MSCs were co-culture with PC12 to investigate its protective effects against hypoxia pretreatment, and the Lactate dehydrogenase (LDH) release assay, MTT and Anexin V staining were performed to analysis the cellular damage or apoptotic. RT-PCR and Western blotting were further used to investigate the underlying mechanism. The results indicate that hypoxia treatment results in the decrease of PC12 cell viability, yet co-culture with MSC could protect the PC12 from hypoxia induced damage. Hypoxia pre-activated or EPO transduced MSC with up-regulated erythropoietin (EPO) expression could further enhance MSC's protective effect against hypoxia induced cell damage, which was associated with high level of anti-apoptotic p-Akt and ration Bcl-2/Bax, and decreased Caspase 3 in PC12. Taken together, these data suggests high levels of MSC-mediated cyto-protection is closely tied to high gene expression levels of EPO. The up-regulation of EPO for enhanced MSC-mediated cyto-protection may has great potential for the MSC cellular therapy of neural or neuronal injuries induced by hypoxia.

  19. Protective Effect of 25Mg-Porphyrin-Fullerene Nanoparticles on Oxygen-Glucose Deprivation/Reperfusion Injury in PC12 Cells.

    Science.gov (United States)

    Ghasemzadeh, Peivand; Rezayat, Seyed Mahdi; Adeli, Sharareh; Rahbar-Roshandel, Nahid

    2016-08-01

    We investigated the effects of 25Mg-Porphyrin-Fullerene nanoparticles, (25MgPMC16) smart ferroporphyrin nanoparticles, on PC12 cells exposed to oxygen-glucose deprivation/reperfusion. In order to explore its effect on cells under oxygen-glucose deprivation conditions, the cultures were pretreated with 25MgPMC16 24 hours prior to oxygen-glucose deprivation/reperfusion. To initiate the oxygen-glucose deprivation/reperfusion, the cell culture medium was replaced with a glucose-free medium and the cells were transferred to a humidified incubation chamber in a mixture of 95% N2 and 5% CO2 at 37° C for 30, 60 and 120 min. Cell viability was assessed by MTT assay. Exposure of PC12 cells to 30, 60 and 120 min oxygen-glucose deprivation significantly decreased the cell viability. Pretreatment of the cultures with 25MgPMC16 significantly increased cell viability in a concentration-dependent manner. Pretreatment, the cultures with MK-801 (10 µM), a non-competitive NMDA antagonist, has attenuated the cell death after 30 min oxygen-glucose deprivation. We concluded that 25MgPMC16 could protect PC12 cells against oxygen-glucose deprivation/reperfusion-induced cell injury in a concentration-dependent manner. That could be due to the effect of 25MgPMC16 on ATP synthesis and the antioxidant effects of its components.

  20. Induction of neuritogenesis in PC12 cells by a pulsed electromagnetic field via MEK-ERK1/2 signaling.

    Science.gov (United States)

    Kudo, Tada-aki; Kanetaka, Hiroyasu; Shimizu, Yoshinaka; Abe, Toshihiko; Mori, Hitoshi; Mori, Kazumi; Suzuki, Eizaburo; Takagi, Toshiyuki; Izumi, Shin-ichi

    2013-01-01

    We examined the regulation of neuritogenesis by a pulsed electromagnetic field (PEMF) in rat PC12 pheochromocytoma cells, which can be induced to differentiate into neuron-like cells with elongated neurites by inducers such as nerve growth factor (NGF). Plated PC12 cells were exposed to a single PEMF (central magnetic flux density, 700 mT; frequency, 0.172 Hz) for up to 12 h per day and were then evaluated for extent of neuritogenesis or acetylcholine esterase (AChE) activity. To analyze the mechanism underlying the effect of the PEMF on the cells, its effects on intracellular signaling were examined using the ERK kinase (MEK) inhibitors PD098059 and U0126 (U0124 was used as a negative control for U0126). The number of neurite-bearing PC12 cells and AChE activity increased after PEMF exposure without the addition of other inducers of neuritogenesis. Additionally, PEMF exposure induced sustained activation of ERK1/2 in PC12 cells, but not in NR8383 rat alveolar macrophages. Furthermore, U0126 strongly inhibited PEMF-dependent ERK1/2 activation and neuritogenesis. The PEMF-dependent neuritogenesis was also suppressed by PD098059, but not U0124. These results suggest that PEMF stimulation independently induced neuritogenesis and that activation of MEK-ERK1/2 signaling was induced by a cell-type-dependent mechanism required for PEMF-dependent neuritogenesis in PC12 cells.

  1. The cannabinoid beta-caryophyllene (BCP) induces neuritogenesis in PC12 cells by a cannabinoid-receptor-independent mechanism.

    Science.gov (United States)

    Santos, Neife Aparecida Guinaim; Martins, Nádia Maria; Sisti, Flávia Malvestio; Fernandes, Laís Silva; Ferreira, Rafaela Scalco; de Freitas, Osvaldo; Santos, Antônio Cardozo

    2017-01-05

    Beta-caryophyllene (BCP) is a phytocannabinoid whose neuroprotective activity has been mainly associated with selective activation of cannabinoid-type-2 (CB2) receptors, inhibition of microglial activation and decrease of inflammation. Here, we addressed the potential of BCP to induce neuritogenesis in PC12 cells, a model system for primary neuronal cells that express trkA receptors, respond to NGF and do not express CB2 receptors. We demonstrated that BCP increases the survival and activates the NGF-specific receptor trkA in NGF-deprived PC12 cells, without increasing the expression of NGF itself. The neuritogenic effect of BCP in PC12 cells was abolished by k252a, an inhibitor of the NGF-specific receptor trkA. Accordingly, BCP did not induce neuritogenesis in SH-SY5Y neuroblastoma cells, a neuronal model that does not express trkA receptors and do not respond to NGF. Additionally, we demonstrated that BCP increases the expression of axonal-plasticity-associated proteins (GAP-43, synapsin and synaptophysin) in PC12 cells. It is known that these proteins are up-regulated by NGF in neurons and neuron-like cells, such as PC12 cells. Altogether, these findings suggest that BCP activates trka receptors and induces neuritogenesis by a mechanism independent of NGF or cannabinoid receptors. This is the first study to show such effects of BCP and their beneficial role in neurodegenerative processes should be further investigated.

  2. Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Synaptotagmin VII (Syt VII), which has a higher Ca2+ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca2+-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca2+ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.

  3. Developmental neurotoxicity of different pesticides in PC-12 cells in vitro.

    Science.gov (United States)

    Christen, Verena; Rusconi, Manuel; Crettaz, Pierre; Fent, Karl

    2017-06-15

    The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals

  4. 苯甲酰化褐藻多糖对PC12细胞存活率的影响%EFFECT OF PHTHALANDIONE FUCOIDAN ON CELL VIABILITY OF PC12 CELLS

    Institute of Scientific and Technical Information of China (English)

    王维维; 贾凤菊; 宋宁; 谢俊霞; 姜宏

    2013-01-01

    Objective To investigate the influence of phthalandione fucoidan (PHF) on PC12 cell viability and the action of PHF on the cells.Methods MTT assay was used to detect the effect of different-concentration PHF on survival rate of PC12 cells.Results Lower doses of PHF (0.001 g/L and 0.010 g/L) had no effect on the cell viability.However,higher doses of PHF (0.100,1.000,10.000 g/L) could significantly reduce the cell viability,the most reduction was noted in the group of 10.000 g/L PHF.Conclusion Low doses of PHF do not affect the survival of PC 12 cells,and high doses result in dose-dependent toxic effect.%目的 通过观察苯甲酰化褐藻多糖(PHF)对PC12细胞存活率的影响,探讨PHF对神经细胞的作用.方法 采用四甲基偶氮唑盐(MTT)方法,检测不同浓度PHF作用下PC12细胞的存活率.结果 低剂量的PHF(0.001、0.010 g/L)对PC12细胞的存活率没有影响;较高剂量的PHF(0.100、1.000、10.000 g/L)可以显著降低PC12细胞的存活率,10.000 g/L PHF处理组细胞的存活率下降最明显.结论 低剂量的PHF不影响PC12细胞的存活率,而较高浓度的PHF能显著降低PC12细胞的存活率,产生剂量依赖性的毒性作用.

  5. Inhibition of Hydrogen Peroxide-induced PC12 Cell Apoptosis by Modafinil%莫达非尼抑制过氧化氢诱导的PC12细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    易宏伟; 刘国卿

    2004-01-01

    目的:研究莫达非尼(modafinil)对过氧化氢诱导的PC12细胞凋亡的抑制作用,并对其机制进行探讨.方法:以噻唑兰(MTT)法测定PC12细胞存活率;用流式细胞术检测PC12细胞凋亡的百分率;测定细胞内罗丹明123(Rhodamine123)的荧光强度,反映细胞线粒体膜电位的改变.结果:莫达非尼能抑制过氧化氢(200 μmol/L)、硝普钠(500μmol/L)和连二亚硫酸钠(2 mmol/L)对PC12细胞的损伤.过氧化氢(200 μmol/L)24h可以诱导PC12细胞凋亡(凋亡率为32.65%).莫达非尼(15,7.5 μmol/L)显著降低凋亡细胞的百分率(凋亡百分率分别为7.95%、15.46%).并且莫达非尼可以抑制过氧化氢引起的线粒体膜电位降低.结论:莫达非尼对PC12细胞有保护作用.能抑制过氧化氢诱导的PC12细胞凋亡,这一作用可能与其抑制过氧化氢引起的线粒体膜电位降低有关.%AIM: To study the inhibition of hydrogen peroxide-induced PC12 cell apoptosis by modafinil and its mechanism. METHOD: PC12 cell survival was measured by MTT assay. The percentage of apoptosis was monitored with flow cytometry. By measuring the intracellular rhodamine123(Rh123)fluorescence density,we evaluated the change of PC12 cell mitochondrial membrane potential (mmp) induced by hydrogen peroxide. RESULT:Modafinil attenuated the cytotoxic effect of hydrogen peroxide (200 μmol/L),sodium nitoprusside (500 μmol/L) and sodium dithionite (2mmol/L). Hydrogen peroxide (200 μmol/L,24 h) induced apoptosis in PC12 cells.PC12 cells were preconditioned with modafinil, then the percentage of apoptosis in the cells was decreased significantly to 7.95%,15.46%,and 17.52% from 32.65%. Modafinil inhibited the mmp decrease in PC12 cells induced by hydrogen peroxide. CONCLUSION:Modafinil prevented PC12 cell from apoptosis induced by hydrogen peroxide,and the mechanism might be associated with its effect of increasing the mitochondrial membrane potential of PC12 cell.

  6. Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion.

    Science.gov (United States)

    Cerna, Joel; Cerecedo, Doris; Ortega, Arturo; García-Sierra, Francisco; Centeno, Federico; Garrido, Efrain; Mornet, Dominique; Cisneros, Bulmaro

    2006-10-01

    Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of beta1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisense-Dp71 clones to analyze in detail the potential involvement of Dp71f isoform with the beta1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell beta1-integrin adhesion complex is composed of beta1-integrin, talin, paxillin, alpha-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the beta1-integrin complex components (beta1-integrin, FAK, alpha-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the beta1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and beta1-integrin. Our data indicate that Dp71f is a structural component of the beta1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance.

  7. Partitioning and Exocytosis of Secretory Granules during Division of PC12 Cells

    Directory of Open Access Journals (Sweden)

    Nickolay Vassilev Bukoreshtliev

    2012-01-01

    Full Text Available The biogenesis, maturation, and exocytosis of secretory granules in interphase cells have been well documented, whereas the distribution and exocytosis of these hormone-storing organelles during cell division have received little attention. By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells. During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules. During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge. Furthermore, by measuring exocytosis at the single granule level, we showed, that during all stages of cell division, secretory granules were competent for regulated exocytosis. In conclusion, our data shed new light on the complex molecular machinery of secretory granule redistribution during cell division, which facilitates their release from the F-actin-rich cortex and active transport along spindle microtubules.

  8. Effect of Tinospora cordifolia on the reduction of ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells

    OpenAIRE

    Masuma, Runa; Okuno, Tsutomu; Choudhuri, Mohammad Shahabuddin Kabir; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    The safety of Tinospora cordifolia and its potential to protect against ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells were investigated. To evaluate the safety of T. cordifolia, cell viability and agarose gel electrophoresis were carried out using PC12 cells treated with 0 to 100g mL(-1) of methanol extract of T. cordifolia. T. cordifolia extracts did not show cytotoxicity ranging 0 to 100g mL(-1). In addition, T. cordifolia extracts significantly increased cell viab...

  9. Neurite Outgrowth in PC12 Cells Stimulated by Components from Dendranthema × grandiflorum cv. “Mottenohoka” Is Enhanced by Suppressing Phosphorylation of p38MAPK

    Directory of Open Access Journals (Sweden)

    Atsuyoshi Nishina

    2013-01-01

    Full Text Available Components from Dendranthema × grandiflorum cv. “Mottenohoka” that promote neurite outgrowth of PC12 cells were identified and the mechanism of neurite outgrowth stimulated by isolated components was studied. Components that promoted the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2 of PC12 cells were isolated. From various structural analyses, the active components were identified as acacetin and luteolin. The effects of acacetin or luteolin on PC12 cells were evaluated by electro-blotting and immunostaining. Slight neurite outgrowth in PC12 cells was observed within 2 days of culture after stimulation by luteolin or acacetin. However, NGF-stimulation induced remarkable neurite outgrowth in comparison. Neurite outgrowth by luteolin or acacetin was significantly enhanced by pretreatment with SB203580 (a p38MAPK inhibitor. The results of this study into the phosphorylation of ERK 1/2 and p38MAPK by flavonoids suggest that the inhibition of p38MAPK phosphorylation may effectively enhance neurite outgrowth.

  10. Cerebrolysin protects PC12 cells from CoCl2-induced hypoxia employing GSK3β signaling.

    Science.gov (United States)

    Hartwig, Kerstin; Fackler, Viktoria; Jaksch-Bogensperger, Heidi; Winter, Stefan; Furtner, Tanja; Couillard-Despres, Sebastien; Meier, Dieter; Moessler, Herbert; Aigner, Ludwig

    2014-11-01

    Cerebrolysin (EVER Neuro Pharma GmbH, Austria) is a peptidergic drug indicated for clinical use in stroke, traumatic brain injury and dementia. The therapeutic effect of Cerebrolysin is thought to ensure from its neurotrophic activity, which shares some properties with naturally occurring neurotrophic factors. However, the exact mechanism of action of Cerebrolysin is yet to be fully deciphered. This study aimed to investigate the neuroprotective effect of Cerebrolysin in a widely used in vitro model of hypoxia-induced neuronal cytotoxicity, namely cobalt chloride (CoCl2)-treatment of PC12 cells. CoCl2-cytotoxicity was indicated by a reduced cell-diameter, cell shrinkage, increased pro-apoptotic Caspase-activities and a decreased metabolic activity. Cerebrolysin maintained the cell-diameter of CoCl2-treated naïve PC12 cells, decreased the activation of Caspase 3/7 in CoCl2-stressed naïve PC12 cells and restored the cells' metabolic activity in CoCl2-impaired naïve and differentiated PC12 cells. Cerebrolysin treatment also decreased the levels of superoxide observed after exposure to CoCl2. Investigating the mechanism of action, we could demonstrate that Cerebrolysin application to CoCl2-stressed PC12 cells increased the phosphorylation of GSK3β, resulting in the inhibition of GSK3β. This might become clinically relevant for Alzheimer's disease, since GSK3β activity has been linked to the production of amyloid beta. Taken together, Cerebrolysin was found to have neuroprotective effects in CoCl2-induced cytotoxicity in PC12 cells.

  11. Lysophosphatidylinositol causes neurite retraction via GPR55, G13 and RhoA in PC12 cells.

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    Yutaro Obara

    Full Text Available GPR55 was recently identified as a putative receptor for certain cannabinoids, and lysophosphatidylinositol (LPI. Recently, the role of cannabinoids as GPR55 agonists has been disputed by a number of reports, in part, because studies investigating GPR55 often utilized overexpression systems, such as the GPR55-overexpressing HEK293 cells, which make it difficult to deduce the physiological role of endogenous GPR55. In the present study, we found that PC12 cells, a neural model cell line, express endogenous GPR55, and by using these cells, we were able to examine the role of endogenous GPR55. Although GPR55 mRNA and protein were expressed in PC12 cells, neither CB(1 nor CB(2 mRNA was expressed in these cells. GPR55 was predominantly localized on the plasma membrane in undifferentiated PC12 cells. However, GPR55 was also localized in the growth cones or the ruffled border in differentiated PC12 cells, suggesting a potential role for GPR55 in the regulation of neurite elongation. LPI increased intracellular Ca(2+ concentration and RhoA activity, and induced ERK1/2 phosphorylation, whereas endogenous and synthetic cannabinoids did not, thereby suggesting that cannabinoids are not GPR55 agonists. LPI also caused neurite retraction in a time-dependent manner accompanied by the loss of neurofilament light chain and redistribution of actin in PC12 cells differentiated by NGF. This LPI-induced neurite retraction was found to be G(q-independent and G(13-dependent. Furthermore, inactivation of RhoA function via C3 toxin and GPR55 siRNA knockdown prevented LPI-induced neurite retraction. These results suggest that LPI, and not cannabinoids, causes neurite retraction in differentiated PC12 cells via a GPR55, G(13 and RhoA signaling pathway.

  12. Protective effect of Nigella sativa and thymoquinone on serum/glucose deprivation-induced DNA damage in PC12 cells

    Directory of Open Access Journals (Sweden)

    Beheshteh Babazadeh

    2012-06-01

    Full Text Available Objective: The discovery and development of natural products with potent antioxidant properties has been one of the most interesting and promising approaches in the search for treatment of CNS injuries. The most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability resulting cellular dysfunction. Serum/glucose deprivation (SGD has served as an excellent in vitro model for the understanding of the molecular mechanisms of neuronal damage during ischemia and for the development of neuroprotective drugs against ischemia-induced brain injury. Nigella sativa (N. sativa seeds and thymoquinone (TQ, its most abundant constituent, have been shown to possess anti-inflammatory, antioxidant, chemopreventive and anti-neoplastic effects both in vitro and in vivo. Therefore, in this study we investigated genoprotective effects of N. sativa and TQ on DNA damage of PC12 cells under SGD condition. Materials and Methods: PC12 cells were cultured in DMEM medium containing 10% (v/v fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Initially cells were pretreated with different concentrations of N. sativa extract (NSE, (10, 50, 250 µg/ml and TQ (1, 5, 10 µg/ml for 6 h and then deprived of serum/glucose (SGD for 18 h. The alkaline comet assay was used to evaluate the effect of these compounds on DNA damage following ischemic insult. The amount of DNA in the comet tail (% tail DNA was measured as an indicator of DNA damage. Results: A significant increase in the % tail DNA was seen in nuclei of cells following SGD induced DNA damage (p0.05. NSE and TQ pretreatment resulted in a significant decrease in DNA damage following ischemic insult (p<0.001. This suppression of DNA damage by NSE and TQ was found to be dose-dependent.Conclusion: These data indicate that NSE and TQ have a genoprotective property, as revealed by

  13. The Neuroprotective Effect of Cannabinoid Receptor Agonist (WIN55,212-2 in Paraoxon Induced Neurotoxicity in PC12 Cells and N-methyl-D-aspartate Receptor Interaction

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    Hedayat Sahraei

    2010-01-01

    Full Text Available Objective: Considering that cannabinoids protect neurons against neurodegeneration, inthis study, the neuroprotective effect of WIN55,212-2 in paraoxon induced neurotoxicity inPC12 cells and the role of the N-methyl-D-aspartate (NMDA receptor were evaluated.Materials and Methods: In this study PC12 cells were maintained in Dulbecco's modifiedeagle’s medium (DMEM+F12 culture medium supplemented with 10% fetal bovineserum. The cells were treated with paraoxon (200 μM in the presence or absence ofWIN55,212-2 (0.1 μM, NMDA receptor agonist NMDA (100 μM, cannabinoid receptorantagonist AM251 and NMDA receptor antagonist MK801 (1 μM at 15 minutes intervals.After 48 hours of exposure, cellular viability and protein expression of the CB1 receptorwere evaluated in PC12 cells.Results: Following the exposure of PC12 cells to paraoxon (200 μM, a reduction in cellsurvival and protein level of the CB1 receptor was observed (p<0.01. Treatment of thecells with WIN55,212-2 (0.1 μM and NMDA (100 μM prior to paraoxon exposure significantlyelevated cell survival and protein level of the CB1 receptor (p<0.01. Also, AM251(1μM did not inhibit the cell survival and protein level of the CB1 receptor increase inducedby WIN55,212-2 (p<0.001. However, MK801 (1 μM did inhibit cell survival andprotein expression of the CB1 receptor increase induced by NMDA (p<0.001.Conclusion: The results indicate that WIN55,212-2 and NMDA protect PC12 cellsagainst paraoxon induced toxicity. In addition, the neuroprotective effect of WIN55,212-2and NMDA was cannabinoid receptor-independent and NMDA receptor dependent, respectively.

  14. Preparation, characterisation, and in vitro evaluation of electrically conducting poly(ɛ-caprolactone)-based nanocomposite scaffolds using PC12 cells.

    Science.gov (United States)

    Gopinathan, Janarthanan; Quigley, Anita F; Bhattacharyya, Amitava; Padhye, Rajiv; Kapsa, Robert M I; Nayak, Rajkishore; Shanks, Robert A; Houshyar, Shadi

    2016-04-01

    In the current study, we describe the synthesis, material characteristics, and cytocompatibility of conducting poly (ɛ-caprolactone) (PCL)-based nano-composite films. Electrically conducting carbon nano-fillers (carbon nano-fiber (CNF), nano-graphite (NG), and liquid exfoliated graphite (G)) were used to prepare porous film type scaffolds using modified solvent casting methods. The electrical conductivity of the nano-composite films was increased when carbon nano-fillers were incorporated in the PCL matrix. CNF-based nano-composite films showed the highest increase in electrical conductivity. The presence of an ionic solution significantly improved the conductivity of some of the polymers, however at least 24 h was required to absorb the simulated ion solutions. CNF-based nano-composite films were found to have good thermo-mechanical properties compared to other conducting polymer films due to better dispersion and alignment in the critical direction. Increased nano-filler content increased the crystallisation temperature. Analysis of cell viability revealed no increase in cell death on any of the polymers compared to tissue culture plastic controls, or compared to PCL polymer without nano-composites. The scaffolds showed some variation when tested for PC12 cell attachment and proliferation, however all the polymers supported PC12 attachment and differentiation in the absence of cell adhesion molecules. In general, CNF-based nano-composite films with highest electrical conductivity and moderate roughness showed highest cell attachment and proliferation. These polymers are promising candidates for use in neural applications in the area of bionics and tissue engineering due to their unique properties.

  15. Schisandrin B protects PC12 cells against oxidative stress of neurodegenerative diseases.

    Science.gov (United States)

    Jiang, En-Ping; Li, He; Yu, Chun-Rong; Yu, Chun-Yan; Jing, Shu; Sun, Hong-Xia; Wang, Chun-Mei; Fan, Xin-Tian; Chen, Jian-Guang; Wang, Sen

    2015-04-15

    Increasing evidence places Schisandrin B (Sch B) at an important position in nerve protection, indicating that Sch B might play a positive role in the therapy of neurodegenerative diseases. However, there is little information on it. Our studies showed that pretreatment with Sch B could reduce lactate dehydrogenase, malondialdehyde, and reactive oxygen species release and significantly increase the cell viability and the superoxide dismutase level. Sch B (10 μM) markedly inhibited cell apoptosis, whereas LY294002 (20 μM), a phosphatidylinositol-3 kinase inhibitor, blocked the antiapoptotic effect. More importantly, Sch B (10 μM) increased the phosphoprotein kinase B/protein kinase B (Akt) and B-cell lymphoma-2/Bcl-2 associated X protein ratios on preincubation with cells for 2 h, which was then inhibited by LY294002 (20 μM). Results indicate that Sch B can protect PC12 cells from apoptosis by activating the phosphatidylinositol-3 kinase/Akt signaling pathway and may emerge as a potential drug for neurodegenerative diseases.

  16. Real-time monitoring of intracellular signal transduction in PC12 cells by non-adiabatic tapered optical fiber biosensor

    Science.gov (United States)

    Zibaii, M. I.; Latifi, H.; Asadollahi, A.; Noraeipoor, Z.; Dargahi, L.

    2014-05-01

    Real-time observation of intracellular process of signal transduction is very useful for biomedical and pharmaceutical applications as well as for basic research work of cell biology. For feasible and reagentless observation of intracellular alterations in real time, we examined the use of a nonadiabatic tapered optical fiber (NATOF) biosensor for monitoring of intracellular signal transduction that was mainly translocation of protein kinase C via refractive index change in PC12 cells adhered on tapered fiber sensor without any indicator reagent. PC12 cells were stimulated with KCl . Our results suggest that complex intracellular reactions could be real-time monitored and characterized by NATOF biosensor.

  17. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    OpenAIRE

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; PAN, GAOFENG; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical...

  18. The effect of an electrically conductive carbon nanotube/collagen composite on neurite outgrowth of PC12 cells.

    Science.gov (United States)

    Cho, Youngnam; Borgens, Richard Ben

    2010-11-01

    We report the preparation of an electrically conductive composite composed of collagen and carbon nanotubes (CNTs) and its use as a substrate for the in vitro growth of PC12 cells. Morphological observation by scanning electron microscopy (SEM) indicated the homogenous dispersion of CNTs in the collagen matrix. Four-point probe and cyclic voltammogram studies demonstrated the enhanced electroactivity and a lowered electrical resistivity of the resulting composites even at low loadings (collagen matrix. SEM and immunofluorescent images have indicated that the morphological features of PC12 cells were dominantly influenced by electrical potential. Greater neurite extension was preferentially induced on the exposure of electrical stimulation by facilitating the differentiation of PC12 cells into neurons indicated by more significant filopodium extension. These electrically conductive, biocompatible CNT/collagen composites could be of benefit for the development of novel neural electrodes, enhancing the growth, differentiation, and branching of neurons in an electrically driven way.

  19. Protective effect of Nigella sativa and thymoquinone on serum/glucose deprivation-induced DNA damage in PC12 cells

    Directory of Open Access Journals (Sweden)

    Beheshteh Babazadeh

    2012-06-01

    Full Text Available Objective: The discovery and development of natural products with potent antioxidant properties has been one of the most interesting and promising approaches in the search for treatment of CNS injuries. The most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability resulting cellular dysfunction. Serum/glucose deprivation (SGD has served as an excellent in vitro model for the understanding of the molecular mechanisms of neuronal damage during ischemia and for the development of neuroprotective drugs against ischemia-induced brain injury. Nigella sativa (N. sativa seeds and thymoquinone (TQ, its most abundant constituent, have been shown to possess anti-inflammatory, antioxidant, chemopreventive and anti-neoplastic effects both in vitro and in vivo. Therefore, in this study we investigated genoprotective effects of N. sativa and TQ on DNA damage of PC12 cells under SGD condition. Materials and Methods: PC12 cells were cultured in DMEM medium containing 10% (v/v fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Initially cells were pretreated with different concentrations of N. sativa extract (NSE, (10, 50, 250 µg/ml and TQ (1, 5, 10 µg/ml for 6 h and then deprived of serum/glucose (SGD for 18 h. The alkaline comet assay was used to evaluate the effect of these compounds on DNA damage following ischemic insult. The amount of DNA in the comet tail (% tail DNA was measured as an indicator of DNA damage. Results: A significant increase in the % tail DNA was seen in nuclei of cells following SGD induced  DNA damage (p0.05. NSE and TQ pretreatment resulted in a significant decrease in DNA damage following ischemic insult (p

  20. The co-culture system of MSCs and injured PC12 in vitro could inhibit the apoptosis of PC12%骨髓基质干细胞与受损PC12细胞的共育体系减少PC12的凋亡

    Institute of Scientific and Technical Information of China (English)

    周进; 罗晓光; 张朝东

    2006-01-01

    目的 建立体外骨髓基质干细胞(MSCs)与Aβ1-40损伤PC12的共育体系,探讨共育体系抑制Aβ1-40致PC12凋亡的效应与可能机制.方法 分别培养MSCs与PC12,以Aβ1-40刺激PC12后,用转移筛网转移PC12.实验分A组:正常培养的PC12+MSCs共育;B组:Aβ1-40刺激的PC12+MSCs共育;C组:正常PC12的培养上清+MSCs;D组:受损PC12上清+MSCs;E组:普通1640培养的MSCs.用PI和Annexin-V进行细胞的双染凋亡检测及电镜检测PC12凋亡,以ELISA方法检测各组中上清液的TGF-β、NGF、BDNF、bFGF含量.结果 骨髓基质干细胞与Aβ1-40损伤PC12共育组即B组凋亡细胞数最少(B组46.17%±8.28%,对照组86.39%±9.34%,F=61.637,P<0.01),ELISA结果显示各组均能检测到bFGF,B组上清中bFGF分泌最高[B组(598.76±41.32)pg/ml,对照组(296.43±47.86)pg/ml,F=24.15,P<0.01)],各组均检测到TGF-β、NGF和BDNF分泌,但差异无统计学意义.结论 MSCs与Aβ1-40刺激的PC12的共育体系能减少受损PC12的凋亡.

  1. 三七总皂苷保护PC12细胞对抗过氧化氢损伤的作用%Effect of Panax notoginseng Saponins Protect PC12 Cell Against Hydrogen Peroxide Induced Damage

    Institute of Scientific and Technical Information of China (English)

    夏星; 钟振国; 冯丹霞

    2013-01-01

    目的:考察三七总皂苷在PC12细胞中对过氧化氢引起损伤的细胞保护作用.方法:采用MTT法考察0.01~100mg·L-1三七总皂苷对正常PC12细胞活力的影响,并在H2O2刺激的PC12细胞中考察0.1~10 mg·L-1的三七总皂苷对细胞活力的影响,通过对细胞活力的考察选择三七总皂苷合适的实验剂量,测定三七总皂苷0.1,1 mg·L-1对H2O2刺激的PC12细胞中活性氧水平、过氧化氢酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力的影响以及对丙二醛(MDA)含量的影响.结果:三七总皂苷在0.1~10 mg·L-1浓度能提高正常PC12细胞活力,并能显著提高H202刺激的PC12细胞活力(P<0.01).0.1,1mg·L-12个剂量的三七总皂苷均能显著减少H2O2刺激的PC12细胞中活性氧水平,提高SOD活力(P <0.001)和GSH-Px活力(P <0.001),并减少MDA生成(P <0.001).结论:三七总皂苷能通过增强细胞内抗氧化酶活力,保护PC12细胞对抗氧化损伤.%Objective: To investigate the protective effect of Panax notoginseng saponins ( PNS) on hydrogen peroxide ( H2O2 ) induced oxidative damage in PC 12 cell. Method: MTT method was employed to determine the viability in PNS (0.01-100 mg·L1) treated PC12 cell and PNS (0. 1-10 mg ·L-1) plus H2O2 treated PC12 cell. Thereafter, intracellular reactive oxygen species amounts, activity of SOD, GSH-Px, as well as MDA concentration were determined at optimal dosage of PNS selected based on MTT experiment. Result; PC12 cell viability was increased by PNS at 0. 1-10 mg ·L-1, and the viability of H2O2 treated PC12 cell was increased by PNS at 0. 1-10 mg ·L-1. Furthermore, PNS at 0. 1 -1. 0 mg ·L-1 significantly decreased intracellular reactive oxygen species levels, as well as enhanced SOD activity (P < 0. 001) and GSH-Px activity ( P < 0. 001). But MDA concentration was decreased significantly (P < 0. 05 ). Conclusion: PNS can protect PC12 cell against H2O2 induced damage via enhancement of intracellular antioxidant

  2. microRNA regulatory mechanism by which PLLA aligned nanofibers influence PC12 cell differentiation

    Science.gov (United States)

    Yu, Yadong; Lü, Xiaoying; Ding, Fei

    2015-08-01

    Objective. Aligned nanofibers (AFs) are regarded as promising biomaterials in nerve tissue engineering. However, a full understanding of the biocompatibility of AFs at the molecular level is still challenging. Therefore, the present study focused on identifying the microRNA (miRNA)-mediated regulatory mechanism by which poly-L-lactic acid (PLLA) AFs influence PC12 cell differentiation. Approach. Firstly, the effects of PLLA random nanofibers (RFs)/AFs and PLLA films (control) on the biological responses of PC12 cells that are associated with neuronal differentiation were examined. Then, SOLiD sequencing and cDNA microarray were employed to profile the expressions of miRNAs and mRNAs. The target genes of the misregulated miRNAs were predicted and compared with the mRNA profile data. Functions of the matched target genes (the intersection between the predicted target genes and the experimentally-determined, misregulated genes) were analyzed. Main results. The results revealed that neurites spread in various directions in control and RF groups. In the AF group, most neurites extended in parallel with each other. The glucose consumption and lactic acid production in the RF and AF groups were higher than those in the control group. Compared with the control group, 42 and 94 miRNAs were significantly dysregulated in the RF and AF groups, respectively. By comparing the predicted target genes with the mRNA profile data, five and 87 matched target genes were found in the RF and AF groups, respectively. Three of the matched target genes in the AF group were found to be associated with neuronal differentiation, whereas none had this association in the RF group. The PLLA AFs induced the dysregulation of miRNAs that regulate many biological functions, including axonal guidance, lipid metabolism and long-term potentiation. In particular, two miRNA-matched target gene-biological function modules associated with neuronal differentiation were identified as follows: (1) miR-23b, mi

  3. Beneficial effect of (-)schisandrin B against 3-nitropropionic acid-induced cell death in PC12 cells.

    Science.gov (United States)

    Lam, Philip Y; Ko, Kam Ming

    2012-01-01

    Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 μM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells.

  4. [EFFECT OF FUCOIDANS ISOLATED FROM SEAWEEDS LAMINARIA DIGITATA AND FUCUS VESICULOSUS ON CELL LINES HELA G-63, ECV 304 AND PC 12].

    Science.gov (United States)

    Zhurishkina, E V; Lapina, I M; Ivanen, D R; Stepanov, S I; Shvetsova, S V; Shavarda, A L; Giliano, N Ya; Kulminskaya, A A

    2015-01-01

    The aim of the research was to investigate cytotoxicity of fucoidans on mammals cells. Three different samples of fucoidans were isolated from mechanically grounded brown algae Laminaria digitata and Fucus ve- siculosus. The sample F2 that differed from the others by higher sulfatation level and suppression of HeLa G-63 line culture growth was taken for further study in cell lines HeLa G-63, ECV 304 and PC 12. We have shown that fucoidan preparation F2 inhibits proliferation and induces cell death in a dose- and time-dependent manner for all investigated cell lines. Neuroendocrine tumor rat cell line PC 12 appeared to be the most sensitive to fucoidan treatment whereas endothelial human cells ECV 304 were the least sensitive.

  5. Mercury induced the Accumulation of Amyloid Beta (Aβ) in PC12 Cells: The Role of Production and Degradation of Aβ

    OpenAIRE

    Song, Ji-Won; Choi, Byung-Sun

    2013-01-01

    Extracellular accumulation of amyloid beta protein (Aβ) plays a central role in Alzheimer’s disease (AD). Some metals, such as copper, lead, and aluminum can affect the Aβ accumulation in the brain. However, the effect of mercury on Aβ accumulation in the brain is not clear. Thus, this study was proposed to estimate whether mercury concentration affects Aβ accumulation in PC12 cells. We treated 10, 100, and 1000 nM HgCl2 (Hg) or CH3HgCl2 (MeHg) for 48 hr in PC12 cells. After treatment, Aβ40 i...

  6. Roscovitine increases intracellular calcium release and capacitative calcium entry in PC12 cells.

    Science.gov (United States)

    Choi, Ho Sook; Chung, Sul-Hee

    2010-01-18

    Cyclin-dependent kinase 5 (Cdk5), which is activated by the non-cyclin regulator p35 or p39, is a proline-directed serine/threonine kinase that is implicated in many physiological and pathological processes. Here, we studied calcium signaling using the fluorescent cytosolic calcium indicator, Fura-4, in NGF-differentiated PC12 cells treated with roscovitine, a Cdk5 inhibitor. As compared to the control cells, the roscovitine-treated cells significantly potentiated intracellular calcium release by membrane depolarization (high K(+)) or through thapsigargin. In addition, roscovitine increased the magnitude of capacitative calcium entry (CCE), i.e., a calcium influx mechanism triggered by the depletion of intracellular calcium stores. Notably, roscovitine markedly slowed the rate of Ca(2+) removal from the plasma membrane. These results suggest that Cdk5 regulates intracellular calcium homeostasis and that the dysregulation of Cdk5 may contribute to disease pathogenesis by perturbing cellular Ca(2+) signaling. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

  7. Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells.

    Science.gov (United States)

    Shacka, J J; Sahawneh, M A; Gonzalez, J D; Ye, Y-Z; D'Alessandro, T L; Estévez, A G

    2006-09-01

    The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

  8. 通络化痰胶囊对H2O2所致的PC12细胞凋亡的保护作用及机制%Protective Effects of Tongluo Huatan Capsule on H2O2-induced Apoptosis in PC12 Cells

    Institute of Scientific and Technical Information of China (English)

    富苏; 范吉平; 王新志

    2013-01-01

    Objective:To investigate the protective effect and mechanism of Tongluo Huatan capsule on H2O2-induced apoptosis in PC12 cells.Method:PC12 cells were cultured,and thecell viability of PC12 cells injured by different H2O2 concentrations and proliferated by different Tongluo Huatan Capsule were observed by MTT assay to determine the concentration that will cause a H2O2-induced apoptosis model of PC12 cells and the security concentrations of Tongluo Huatan Capsule for PC12 cells.PC12 cells in the logarithmic growth phase were divided into control group,model group and low,medium and high dose Tongluo Huatan capsule treatment groups.Apoptosis of PC12 cells were induced by H2O2 at the concentration of 200 μmol ·L-1,MTT assay was used to detect the cell viability of PC12 cells protected by Tongluo Huatan capsule at different concentarions,Hoechst33342 fluorescent staining was used to observe morphological changes of cell apoptosis,the flow cytometry were used to detect the rate of apoptosis in PC12 cells,and the Western blot technology was used to detect the expressios of protein CytC and Caspase-3.Result:PC12 cells were injured by H2O2 at 200 μmol ·L-1 concentration,while Tongluo Huatan capsule at less than or equal to the concentration of 100 μmol ·L-1 did not inhibit the cell viability of PC12 cells.Compared with the model group,Tongluo Huatan capsule groups improved cell viability (P < 0.01),decreased apoptotic cells and apoptosis rate (P < 0.01),and decreased the expression of Cytc and Caspase-3 (P < 0.01) which was related to doses Tongluohuatan capsule.Conclusion:Tongluo Huatan capsule can inhibit H2O2-induced apoptosis in PC12 cells,while the mechanism may be related to the inhibition of the expression of apoptosis-related protein CytC and caspase-3 in the mitochondrial pathway%目的:探讨通络化痰胶囊对过氧化氢(H2O2)所致的大鼠嗜铬瘤细胞(PC12)细胞凋亡的保护作用及机制.方法:PC12细胞常规培养后,首先通过MTT

  9. Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

    Directory of Open Access Journals (Sweden)

    Andersen Søren S.L.

    2004-01-01

    Full Text Available In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100-150 mM. It was recently discovered that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI with 9-11 predicted transmembrane spanning domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12 cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes. The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake in vitro using lipid-detergent vesicles.

  10. 曲古抑菌素A对缺糖/缺氧损伤PC12细胞的保护作用%Protective effect of TSA on PC12 cell injury induced by the deprivation of oxygen/glucose

    Institute of Scientific and Technical Information of China (English)

    越茂松; 黄琼; 王进京; 季秋虹; 季煜华

    2013-01-01

    Aim To study the protective effects of his-tone deacetylase inhibitor ( HDACi ) , trichostatin A ( TSA ), on oxygen/glucose deprivation ( OGD ) inju-ried PC 12 cells and its underlying mechanism. Methods PC 12 OGD injury model was established by using cobalt chloride ( CoCl2 ) and glucose-free medium , and treated by 1-640 nmol · L-1 TSA. Cell viability was measured by MTT assay, cell apoptosis and necrosis was observed by PI and Hoechst staining, and flow cytometry detection and fluorescence microscope were applied to determine the reactive oxygen species in each group. Results Compared with control group, 80 nmol · L-1 TSA significantly improved cell viability and reduced intracellular reactive oxygen species ( ROS ). Conclusions TSA protects the OGD injured PC 12 cells. The possible underlying mechanisms may be related to maintaining or increasing the acetylation level of the energy metabolism enzymes, whereby keeping the PC12 cells from the injury induced by OGD.%目的 研究组蛋白去乙酰化酶抑制剂曲古抑菌素A(trichostatin A,TSA)对PC12大鼠肾上腺嗜铬细胞瘤细胞缺糖/缺氧损伤的保护作用及其可能的作用机制.方法 建立PC12细胞缺糖/缺氧 (oxygen and glucose deprivation,OGD) 损伤模型,1-640 nmol·L-1 TSA处理细胞,噻唑蓝(MTT)检测对PC12细胞活性的影响,Propidium iodide (PI)和Hoechst 33258染色检测细胞凋亡与坏死,荧光显微镜和流式细胞仪检测各组细胞内活性氧(ROS)的含量.结果 与模型组相比,80 nmol·L-1 TSA可明显提高ODG PC12的细胞存活率,降低细胞内的ROS含量(P<0.05).结论 TSA对OGD损伤的PC12细胞具有保护作用,其机制可能是通过增加细胞内能量代谢中各种酶的乙酰化水平来应对缺血损伤.

  11. Luteolin modulates 6-hydroxydopamine-induced transcriptional changes of stress response pathways in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Ling-Wei Hu

    Full Text Available The neurotoxin 6-hydroxydopamine (6-OHDA, which causes transcriptional changes associated with oxidative and proteotoxic stress, has been widely used to generate an experimental model of Parkinson's disease. The food-derived compound luteolin has multi-target actions including antioxidant, anti-inflammatory and neurotrophic activities. The aim of this study is to investigate how luteolin affects 6-OHDA-mediated stress response pathways. The results showed that when PC12 cells were pre-treated with luteolin (20 µM 30 min prior to 6-OHDA (100 µM exposure, 6-OHDA-induced ROS overproduction, cytotoxicity, caspase-3 activation, and mRNA expression of BIM, TRB3 and GADD34 were significantly attenuated. Moreover, 6-OHDA-mediated cell cycle arrest and transcription of p53 target genes, p21, GADD45α and PUMA, were reduced by luteolin. Luteolin also significantly down-regulated 6-OHDA-mediated unfolded protein response (UPR, leading to decreases in phospho-eIF2α, ATF4, GRP78 and CHOP. In addition, luteolin attenuated 6-OHDA-induced Nrf2-mediated HO-1 and GCLC. Taken together, these results suggest that diminishing intracellular ROS formation and down-regulation of p53, UPR and Nrf2-ARE pathways may be involved in the neuroprotective effect of luteolin.

  12. Dynamics of dynamin during clathrin mediated endocytosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Joshua Z Rappoport

    Full Text Available BACKGROUND: Members of the dynamin super-family of GTPases are involved in disparate cellular pathways. Dynamin1 and dynamin2 have been implicated in clathrin-mediated endocytosis. While some models suggest that dynamin functions specifically at the point of vesicle fission, evidence also exists for a role prior to fission during vesicle formation and it is unknown if there is a role for dynamin after vesicle fission. Although dynamin2 is ubiquitously expressed, dynamin1 is restricted to the nervous system. These two structurally similar endocytic accessory proteins have not been studied in cells that endogenously express both. METHODOLOGY/PRINCIPAL FINDINGS: The present study quantitatively assesses the dynamics of dynamin1 and dynamin2 during clathrin-mediated endocytosis in PC12 cells, which endogenously express both proteins. Both dynamin isoforms co-localized with clathrin and showed sharp increases in fluorescence intensity immediately prior to internalization of the nascent clathrin-coated vesicle. The fluorescence intensity of both proteins then decreased with two time constants. The slower time constant closely matched the time constant for the decrease of clathrin intensity and likely represents vesicle movement away from the membrane. The faster rate may reflect release of dynamin at the neck of nascent vesicle following GTP hydrolysis. CONCLUSIONS/SIGNIFICANCE: This study analyses the role of dynamin in clathrin-mediated endocytosis in a model for cellular neuroscience and these results may provide direct evidence for the existence of two populations of dynamin associated with nascent clathrin-coated vesicles.

  13. Chemically-induced oxidative stress increases the vulnerability of PC12 cells to rotenone-induced toxicity

    NARCIS (Netherlands)

    de Groot, Martje W G D M; Westerink, Remco H S

    2014-01-01

    In vitro models, including the widely used PC12 cell line, can increase insight into cellular and molecular mechanisms underlying neurodegenerative processes. An important determinant for the vulnerability of cells for chemical insults may be the endogenous level of oxidative stress. To test this hy

  14. Chemically-induced oxidative stress increases the vulnerability of PC12 cells to rotenone-induced toxicity

    NARCIS (Netherlands)

    de Groot, Martje W G D M|info:eu-repo/dai/nl/411965328; Westerink, Remco H S|info:eu-repo/dai/nl/239425952

    In vitro models, including the widely used PC12 cell line, can increase insight into cellular and molecular mechanisms underlying neurodegenerative processes. An important determinant for the vulnerability of cells for chemical insults may be the endogenous level of oxidative stress. To test this

  15. Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

    Science.gov (United States)

    Dispersyn, G; Nuydens, R; Connors, R; Borgers, M; Geerts, H

    1999-08-05

    This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).

  16. Lead Intoxication Synergies of the Ethanol-Induced Toxic Responses in Neuronal Cells--PC12.

    Science.gov (United States)

    Kumar, V; Tripathi, V K; Jahan, S; Agrawal, M; Pandey, A; Khanna, V K; Pant, A B

    2015-12-01

    Lead (Pb)-induced neurodegeneration and its link with widespread neurobehavioral changes are well documented. Experimental evidences suggest that ethanol could enhance the absorption of metals in the body, and alcohol consumption may increase the susceptibility to metal intoxication in the brain. However, the underlying mechanism of ethanol action in affecting metal toxicity in brain cells is poorly understood. Thus, an attempt was made to investigate the modulatory effect of ethanol on Pb intoxication in PC12 cells, a rat pheochromocytoma. Cells were co-exposed to biological safe doses of Pb (10 μM) and ethanol (200 mM), and data were compared to the response of cells which received independent exposure to these chemicals at similar doses. Ethanol (200 mM) exposure significantly aggravated the Pb-induced alterations in the end points associated with oxidative stress and apoptosis. The finding confirms the involvement of reactive oxygen species (ROS)-mediated oxidative stress, and impairment of mitochondrial membrane potential, which subsequently facilitate the translocation of triggering proteins between cytoplasm and mitochondria. We further confirmed the apoptotic changes due to induction of mitochondria-mediated caspase cascade. These cellular changes were found to recover significantly, if the cells are exposed to N-acetyl cysteine (NAC), a known antioxidant. Our data suggest that ethanol may potentiate Pb-induced cellular damage in brain cells, but such damaging effects could be recovered by inhibition of ROS generation. These results open up further possibilities for the design of new therapeutics based on antioxidants to prevent neurodegeneration and associated health problems.

  17. Inhibition of Corydalis decumbens Alkaloids on Hydrogen Peroxideinduced Apoptosis of PC12 Cells through Down-regulating Caspase-3 Expression

    Institute of Scientific and Technical Information of China (English)

    YAN Ren-jie; YANG Yi-fang; LUO Yong-ming; WU Chun-zhen

    2011-01-01

    Objective To extract alkaloids from Corydalis decumbens (AsCD) by supercritical CO2 fluid extraction (SFE) and to evaluate protective effects of AsCD against hydrogen peroxide (H2O2)-induced apoptosis in rat PC12 cells.Methods AsCD were extracted by SFE and oxidative damage PC12 cells model was induced by H2O2.The survival rate of the cells was determined by MTT assay; Lactate dehydrogenase release was determined by ultraviolet spectrophotometry; Flow cytometry was used to detect apoptosis; Caspase-3 mRNA and protein were determined by real-time PCR and Western blotting assay,respectively.Results AsCD remarkably reduced the cytotoxicity,prevented membrane damage,and inhibited cell apoptosis.AsCD inhibited Caspase-3 mRNA and protein expression induced by H2O2 in PC12 cells.Conclusion AsCD possess protective effects against H2O2-induced apoptosis in PC12 cells,and the mechanism of AsCD responsible to the inhibition of apoptosis is possibly attributed to thedown-regulating Caspase-3 expression.AsCD might be useful in the treatment of oxidative stress-related neurodegenerative diseases.

  18. Evidence of increased reactive species formation by retinol, but not retinoic acid, in PC12 cells.

    Science.gov (United States)

    Gelain, Daniel Pens; Moreira, José Claudio Fonseca

    2008-04-01

    The biological effects of vitamin A (retinol) are generally ascribed to the activation of nuclear retinoid receptors by retinoic acid (RA), considered the most biologically active retinoid. However, it is not established whether the cytotoxic effects of vitamin A are due to retinoid receptors activation by RA. Vitamin A-related toxicity is associated with cellular redox modifications, often leading to severe oxidative damage, but the role of RA in this effect is also uncertain. We therefore studied the formation of intracellular reactive species induced by retinol and retinoic acid in PC12 cells, using an in vitro dichlorofluorescein (DCFH) fluorescence real-time assay. We observed that retinol, but not retinoic acid, induced a steady increase in DCF-based fluorescence over 60 min of incubation, and this increase was reversed by antioxidant (N-acetyl-cysteine and alpha-tocopherol) pre-treatment. This effect was also inhibited by the iron chelator 1,10-phenantroline and the impermeable calcium chelator EGTA. These results suggest that vitamin A-associated cytotoxicity is probably related to an oxidant mechanism dependent on iron and calcium, and the formation of intracellular reactive species is related to retinol, but not to RA.

  19. Toosendanin interferes with pore formation of botulinum toxin type A in PC12 cell membrane

    Institute of Scientific and Technical Information of China (English)

    Mu-feng LI; Yu-liang SHI

    2006-01-01

    Aim: Botulinum neurotoxins (BoNT) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. The ability of botulinum toxin to produce its effect is dependent on the ability of the light chain to cleave the SNARE proteins associated with transmitter release. Translocation of the light chain protease through the heavy chain-formed channel is a pivotal step in the intoxication process. Toosendanin (TSN), a triterpenoid derivative extracted from a Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study was designed to explore the antibotulismic mechanisms of toosendanin. Methods: The inside-out singlechannel recording patch-clamp technique was used to record the BoNT/A-induced currents in the presence and absence of TSN. Results: Channel formation was delayed and the sizes of the channels were reduced in the TSN-treated PC12cell membrane. Conclusion: The antibotulismic effect of TSN might occur via interference with toxin translocation.

  20. Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats.

    Science.gov (United States)

    Lin, Han-Chen; Narasimhan, Purnima; Liu, Shin-Yun; Chan, Pak H; Lai, I-Rue

    2015-01-01

    Postconditioning mitigates ischemia-induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen-glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10-min reoxygenation/10-min rehypoxia after OGD. The MPT inhibitor N-methyl-4-isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC-1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψm ). Transient global cerebral ischemia (tGCI) was induced via the two-vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10-sec reperfusion/10-sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase-3 activity and maintained △Ψm of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase-9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia.

  1. 工频磁场急性暴露对PC12细胞生长和凋亡的影响%Effects of Power Frequency Magnetic Fields Acute Exposure on Growth and Apoptosis of PC12 Cells

    Institute of Scientific and Technical Information of China (English)

    刘肖; 左红艳; 王德文; 彭瑞云; 王水明; 徐新萍; 高亚兵; 吴惠; 王少霞

    2012-01-01

    将体外传代培养的PC12细胞,经50Hz、100 μT的工频磁场暴露24 h.采用噻唑蓝比色法检测细胞增殖活力,流式细胞术检测细胞周期,吖啶橙/溴化乙锭免疫荧光双染色法检测细胞凋亡.结果表明:1)细胞增殖活力于磁场暴露终止后0h明显下降(P<0.01); 4h未见著变;8 h (P<0.05)和12 h(P<0.01)显著升高.2)磁场暴露终止后0h,G0/G1期细胞百分比显著增高(P<0.01),S期细胞百分比显著下降(P<0.05);6 h的G2/M期细胞百分比显著增高(P<0.05);12 h的G0/G1期细胞百分比明显下降(P<0.01),S期细胞百分比显著升高(P<0.05);24 h未见著变.3)磁场暴露期间,6h细胞凋亡率未见显著改变,12、18和24 h凋亡明显增加,至暴露终止后4、8、12和24 h均显著升高,未见恢复.以上结果说明50Hz、100 μT的工频磁场急性暴露,可导致PC12细胞周期和增殖活力的改变,以及细胞凋亡增多.%PC12 cells were cultured in vitro, then exposed to 50 Hz and 100 |xT extremely low frequency electromagnetic fields for 24 h. The proliferation activity, cell cycle and apoptosis was detected respectively by methyl thiazolyl tetrazolium colorimetry, flow cytometry and acridine orange/ethidium bromide, double label immunofluorescence. It was found that: 1) After exposure, proliferation activity was decreased at 0 h (P< 0.01), but increased at 8 h (P<0.05) and 12 h (P<0.01), and there were no significant changes at 4 h. 2) After exposure, G0/G1 cells were increased (P<0.01) and S cells were decreased (P<0.05) at 0h, G2/M cells were increased (P<0.05) at 6 h, G0/G1 cells were decreased(P<0.01) and S cells were increased (P< 0.05) at 12 h, there were no significant changes at 24 h. 3) The apoptotic rates were increased significantly at 12 h (P<0.05), 18 h (P<0.01) and 24 h (P<0.01) during exposure, and 6 h, 12 h, 18 h and 24 h after exposure (P<0.01). The results indicated that 50 Hz and 100 μT extremely low frequency electromagnetic fields

  2. Azole fungicides disturb intracellular Ca2+ in an additive manner in dopaminergic PC12 cells.

    Science.gov (United States)

    Heusinkveld, Harm J; Molendijk, Jeffrey; van den Berg, Martin; Westerink, Remco H S

    2013-08-01

    Humans are exposed to complex mixtures of pesticides and other compounds, mainly via food. Azole fungicides are broad spectrum antifungal compounds used in agriculture and in human and veterinary medicine. The mechanism of antifungal action relies on inhibition of CYP51, resulting in inhibition of fungal cell growth. Known adverse health effects of azole fungicides are mainly linked to CYP inhibition. Additionally, azole fungicide-induced neurotoxicity has been reported, though the underlying mechanism(s) are largely unknown. We therefore investigated the effects of a group of six azole fungicides (imazalil, flusilazole, fluconazole, tebuconazole, triadimefon, and cyproconazole) on cell viability using a combined alamar Blue/CFDA-AM assay and on oxidative stress using a H2-DCFDA fluorescent assay. As calcium plays a pivotal role in neuronal survival and functioning, effects of these six azole fungicides and binary and quaternary mixtures of azole fungicides on the intracellular calcium concentration ([Ca(2+)]i) were investigated using single-cell fluorescence microscopy in dopaminergic PC12 cells loaded with the calcium-sensitive fluorescent dye Fura-2. Only modest changes in cell viability and ROS production were observed. However, five out of six azole fungicides induced a nonspecific inhibition of voltage-gated calcium channels (VGCCs), though with varying potency. Experiments using binary IC20 and quaternary IC10 mixtures indicated that the inhibitory effects on VGCCs are additive. The combined findings demonstrate modulation of intracellular Ca(2+) via inhibition of VGCCs as a novel mode of action of azole fungicides. Furthermore, mixtures of azole fungicides display additivity, illustrating the need to take mixture effects into account in human risk assessment.

  3. 益母草碱对缺糖/缺氧损伤PC12细胞的保护作用%Protective effect of leonurine on PC12 cell injury induced by the deprivation of oxygen/glucose

    Institute of Scientific and Technical Information of China (English)

    袁楠; 王训翠; 李晓祥; 李庆林

    2012-01-01

    Objective To study the protective effect of the hydrochloride leonurine on deprivation of oxygen/glucose injury in rat adrenal chromium tumor cell line ( PC12 ). Methods We used the sodium dithionite ( Na2S2O4 ) + sugar-free in PC12 cells to bulid glucose deprivation/hypoxia injury model ( the oxygen and glucose,deprivation,OGD ). Cell viability was measured by MTT assay,flow cytometry detection was used to determine the oxygen content of active cells in each group. Results Compared with model group, the 25 μmol · L ~' hydrochloride leonurine significantly improved cells viability, reducing intracellular reactive oxygen species. Conclusion The hydrochloric leonurine has a significant protective effect on PC12 cell hypoglycemia/hypoxia injury model .%目的 研究盐酸益母草碱对缺糖/缺氧损伤大鼠肾上腺嗜铬瘤细胞株(PC12)的保护作用.方法 采用连二亚硫酸钠(Na2S2O4)+缺糖建立PC12细胞缺糖/缺氧损伤模型(oxygen and glucose deprivation,OGD),MTT法检测各组细胞存活率,流式细胞仪检测各组细胞内活性氧含量.结果 与模型组相比,25 μmol·L-1盐酸益母草碱显著提高细胞存活率,降低细胞内的活性氧含量.结论 盐酸益母草碱对PC12 细胞缺糖/缺氧损伤模型有明显的保护作用.

  4. Neuroprotection of geniposide against hydrogen peroxide induced PC12 cells injury: involvement of PI3 kinase signal pathway

    Institute of Scientific and Technical Information of China (English)

    Jianhui LIU; Fei YIN; Lixia GUO; Xiaohong DENG; Yinhe HU

    2009-01-01

    Aim:Oxidative stress plays a critical role in the pathogenic cascade leading to neuronal degeneration in AD.Consequently,the induction of endogenous antioxidative proteins by antioxidants seems to be a very reasonable strategy for delaying the disease's progression.In previous work,we identified the neurotrophic and neuroprotective effects of geniposide,which result from the activation of glucagon-like peptide 1 receptor (GLP-1R).In this study,we explore the role of PI3 kinase sig-naling pathway in the neuroprotection of geniposide in PC12 cells.Methods: Cell viability was determined by MTr assay.Apoptosis was detected by Hoechst and PI double staining.The protein expression of Bcl-2 and phosphorylation of Akt308,Akt473,GSK-3β,and PDK1 was measured by Western blot.Results: Geniposide induced the expression of the antiapoptotic protein Bcl-2,which inhibited apoptosis in PC12 cells induced by H2O2,and this effect could be inhibited by preincubation with LY294002,a selective inhibitor of PI3K.Further-more,geniposide enhanced the phosphorylation of Akt308,Akt473,GSK-3β and PDK1 under conditions of oxidative stress.Conclusion: These results demonstrate that the PI3K signaling pathway is involved in the neuroprotection of geniposide in PC12 cells against the oxidative damage induced by H202 in PC12 cells.

  5. Neuroprotective effects of the citrus flavanones against H2O2-induced cytotoxicity in PC12 cells.

    Science.gov (United States)

    Hwang, Sam-Long; Yen, Gow-Chin

    2008-02-13

    The citrus flavanones hesperidin, hesperetin, and neohesperidin are known to exhibit antioxidant activities and could traverse the blood-brain barrier. H2O2 formation induces cellular oxidative stress associated with neurodegenerative diseases. In this study, protective effects of pretreatments (6 h) with hesperidin, hesperetin, and neohesperidin (0.8, 4, 20, and 50 microM) on H2O2-induced (400 microM, 16 h) neurotoxicity in PC12 cells were evaluated. The results showed that hesperetin, hesperidin, and neohesperidin, at all test concentrations, significantly ( p neohesperidin-treated cells) and the increase of caspase-3 activity in H2O2-induced PC12 cells. Meanwhile, hesperidin and hesperetin attenuated decreases of glutathione peroxidase and glutathione reductase activities and decreased DNA damage in H2O2-induced PC12 cells. These results first demonstrate that the citrus flavanones hesperidin, hesperetin, and neohesperidin, even at physiological concentrations, have neuroprotective effects against H2O2-induced cytotoxicity in PC12 cells. These dietary antioxidants are potential candidates for use in the intervention for neurodegenerative diseases.

  6. Mechanism of morphine on proliferation of pheochromocytoma cells%吗啡对 PC12细胞增殖的抑制作用及机制初探

    Institute of Scientific and Technical Information of China (English)

    陈伟强; 刘付宁; 刘玲; 何惠燕; 曹铭辉

    2014-01-01

    Objective To investigate the effects of morphine on roliferation of pheochromocytoma cell and its mechanism. Methods PC12 cell cultures were exposed to 10,20,30 μmol/L morphine. The inhibition of the cell proliferation was determined by MTT assay. The level of p38MAPK was detected by immunoblotting. Results The proliferation of PC12 cells were inhibited after morphine exposure for 48 h.Immunoblotting showed that morphine increased the phosphorylation of p38MAPK was increased in stimulation of monphine. SB203580 , the inhibitor of p38MAPK , reduced inhibition the proliferation of PC12 cells by morphine. Conclusion These results suggest that morphine can significantly inhibits the proliferation of PC12 cells by an increase in p38MAPK phosphorylation.%目的:探讨吗啡对大鼠嗜铬细胞瘤 PC12细胞增殖的抑制作用及其分子机制。方法 PC12细胞体外培养,将吗啡加入细胞培养液中使吗啡浓度分别稀释至10、20、30μmol/L ,然后分别与 PC12细胞一同孵育,四甲基偶氮噻唑蓝法(MTT)观察吗啡对细胞增殖的抑制作用;免疫印迹法检测PC12细胞p38MAPK 表达及磷酸化水平。结果不同浓度吗啡对PC12细胞增殖有明显的抑制作用,抑制率为17.66%~31.05%;并且吗啡可以诱导PC12细胞p38MAPK 磷酸化水平升高,而p38MAPK 通路阻断剂SB203580可以抑制吗啡诱导 PC12细胞 p38MAPK 磷酸化水平升高,减轻吗啡对 PC12细胞增殖的抑制。结论吗啡可以明显抑制 PC12细胞的增殖,升高p38MAPK 的磷酸化水平可能是其重要分子机制之一。

  7. Hydrogen sulfide inhibits beta-amyloid peptide-induced apoptosis in PC12 cells and the underlying mechanisms

    Institute of Scientific and Technical Information of China (English)

    Xiuqin Chen; Jingtian Li; Jinhui Zou; Bailing Li; Meng Wang

    2008-01-01

    BACKGROUND: Studies have demonstrated that hydrogen sulfide (H2S) levels are 55% lower in brains of Alzheimer's disease (AD) patients than in age-matched normal individuals, which suggests that H2S might be involved in some aspects of AD pathogenesis.OBJECTIVE: To observe the protective mechanisms of varied concentrations of H2S against β -amyloid-peptide (A β) induced apoptosis in pheochromoytoma (PC12) cells, and to analyze the pathway of action.DESIGN, TIME AND SETTING: A controlled, observational, in vitro experiment was performed at Nenrophysiology Laboratory in Zhougshan Medical School, Sun Yat-sen University between July 2006 and May 2007.MATERIALS: PC12 cells were provided by the Animal Experimental Center of Medical School of Sun Yat-sen University. Glybenclamide, rhodamine123, and dihydrorhodamine123 were purchased from Sigma (USA).METHODS: PCI2 cells were incubated at 37℃ in a 5% CO2-enriched incubator with RPMI-1640 medium, supplemented with 5% horse-serum and 10% fetal bovine serum. Cells in logarithmic growth curves received different treatment: The PC12 cells were maintains at 37℃ with the original medium, then incubated in A β 25-35, sodium hydrosulfide (NariS), glybenclamide, NailS+ A β 25-35, or pretreated with glybenelamide 30 minutes prior to administration of and A β 25-35, respectively. MAIN OUTCOME MEASURES: (1) The survival rate of PC12 cells was detected by MTT assay and Hoechst staining. (2) The apoptosis rate of PC12 cells was detected utilizing flow cytometry with propidium iodide staining, and morphological changes of apoptotic cells were observed. (3) The mitochondrial membrane potential was detected by Rhodamine 123-combined flow cytometry. (4) The intracellular reactive oxygen species content was detected by dihydrorhodamine123-combined flow cytometry. RESULTS: A β 25-35 induced significantly decreased viability and increased percentage of apoptosis in PC 12 cells, as well as dissipated mitochondrial membrane potential

  8. CEPO对6-OHDA诱导PC12细胞损伤的保护作用%Experimental exploration on the protective effect of CEPO on 6-hydroxydopamine-induced injury of PC12 cells

    Institute of Scientific and Technical Information of China (English)

    贾钰; 雷万龙; 欧阳丽斯; 马宇昕; 陈嘉昌; 阳桂香; 李幽兰; 刘冰冰; 穆淑花; 陈思

    2013-01-01

    Objective To investigate the effect and possible mechanisms of carbamylated erythropoietin (CEPO) in inhibiting the injury of PC12 cells induced by 6-hydroxydopamine (6-OHDA). Methods PC12 cells were divided into the three groups: control group, 6-OHDA group and 6-OHDA + CEPO group. The PC12 cells viability was measured by CCK8 assay. Flow cytometry (FCM) was used to determine apoptosis rate of PC12 cells. The expression of cleaved caspase-3 was detected by western-blotting. The expression of Bcl-2 and Bax mRNA in PC12 cells were measured by Reverse transcriptase polymerase chain reaction (RT-PCR). Results CCK8 assay showed that the cell viability of PC12 cells treated with 200μmol 6-OHDA decreased to 56.70±7.86%, while 40U CEPO treatment increased the cell viability to (87.9±5.3)% (P<0.05). Flow cytometry demonstrated that CEPO treatment significantly inhibited the apoptosis of PC12 cells induced by 6-OHDA (P<0.05). RT-PCR results showed that the expression of Bcl-2 and Bax mRNA in PC12 cells were significantly upregulated and downregulated by CEPO as compared with 6-OHDA treated group (P< 0.05 ). Western-blotting showed CEPO treatment markedly induced the downregulation of cleaved caspase-3 expression compared to 6-OHDA treatment alone (P<0.05). Conclusion CEPO protects PC12 cells from injury and apoptosis induced by 6-OHDA. The protective effect of CEPO might be executed by upregulation of Bcl-2 and downregulation of Bax and Caspase-3 expression in PC12 cells.%目的 探讨氨甲酰化促红细胞生成素(CEPO)对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导PC12细胞损伤及凋亡的保护作用及其可能机制.方法 借助CCK8、流式细胞(Flow cytometry,FCM)、Western-blotting和逆转录PCR(RT-PCR)技术检测PC12模型细胞相关指标的变化,实验数据以SPSS15软件统计分析.结果 CCK8结果显示6-OHDA处理能够显著降低PC12模型细胞的存活率,而CEPO处理对其变化显示抑制作用;FCM技术探察结果显示,6

  9. Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

    Science.gov (United States)

    Barbakadze, Tamar; Goloshvili, Galina; Narmania, Nana; Zhuravliova, Elene; Mikeladze, David

    2017-10-01

    Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S

  10. 生物发光法测定PC12细胞微量ATP含量%Detection of ATP content in PC12 cells by bioluminescence method

    Institute of Scientific and Technical Information of China (English)

    邓晓红; 许予明; 张苏明; 许康

    2004-01-01

    目的研究生物发光法测定PC12细胞内腺苷三磷酸(ATP)含量的可行性及短暂缺氧血清剥夺再灌注后ATP含量的动态变化.方法将PC12细胞随机分为正常对照组、缺氧组, 测定短暂缺氧血清剥夺再灌注后不同时间点的ATP含量和细胞活性, 对缺氧组ATP含量与细胞活性进行相关性分析.结果与正常对照组相比, 缺氧血清剥夺15 min再灌注1 h后的ATP含量、细胞活性显著降低, 差异具有显著性意义(P<0.05);缺氧血清剥夺15 min再灌注6 h后基本恢复至正常水平.缺氧组ATP含量与细胞活性呈显著正相关(r=0.904, P<0.05).结论生物发光法测定PC12细胞内ATP含量有较高的准确性.短暂缺氧血清剥夺再灌注后, 存在低能量状态, 且可完全恢复.

  11. Multipotent Neural Crest Stem Cell-Like Cells from Rat Vibrissa Dermal Papilla Induce Neuronal Differentiation of PC12 Cells

    Directory of Open Access Journals (Sweden)

    Meiying Li

    2014-01-01

    Full Text Available Bone marrow mesenchymal stem cells (BMSCs transplants have been approved for treating central nervous system (CNS injuries and diseases; however, their clinical applications are limited. Here, we model the therapeutic potential of dermal papilla cells (DPCs in vitro. DPCs were isolated from rat vibrissae and characterized by immunocytofluorescence, RT-PCR, and multidifferentiation assays. We examined whether these cells could secrete neurotrophic factors (NTFs by using cocultures of rat pheochromocytoma cells (PC12 with conditioned medium and ELISA assay. DPCs expressed Sox10, P75, Nestin, Sox9, and differentiated into adipocytes, osteoblasts, smooth muscle cells, and neurons under specific inducing conditions. The DPC-conditioned medium (DPC-CM induced neuronal differentiation of PC12 cells and promoted neurite outgrowth. Results of ELISA assay showed that compared to BMSCs, DPCs secreted more brain-derived neurotrophic factor (BDNF and glial cell line-derived neurotrophic factor (GDNF. Moreover, we observed that, compared with the total DPC population, sphere-forming DPCs expressed higher levels of Nestin and P75 and secreted greater amounts of GDNF. The DPCs from craniofacial hair follicle papilla may be a new and promising source for treating CNS injuries and diseases.

  12. Protective effects of veskamide, enferamide, becatamide, and oretamide on H2O2-induced apoptosis of PC-12 cells

    Science.gov (United States)

    Veskamide, enferamide, becatamide, and oretamide are phenolic amides whose analogues are found in plants. In this study, the four amides were prepared by chemical synthesis and their protective effects on H(2)O(2)-induced apoptosis in PC-12 cells were investigated. The syntheses were relatively si...

  13. MELATONIN-INDUCED SUPPRESSION OF PC12 CELL GROWTH IS MEDIATED BY ITS GI COUPLED TRANSMEMBRANE RECEPTORS. (R826248)

    Science.gov (United States)

    The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the mela...

  14. MAGNETIC FIELD INFLUENCE ON NGF-STIMULATED NEURITE OUTGROWTH IN PC-12 CELLS: EFFECT OF PAINT FUMES

    Science.gov (United States)

    MAGNETIC FIELD INFLUENCE ON NGF-STIMULATED NEURITE OUTGROWTH IN PC-12 CELLS: EFFECT OF PAINT FUMES. C. F. Blackman1, D. E. House2*, S. G. Benane3*, A. Ubeda4, M.A. TrilIo4. 1 National Health and Environmental Effects Research Laboratory, EPA,Research Triangle Park, North Caro...

  15. Protective Effect of Ecdysterone on PC12 Cells Cytotoxicity Induced by Beta-amyloid25-35

    Institute of Scientific and Technical Information of China (English)

    YANG Su-fen; WU Zhong-jun; YANG Zheng-qin; WU Qin; GONG Qi-hai; ZHOU Qi-xin; SHI Jing-shan

    2005-01-01

    Objective: To examine the protective effect of ecdysterone (ECR) against beta-amyloid peptide fragment25-35 (Aβ25-35)-induced PC12 cells cytotoxicity, and to further explore its mechanism. Methods:Experimental PC12 cells were divided into the Aβ group (treated by Aβ25-35 100 μmol/L), the blank group (untreated), the positive control group (treated by Vit E 100 μmol/L after induction) and the ECR treated groups (treated by ECR with different concentrations of 1, 50 and 100 μmol/L). The damaged and survival condition of PC12 cells in various groups was monitored by lactate dehydrogenase (LDH) release and MTT assay. The content of malondialdehyde (MDA) was measured by fluorometric assay to indicate the lipid peroxidation. And the antioxidant enzymes activities in PC12 cells, including superoxide dismutases(SOD), catalase (CAT) and glutathione peroxidase(GSH-Px), were detected respectively. Results: After PC12 cells were treated with Aβ25-35 ( 100 μmol/L) for 24 hrs, they revealed a great decrease in MTT absorbance and activity of antioxidant enzymes, including SOD, CAT and GSH-Px as well as a significant increase of LDH activity and MDA content in PC12 cells (P<0.01). When the cells was pretreated with 1-100 μmol/L ECR for 24 hrs before Aβ25-35 treatment, the above-mentioned cytotoxic effect of Aβ25-35 could be significantly attenuated dose-dependently, for ECR 50 μmol/L, P<0.05 and for ECR 100 μmol/L, P<0.01. Moreover, ECR also showed significant inhibition on the Aβ25-35 induced decrease of SOD and GSH-Px activity, but not on that of CAT. Conclusion: ECR could protect PC12 cells from cytotoxicity of Aβ25-35, and the protective mechanism might be related to the increase of SOD and GSH-Px activities and the decrease of MDA resulting from the ECR-pretreatment.

  16. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Park, Jae Hyeon [Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2012-09-01

    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  17. Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Yan Dong

    Full Text Available A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD. Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM. PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising

  18. Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells.

    Science.gov (United States)

    Yin, Fei; Sancheti, Harsh; Cadenas, Enrique

    2012-03-01

    Mitochondrial NADPH generation is largely dependent on the inner-membrane nicotinamide nucleotide transhydrogenase (NNT), which catalyzes the reduction of NADP(+) to NADPH utilizing the proton gradient as the driving force and NADH as the electron donor. Small interfering RNA (siRNA) silencing of NNT in PC12 cells results in decreased cellular NADPH levels, altered redox status of the cell in terms of decreased GSH/GSSG ratios and increased H(2)O(2) levels, thus leading to an increased redox potential (a more oxidized redox state). NNT knockdown results in a decrease of oxidative phosphorylation while anaerobic glycolysis levels remain unchanged. Decreased oxidative phosphorylation was associated with a) inhibition of mitochondrial pyruvate dehydrogenase (PDH) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) activity; b) reduction of NADH availability, c) decline of mitochondrial membrane potential, and d) decrease of ATP levels. Moreover, the alteration of redox status actually precedes the impairment of mitochondrial bioenergetics. A possible mechanism could be that the activation of the redox-sensitive c-Jun N-terminal kinase (JNK) and its translocation to the mitochondrion leads to the inhibition of PDH (upon phosphorylation) and induction of intrinsic apoptosis, resulting in decreased cell viability. This study supports the notion that oxidized cellular redox state and decline in cellular bioenergetics - as a consequence of NNT knockdown - cannot be viewed as independent events, but rather as an interdependent relationship coordinated by the mitochondrial energy-redox axis. Disruption of electron flux from fuel substrates to redox components due to NNT suppression induces not only mitochondrial dysfunction but also cellular disorders through redox-sensitive signaling.

  19. Photosensitizer-induced fluorescence of the rat adrenal gland and rat pheochromocytoma cells (PC 12) by meso-tetra(hydroxyphenyl)chlorin (mTHPC)

    Science.gov (United States)

    Colombo-Benkmann, Mario; Muhm, Markus; Gahlen, Johannes; Heym, Christine; Senninger, Norbert

    1997-12-01

    Rat adrenal glands exhibit an intense mTHPC-induced fluorescence. The objective of our study was the identification of adrenal cells exhibiting mTHPC-induced fluorescence under normal conditions and under stimulation of adrenal proliferation by reserpine. Furthermore mTHPC-uptake of rat pheochromocytoma (PC 12) cells was investigated. Four male Wistar rats received 0.5 mg mTHPC/kg iv 48 hours before perfusion. Furthermore four rats received reserpine (2 mg/kg im od), bromo-deoxy-uridine (BrdU; 50 mg/kg ip od) each for one week and mTHPC (0.5 mg/kg) 48 hours before perfusion. BrdU was detected immunohistochemically. PC 12-cells were incubated with 0.5 mg mTHPC/l culture medium for 24 or 48 hours. Cells and tissues were examined by fluorescence microscopy. The adrenal cortex exhibited an intense mTHPC-induced fluorescence. The adrenal medulla fluoresced faintly. Reserpine increased fluorescence of intramedullary cells, not coinciding with adrenal proliferation. Cortical fluorescence remained unchanged. PC 12-cells lying singly or in small groups and differentiating cells showed a more intense mTHPC- induced fluorescence than confluent cells. Differences of cortical and medullary uptake of mTHPC are independent of proliferation and may be explained by lipophilia of mTHPC, since adrenocytes have an uptake mechanism for cholesterol. The difference of mTHPC-uptake between PC 12-cells and chromaffin cells implicate the possibility of photodynamic applications for medullary neoplasia.

  20. Hydrogen peroxide preconditioning protects PC12 cells against apoptosis induced by oxidative stress%过氧化氢预处理对抗氧化应激诱导的PC12细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    唐小卿; 陈静; 唐二虎; 冯鉴强; 陈培熹

    2005-01-01

    Oxidative stress can induce significant cell death by apoptosis. We explore whether prior exposure to H2O2(H2O2 preconditioning) protects PC12 cells against the apoptotic consequences of subsequent oxidative damages and what role the ATP sensitive potassium (KATP) channels play in the preconditioning protection. PC12 cells were preconditioned with 90 min exposure to H2O2 at 10 μmol/L, followed by 24-h recovery and subsequent exposures to different concentrations (20, 30, 50 and 100 μmol/L) of H2O2 for 24 h respectively. We used PI staining flow cytometry (FCM) to observe the apoptosis of PC12 cells. It was shown that 24-h exposures to H2O2 at 20, 30, 50 and 100 μmol/L respectively induced substantial cell apoptosis, which was greatly prevented in the preconditioning cells, indicating that H2O2 preconditioning protected PC 12 cells against apoptosis induced by H2O2. Administration of pinacidil (10 μmol/L), an KATP channel activator, significantly attenuated the apoptosis of PC12 cells induced by H2O2 at 30 and 50μmol/L for 24 h respectively. Glybenclamide (10 μmol/L), a KATP channel inhibitor, significantly suppressed or abolished the protective effects caused by the pinacidil but not by H2O2 preconditioning. However, when both H2O2 preconditioning and pinacidil were coapplied, their protection against the apoptosis of PC12 cells was much stronger than that of the individual one of them. These results suggest that H2O2 preconditioning protects PC12 cells against apoptosis and that the activation of KATP channels is not involved in, but synergetically enhances adaptive protection of H2O2 preconditioning.%氧化应激可明显地诱导细胞凋亡.本研究旨在探讨H2O2预处理能否对H2O2诱导的PC12细胞凋亡产生保护作用及ATP敏感性K+(ATP-sensitive potassium,KATP)通道在其中的作用.采用PI染色流式细胞仪(flow cytometry,FCM)检测PC12细胞凋亡.结果表明,经10μmol/L H2O2预处理90 min的PC12细胞,分别在20、30、50和100

  1. 6-OHDA Induces Cycle Reentry and Apoetosis of PC12 Cells through Activation of ERK1/2 Signaling Pathway

    Institute of Scientific and Technical Information of China (English)

    Zhentao ZHANG; Tao WANG; Xuebing CAO; Shenggang SUN; Lan WANG

    2009-01-01

    This study investigated the effect and mechanism of cell cycle reentry induced by 6-hydrodopamine (6-OHDA) in PCI2 cells.By using neural differentiated PCI2 cells treated with 6-OHDA,the apoptosis model of dopaminergic neurons was established.Cell viability was measured by MTT.Cell apoptosis and the distribution of cell cycle were assessed by flow cytometry.Western blot was used to detect the activation of extracellular regulator kinasel/2 (ERK1/2) pathway and the phosphorylation of retinoblastoma protein (RB).Our results showed that after PC12 cells were treated wtih 6-OHDA,the viability of PC12 cells was declined in a concentration-dependent manner.Flow cytometry revealed that 6-OHDA could increase the apoptosis ratio of PC12 cells in a time-dependent manner.The percentage of cells in G0/G1 phase of cell cycle was decreased and that in S phase and G2/M phase increased.Simultaneously,ERK1/2 pathway was activated and phos- phorylated RB increased.It was concluded that 6-OHDA could induce cell cycle reentry of dopa-minergic neurons through the activation of ERK1/2 pathway and RB phosphorylation.The aberrant cell cycle reentry contributes to the apoptosis of dopaminergic neurons.

  2. Protective Effect of 25Mg-Porphyrin-Fullerene Nanoparticles on Oxygen-Glucose Deprivation/Reperfusion Injury in PC12 Cells

    OpenAIRE

    Peivand Ghasemzadeh; Seyed Mahdi Rezayat; Sharareh Adeli; Nahid Rahbar-Roshandel

    2016-01-01

    We investigated the effects of 25Mg-Porphyrin-Fullerene nanoparticles, (25MgPMC16) smart ferroporphyrin nanoparticles, on PC12 cells exposed to oxygen-glucose deprivation/reperfusion. In order to explore its effect on cells under oxygen-glucose deprivation conditions, the cultures were pretreated with 25MgPMC16 24 hours prior to oxygen-glucose deprivation/reperfusion. To initiate the oxygen-glucose deprivation/reperfusion, the cell culture medium was replaced with a glucose-free medium and th...

  3. 红藻氨酸诱导PC12细胞凋亡及阿魏酸对神经元的保护作用%Ferulic acid protects against apoptosis of PC12 cells induced by kainic acid

    Institute of Scientific and Technical Information of China (English)

    陈勤; 叶海燕; 陈逸青; 余嗣明

    2013-01-01

    AIM:To investigate the effect of ferulic acid (FA) on the apoptosis of PC12 cells induced by kainic acid (KA) in vitro.METHODS:In order to establish an Alzheimer disease neuronal cell model,the rat pheochromocytoma cell line PC12 was treated with KA at a concentration of 50 μmol/L.These model neurons were divided into KA model group and3 groups treated with FA at doses of 25,50 and 100 μmol/L,respectively.At the same time,normal group was established without KA pretreatment.The viability of the PC12 cells was detected by MTT assay.The expression of Bcl-2,Bax and cytochrome C (Cyt C) was determined by immunocytochemical method.Apoptotic rate of the PC12 cells was measured by flow cytometry with annexin V/PI double staining.The protein levels of Bcl-2,Bax and Cyt C were analyzed by Western blotting.RESULTS:The cell survival rate,the expression of Bcl-2 and the ratio of Bcl-2 to Bax in KA model group were significantly decreased (P <0.01),while the expression of Bax and Cyt C was obviously increased com pared with normal control group (P <0.01).The apoptotie rate in KA model group was obviously increased compared with normal control group (P <0.01) After the intervention of FA,the cell survival rates were increased and the apoptotic rates were decreased.Furthermore,the positive rate and expression of Bcl-2,and the ratio of Bcl-2 to Bax in each dose of FA treatment group were significantly increased,while the expression of Bax and Cyt C in each dose group was significantly reduced as compared with KA model group (P < 0.05 or P < 0.01).CONCLUSION:KA obviously induces apoptosis of PC12 cells.FA had obvious protective effect on PC12 cells against the toxicity of KA.FA blocks endogenous apoptic pathway through inhibiting the expression of Bax and Cyt C and increasing the expression of Bcl-2 and the ratio of Bcl-2/Bax,thus improving the survival rate of PC12 cells.%目的:探讨阿魏酸(ferulic acid,FA)对红藻氨酸(kainic acid,KA)诱导的PC12细胞凋亡

  4. Protective Effects of Some Medicinal Plants from Lamiaceae Family Against Beta-Amyloid Induced Toxicity in PC12 Cell

    Directory of Open Access Journals (Sweden)

    S Saeidnia

    2012-10-01

    Full Text Available Background: Excessive accumulation of beta-amyliod peptide (Aβ, the major component of senile plaques in Alzheimer's disease (AD, causes neuronal cell death through induction of oxidative stress. Therefore, antioxidants may be of use in the treatment of AD. The medicinal plants from the Lamiaceae family have been widely used in Iranian traditional medicine. These plants contain compounds with antioxidant activity and some species in this family have been reported to have neuroprotective properties. In the present study, methanolic extract of seven plants from salvia and satureja species were evaluated for their protective effects against beta-amyloid induced neurotoxicity.Methods: Aerial parts of the plants were extracted with ethyl acetate and methanol, respectively, by percolation at room temperature and subsequently, methanolic extracts of the plants were prepared. PC12 cells were incubated with different concentrations of the extracts in culture medium 1h prior to incubation with Aβ. Cell toxicity was assessed 24h after addition of Aβ by MTT assay.Results: Satureja bachtiarica, Salvia officinalis and Salvia macrosiphon methanolic extracts exhibited high protective effects against Aβ induced toxicity (P<0.001. Protective effects of Satureja bachtiarica and Salvia officinalis were dose-dependent.Conclusion: The main constituents of these extracts are polyphenolic and flavonoid compounds such as rosmarinic acid, naringenin, apigenin and luteolin which have antioxidant properties and may have a role in neuroprotection. Based on neuroprotective effect of these plants against Aβ induced toxicity, we recommend greater attention to their use in the treatment of Alzheimer disease.

  5. Role of mitochondrial dysfunction in neurotoxicity of MPP+: partial protection of PC12 cells by acetyl-L-carnitine.

    Science.gov (United States)

    Virmani, Ashraf; Gaetani, Franco; Binienda, Zbigniew; Xu, Alex; Duhart, Helen; Ali, Syed F

    2004-10-01

    The damage to the central nervous system that is observed after administration of either methamphetamine (METH) or 1-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is known to be linked to dopamine (DA). The underlying neurotoxicity mechanism for both METH and MPP+ seem to involve free radical formation and impaired mitochondrial function. The MPP+ is thought to selectively kill nigrostriatal dopaminergic neurons by inhibiting mitochondrial complex I, with cell death being attributed to oxidative stress damage to these vulnerable DA neurons. In the present study, MPP+ was shown to significantly inhibit the response to MTT by cultured PC12 cells. This inhibitory action of MPP+ could be partially reversed by the co-incubation of the cells with the acetylated form of carnitine, acetyl-L-carnitine (ALC). Since at least part of the toxic action of MPP+ is related to mitochondrial inhibition, the partial reversal of the inhibition of MTT response by ALC could involve a partial restoration of mitochondrial function. The role carnitine derivatives, such as ALC, play in attenuating MPP+ and METH-evoked toxicity is still under investigation to elucidate the contribution of mitochondrial dysfunction in mechanisms of neurotoxicity.

  6. Oxidative and excitatory mechanisms of developmental neurotoxicity: transcriptional profiles for chlorpyrifos, diazinon, dieldrin, and divalent nickel in PC12 cells.

    Science.gov (United States)

    Slotkin, Theodore A; Seidler, Frederic J

    2009-04-01

    Oxidative stress and excitotoxicity underlie the developmental neurotoxicity of numerous chemicals. We compared the effects of organophosphates (chlorpyrifos and diazinon), an organo-chlorine (dieldrin), and a metal [divalent nickel (Ni2+)] to determine how these mechanisms contribute to similar or dissimilar neurotoxic outcomes. We used PC12 cells as a model of developing neurons and evaluated transcriptional profiles for genes for oxidative stress responses and glutamate receptors. Chlorpyrifos had a greater effect on oxidative-stress-related genes in differentiating cells compared with the undifferentiated state. Chlorpyrifos and diazinon showed significant concordance in their effects on glutathione-related genes, but they were negatively correlated for effects on catalase and superoxide dismutase isoforms and had no concordance for effects on ionotropic glutamate receptors. Surprisingly, the correlations were stronger between diazinon and dieldrin than between the two organophosphates. The effects of Ni2+ were the least similar for genes related to oxidative stress but had significant concordance with dieldrin for effects on glutamate receptors. Our results point to underlying mechanisms by which different organophosphates produce disparate neurotoxic outcomes despite their shared property as cholinesterase inhibitors. Further, apparently unrelated neurotoxicants may produce similar outcomes because of convergence on oxidative stress and excitotoxicity. The combined use of cell cultures and microarrays points to specific end points that can distinguish similarities and disparities in the effects of diverse developmental neurotoxicants.

  7. A study of the effects of flux density and frequency of pulsed electromagnetic field on neurite outgrowth in PC12 cells.

    Science.gov (United States)

    Zhang, Yang; Ding, Jun; Duan, Wei

    2006-01-01

    The aim of this study was to investigate the influence of pulsed electromagnetic fields with various flux densities and frequencies on neurite outgrowth in PC12 rat pheochromocytoma cells. We have studied the percentage of neurite-bearing cells, average length of neurites and directivity of neurite outgrowth in PC12 cells cultured for 96 hours in the presence of nerve growth factor (NGF). PC12 cells were exposed to 50 Hz pulsed electromagnetic fields with a flux density of 1.37 mT, 0.19 mT and 0.016 mT respectively. The field was generated through a Helmholtz coil pair housed in one incubator and the control samples were placed in another identical incubator. It was found that exposure to both a relatively high flux density (1.37 mT) and a medium flux density (0.19 mT) inhibited the percentage of neurite-bearing cells and promoted neurite length significantly. Exposure to high flux density (1.37 mT) also resulted in nearly 20% enhancement of neurite directivity along the field direction. However, exposure to low flux density field (0.016 mT) had no detectable effect on neurite outgrowth. We also studied the effect of frequency at the constant flux density of 1.37 mT. In the range from 1 approximately 100 Hz, only 50 and 70 Hz pulse frequencies had significant effects on neurite outgrowth. Our study has shown that neurite outgrowth in PC12 cells is sensitive to flux density and frequency of pulsed electromagnetic field.

  8. A genome-wide signature of glucocorticoid receptor binding in neuronal PC12 cells

    Directory of Open Access Journals (Sweden)

    Polman J Annelies E

    2012-10-01

    Full Text Available Abstract Background Glucocorticoids, secreted by the adrenals in response to stress, profoundly affect structure and plasticity of neurons. Glucocorticoid action in neurons is mediated by glucocorticoid receptors (GR that operate as transcription factors in the regulation of gene expression and either bind directly to genomic glucocorticoid response elements (GREs or indirectly to the genome via interactions with bound transcription factors. These two modes of action, respectively called transactivation and transrepression, result in the regulation of a wide variety of genes important for neuronal function. The objective of the present study was to identify genome-wide glucocorticoid receptor binding sites in neuronal PC12 cells using Chromatin ImmunoPrecipitation combined with next generation sequencing (ChIP-Seq. Results In total we identified 1183 genomic binding sites of GR, the majority of which were novel and not identified in other ChIP-Seq studies on GR binding. More than half (58% of the binding sites contained a GRE. The remaining 42% of the GBS did not harbour a GRE and therefore likely bind GR via an intermediate transcription factor tethering GR to the DNA. While the GRE-containing binding sites were more often located nearby genes involved in general cell functions and processes such as apoptosis, cell motion, protein dimerization activity and vasculature development, the binding sites without a GRE were located nearby genes with a clear role in neuronal processes such as neuron projection morphogenesis, neuron projection regeneration, synaptic transmission and catecholamine biosynthetic process. A closer look at the sequence of the GR binding sites revealed the presence of several motifs for transcription factors that are highly divergent from those previously linked to GR-signaling, including Gabpa, Prrx2, Zfp281, Gata1 and Zbtb3. These transcription factors may represent novel crosstalk partners of GR in a neuronal context

  9. Cytoprotection of 13 Pakistan Medicinal Plants on Neurotoxicity of PC12 Cells%13种巴基斯坦草药对PC12细胞保护作用的研究

    Institute of Scientific and Technical Information of China (English)

    梁岚; 刘新民; 孙丽华; 徐淑萍; 杨艳艳; 赵明耀; Ahsana Dar; Iqbal Choudhary; Dr. Bina; Dr. Shaheen

    2011-01-01

    目的:选择13种巴基斯坦传统草药,观察其对PC12细胞增殖的影响,并探讨其对H202、谷氨酸诱导PC12细胞损伤的保护作用,为寻找神经细胞保护作用新药提供实验依据.方法:体外培养PC12细胞,应用不同浓度的H202、GLU,分别建立神经细胞损伤模型,采用MTT法检测细胞存活率.结果:虎爪豆、诃子等能明显促进PC12细胞增殖作用(P<0.05);在H202 150μmol· L-1损伤模型中,药物组和模型组相比,罗勒、仙人掌、芦笋、灯油藤、诃子等能显著减轻H202的细胞损伤作用(P<0.05);在GLU 30mmol·L-1诱导PC12细胞损伤,与模型组比较,凤仙花、仙人掌、虎爪豆、诃子等均显示出损伤保护作用(P<0.05).其中,诃子不仅能促进PC12细胞增殖,且在两种PC12细胞损伤模型中均具有较强的损伤保护作用.结论:具有促进PC12细胞增殖的药物为诃子、罗勒、虎爪豆;具有对抗H202损伤作用的药物有:诃子、凤仙花、仙人掌、芦笋、灯油藤、罗勒;具有减弱GLU诱导PC12细胞损伤的药物为:虎爪豆、诃子、凤仙花、仙人掌、芦笋、甘草;诃子在3种模型中均表现出较强的细胞损伤保护作用.本研究结果为神经退行性疾病药物的开发和选择提供了初步的实验依据.%This study aimed to investigate the cytoprotection of 20 extracts from 13 Pakistan medicinal plants on PC12 cells injured by H2O2 and glutamate (GLU). Different concentrations of H2O2 and GLU were used to establish the toxicity models on PC12 cells. Cell viability was determined by MTT assay. The results showed that the extracts of Mucuna pruriens L and Terminalia chebula had significantly proliferative effects on PC12 cells (P<0.05). Additionally, extracts of Lawsonia alba, Opuntia dillenii Haw., Celastrus panic ulata, Terminalia chebula showed markedly cytoprotection against H2O2-induced cell death (P<0.05). Similar results were observed in GLU injured model, which showed that

  10. Effect of Tinospora cordifolia on the reduction of ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells.

    Science.gov (United States)

    Masuma, Runa; Okuno, Tsutomu; Kabir Choudhuri, Mohammad Shahabuddin; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    The safety of Tinospora cordifolia and its potential to protect against ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells were investigated. To evaluate the safety of T. cordifolia, cell viability and agarose gel electrophoresis were carried out using PC12 cells treated with 0 to 100 μg mL(-1) of methanol extract of T. cordifolia. T. cordifolia extracts did not show cytotoxicity ranging 0 to 100 μg mL(-1). In addition, T. cordifolia extracts significantly increased cell viability at 1 ng, 10 ng and 1 μg mL(-1) concentrations in serum-deprived medium compared to control. To confirm the protective role against UV-induced damage, PC12 cells alone or in the presence of 10 ng, 100 ng, or 1 μg mL(-1) of T. cordifolia extract were exposed to 250, 270 and 290 nm of UV radiation, which corresponded to doses of 120, 150 and 300 mJ cm(-2), respectively. Treatment with T. cordifolia extracts significantly increased the cell survival rate irradiated at 290 nm. In addition, T. cordifolia extracts significantly reduced cyclobutane pyrimidine dimer formation induced by UV irradiation at all wavelengths. In conclusion, T. cordifolia is not toxic and safe for cells. Our findings can support its application as phototherapy in the medical sector.

  11. Erythropoietin prevents PC12 cells from beta-amyloid-induced apoptosis via PI3K⁄Akt pathway

    Directory of Open Access Journals (Sweden)

    Zhi-Kun Sun

    2012-02-01

    Full Text Available Abstract Background Several studies indicated that Erythropoietin (Epo may provide remarkable neuroprotection in some neurological diseases. It also showed the significant decrease of Epo immunoreactivity in the cerebral cortex and hippocampus in aged rats, suggesting the role of Epo in the pathogenesis of age-related neurodegenerative diseases such as AD. Methods The protective effect of Epo was studied in differentiated PC12 cells treated with Abeta. The viability of the cells, the apoptosis of the cells and the level of Bax, Bcl-2, cleaved caspase-3 and cleaved PARP expression were detected by MTT, Hoechst 33258 staining and Western blotting respectively. Results 20 μM Abeta (25-35 could induce a decreased viability and a increased apoptosis in PC12 cell in a time-dependent manner. However, 20 μM Abeta (35-25 had no effect on cell viability and apoptosis. Western blot analysis also showed that Abeta(25-35 treatment could decrease the expression of Bcl-2 (P P P P (25-35 (P P Conclusions Epo prevented cell injuries in PC12 cells exposed to the Abeta(25-35 and this effect may depend on the PI3K⁄Akt pathway. Our study provided an important evidence for the potential application of Epo in the therapy of Alzheimer's disease.

  12. Early Cellular Responses of Purine Nucleoside-mediated Protection of Hypoxia-induced Injuries of Neuronal PC12 Cells

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    Bettina Tomaselli

    2005-01-01

    Full Text Available Hypoxia in brain may lead to cell death by apoptosis and necrosis. In parallel adenosine, a powerful endogenous neuroprotectant is formed. We wanted to investigate the effect of adenosine and its purine nucleoside relatives, inosine and guanosine on early cellular responses to hypoxia. O2-sensitive neuronal PC12-cells were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Loss of viability after hypoxic insult was impressively rescued by adenosine, guanosine and inosine. PC12-cells mainly express the A2A adenosine receptor. Its inhibition with a specific antagonist (CSC induced cell death of PC12-cells, which could be salvaged by adenosine but not with guanosine or inosine. We have previously demonstrated the important role of mitogen activated protein kinases 1/2 (p42/44 MAPK in purine-mediated rescue. In this study we were interested in the involvement of protein kinases whose activities mediate these processes, including protein kinase A (PKA, phosphoinositide 3-kinase (PI3-K and protein kinase C-related kinases (PRK 1/2. Pharmacological inhibition of PKA and PI3-K increased hypoxia-induced toxicity and likewise also affected the rescue by purine nucleosides. Nerve growth factor (NGF and purine nucleosides induced an activation of PRK 1/2, which to our knowledge indicates for the first time that these kinases are potentially involved in purine nucleoside-mediated rescue of hypoxic neuronal cells. Results suggest that A2A receptor expressing cells are mainly dependent on the purine nucleoside adenosine for their rescue after hypoxic insult. In addition to PKA, PI3-K is an important effector molecule in A2A-mediated signaling and for the rescue of PC12-cells after hypoxic insult.

  13. Synthesis of Functional Polyester Based on Polylactic Acid and Its Effect on PC12 Cells after Coupling with Small Peptides

    Directory of Open Access Journals (Sweden)

    Na Qiang

    2016-01-01

    Full Text Available Polyesters containing functional groups are a suitable candidate matrix for cell culture in tissue engineering. Three types of semicrystalline copolymer poly(L-lactide-co-β-malic acid [P(LA-co-BMD] with pendent carboxyl groups were synthesized in this study. The functional monomer 3(S-[(benzyloxycarbonylmethyl]-1,4-dioxane-2,5-dione (BMD was synthesized using L-aspartic acid. The copolymer P(LA-co-BMD was then synthesized through ring-opening copolymerization of L-LA and BMD, with dodecanol as initiator and stannous octoate as catalyst. Copolymer structure was characterized by 1H nuclear magnetic resonance (1H NMR, gel permeation chromatography (GPC, and differential scanning calorimetry (DSC analyses. Results of 1H NMR and GPC analyses showed that the copolymers were synthesized successfully. DSC curves showed that the crystal melting peak and enthalpy decreased with increased BMD. The crystallinity of the copolymer was destroyed by the presence of the functional monomer. After deprotection, carboxyl groups were coupled with the isoleucine-lysine-valine-alanine-valine peptide through N-hydroxysuccinimide/dicyclohexylcarbodiimide method. The small peptide was beneficial to the axon growth of PC12 cells.

  14. Common increase of GATA-3 level in PC-12 cells by three teratogens causing autism spectrum disorders.

    Science.gov (United States)

    Rout, Ujjwal K; Clausen, Pete

    2009-06-01

    Autism spectrum disorder (ASD) is a disease of neuro-developmental origin of uncertain etiology. The current understanding is that both genetic and environmental factors contribute to the development of ASD. Exposure to valproate, thalidomide and alcohol during gestation are amongst the environmental triggers that are associated with the development of ASD. These teratogens may disturb the ontogeny of the brain by altering the expression pattern of genes that regulate the normal development of the brain. In this study, a neuron-like PC-12 cell model was used to examine the effects of these compounds on the binding potential of 50 different transcription factors to understand the molecular mechanism/s that may be involved in the teratogenesis caused by these agents. Cells in culture were treated with low or high concentrations of teratogens within a range that are reported in the blood of individuals. A pronounced increase in GATA transcription factor binding was observed for all three teratogens. Furthermore, Western blot analysis showed that GATA-3 level in the nuclear fractions was enhanced by each of the three teratogens. Results suggest that altered gene expression pattern due to heightened GATA-3 activities in the fetral brains following exposure to these teratogens may contribute to the development of ASD.

  15. Protective effect of whey protein hydrolysates against hydrogen peroxide-induced oxidative stress on PC12 cells.

    Science.gov (United States)

    Zhang, Qiu-Xiang; Ling, Yu-Fang; Sun, Zhen; Zhang, Li; Yu, Hui-Xin; Kamau, Samuel Mburu; Lu, Rong-Rong

    2012-11-01

    The protective effect of whey protein hydrolysates (WPHs) against H(2)O(2)-induced oxidative damage on rat pheochromocytoma line 12 (PC12) cells was studied. Whey protein was hydrolyzed by pepsin and trypsin and purified by macrospore absorption resins. PC12 cells were pretreated with WPHs (from 369 to 1,980 Da) at different concentrations for 2 h, then washed and incubated with 100 μM H(2)O(2) in the presence of WPHs for another 24 h. With 100-400 μg WPH/ml the viable cells increased by 20-30 % when incubated with H(2)O(2) suggesting that they may play a role as antioxidant in foods.

  16. The effects of functional magnetic nanotubes with incorporated nerve growth factor in neuronal differentiation of PC12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Xie Jining; Chen Linfeng; Varadan, Vijay K [Nanomaterials and Nanotubes Research Laboratory, College of Engineering, University of Arkansas, Fayetteville, AR 72701 (United States); Yancey, Justin; Srivatsan, Malathi [Department of Biological Sciences, Arkansas State University, State University, AR 72467 (United States)], E-mail: jxie@uark.edu, E-mail: msrivatsan@astate.edu

    2008-03-12

    In this in vitro study the efficiency of magnetic nanotubes to bind with nerve growth factor (NGF) and the ability of NGF-incorporated magnetic nanotubes to release the bound NGF are investigated using rat pheochromocytoma cells (PC12 cells). It is found that functional magnetic nanotubes with NGF incorporation enabled the differentiation of PC12 cells into neurons exhibiting growth cones and neurite outgrowth. Microscope observations show that filopodia extending from neuron growth cones were in close proximity to the NGF-incorporated magnetic nanotubes, at times appearing to extend towards or into them. These results show that magnetic nanotubes can be used as a delivery vehicle for NGF and thus may be exploited in attempts to treat neurodegenerative disorders such as Parkinson's disease with neurotrophins. Further neurite outgrowth can be controlled by manipulating magnetic nanotubes with external magnetic fields, thus helping in directed regeneration.

  17. Combined Effects of 50 Hz Magnetic Field and Magnetic Nanoparticles on the Proliferation and Apoptosis of PC12 Cells

    Institute of Scientific and Technical Information of China (English)

    JIA Hong Li; WANG Chao; LI Yue; LU Yan; WANG Ping Ping; PAN Wei Dong; SONG Tao

    2014-01-01

    ObjectiveTo investigate the bioeffects of extremely low frequency (ELF) magnetic field (MF) (50 Hz, 400μT) and magnetic nanoparticles (MNPs) via cytotoxicity and apoptosis assays on PC12 cells. MethodsMNPs modified by SiO2 (MNP-SiO2) were characterized by transmission electron microscopy (TEM), dynamic light scattering and hysteresis loop measurement.PC12 cells were administrated with MNP-SiO2 with or without MF exposure for 48 h. Cytotoxicity and apoptosis were evaluated with MTT assay and annexin V-FITC/PI staining, respectively. The morphology and uptake of MNP-SiO2 were determined by TEM. MF simulation was performed by Ansoft Maxwell based on the finite element method. ResultsMNP-SiO2 were identified as~20nm (diameter) ferromagnetic particles. MNP-SiO2reduced cell viability in a dose-dependent manner. MF also reduced cell viability with increasing concentrations of MNP-SiO2. MNP-SiO2 alone did not cause apoptosis in PC12 cells; instead, the proportion of apoptotic cells increased significantly under MF exposure and increasing doses of MNP-SiO2. MNP-SiO2 could be ingested andthen cause a slight change in cellmorphology. ConclusionCombined exposure of MF and MNP-SiO2 resulted in remarkable cytotoxicity and increased apoptosis in PC12 cells. The results suggested that MF exposure couldstrengthen the MF of MNPs, which may enhance the bioeffects of ELF MF.

  18. Urocortin stimulates tyrosine hydroxylase activity via the cAMP/protein kinase a pathway in rat Pheochromocytoma PC12 cells.

    Science.gov (United States)

    Nanmoku, Toru; Takekoshi, Kazuhiro; Fukuda, Toshiyuki; Isobe, Kazumasa; Shibuya, Shunsuke; Kawakami, Yasushi

    Urocortin is a novel mammalian member of the corticotrophin releasing factor (CRF)-related peptides. We have investigated the expression, mechanism of action and second messenger for urocortin in rat pheochromocytoma PC12 cells. We initially confirmed the expression of urocortin and CRF-R2beta, which is thought to be an endogenous receptor for urocortin, in PC12 cells. We also demonstrate that urocortin (> or = 1 nM) significantly elevates the level of cAMP in these cells. Moreover, alpha-helical CRF-(9-41), a more specific antagonist of CRF-R2 than CRF-R1 and the adenylate cyclase inhibitor SQ22536, inhibited the urocortin-induced increase in the level of cAMP. Thus, urocortin may exert its physiological role in chromaffin cells via CRF-R2beta coupling to adenylate cyclase. Urocortin (> or = 1 nM) significantly increased the mRNA level and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme in the biosynthesis of catecholamine. Furthermore, urocortin-induced changes in TH-mRNA and activity were inhibited by H89 (a PKA inhibitor) and SQ22536 as well as alpha-helical CRF-(9-41). However, urocortin did not affect DNA synthesis or catecholamine secretion in these cells. In conclusion, we have demonstrated that urocortin stimulates catecholamine biosynthesis via the cAMP/protein kinase A pathway in PC12 cells, where both urocortin and its receptor, CRF-R2, are expressed.

  19. Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

    Directory of Open Access Journals (Sweden)

    Becker Walter

    2011-09-01

    Full Text Available Abstract Background Treatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells. We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells. Results The activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 μM. UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS. This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin and BDNF (brain derived neurotrophic factor. Conclusion The present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein

  20. Radiofrequency radiation at 1950 MHz (UMTS) does not affect key cellular endpoints in neuron-like PC12 cells.

    Science.gov (United States)

    Zeni, Olga; Sannino, Anna; Sarti, Maurizio; Romeo, Stefania; Massa, Rita; Scarfì, Maria R

    2012-09-01

    In this study, rat pheochromocytoma (PC12) cells were exposed, as a model of neuron-like cells, to 1950 MHz radiofrequency (RF) radiation with a signal used by the 3G wireless technology of the Universal Mobile Telecommunications System (UMTS) to assess possible adverse effects. RF exposure for 24 h at a specific absorption rate (SAR) of 10 W/kg was carried out in a waveguide system under accurately controlled environmental and dosimetric parameters. DNA integrity, cell viability, and apoptosis were investigated as cellular endpoints relevant for carcinogenesis and other diseases of the central nervous system. Very sensitive biological assays were employed to assess the effects immediately after RF exposure and 24 h later, as demonstrated by the cellular response elicited in PC12 cells using positive control treatments provided for each assay. In our experimental conditions, 24 h of RF exposure at a carrier frequency and modulation scheme typical of a UMTS signal was not able to elicit any effect in the selected cellular endpoints in undifferentiated PC12 cells, despite the application of a higher SAR value than those applied in the majority of the studies reported in the literature. Copyright © 2012 Wiley Periodicals, Inc.

  1. Neuroprotective effect of Eleutheroside B on 1-methyl-4-phenylpyridinium ion-induced apoptosis in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Fang Lu; Yang Dong; Laijun Deng; Shumin Liu; Shihui Zhou; Lifeng An; Bo Tang

    2011-01-01

    Apoptosis and viability of PC12 cells following 1-methyl-4-phenylpyridinium ion (MPP+)-induced injury were monitored by flow cytometry, following Annexin V-propidium iodide double labeling, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. The release of lactate dehydrogenase, superoxide dismutase activity and levels of malondialdehyde were determined by UV spectrophotometry. The changes in mitochondrial membrane potential and the intracellular concentration of calcium were determined by flow cytometry, and the activity of caspase-3 was monitored by western blot. According to cell viability and apoptosis studies, MPP+-induced apoptosis in PC12 cells was inhibited in the presence of 10 μg/mL of Eleutheroside B. Our results indicate that the neuroprotective effect of Eleutheroside B, following MPP+-induced apoptosis in PC12 cells, involves increasing the anti-oxidative stress capacity of cells, maintaining the high-energy state of mitochondrial membrane potential, reducing intracellular calcium concentration and inhibiting caspase-3 activity.

  2. Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

    Science.gov (United States)

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais ML; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-01-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells. PMID:19183502

  3. Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

    Directory of Open Access Journals (Sweden)

    Lepsch Lucilia B

    2009-02-01

    Full Text Available Abstract Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

  4. Epidermal growth factor-receptor interaction in rat pheochromocytoma (PC12) and human epidermoid A431 cells: Biochemical and ultrastructural studies

    NARCIS (Netherlands)

    Laat, S.W. de; Boonstra, J.; Mummery, C.L.; Defize, L.; Leunissen, J.; Verkleij, A.J.

    1984-01-01

    Pheochromocytoma cells (clone PC12) have specific plasmamembrane receptors for both epidermal growth factor (EGF) and nerve growth factor (NGF). These growth factors have however, opposite biological effects in PC12 cells; EGF acts mitogenically, while NGF induces differentiation and causes arrest o

  5. PC12 differentiation on biopolymer nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Cecchini, Marco [Scuola Normale Superiore and NEST-CNR-INFM, Pisa (Italy); Bumma, Giorgia [Scuola Normale Superiore and Italian Institute of Technology, Pisa (Italy); Serresi, Michela [Scuola Normale Superiore and Italian Institute of Technology, Pisa (Italy); Beltram, Fabio [Scuola Normale Superiore and NEST-CNR-INFM, Pisa (Italy)

    2007-12-19

    The study of nervous system regeneration and axonal outgrowth control are relevant in several research areas, like neurophysiology or biomedical engineering. Among the elements that control neuron dynamics, the host substrate topography is a key parameter in determining cell differentiation. We present time-lapse experiments to analyze the differentiation dynamics of PC12 cells on nanopatterned biocompatible substrates. 200 nm depth gratings were fabricated on tissue-culture polystyrene substrates by nanoimprint lithography; different linewidths and pitches were compared down to 500 nm and 1000 nm, respectively. PC12 cells were cultured on these substrates and, following NGF administration to the medium, body morphology, cell movement and neuritogenesis were monitored at different time periods. In addition to demonstrating guided differentiation, our studies show complex time variations in body morphology and axon length, and guided cell movement. We show unstable synaptic connections and cell-body polarization, and the competition between topographical guidance and cell-cell interactions.

  6. PC12 differentiation on biopolymer nanostructures

    Science.gov (United States)

    Cecchini, Marco; Bumma, Giorgia; Serresi, Michela; Beltram, Fabio

    2007-12-01

    The study of nervous system regeneration and axonal outgrowth control are relevant in several research areas, like neurophysiology or biomedical engineering. Among the elements that control neuron dynamics, the host substrate topography is a key parameter in determining cell differentiation. We present time-lapse experiments to analyze the differentiation dynamics of PC12 cells on nanopatterned biocompatible substrates. 200 nm depth gratings were fabricated on tissue-culture polystyrene substrates by nanoimprint lithography; different linewidths and pitches were compared down to 500 nm and 1000 nm, respectively. PC12 cells were cultured on these substrates and, following NGF administration to the medium, body morphology, cell movement and neuritogenesis were monitored at different time periods. In addition to demonstrating guided differentiation, our studies show complex time variations in body morphology and axon length, and guided cell movement. We show unstable synaptic connections and cell-body polarization, and the competition between topographical guidance and cell-cell interactions.

  7. Deep hypothermia-enhanced autophagy protects PC12 cells against oxygen glucose deprivation via a mitochondrial pathway.

    Science.gov (United States)

    Tang, Dang; Wang, Cheng; Gao, Yongjun; Pu, Jun; Long, Jiang; Xu, Wei

    2016-10-06

    Deep hypothermia is known for its organ-preservation properties, which is introduced into surgical operations on the brain and heart, providing both safety in stopping circulation as well as an attractive bloodless operative field. However, the molecular mechanisms have not been clearly identified. This study was undertaken to determine the influence of deep hypothermia on neural apoptosis and the potential mechanism of these effects in PC12 cells following oxygen-glucose deprivation. Deep hypothermia (18°C) was given to PC12 cells while the model of oxygen-glucose deprivation (OGD) induction for 1h. After 24h of reperfusion, the results showed that deep hypothermia decreased the neural apoptosis, and significantly suppressed overexpression of Bax, CytC, Caspase 3, Caspase 9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression. While deep hypothermia increased the LC3II/LC3I and Beclin 1, an autophagy marker, which can be inhibited by 3-methyladenine (3-MA), indicating that deep hypothermia-enhanced autophagy ameliorated apoptotic cell death in PC12 cells subjected to OGD. Based on these findings we propose that deep hypothermia protects against neural apoptosis after the induction of OGD by attenuating the mitochondrial apoptosis pathway, moreover, the mechanism of these antiapoptosis effects is related to the enhancement of autophagy, which autophagy might provide a means of neuroprotection against OGD.

  8. Nitric oxide enhances increase in cytosolic Ca(2+) and promotes nicotine-triggered MAPK pathway in PC12 cells.

    Science.gov (United States)

    Kajiwara, Aya; Tsuchiya, Yukihiro; Takata, Tsuyoshi; Nyunoya, Mayumi; Nozaki, Naohito; Ihara, Hideshi; Watanabe, Yasuo

    2013-11-01

    The purpose of this study was to investigate the roles of neuronal nitric oxide synthase (nNOS), Ca(2+)/calmodulin (CaM)-dependent protein kinases (CaMKs), and protein kinase C (PKC) in nicotine-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) activation. Treatment with nicotine stimulated ERK1/2 and p38 MAPK phosphorylation in the PC12 cells expressing nNOS (NPC12 cells) as compared with that in control PC12 cells. An inhibitor of L-type voltage-sensitive Ca(2+) channel suppressed the nicotine-induced phosphorylation of p38 MAPK. The inhibition of CaMK-kinase, the upstream activator of CaMKI and CaMKIV, did not inhibit the enhanced their phosphorylation. ERK1/2 phosphorylation was attenuated by inhibitors of p38 MAPK, PKC, and MAPK-kinase 1/2, indicating the involvement of these protein kinases upstream of ERK1/2. Furthermore, we found that nNOS expression enhances the nicotine-induced increase in the intracellular concentration of Ca(2+), using the Ca(2+)-sensitive fluorescent probe Fura2. These data suggest that NO promotes nicotine-triggered Ca(2+) transient in PC12 cells to activate possibly CaMKII, leading to sequential phosphorylation of p38 MAPK and ERK1/2.

  9. Stabilization of Nrf2 protein by D3T provides protection against ethanol-induced apoptosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Jian Dong

    Full Text Available Previous studies have demonstrated that maternal ethanol exposure induces a moderate increase in Nrf2 protein expression in mouse embryos. Pretreatment with the Nrf2 inducer, 3H-1, 2-dithiole-3-thione (D3T, significantly increases the Nrf2 protein levels and prevents apoptosis in ethanol-exposed embryos. The present study, using PC12 cells, was designed to determine whether increased Nrf2 stability is a mechanism by which D3T enhances Nrf2 activation and subsequent antioxidant protection. Ethanol and D3T treatment resulted in a significant accumulation of Nrf2 protein in PC 12 cells. CHX chase analysis has shown that ethanol treatment delayed the degradation of Nrf2 protein in PC12 cells. A significantly greater decrease in Nrf2 protein degradation was observed in the cells treated with D3T alone or with both ethanol and D3T. In addition, D3T treatment significantly reduced ethanol-induced apoptosis. These results demonstrate that the stabilization of Nrf2 protein by D3T confers protection against ethanol-induced apoptosis.

  10. Quercetin and sesamin protect neuronal PC12 cells from high-glucose-induced oxidation, nitrosative stress, and apoptosis.

    Science.gov (United States)

    Bournival, Julie; Francoeur, Marc-André; Renaud, Justine; Martinoli, Maria-Grazia

    2012-06-01

    Complications of diabetes are now well-known to affect sensory, motor, and autonomic nerves. Diabetes is also thought to be involved in neurodegenerative processes characteristic of several neurodegenerative diseases. Indeed, it has been acknowledged recently that hyperglycemia-induced oxidative stress contributes to numerous cellular reactions typical of central nervous system deterioration. The goal of the present study was to evaluate the effects of the polyphenol quercetin and the lignan sesamin on high-glucose (HG)-induced oxidative damage in an in vitro model of dopaminergic neurons, neuronal PC12 cells. When incubated with HG (13.5 mg/mL), neuronal PC12 cells showed a significant increase of cellular death. Our results revealed that quercetin and sesamin defend neuronal PC12 cells from HG-induced cellular demise. An elevated level of reactive oxygen and nitrogen species is a consequence of improved oxidative stress after HG administration, and we demonstrated that this production diminishes with quercetin and sesamin treatment. We also found that quercetin and sesamin elicited an increment of superoxide dismutase activity. DNA fragmentation, Bax/Bcl-2 ratio, nuclear translocation of apoptosis-inducing factor, as well as poly(adenosine diphosphate [ADP]-ribose) polymerase cleavage were significantly reduced by quercetin and sesamin administration, affirming their antiapoptotic features. Also, HG treatment impacted caspase-3 cleavage, supporting caspase-3-dependent pathways as mechanisms of apoptotic death. Our results indicate a powerful role for these natural dietary compounds and emphasize preventive or complementary nutritional strategies for diabetes control.

  11. The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation.

    Science.gov (United States)

    Leiser, Yoav; Silverstein, Nechama; Blumenfeld, Anat; Shilo, Dekel; Haze, Amir; Rosenfeld, Eli; Shay, Boaz; Tabakman, Rinat; Lecht, Shimon; Lazarovici, Philip; Deutsch, Dan

    2011-01-01

    The tuftelin protein isoforms undergo post-translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down-regulation of HIF1α mRNA blocked hypoxia-induced HIF1α expression, and reduced by 89% hypoxia-induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF-mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA-signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the "hypoxic genome," and NGF-induced neurotrophic and angiogenic effects.

  12. Nuclear translocation of phosphatidylinositol 3-kinase in rat pheochromocytoma PC 12 cells after treatment with nerve growth factor.

    Science.gov (United States)

    Neri, L M; Milani, D; Bertolaso, L; Stroscio, M; Bertagnolo, V; Capitani, S

    1994-07-01

    Immunocytochemical analysis of PI 3-kinase localization in PC 12 cells demonstrates that the enzyme translocates to the nucleus after cell treatment with differentiating doses of NGF. The association of PI 3-kinase to the nucleus occurs rapidly (within minutes) and increases with the time of exposure of NGF. We suggest that PI-3 kinase specific localization may determine the production of novel phosphoinositides in cell compartments targeted to effect diverse cell responses. The nuclear translocation is consistent with accumulating data on the existence of a nuclear inositol lipid cycle which could also include 3-phosphorylated inositides, participating to the modulation of the cell response to extracellular stimuli.

  13. Experimental Study on the Inhibitory Effects of Vitamin E Against Manganese-induced Cytotoxicity in PC12 Cells%维生素E抑制锰诱导的PC12细胞毒性的实验研究

    Institute of Scientific and Technical Information of China (English)

    王迪雅; 蔡同建; 赵芳; 姚婷; 陈景元; 骆文静

    2012-01-01

    目的 观察维生素E对锰诱导的PC12细胞毒性的抑制效应,从而为锰神经毒性的防护提供可能的途径.方法 MTT、TUNEL、姬姆萨染色、流式细胞仪检测细胞毒性;试剂盒检测MDA含量以及T-SOD、MnSOD活性的变化. 结果 锰可以诱导PC12细胞氧化应激水平显著增高,显示为MDA含量的增高和T-SOD、MnSOD活性代偿性升高.维生素E可以抑制锰诱导的PC12细胞毒性,表现为其可抑制锰诱导的细胞活力的下降以及凋亡率的升高. 结论 维生素E作为抗氧化剂可以抑制由锰诱导的PC12细胞的一系列细胞毒性,补充维生素可作为一种防护锰毒性的有效手段.%Objective To study the inhibitory effects of vitamin E on manganese- induced cytotoxicity in PC12 cells and to provide a potential new way against manganese — induced neurotoxicity. Methods The cytotoxicity was determined by MTT assay, TUNEL, Giemsa staining and flow cytometry, while the MDA content, T- SOD and MnSOD activities were detected by kits. Results Manganese induced enhanced oxidative stress, as shown by the elevation of MDA content and compensatory increase of T— SOD and MnSOD activities. The supplement of vitamin E could inhibit manganese- induced cytotoxicity in that it could antagonize the decrease of cell viability and increase of cell apoptosis rate caused by manganese. Conclusions As a typical antioxidant, vitamin E can inhibit manganese- induced cytotoxicity in PC12 cells. Vitamin E supplement can be an effective way against manganese toxicity.

  14. Protective effect of cyclophilin A against Alzheimer's amyloid beta-peptide (25-35)-induced oxidative stress in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    GE Yu-song; TENG Wei-yu; ZHANG Chao-dong

    2009-01-01

    Background β-amyloid peptide (Aβ) is considered responsible for the pathogenesis of Alzheimer's disease (AD). Possible mechanisms underlying Aβ-induced neuronal cytotoxicity include excessive production of reactive oxidative species (ROS) and apoptosis. Cyclophilin A (CypA), exhibits antioxidant properties and protects neurons against oxidative stress induced injury. This study was conducted to demonstrate whether CyPA added to cultured PC12 cells could alleviate Aβ-induced oxidative stress and protect them from apoptosis.Methods PC12 cells were pre-incubated for 30 minutes with recombinant human cyclophilin A (rhCyPA) in 0.1 nmol/L, 1.0 nmol/L, 10 nmol/L and 100 nmol/L and then incubated with 10 umol/L Aβ25-35. In every group, cell viability, apoptotic morphology, apoptotic rate, intracellular ROS accumulation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of PC12 cells and mitochondrial transmembrane potential were detected. Subsequently, the expression of the active form of caspase-3 was determined by Western blotting.Results It was shown that cultures treated with 1.0 nmol/L, 10 nmol/L or 100 nmol/L rhCyPA + Aβ25-35 had significantly higher cell viability and a lower rate of apoptosis compared with the cultures exposed only to Aβ25-35. In addition, rhCyPA attenuated Aβ25-35-induced overproduction of intracellular ROS and Aβ25-35-induced a decrease in activity of the key antioxidant enzymes SOD and GSH-Px. Furthermore, rhCyPA also attenuated Aβ25-35-induced mitochondrial dysfunction and the activation of caspase-3.Conclusion CyPA may act as an ROS scavenger, and prevent Aβ25-35-induced neurotoxicity through attenuating oxidative stress induced by Aβ25-35.

  15. BDNF-TrkB pathway mediates neuroprotection of hydrogen sulfide against formaldehyde-induced toxicity to PC12 cells.

    Directory of Open Access Journals (Sweden)

    Jia-Mei Jiang

    Full Text Available Formaldehyde (FA is a common environmental contaminant that has toxic effects on the central nervous system (CNS. Our previous data demonstrated that hydrogen sulfide (H2S, the third endogenous gaseous mediator, has protective effects against FA-induced neurotoxicity. As is known to all, Brain-derived neurotropic factor (BDNF, a member of the neurotrophin gene family, mediates its neuroprotective properties via various intracellular signaling pathways triggered by activating the tyrosine kinase receptor B (TrkB. Intriguingly, our previous data have illustrated the upregulatory role of H2S on BDNF protein expression in the hippocampus of rats. Therefore, in this study, we hypothesized that H2S provides neuroprotection against FA toxicity by regulating BDNF-TrkB pathway. In the present study, we found that NaHS, a donor of H2S, upregulated the level of BDNF protein in PC12 cells, and significantly rescued FA-induced downregulation of BDNF levels. Furthermore, we found that pretreatment of PC12 cells with K252a, an inhibitor of the BDNF receptor TrkB, markedly reversed the inhibition of NaHS on FA-induced cytotoxicity and ablated the protective effects of NaHS on FA-induced oxidative stress, including the accumulation of intracellular reactive oxygen species (ROS, 4-hydroxy-2-trans-nonenal (4-HNE, and malondialdehyde (MDA. We also showed that K252a abolished the inhibition of NaHS on FA-induced apoptosis, as well as the activation of caspase-3 in PC12 cells. In addition, K252a reversed the protection of H2S against FA-induced downregulation of Bcl-2 protein expression and upregulation of Bax protein expression in PC12 cells. These data indicate that the BDNF-TrkB pathway mediates the neuroprotection of H2S against FA-induced cytotoxicity, oxidative stress and apoptosis in PC12 cells. These findings provide a novel mechanism underlying the protection of H2S against FA-induced neurotoxicity.

  16. Repeated Glucose Deprivation/Reperfusion Induced PC-12 Cell Death through the Involvement of FOXO Transcription Factor

    Science.gov (United States)

    Han, Na; Kim, You Jeong; Park, Su Min; Kim, Seung Man; Lee, Ji Suk; Jung, Hye Sook; Lee, Eun Ju; Kim, Tae Kyoon; Kim, Tae Nyun; Kwon, Min Jeong; Lee, Soon Hee; Rhee, Byoung Doo

    2016-01-01

    Background Cognitive impairment and brain damage in diabetes is suggested to be associated with hypoglycemia. The mechanisms of hypoglycemia-induced neural death and apoptosis are not clear and reperfusion injury may be involved. Recent studies show that glucose deprivation/reperfusion induced more neuronal cell death than glucose deprivation itself. The forkhead box O (FOXO) transcription factors are implicated in the regulation of cell apoptosis and survival, but their role in neuronal cells remains unclear. We examined the role of FOXO transcription factors and the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt and apoptosis-related signaling pathways in PC-12 cells exposed to repeated glucose deprivation/reperfusion. Methods PC-12 cells were exposed to control (Dulbecco's Modified Eagle Medium [DMEM] containing 25 mM glucose) or glucose deprivation/reperfusion (DMEM with 0 mM glucose for 6 hours and then DMEM with 25 mM glucose for 18 hours) for 5 days. MTT assay and Western blot analysis were performed for cell viability, apoptosis, and the expression of survival signaling pathways. FOXO3/4',6-diamidino-2-phenylindole staining was done to ascertain the involvement of FOXO transcription factors in glucose deprivation/reperfusion conditions. Results Compared to PC-12 cells not exposed to hypoglycemia, cells exposed to glucose deprivation/reperfusion showed a reduction of cell viability, decreased expression of phosphorylated Akt and Bcl-2, and an increase of cleaved caspase-3 expression. Of note, FOXO3 protein was localized in the nuclei of glucose deprivation/reperfusion cells but not in the control cells. Conclusion Repeated glucose deprivation/reperfusion caused the neuronal cell death. Activated FOXO3 via the PI3K/Akt pathway in repeated glucose deprivation/reperfusion was involved in genes related to apoptosis.

  17. 乳清蛋白酶解物对PC12细胞氧化损伤防护作用的体外研究%STUDY ON THE PROTECTIVE EFFECTS OF WHEY PROTEIN HYDROLYSATES TO PC12 CELLS AGAINST OXIDATIVE DAMAGE IN VITRO

    Institute of Scientific and Technical Information of China (English)

    凌玉芳; 卢蓉蓉; 孙震; 赵新灿; 吴慧; 陈潇

    2011-01-01

    目的 研究乳清蛋白酶解物对由过氧化氮引起的PC12细胞氧化损伤的防护作用.方法 采用胃蛋白酶-胰蛋白酶双酶酶解乳清蛋白得到乳源性多肽;通过细胞存活率、细胞形态、细胞抗氧化酶活性、丙二醛等指标,分析探讨乳清蛋白酶解物对细胞损伤的防护作用.结果 双酶酶解所得乳清蛋白抗氧化肽相对分子童主要分布在369~1980u;酶解物浓度100~400 μg/ml时,可使H2O:诱导损伤的PC12细胞存活率提高20%-30%,提高PC12细胞中杭氧化系统(包括酶、非酶系统)水平及细胞膜的完整性.结论 乳清蛋白酶解产物对过氧化氢诱导氧化损伤的PC12细胞具有显著的防护作用,可作为食源性抗氧化剂.%Objective To study the protective effects of whey protein hydrolysates against H2O2-induced oxidative damage in PC12 cells. Method Whey protein was hydrolyzed by pepsin and trypsin. PC12 cells were seeded into culture plates, and treated with whey protein hydrolysates (WPHs) at different concentrations, then incubated together with 100 μmol/L H2O2 for 24 h. The protective effects were studied by measuring the cell viability (MTT assay), photographing the cell morphological changes and detecting the activity of cell antioxidant enzymes. Results The relative molecular mass of WPHs was 369-1980 u. Incubation of 100-400 μg/ml WPHs with H2O2 increased the percentage of viable cells,improved the antioxidant system level of cells and kept the integrity of cell membrane. Conclusion WPHs are endowed with significant protective effects against H2O2-induced oxidative stress in PC12 cells by their antioxidant ability, and might be used as a foodborne antioxidant. [ACTA NUTRIMENTA SINICA, 2011, 33(2) : 162-166

  18. 外源性 ATP 诱导 PC12细胞的膜孔形成%Exogenous ATP induces formation of membrane pore in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    沈慧; 尹雅玲; 李超堃; 赵红岗; 马洁; 李东亮

    2014-01-01

    AIM:To investigate the formation of membrane pore in PC 12 cells induced by exogenous adenosine triphosphate ( ATP) and to identify the key molecular targets .METHODS:PC12 cells were treated with different concen-trations of ATP to establish the injury model .The morphological change was observed under an inverted phase -contrast mi-croscope.The viability of the PC12 cells was measured by CCK-8 assay.Fluorescent dye YO-PRO-1 was used to detect the membrane permeability.The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was as-sessed by real-time PCR and Western blotting .RESULTS:After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous , and the number of adherent cells decreased gradually in a dose-dependent man-ner .The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with con-trol group (P0. 05).The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P0.05) when PC12 cells were exposed to ATP for 3 h.CONCLUSION:Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P 2X7 receptor in PC12 cells.%目的:探讨外源性三磷酸腺苷( ATP)诱导PC12细胞的膜孔形成及关键分子靶标。方法:用不同浓度的ATP处理培养的PC12细胞,采用倒置相差显微镜观察形态,CCK-8法检测细胞存活率,YO-PRO-1染色检测细胞膜通透性,Western blotting和real-time PCR检测P2X7受体和pannexin 1(Panx1)表达的变化。结果:(1)ATP(1 mmol/L、3 mmol/L、5 mmol/L)作用3 h,可见随着ATP浓度升高,PC12细胞变圆,脱壁细胞增多;当ATP浓度为3 mmol/L或5 mmol/L时,PC12细胞活力较对照组显著下降(P<0.05)。(2)不同浓度的ATP(0、1、3、5 mmol/L)作用1 h,PC12细胞摄入YO-PRO-1的荧光强度随着浓度增加而增加;同一浓度的ATP作用不同时间(15

  19. Effects of an autophagy/lysosomal pathway induced by 6-hydroxydopamine in PC12 cells%6-羟基多巴胺致PC12细胞损伤机制中自噬的影响

    Institute of Scientific and Technical Information of China (English)

    周生奎; 程言博; 耿润潼; 陈浩; 刘涵; 徐兴顺; 耿德勤

    2011-01-01

    Objective To investigated the role of the autophagy lysosomal pathway in PD cells and the possible molecular mechanisms. Methods A dopaminergic neuronal injury model was induced by 6-OHDA in PC12 cells . Autophagosomes in PC12 cells were examined by transmission electronmicro-scopy( TEM ). The expression of LC3- Ⅱ , Cathepsin B were assayed by western blot analysis. Results TEM revealed that the autophagosomes were increased in PC12 cells after 6-OHDA treatment and appeared apoptosis. The LC3-Ⅱ (2h:52.57 ±2.27,4h:56.83 ±3.51,6h:73.43 ±5.41,12h:103.90 ±2.57,24h: 100.40 ±3.91 )and Cathepsin B expression ( model group: 113.80 ± 4.46; normal group 35.89 ± 3.40) were increased after 6-OH DA treatments (P < 0.05 or P < 0.01 ). Conclusion The results indicate that autophagy lysosome pathway is involved in 6-OHDA-induced cell death in PC12 cells.%目的 研究自噬在帕金森病(PD)细胞模型中的作用及可能的机制.方法 体外培养的PC12细胞加入6-羟基多巴(6-OHDA)诱导多巴胺能神经元损伤模型.利用透射电镜观察PC12细胞中自噬的激活,免疫印迹法检测LC3-Ⅱ、Cathepsin B蛋白的表达.结果 电镜下观察到6-OHDA可使PC12细胞内自噬体增多,并出现了凋亡特征.6-OHDA作用2h(灰度比:52.57±2.27),4h(灰度比:56.83±3.51),6h(灰度比:73.43±5.41),12h(灰度比:103.90±2.57),24h(灰度比:100.40±3.91)时LC3-Ⅱ表达逐渐升高,与正常对照组(42.10±2.05)比较差异有统计学意义(P<0.05),模型组Cathepsin B(113.80±4.46)表达与正常对照组(35.89±3.40)比较明显增加(P<0.01),与模型组相比,广谱蛋白酶抑制剂UTI组(57.69±4.24)降低Cathepsin B表达(P<0.01).结论 自噬/溶酶体途径参与PC12细胞的死亡过程:6-OHDA诱导自噬过度激活,LC3-Ⅱ与Cathepsin B表达增加,促进细胞死亡.

  20. Endosomes and lysosomes are involved in early steps of Tl(III)-mediated apoptosis in rat pheochromocytoma (PC12) cells.

    Science.gov (United States)

    Hanzel, Cecilia E; Almeira Gubiani, María F; Verstraeten, Sandra V

    2012-11-01

    The mechanisms that mediate thallium (Tl) toxicity are still not completely understood. The exposure of rat pheochromocytoma (PC12) cells to Tl(I) or Tl(III) activates both mitochondrial (Tl(I) and Tl(III)) and extrinsic (Tl(III)) pathways of apoptosis. In this work we evaluated the hypothesis that the effects of Tl(III) may be mediated by the damage to lysosomes, where it might be incorporated following the route of iron uptake. PC12 cells exposed for 3 h to 100 μM Tl(III) presented marked endosomal acidification, effect that was absent when cells were incubated in a serum-free medium and that was fully recovered when the latter was supplemented with transferrin. After 6 h of incubation the colocalization of cathepsins D and B with the lysosomal marker Lamp-1 was decreased together with an increase in the total activity of the enzymes. A permanent damage to lysosomes after 18 h of exposure was evidenced from the impairment of acridine orange uptake. Cathepsin D caused the cleavage of pro-apoptotic protein BID that is involved in the activation of the intrinsic pathway of apoptosis. Supporting that, BID cleavage and the activation of caspase 3 by Tl(III) were fully prevented when cells were preincubated with cathepsin D inhibitor (pepstatin A) and only partially prevented when cathepsin B inhibitor (E64d) was used. None of these inhibitors affected BID cleavage or caspase 3 activation in Tl(I)-treated cells. Together, experimental results support the role of Tl(III) uptake by the acidic cell compartments and their involvement in the early steps of Tl(III)-mediated PC12 cells apoptosis.

  1. Reduction of trophic support enhances apoptosis in PC12 cells expressing Alzheimer's APP mutation and sensitizes cells to staurosporine-induced cell death.

    Science.gov (United States)

    Leutz, Steffen; Steiner, Barbara; Marques, Celio A; Haass, Christian; Müller, Walter E; Eckert, Anne

    2002-06-01

    Mutations in the amyloid precursor protein (APP) gene are known as causative factors in the pathogenesis of early-onset familial Alzheimer's disease (FAD). In this study, the influence of the Swedish double-mutation form of APP (APPsw; KM670/671NL) on apoptosis regulation in PC12 cells was investigated. APPsw-transfected PC12 cells were compared with wild-type APP (APPwt)-expressing and vector-transfected PC12 cells with regard to their susceptibility to cell death induced by the reduction of trophic support or by additional treatment with staurosporine. Expression of APPsw markedly enhanced the level of apoptotic PC12 cells induced by serum reduction. A similar hypersensitivity of APPsw-expressing PC12 cells could be detected after differentiation with nerve growth factor under serum-reduced conditions. Likewise, the expression of APPsw rendered PC12 cells more vulnerable to staurosporine but only under serum-reduced conditions. This APPsw-effect disappeared in high serum-containing medium. Thus, expression of APPsw seems to enhance cellular sensitivity not in general but after the reduction of trophic factors probably by causing oxidative stress. This, in turn, may sensitize cells to secondary apoptotic stimuli. Moreover, the mutation-specific increase in vulnerability to cell death was only seen at the stage of apoptotic nuclei, but not using methods measuring cell death by determining metabolic activity or membrane integrity. Therefore, the expression of APPsw seems to affect specifically apoptotic cell death rather than overall cell death in vitro. Our study further emphasizes the pathogenic role of mutant APP and may provide new insights in the mechanisms underlying the massive neurodegeneration in brain from patients bearing the APPsw mutation.

  2. 利福平对鱼藤酮诱导的分化PC12细胞线粒体损伤的保护作用%Rifampicin protects rotenone-induced mitochondrial damage in differentiated PC12 cells

    Institute of Scientific and Technical Information of China (English)

    陈世文; 孙元林; 曾志芬; 陶恩祥

    2009-01-01

    Objective To explore the effects ofrifampicin(RFP)on the cell viability.reactive oxygen species(ROS)formation,the change of mitochondrial transmembrance potential(△ψm)and cell apoptosis induced by rotenone(Rot)in differentiated PC12 cells.Methods Rot was added to make a model ofParkinson's disease in rat pheochromocytoma(PCI2)cells in the presence of RFP.Cell viability was determined by MTT assay.Change of △ψm and cell apoptosis were measured by fluorescence microscope and flow cytometry respectively.Results Compared with control group and 300 μmol/L RFP group,cell viability was significantly decreased but depolarization of △ψm,ROS formation and cell apoptosis rate were significantly increased in 2.5 μmol/L Rot group.Compared with 2.5 μmol/L Rot group,RFP(100,200 and 300 μmol/L)pretreated groups,cell viability was significantly increased,but depolarization of △ψm、ROS formation and cell apoptosis rate were significantly decreased in a dose-dependent manner.Conclusion RFP may protect the damage induced by Rot in differentiated PC12 by reducing depolarization of △ψm and ROS formation in a dose-dependent manner.%目的 探讨利福平(RFP)对鱼藤酮(Rot)诱导的分化PC12细胞活性、胞内活性氧(ROS)水平、线粒体膜电位(AWm)及凋亡的影响. 方法 利用Rot诱导的分化PC12细胞建立帕金森病(PD)细胞模型,应用不同浓度RFP(100、200、300 μmol/L)预先干预,分别采用MTT法检测PC12细胞活性、流式细胞仪检测胞内ROS生成量、荧光显微镜和流式细胞仪检测△ψm及凋亡的变化.结果 2.5 μmol/L Rot可使PC12细胞活性降低.ROS生成、△ψm去极化程度和细胞凋亡率增加;100、200、300 μmol/L RFP预处理对上述变化有抑制作用,且浓度越大,作用越明显. 结论 RFP可能通过稳定△ψm、降低细胞内ROS生成来对抗Rot对分化PC12细胞的损伤,且这种作用呈浓度依赖性.

  3. Protective effect of curcumin against Aβ25-35-induced neurotoxicity in differentiated PC12 cells%姜黄素对Aβ25-35致PC12细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    张英; 雍慧媛; 史小婷; 周俊; 马千; 连庆泉; 曹红; 李军

    2013-01-01

    目的 探讨姜黄素(Cur)对AD细胞活力及HMGB1表达的影响.方法 对数期生长的PC12细胞分为5组:空白组(A组)不做处理,Aβ 25-35模型组(B组)加入20μmol/L的Aβ25-35,Aβ25-35+Cur治疗组(C组)加入20 μmol/L的Aβ25-35和1μmol/L的Cur,Aβ25-35+ rHMG1损伤组(D组)加入20 μmol/L的Aβ25-35和500 ng/ml的HMGB1,Aβ25-35+溶剂对照组(E组)加入20 μmol/L的Aβ25-35和1μl/ml的DMSO,孵育24h后行细胞形态学观察、免疫荧光定性和免疫蛋白印迹(Western blot)法检测HMGB1蛋白的表达情况.结果 与A组相比,B、D、E组细胞活力明显下降(0.76±0.06、0.63±0.02、0.75±0.03比1.22 ±0.06,P<0.05)、胞内HMGB1表达明显升高(1.19±0.14、1.12±0.16、1.16±0.09比0.85±0.04,P<0.05);与B组相比,C组细胞活力上升33%、HMGB1表达下降31% (P <0.05).B组较A组存在大量的HMGB1核外释放,而C组HMGB1的核外释放较B组减少.结论 姜黄素可减轻Aβ25-35引起的PC12细胞毒性,其机制与下调HMGB1的表达、抑制HMGB1的核外释放有关.%Objective To explore the effects of curcumin on the expression of high mobility group box1 (HMGB1),cell viability and morphology in a cellular model of Alzheimer's disease (AD).Methods Cultured PC12 cells in logarithmic growth phase were divided into 5 groups:normal cell group (A,nontreatment),model control group (B,20 μmol/L Aβ25-35),curcumin treatment group (C,20 μmol/LAβ25-35 + 1 μmol/L Cur),Aβ25-35 + rHMG1 (D,20 μ mol/L Aβ25-35 + 500 ng/ml HMGB1) and solvent control group (E,20 μ mol/L Aβ25-35 + 1 μl/ml DMSO).Cell viability was examined by methyl thiazolyl tetrazolium (MTT).And the cellular expression and distribution of HMGB1 were detected by immunofluorescence and Western blot 24 hours later.Results Compared with group A,the levels of cell viability in groups B,D and E significantly declined (0.76 ± 0.06,0.63 ± 0.02,0.75 ± 0.03 vs 1.22 ±0.06,P<0.05) while the expression of HMGB1 increased (1.19 ±0.14,1.12 ±0

  4. Eriocaulon buergerianum extract protects PC12 cells and neurons in zebrafish against 6-hydroxydopamine-induced damage

    Directory of Open Access Journals (Sweden)

    Lin Zhixiu

    2011-04-01

    Full Text Available Abstract Background Ericaulon buergerianum (Gujingcao is an ophthalmic, anti-inflammatory and antimicrobial Chinese medicinal herb. This study aims to investigate the neuroprotective effects of Ericaulon buergerianum ethanol extract (EBE and to elucidate its underlying action mechanism. Methods The viability of dopaminergic (DA neuron in zebrafish was examined by anti-tyrosine hydroxylase (TH immunostaining. The locomotor activity of zebrafish was assessed with a digital video tracking system. The viability and cellular damage of the PC12 cells were determined by MTT and LDH assays respectively. The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33342 dye. Intracellular nitric oxide (NO was quantified by DAF-FM diacetate staining. The expression of inducible nitric oxide synthase (iNOS was determined by Western blot. Results EBE inhibited the 6-OHDA-induced decrease in total distance of movement in zebrafish. Pretreatments of EBE (25, 50, 100 and 200 μg/ml increased the viability of 6-OHDA-damaged PC12 cells in a dose dependent manner. Protection against 6-OHDA-induced nuclear fragmentation and accumulation of apoptotic bodies was also observed in EBE pretreated cells. Anti-oxidative (inhibition of NO production and iNOS expression in PC12 cells in vitro activities of EBE are related to its neuroprotective effects in 6-OHDA-induced DA neuron damage. Conclusion EBE exhibited significant neuroprotective activities in zebrafish, including recovery of dopaminergic neuron loss caused by 6-OHDA in a dose-dependent manner in vivo, inhibition of 6-OHDA-induced decrease of total distance in movement in zebrafish. The iNOS-NO pathway may be involved.

  5. Cajaninstilbene Acid Prevents Corticosterone-Induced Apoptosis in PC12 Cells by Inhibiting the Mitochondrial Apoptotic Pathway

    Directory of Open Access Journals (Sweden)

    Bao-Ping Jiang

    2014-09-01

    Full Text Available Background/Aims: Cajaninstilbene acid (3-hydroxy-4-prenyl-5-methoxystilben-2 -carboxylic acid, CSA, a natural stilbene isolated from the leaves of Cajanus cajan, has attracted considerable attention for its wide range of pharmacological activities. This study investigated whether CSA protects against corticosterone (CORT-induced injury in PC12 cells and examined the potential mechanisms underlying this protective effect. Methods: Cell viability and cytotoxicity were detected using a 3-(4,5-desethyithiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and a lactate dehydrogenase (LDH assay kit, respectively. PC12 cell apoptosis was measured using Hoechst 33342 staining and a DNA fragmentation assay kit, and intracellular Ca2+ concentrations were assessed by fluorescent labelling. Next, the mitochondrial permeability transition pores (mPTPs and mitochondrial membrane potentials (∆Ψm were detected using a colorimetric mPTP detection kit and a 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolyl-carbocyanine iodide (JC-1 kit, respectively. Finally, cytochrome c, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD expression levels were monitored by western blot analysis. Results: Treatment with 100 µmol/l CORT induced cytotoxicity in PC12 cells. However, CSA dose-dependently increased cell viability and decreased LDH release as well as CORT-induced apoptosis. Mechanistically, compared with the CORT-treated group, CSA strongly attenuated intracellular Ca2+ overload and restored mitochondrial functions, including mPTPs and ∆Ψm. Furthermore, the down-regulation of cytochrome c and ICAD protein expression and the blockage of caspase-3 activity were observed upon CSA treatment. Conclusions: In summary, our data are the first to show that the in vitro antidepressant-like effect of CSA may be attributed to the cytoprotection of neurons and that such neuroprotective mechanisms are correlated with intracellular Ca2+ homeostasis

  6. Toxic effect of zinc nanoscale metal-organic frameworks on rat pheochromocytoma (PC12) cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Fei, E-mail: paper_mail@126.com [Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Yang, Baochun; Cai, Jing [Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Jiang, Yaodong [Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Xu, Jun [Department of Health Economy Administration, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Wang, Shan [Department of Pharmacy, Winthrop University Hospital, Mineola, NY 11501 (United States)

    2014-04-01

    Highlights: • Metal-organic frameworks (MOFs) represent a newborn family of hybrid materials. • MOFs have already shown promise in a number of biological applications. • The biological applications of MOFs raise concerns for potential cytotoxicity. • Substantial information about MOF's neurotoxicity is still quite scarce. • This study reveals for the first time the interaction of MOFs with neural cells. - Abstract: Metal-organic frameworks (MOFs) possess unique properties desirable for delivery of drugs and gaseous therapeutics, but their uncharacterized interactions with cells raise increasing concerns of their safety in such biomedical applications. We evaluated the adverse effects of zinc nanoscale MOFs on the cell morphology, cytoskeleton, cell viability and expression of neurotrophin signaling pathway-associated GAP-43 protein in rat pheochromocytoma PC12 cells. At the concentration of 25 μg/ml, zinc MOFs did not significantly affect morphology, viability and membrane integrity of the cells. But at higher concentrations (over 100 μg/ml), MOFs exhibited a time- and concentration-dependent cytotoxicity, indicating their entry into the cells via endocytosis where they release Zn{sup 2+} into the cytosol to cause increased intracellular concentration of Zn{sup 2+}. We demonstrated that the toxicity of MOFs was associated with a disrupted cellular zinc homeostasis and down-regulation of GAP-43 protein, which might be the underlying mechanism for the improved differentiation in PC12 cells. These findings highlight the importance of cytotoxic evaluation of the MOFs before their biomedical application.

  7. Toxic effect of zinc nanoscale metal-organic frameworks on rat pheochromocytoma (PC12) cells in vitro.

    Science.gov (United States)

    Ren, Fei; Yang, Baochun; Cai, Jing; Jiang, Yaodong; Xu, Jun; Wang, Shan

    2014-04-30

    Metal-organic frameworks (MOFs) possess unique properties desirable for delivery of drugs and gaseous therapeutics, but their uncharacterized interactions with cells raise increasing concerns of their safety in such biomedical applications. We evaluated the adverse effects of zinc nanoscale MOFs on the cell morphology, cytoskeleton, cell viability and expression of neurotrophin signaling pathway-associated GAP-43 protein in rat pheochromocytoma PC12 cells. At the concentration of 25 μg/ml, zinc MOFs did not significantly affect morphology, viability and membrane integrity of the cells. But at higher concentrations (over 100 μg/ml), MOFs exhibited a time- and concentration-dependent cytotoxicity, indicating their entry into the cells via endocytosis where they release Zn(2+) into the cytosol to cause increased intracellular concentration of Zn(2+). We demonstrated that the toxicity of MOFs was associated with a disrupted cellular zinc homeostasis and down-regulation of GAP-43 protein, which might be the underlying mechanism for the improved differentiation in PC12 cells. These findings highlight the importance of cytotoxic evaluation of the MOFs before their biomedical application.

  8. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

    Institute of Scientific and Technical Information of China (English)

    Mingmin Yan; Shanping Mao; Huimin Dong; Baohui Liu; Qian Zhang; Gaofeng Pan; Zhiping Fu

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer's disease.The cells were then treated with 5, 10, and 25 μM Schisandrin B.Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased.Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased.Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change.These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner.This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer's disease.The cells were then treated with 5, 10, and 25 μM Schisandrin B.Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased.Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased.Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change.These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25

  9. Neuroprotective effects of Activin A on endoplasmic reticulum stress-mediated apoptotic and autophagic PC12 cell death

    Directory of Open Access Journals (Sweden)

    Long-xing Xue

    2017-01-01

    Full Text Available Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum (ER stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein (CHOP and cleaved-caspase-3] and biomarkers of autophagy (Beclin-1 and light chain 3, and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1 (IRE1, tumor necrosis factor receptor-associated factor 2 (TRAF2, apoptosis signal-regulating kinase 1 (ASK1, c-Jun N-terminal kinase (JNK and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.

  10. Inhibition of Voltage-Gated Calcium Channels After Subchronic and Repeated Exposure of PC12 Cells to Different Classes of Insecticides.

    Science.gov (United States)

    Meijer, Marieke; Brandsema, Joske A R; Nieuwenhuis, Desirée; Wijnolts, Fiona M J; Dingemans, Milou M L; Westerink, Remco H S

    2015-10-01

    We previously demonstrated that acute inhibition of voltage-gated calcium channels (VGCCs) is a common mode of action for (sub)micromolar concentrations of chemicals, including insecticides. However, because human exposure to chemicals is usually chronic and repeated, we investigated if selected insecticides from different chemical classes (organochlorines, organophosphates, pyrethroids, carbamates, and neonicotinoids) also disturb calcium homeostasis after subchronic (24 h) exposure and after a subsequent (repeated) acute exposure. Effects on calcium homeostasis were investigated with single-cell fluorescence (Fura-2) imaging of PC12 cells. Cells were depolarized with high-K(+) saline to study effects of subchronic or repeated exposure on VGCC-mediated Ca(2+) influx. The results demonstrate that except for carbaryl and imidacloprid, all selected insecticides inhibited depolarization (K(+))-evoked Ca(2+) influx after subchronic exposure (IC50's: approximately 1-10 µM) in PC12 cells. These inhibitory effects were not or only slowly reversible. Moreover, repeated exposure augmented the inhibition of the K(+)-evoked increase in intracellular calcium concentration induced by subchronic exposure to cypermethrin, chlorpyrifos, chlorpyrifos-oxon, and endosulfan (IC50's: approximately 0.1-4 µM). In rat primary cortical cultures, acute and repeated chlorpyrifos exposure also augmented inhibition of VGCCs compared with subchronic exposure. In conclusion, compared with subchronic exposure, repeated exposure increases the potency of insecticides to inhibit VGCCs. However, the potency of insecticides to inhibit VGCCs upon repeated exposure was comparable with the inhibition previously observed following acute exposure, with the exception of chlorpyrifos. The data suggest that an acute exposure paradigm is sufficient for screening chemicals for effects on VGCCs and that PC12 cells are a sensitive model for detection of effects on VGCCs. © The Author 2015. Published by Oxford

  11. Expression changes of dopaminergic system-related genes in PC12 cells induced by manganese, silver, or copper nanoparticles.

    Science.gov (United States)

    Wang, Jianyong; Rahman, Mohammed F; Duhart, Helen M; Newport, Glenn D; Patterson, Tucker A; Murdock, Richard C; Hussain, Saber M; Schlager, John J; Ali, Syed F

    2009-11-01

    Nanoparticles have received a great deal of attention for producing new engineering applications due to their novel physicochemical characteristics. However, the broad application of nanomaterials has also produced concern for nanoparticle toxicity due to increased exposure from large-scale industry production. This study was conducted to investigate the potential neurotoxicity of manganese (Mn), silver (Ag), and copper (Cu) nanoparticles using the dopaminergic neuronal cell line, PC12. Selective genes associated with the dopaminergic system were investigated for expression changes and their correlation with dopamine depletion. PC12 cells were treated with 10 microg/ml Mn-40 nm, Ag-15 nm, or Cu-90 nm nanoparticles for 24 h. Cu-90 nanoparticles induced dopamine depletion in PC12 cells, which is similar to the effect induced by Mn-40 shown in a previous study. The expression of 11 genes associated with the dopaminergic system was examined using real-time RT-PCR. The expression of Txnrd1 was up-regulated after the Cu-90 treatment and the expression of Gpx1 was down-regulated after Ag-15 or Cu-90 treatment. These alterations are consistent with the oxidative stress induced by metal nanoparticles. Mn-40 induced a down-regulation of the expression of Th; Cu-90 induced an up-regulation of the expression of Maoa. This indicates that besides the oxidation mechanism, enzymatic alterations may also play important roles in the induced dopamine depletion. Mn-40 also induced a down-regulation of the expression of Park2; while the expression of Snca was up-regulated after Mn-40 or Cu-90 treatment. These data suggest that Mn and Cu nanoparticles-induced dopaminergic neurotoxicity may share some common mechanisms associated with neurodegeneration.

  12. Lack of effects of typical and atypical antipsychotics in DARPP-32 and NCS-1 levels in PC12 cells overexpressing NCS-1

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    Reis Helton J

    2010-06-01

    Full Text Available Abstract Background Schizophrenia is the major psychiatry disorder, which the exact cause remains unknown. However, it is well known that dopamine-mediated neurotransmission imbalance is associated with this pathology and the main target of antipsychotics is the dopamine receptor D2. Recently, it was described alteration in levels of two dopamine signaling related proteins in schizophrenic prefrontal cortex (PFC: Neuronal Calcium Sensor-1 (NCS-1 and DARPP-32. NCS-1, which is upregulated in PFC of schizophrenics, inhibits D2 internalization. DARPP-32, which is decreased in PFC of schizophrenics, is a key downstream effector in transducing dopamine signaling. We previously demonstrated that antipsychotics do not change levels of both proteins in rat's brain. However, since NCS-1 and DARPP-32 levels are not altered in wild type rats, we treated wild type PC12 cells (PC12 WT and PC12 cells stably overexpressing NCS-1 (PC12 Clone with antipsychotics to investigate if NCS-1 upregulation modulates DARPP-32 expression in response to antipsychotics treatment. Results We chronically treated both PC12 WT and PC12 Clone cells with typical (Haloperidol or atypical (Clozapine and Risperidone antipsychotics for 14 days. Using western blot technique we observed that there is no change in NCS-1 and DARPP-32 protein levels in both PC12 WT and PC12 Clone cells after typical and atypical antipsychotic treatments. Conclusions Because we observed no alteration in NCS-1 and DARPP-32 levels in both PC12 WT and Clone cells treated with typical or atypical antipsychotics, we suggest that the alteration in levels of both proteins in schizophrenic's PFC is related to psychopathology but not with antipsychotic treatment.

  13. The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells

    OpenAIRE

    1986-01-01

    Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differentia...

  14. Protective Effect of Tetramethylpyrazine on Caffeine-induced PC12 Cell Injury%川芎嗪对咖啡因引起的PC12细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    王佳; 高峰; 张春兵

    2014-01-01

    目的:分析川芎嗪对咖啡因引起大鼠肾上腺嗜铬细胞瘤克隆化细胞株PC12细胞损伤的保护作用,探讨川芎嗪治疗脑缺血-再灌注损伤的机制。方法制备咖啡因细胞损伤模型,通过CCK-8法活细胞检测、流式细胞术线粒体膜电位测定、Western-blot检测高迁移率族蛋白B1(HMGB1)、酶联免疫吸附测定(ELISA)检测氧化应激指标观察咖啡因的毒性及川芎嗪的保护作用。结果川芎嗪预处理后,PC12细胞的存活数显著提高,细胞线粒体膜电位提高,HMGB1表达显著降低,超氧化物歧化酶( SOD)上调,乳酸脱氢酶( LDH)和丙二醛( MDA)下调,谷胱甘肽( GSH)升高。结论川芎嗪对咖啡因引起的PC12细胞损伤有显著的保护作用,其保护作用可能与川芎嗪抑制细胞凋亡、调节炎症性递质表达水平及氧化应激反应相关。%Objective To analyze whether tetramethylpyrazine could protect PC12 cells from injuries induced by caffeine,and to explore the mechanism of tetramethyipyrazine in the treatment of cerebral ischemia-reperfusion injury. Methods Caffeine was added to induce apoptosis of PC12 cells. Cytotoxicity was detected by CCK-8 assay. The electric potential of mitochondrial membrane was determined by flow cytometry. HMGB1 was detected by Western blotting. Oxidative stress was detected by ELISA. We observed toxicity of caffeine and the protective effects of tetramethylpyrazine. Results After the pre-treatment,tetramethylpyrazine significantly improved PC12 cell survival. Mitochondrial membrane potential was increased,the expression of HMGB1 decreased,SOD increased,LDH and MDA decreased,and GSH elevated. Conclusion Tetramethylpyrazine exerts a significant protective effect on PC12 cell injury caused by caffeine. The protective effect may be related to inhibition of apoptosis and regulation of the expression level of mediators involved in inflammation and oxidative stress.

  15. Lycium chinensis Mill attenuates glutamate induced oxidative toxicity in PC12 cells by increasing antioxidant defense enzymes and down regulating ROS and Ca(2+) generation.

    Science.gov (United States)

    Olatunji, Opeyemi J; Chen, Hongxia; Zhou, Yifeng

    2016-03-11

    Lycium chinensis Mill is a famous traditional Chinese medicine which displays several medicinal activities including antioxidant and neuroprotective activities. However, the mechanism of action towards the neuroprotective action has not been fully elucidated. This work was aimed at investigating the neuroprotective effects of L. chinensis Mill against glutamate-induced oxidative neurotoxicity in PC12 cells. Oxidative cell death was induced with 5mM glutamate in PC12 cells. Cell viability, LDH release, intracellular Ca(2+) concentration, reactive oxygen species (ROS) accumulation, GSH-Px, CAT and SOD antioxidant enzyme levels were measured. Our results indicated that pretreatment of PC12 cells with L. chinensis Mill extracts markedly attenuated the loss of cell viability, the release of lactate dehydrogenase (LDH), Ca(2+) overload, ROS generation, and cell apoptosis induced by glutamate toxicity. Furthermore, L. chinensis Mill extracts also significantly increased the levels of innate antioxidant enzymes GSH-Px, SOD and CAT in glutamate-induced PC12 cells. Conclusively, our results provided substantial evidence that L. chinensis Mill protected PC12 cells against glutamate-induced cell death by attenuating ROS generation, Ca(2+) influx, and increased the antioxidant defense capacity of PC12 cells against oxidative stress damages, suggesting the possible potential of extracts from the plant as sources of bioactive molecules in the treatment of neurodegenerative disorders.

  16. PC12 polarity on biopolymer nanogratings

    Energy Technology Data Exchange (ETDEWEB)

    Cecchini, M; Ferrari, A; Beltram, F [Scuola Normale Superiore and NEST-CNR-INFM, Pisa (Italy); Scuola Normale Superiore and Italian Institute of Technology, Pisa (Italy)], E-mail: m.cecchini@sns.it

    2008-03-15

    Cell differentiation properties are strongly entangled with the morphology and physical properties of the extracellular environment. A complete understanding of this interaction needs artificial scaffolds with controlled nano-/micro-topography. We induced specific topographies by nanoimprint lithography (NIL) on tissue culture polystyrene (TCPS) dishes substrates and, using light microscopy and high-magnification scanning-electron-microscopy, quantitatively compared the changes in PC12 differentiation phenotype induced by the periodicity of the nanopatterns. This analysis revealed that nanogratings reduce the number of neurites produced by PC12 cells upon treatment with NGF and that neuronal bipolarity correlated with an increased stretching of the cell body and a reduced length of the cell neuronal protrusions.

  17. Hybrid tetanus toxin C fragment-diphtheria toxin translocation domain allows specific gene transfer into PC12 cells.

    Science.gov (United States)

    Barati, Shahram; Chegini, Fariba; Hurtado, Plinio; Rush, Robert A

    2002-09-01

    To study the mechanism by which genes can efficiently be transferred into specific cell types, we have constructed several novel, single-chain multicomponent proteins by recombining the nontoxic C fragment of tetanus toxin and the translocation domain of diphtheria toxin together with the DNA-binding fragment of GAL4 transcription factor, for transportation of plasmid DNA into neuronal cells. The C fragment of tetanus toxin provided neuronal selectivity, the translocation domain of diphtheria toxin permitted endosomal escape, and the GAL4 domain provided binding to DNA. To assess the cellular tasks of each component in gene transfer, different combinations of these fragments were produced by polymerase chain reaction, expressed in Escherichia coli, and purified under native conditions from the soluble proteins. We show that only fusion proteins bearing the C fragment of tetanus toxin bind to gangliosides and, followed by their specific binding to differentiated PC12 cells, are internalized within 10 min. These proteins delivered the green fluorescence protein gene to PC12 cells, with the highest transfection efficiency achieved with proteins containing both the C fragment and the translocation domain. Addition of chloroquine elevated the transfection efficiency, which was further increased by incorporation of a nuclear localization signal in the delivery system. In addition, the effect of different DNA-condensing materials (poly-L-lysine, protamine, lysine(n=8)-trytophan(n=2)-lysine(n=8)) on gene transfer was investigated.

  18. Tetrahydroxystilbene glucoside attenuates MPP+-induced apoptosis in PC12 cells by inhibiting ROS generation and modulating JNK activation.

    Science.gov (United States)

    Li, Xiaobing; Li, Yan; Chen, Jianzong; Sun, Jing; Li, Xiaofeng; Sun, Xin; Kang, Xiaogang

    2010-10-08

    It is known that oxidative stress plays a major role in the progression of Parkinson's disease (PD). Previous studies have suggested that 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside (TSG), an active component extracted from a traditional Chinese herb Polygonum multiflorum Thunb., has significant antioxidant and free radical-scavenging activities. This is the first study that investigated the protective effects of TSG against MPP(+)-induced apoptosis in PC12 cells and determined the underlying mechanism. The results showed that incubation of PC12 cells with TSG before exposing them to MPP(+) could significantly decrease cell viability loss and reverse cell apoptosis in a dose-dependent manner. The anti-apoptotic effects of TSG were probably mediated via the inhibition of ROS generation and modulation of JNK activation because TSG blocked ROS increase and JNK phosphorylation induced by MPP(+). Taken together, these results indicated that TSG may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases such as PD.

  19. Protective Effect of Diospyros kaki against Glucose-Oxygen-Serum Deprivation-Induced PC12 Cells Injury

    Directory of Open Access Journals (Sweden)

    Fatemeh Forouzanfar

    2016-01-01

    Full Text Available Ischemic cerebrovascular disease is one of the most common causes of death in the world. Recent interests have been focused on natural antioxidants and anti-inflammatory agents as potentially useful neuroprotective agents. Diospyros kaki (persimmon has been shown to exert anti-inflammatory, antioxidant, and antineoplastic effects. However, its effects on ischemic damage have not been evaluated. Here, we used an in vitro model of cerebral ischemia and studied the effects of hydroalcoholic extract of peel (PeHE and fruit pulp (PuHE of persimmon on cell viability and markers of oxidative damage mainly intracellular reactive oxygen species (ROS induced by glucose-oxygen-serum deprivation (GOSD in PC12 cells. GOSD for 6 h produced significant cell death which was accompanied by increased levels of ROS. Pretreatment with different concentrations of PeHE and PuHE (0–500 μg/mL for 2 and 24 h markedly restored these changes only at high concentrations. However, no significant differences were seen in the protection against ischemic insult between different extracts and the time of exposure. The experimental results suggest that persimmon protects the PC12 cells from GOSD-induced injury via antioxidant mechanisms. Our findings might raise the possibility of potential therapeutic application of persimmon for managing cerebral ischemic and other neurodegenerative disorders.

  20. MECHANISMS OF MANGANESE-INDUCED RAT PHEOCHROMOCYTOMA (PC12) CELL DEATH AND CELL DIFFERENTIATION. (R826248)

    Science.gov (United States)

    Mn is a neurotoxin that leads to a syndrome resembling Parkinson's disease after prolonged exposure to high concentrations. Our laboratory has been investigating the mechanism by which Mn induces neuronal cell death. To accomplish this, we have utilized rat pheochromocytom...

  1. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+homeostasis

    Institute of Scientific and Technical Information of China (English)

    Yuan Liu; Xue-chun Wang; Dan Hu; Shu-ran Huang; Qing-shu Li; Zhi Li; Yan Qu

    2016-01-01

    Heat shock protein 70 (HSP70) maintains Ca2+homeostasis in PC12 cells, which may protect against apoptosis;however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overex-pression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R), thereby leading to decreased intracellular Ca2+concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

  2. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2016-01-01

    Full Text Available Heat shock protein 70 (HSP70 maintains Ca2+ homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+ concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R, thereby leading to decreased intracellular Ca2+ concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+ homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

  3. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35.

    Science.gov (United States)

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-03-25

    PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.

  4. ETAS, an enzyme-treated asparagus extract, attenuates amyloid beta-induced cellular disorder in PC12 cells.

    Science.gov (United States)

    Ogasawara, Junetsu; Ito, Tomohiro; Wakame, Koji; Kitadate, Kentaro; Sakurai, Takuya; Sato, Shogo; Ishibashi, Yoshinaga; Izawa, Tetsuya; Takahashi, Kazuto; Ishida, Hitoshi; Takabatake, Ichiro; Kizaki, Takako; Ohno, Hideki

    2014-04-01

    One of the pathological characterizations of Alzheimer's disease (AD) is the deposition of amyloid beta peptide (Abeta) in cerebral cortical cells. The deposition of Abeta in neuronal cells leads to an increase in the production of free radicals that are typified by reactive oxygen species (ROS), thereby inducing cell death. A growing body of evidence now suggests that several plant-derived food ingredients are capable of scavenging ROS in mammalian cells. The purpose of the present study was to investigate whether enzyme-treated asparagus extract (ETAS), which is rich in antioxidants, is one of these ingredients. The pre-incubation of differentiated PC 12 cells with ETAS significantly recovered Abeta-induced reduction of cell viability, which was accompanied by reduced levels of ROS. These results suggest that ETAS may be one of the functional food ingredients with anti-oxidative capacity to help prevent AD.

  5. 阿米洛利通过自噬-溶酶体途径保护PC12细胞%Amiloride protects PC12 cells from MPP + induced injury via autophagy-lysosome pathway

    Institute of Scientific and Technical Information of China (English)

    张润娉; 李建平

    2014-01-01

    Objective:To evaluate the neuroprotective effects of amiloride; a non-selective blockers of acid-sensing ion channels,on PC12 cells and to view the influence of autophagy-lysosome pathway (ALP).Methods:PC12 cells were tested with methy-phenylpyridi(MPP+),while giving amiloride as interventiong:The cells viability was analyzed by MTT assay; The cells injury was assessed by lactate dehydrogenase (LDH) assay;Flow cytometry was used to study the apoptotic;Western blot analysis was used to study the autophagic mechanisms.Results:MPP + medium treatment resulted in significantly lower survival rate,the supernatant of LDH leakage was significantly higher,the apoptosis rate increased,the expression of LC3-Ⅱ was inhibited while induced more expression of LAMP2a ; but amiloride effectively improved the cell survival rate,reduced LDH leakage rate,inhibited apoptosis,and upregulated LC3-Ⅱ protein,which is associated decrease expression of LAMP2a.Conclusion:Amiloride can protect PC12 cells against MPP +-induced cell death by autophagic,through inhibition of acid-sensing ion channels activity.%目的:研究酸敏感离子通道阻断剂阿米洛利对大鼠肾上腺嗜铬细胞瘤PC12细胞株的保护作用及其对自噬-溶酶体通路的影响.方法:在PC12细胞模型上,观察甲基苯基吡啶(methy-phenylpyridi,MPP+)处理对细胞存活率(MTT检测)、乳酸脱氢酶(LDH)漏出率、细胞凋亡率的影响,并观察阿米洛利对MPP+所致细胞死亡的影响;蛋白质印迹法检测大自噬通路标志蛋白LC3和分子伴侣介导的自噬通路标志蛋白LAMP2a表达水平的变化.结果:MPP+导致细胞存活率明显降低,上清液中LDH漏出率明显升高,细胞凋亡率升高,抑制大自噬通路标志蛋白LC3-Ⅱ的表达,同时上调了分子伴侣介导的自噬通路标志性蛋白LAMP2a的表达水平;而阿米洛利可提高细胞存活率,减少上清液中LDH漏出率,降低细胞凋亡率,并能在激活大自噬通路的同时下调分

  6. Microwave irradiation induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Inoue, Shigeki; Motoda, Hirotoshi; Koike, Yoshihisa; Kawamura, Kenji; Hiragami, Fukumi; Kano, Yoshio

    2008-02-13

    The increasing use of mobile phone communication has raised concerns about possible health hazard effects of microwave irradiation. We investigated damage and differentiation caused by microwave irradiation on drug-hypersensitive PC12 cell line (PC12m3). These cells showed enhancement of neurite outgrowth to various stimulants. The frequency of neurite outgrowth induced by 2.45 GHz (200 W) of microwave irradiation was approximately 10-fold greater than that of non-irradiated control cells. Incubation of PC12m3 cells with SB203580, a specific inhibitor of p38 MAPK, resulted in marked inhibition of the microwave radiation-induced neurite outgrowth. Also, activation of the transcription factor CREB induced by microwave irradiation was inhibited by SB203580. Heat shock treatment at 45 degrees C had a strong toxic effect on PC12m3 cells, whereas microwave treatment had no toxic effect on PC12m3 cells. These findings indicate that p38 MAPK is responsible for the survival of PC12m3 cells and might induce neurite outgrowth via a CREB signaling pathway when subjected to microwave irradiation.

  7. Effects of selenium and topiramate on cytosolic Ca(2+) influx and oxidative stress in neuronal PC12 cells.

    Science.gov (United States)

    Demirci, Seden; Kutluhan, Süleyman; Naziroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Yürekli, Vedat Ali; Demirci, Kadir

    2013-01-01

    It has been widely suggested that selenium (Se) deficiency play an important role in the pathophysiology of epilepsy. It has been reported that Se provides protection against the neuronal damage in patients and animals with epilepsy by restoring the antioxidant defense mechanism. The neuroprotective effects of topiramate (TPM) have been reported in several studies but the putative mechanism of action remains elusive. We investigated effects of Se and TPM in neuronal PC12 cell by evaluating Ca(2+) mobilization, lipid peroxidation and antioxidant levels. PC12 cells were divided into eight groups namely control, TPM, Se, H(2)O(2), TPM + H(2)O(2), Se + H(2)O(2), Se + TPM and Se + TPM + H(2)O(2). The toxic doses and times of H(2)O(2), TPM and Se were determined by cell viability assay which is used to evaluate cell viability. Cells were incubated with 0.01 mM TPM for 5 h and 500 nM Se for 10 h. Then, the cells were exposed to 0.1 mM H(2)O(2) for 10 h before analysis. The cells in all groups except control, TPM and Se were exposed to H(2)O(2) for 15 min before analysis. Cytosolic Ca(2+) release and lipid peroxidation levels were higher in H(2)O(2) group than in control, Se and TPM combination groups although their levels were decreased by incubation of Se and TPM combination. However, there is no difference on Ca(2+) release in TPM group. Glutathione peroxidase activity, reduced glutathione and vitamin C levels in the cells were lower in H(2)O(2) group than in control, Se and TPM groups although their values were higher in the cells incubated with Se and TPM groups than in H(2)O(2) groups. In conclusion, these results indicate that Se induced protective effects on oxidative stress in PC12 cells by modulating cytosolic Ca(2+) influx and antioxidant levels. TPM modulated also lipid peroxidation and glutathione and vitamin C concentrations in the cell system.

  8. Protective effect of glutathione proxidase 1 overexpression on amyloid β protein-mediated injury of PC12 cells%谷胱甘肽过氧化物酶1高表达对β淀粉样蛋白介导PC12细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    马琳; 王辉; 王淑荣; 陈志斌; 张海英

    2012-01-01

    Objective To study the molecular mechanism underlying amyloid β(Aβ) protein-mediated Aizheimer's disease and the protective effect of glutathione proxidase 1(GPX1) overexpres-sion on Aβ protein-mediated injury of PC12 cells. Methods PC12 cells, transfected into plticx plasmids and blank vector plasmids containing human GPX1 gene,were divided into PC12 group, GPX1-PC12 group and plncx-PC12 group,and intervened with Aβ25-35 and sodium selenite+Aβ25-35, respectively. Their survival rate was measured by MTT assay. Expression of pCREB was detected by immunohistochemistry. Results No significant difference was found in the survival rate of PC12 ceils between PC1 2 and pincx-PC12 groups(P>0. 05). The survival rate of PC12 cells was significantly higher in GPX1-PC12 group than in plncx-PC12 group(P<0. 01). The positive rate of pCREB protein was significantly lower in plncx-PC12 and GPX1-PC12 groups, especially in plncx-PC12 group than in GPX1-PC12 group after intervention with Aβ25-35 (P<0. 05) and significantly higher in plncx-PC12 and GPX1-PC12 grous,especially in GPX1-PC12 group than in plncx-PC12 group after intervention with sodium selenite+ Aβ25-35 (P<0, 01). Conclusion GPX1 gene overexpression can effectively protect PC12 cells against Aβ25-35-mediated injury. Aβ25-35 can down-regulate the expression of pCREB in PC12 cells. Both GPX1 and pCREB participate in protection of PC12 cells,thus reducing Aβ25-35-induced injury of PC12 cells.%目的 探讨β淀粉样蛋白(Aβ)所致阿尔茨海默病发病的分子机制,以及谷胱甘肽过氧化物酶1(GPX1)高表达时对其所致细胞损伤的保护作用.方法 将PC12细胞转染含人GPX1基因的plncx质粒及空载体plncx质粒,对PC12、GPX1-PC12、plncx-PC123组细胞,分别给予Aβ25-35和亚硒酸钠+Aβ25-35 2种干预方式,MTT法检测细胞的存活情况,免疫细胞化学法观察磷酸化环磷酸腺苷反应元件结合蛋白(pCREB)的表达情况.结果 PC12组与plncX-PC12组细胞存

  9. Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway.

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    Rongcai Yue

    Full Text Available Astragaloside IV (AGS-IV is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43 were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations.

  10. Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

    Science.gov (United States)

    Makuch, Edyta; Kuropatwa, Marianna; Kurowska, Ewa; Ciekot, Jaroslaw; Klopotowska, Dagmara; Matuszyk, Janusz

    2014-07-05

    Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

  11. The urokinase plasminogen activator receptor (UPAR) is preferentially induced by nerve growth factor in PC12 pheochromocytoma cells and is required for NGF-driven differentiation.

    Science.gov (United States)

    Farias-Eisner, R; Vician, L; Silver, A; Reddy, S; Rabbani, S A; Herschman, H R

    2000-01-01

    Nerve growth factor (NGF)-driven differentiation of PC12 pheochromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A "second generation" representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation.

  12. Inhibitory effect of Polygalasaponin F on the apoptosis of PC12 cells induced by sodium dithionite-caused oxygen-glucose deprivation and reperfusion%瓜子金皂苷己对连二亚硫酸钠致氧糖剥夺/复供诱导PC12细胞凋亡的抑制作用

    Institute of Scientific and Technical Information of China (English)

    石瑞丽; 胡金凤; 孔令雷; 牛非; 吴苗苗; 何鑫; 李培锋; 陈乃宏

    2013-01-01

    目的 观察瓜子金皂苷己(polygalasaponin F,PGSF)对氧糖剥夺/复供(oxygen-glucose deprivation and reperfusion,OGD/R) 所诱导的PC12细胞凋亡的作用及其机制.方法 以连二亚硫酸钠合并无糖Earle's液造成氧糖剥夺,继而恢复为完全培养基以建立体外OGD/R模型,以Hoechst33342 / PI双染及流式细胞术观察细胞受损和凋亡情况;以JC-1荧光染色法测定细胞线粒体膜电位(mitochondrial membrane potential,MMP);以Western blot法检测凋亡相关蛋白的表达.结果 PGSF可明显改善细胞形态并降低细胞受损和凋亡百分率,抑制MMP水平的降低,增加Bcl-2/Bax表达比值.结论 PGSF对OGD/R诱导的PC12细胞凋亡具有明显抑制作用,机制与其调节Bcl-2和Bax的表达及稳定MMP有关.%Aim To study the effect of Polygalasapo-nin F ( PGSF ) on apoptosis of PC 12 cells induced by oxygen-glucose deprivation and reperfusion ( OGD/R ) and its mechanism. Methods The OGD/R model was established by first exposing PC 12 cells to sodium di-thionite ( Na2S2O4 ) in glucose-free Earle's solution and then by changing the culture into complete mediums, with the cell injury and apoptosis analyzed by Hoechst 33342 / PI dual staining and flow cytometry assay. The mitochondrial membrane potential ( MMP ) was determined by JC-1 staining, and the expression of Bcl-2 and Bax was detected by Western blot. Results PGSF could significantly improve the morphology of injured PC 12 cells and reduce the percentage of apop-totic cells, reverse the decrease in MMP and up-regu-late the ratio of Bcl-2/Bax proteins. Conclusion PGSF can markedly inhibit the OGD/R-induced apoptosis of PC 12 cells by regulating the expression of Bcl-2 and Bax proteins and maintaining the normal MMP.

  13. Analysis of the role of nerve growth factor in promoting cell survival during endoplasmic reticulum stress in PC12 cells.

    Science.gov (United States)

    Shimoke, Koji; Sasaya, Harue; Ikeuchi, Toshihiko

    2011-01-01

    Nerve growth factor (NGF) was first described by Rita Levi-Montalcini in the early 1960s from her studies of peripheral neurons. It has since been reported that NGF has the potential to elongate neurites or to prevent apoptosis via specific intracellular mechanisms. It has further been reported that as a component of these mechanisms, NGF binds to a specific receptor, TrkA, and thereby contributes to peripheral nerve cell functions or neuronal functions. It is noteworthy in this regard that pheochromocytoma 12 (PC12) cells express TrkA and respond to neurite outgrowth or anti-apoptotic signals by binding to NGF. Hence, PC12 cells have been used as an in vitro model system for the study of neuronal functions. It has been reported that endoplasmic reticulum (ER) stress is involved in neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease. The common link with regard to ER stress is that the neuronal cells die in these pathologies via specific intracellular mechanisms. This type of cell death, if it is apoptotic in nature, is termed ER stress-mediated apoptosis. In the process of ER stress-mediated apoptosis, the cleavage of pro-caspase-12 residing on the ER and the expression of glucose-regulated protein 78 (GRP78) can be observed. The expression of GRP78 protein is a characteristic of an unfolded protein response (UPR) via specific signal transduction pathways mediated by the unfolded protein response element (UPRE) in the upstream region of the grp78 gene so on. In ER stress-mediated apoptosis, a caspase cascade is also observed. To further clarify the mechanisms underlying ER stress-mediated apoptosis, a better understanding of the UPR is therefore important. In our current study, we describe a method for detecting gene induction via the UPR, focusing on GRP78 and caspase activities as the measurement end-points. The information generated by our method will accelerate our understanding of the pathophysiological processes leading

  14. Preparation, characterization of nanoceria and its protection on oxidative damage of PC12 cells%CeO2的制备、表征及对 PC12细胞氧化损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    刘纯青; 申林林; 栾艺; 刘娜

    2015-01-01

    Objective To prepare nanoceria ( CeO2 ) and to investigate the protective effect on oxidative damage of Pheo-chromocytoma cell (PC12).Methods Homogenous precipitation method was used to prepare the nanoceria.XRD and TEM were used to characterize and determine the size of CeO2 .250 mmol/L rough concentration of CeO2 was added into PC12 cell culture fluid.Cells were induced and damaged by 20 μmol/L H2 O2 .And oxidative stress damage model of PC12 cell was set up.The cell survival rate of control group, CeO2 +H2 O group and H2 O2 group was measured by MTT method, re-spectively.The antioxidative protection of CeO2 to the PC12 damaged by H2 O2 was evaluated.Results The size of nanocer-ia was 5-10 nm.According to the results of MTT method, it was shown that the cell survival rate of H2 O2 group was (74.61 ±3.97)%.And the cell survival rate of CeO2 +H2 O group was (99.21 ±4.01)%, which was significantly high-er than H2O2 group (P<0.05).Conclusion Nanoceria would have the protective effect on the PC12 which was oxidative damaged by H2 O2 .%目的:制备纳米氧化铈( nanoceria,CeO2),初步探讨其对肾上腺嗜铬细胞瘤细胞PC12氧化损伤的保护作用。方法采用均相沉淀法制备纳米CeO2,对其晶粒大小和晶粒形貌进行表征。将粗浓度为250 mmol/L的CeO2加入PC12细胞培养液,通过20μmol/L H2 O2诱导细胞损伤,建立PC12细胞的氧化应激损伤模型,采用MTT法检测对照组、CeO2组、CeO2+H2 O2组,H2 O2组的细胞活性,测定CeO2对PC12细胞的保护作用。结果采用均相沉淀的方法合成粒子直径为5~10 nm的CeO2。 MTT实验显示H2 O2组细胞存活率为(74.61±3.97)%,CeO2+H2O2组的细胞存活率为(99.21±4.01)%,明显高于H2O2组(P<0.05)。结论 CeO2对H2O2造成的氧化损伤细胞具有保护作用。

  15. Estrogen receptor ERα36 gene silencing impacts the expression of growth-associated protein in PC12 cells%雌激素受体ERα36基因沉默对PC12细胞生长相关蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    嵇志红; 邹萍; 邹伟

    2014-01-01

    Objective To investigate the effects of ERα36 (a novel subtype of estrogen receptor alpha) on growth and proliferation in PC12 cells via examining the expression of growth-associated protein in differentiated PC12 cells after ERα36 gene silencing.Methods Transfection of ERα36-shRNA plasmid into PC12 cells was performed to establish the ERoα36 gene silencing cells model (PC12-36L1,PC12-36L2).Immunocytofluorescence was used to examine the expression of ERα36,and Western blot was used to analyze the expression of PCNA,cyclinD1 and MAPK in the PC12 cells.Results ① ERα36 was expressed in both cell types(PC12-36C1,PC12-36L1 and PC12-36L2).Compared with PC12-36C1,PC12-36L2 cells(OD value were respectively 0.95±0.05,0.78±0.10),PC12-36L1 cells significantly decreased expression of ERα36(OD value 0.47±0.12,P<0.01).② Compared with PC12 and PC12-36C1 cells,PC12-36L1 cells were significantly higher expression of PCNA,CyclinD1 and p-MAPK(P<0.01)(OD value of PCNA,CyclinD1 and p-MAPK:PC12 cells were respectively 1.00±0.05,1.00± ±0.11,1.00±0.05,PC12-36C1 cells were respectively 1.09±0.15,0.92±0.23,1.12± 0.08,PC12-36L1 cells were respectively 1.74±0.12,2.20±0.25,1.77±0.06).Conclusion ERα36 gene silencing can promote the growth and proliferation in PC12 cells.It suggests that the lower expression of ERα36 may be related to the diseases in nervous system such as brain tumor.%目的 通过检测新型雌激素受体(ERα36)基因沉默后PC12高分化细胞中生长相关蛋白的表达,探讨ERα36对PC12细胞生长增殖的影响.方法 利用ERα36-shRNA质粒转染细胞建立ERα36基因沉默细胞模型(PC12-36L1,PC12-36L2),免疫细胞荧光法检测ERα36的表达,Western blot检测PC12细胞中增殖细胞核抗原(PCNA)、细胞周期蛋白D1(CyclinD1)和丝裂原激活的蛋白激酶(MAPK)等生长相关蛋白的表达变化.结果 ①三个细胞株PC12-36C1(转染空质粒)、PC 12-36L1和PC12-36L2中均有ERα36表达;与PC12-36C1、PC12

  16. Evidence for the Interaction of Endophilin A3 with Endogenous Kca2.3 Channels in PC12 Cells

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    Malika Janbein

    2014-07-01

    Full Text Available Background/Aims: Small-conductance calcium-activated (SK channels play an important role by controlling the after-hyperpolarization of excitable cells. The level of expression and density of these channels is an essential factor for controlling different cellular functions. Several studies showed a co-localization of KCa2.3 channels and Endophilin A3 in different tissues. Endophilin A3 belongs to a family of BAR- and SH3 domain containing proteins that bind to dynamin and are involved in the process of vesicle scission in clathrin-mediated endocytosis. Methods: Using the yeast two-hybrid system and the GST pull down assay we demonstrated that Endophilin A3 interacts with the N-terminal part of KCa2.3 channels. In addition, we studied the impact of this interaction on channel activity by patch clamp measurements in PC12 cells expressing endogenous KCa2.3 channels. KCa2.3 currents were activated by using pipette solutions containing 1 µM free Ca2+. Results: Whole-cell measurements of PC12 cells transfected with Endophilin A3 showed a reduction of KCa2.3 specifc Cs+ currents indicating that the interaction of Endophilin A3 with KCa2.3 channels also occurs in mammalian cells and that this interaction has functional consequences for current flowing through KCa2.3 channels. Since KCa2.3 specific currents could be increased in PC12 cells transfected with Endophilin A3 with DC-EBIO (30 µM, a known SK-channel activator, these data also implicate that Endophilin A3 did not significantly remove KCa2.3 channels from the membrane but changed the sensitivity of the channels to Ca2+ which could be overcome by DC-EBIO. Conclusion: This interaction seems to be important for the function of KCa2.3 channels and might therefore play a significant role in situations where channel activation is pivotal for cellular function.

  17. Salvianolic acid Y: a new protector of PC12 cells against hydrogen peroxide-induced injury from Salvia officinalis.

    Science.gov (United States)

    Gong, Jun; Ju, Aichun; Zhou, Dazheng; Li, Dekun; Zhou, Wei; Geng, Wanli; Li, Bing; Li, Li; Liu, Yanjie; He, Ying; Song, Meizhen; Wang, Yunhua; Ye, Zhengliang; Lin, Ruichao

    2015-01-06

    Salvianolic acid Y (TSL 1), a new phenolic acid with the same planar structure as salvianolic acid B, was isolated from Salvia officinalis. The structural elucidation and stereochemistry determination were achieved by spectroscopic and chemical methods, including 1D, 2D-NMR (1H-1H COSY, HMQC and HMBC) and circular dichroism (CD) experiments. The biosynthesis pathway of salvianolic acid B and salvianolic acid Y (TSL 1) was proposed based on structural analysis. The protection of PC12 cells from injury induced by H2O2 was assessed in vitro using a cell viability assay. Salvianolic acid Y (TSL 1) protected cells from injury by 54.2%, which was significantly higher than salvianolic acid B (35.2%).

  18. Salvianolic Acid Y: A New Protector of PC12 Cells against Hydrogen Peroxide-Induced Injury from Salvia officinalis

    Directory of Open Access Journals (Sweden)

    Jun Gong

    2015-01-01

    Full Text Available Salvianolic acid Y (TSL 1, a new phenolic acid with the same planar structure as salvianolic acid B, was isolated from Salvia officinalis. The structural elucidation and stereochemistry determination were achieved by spectroscopic and chemical methods, including 1D, 2D-NMR (1H-1H COSY, HMQC and HMBC and circular dichroism (CD experiments. The biosynthesis pathway of salvianolic acid B and salvianolic acid Y (TSL 1 was proposed based on structural analysis. The protection of PC12 cells from injury induced by H2O2 was assessed in vitro using a cell viability assay. Salvianolic acid Y (TSL 1 protected cells from injury by 54.2%, which was significantly higher than salvianolic acid B (35.2%.

  19. 海参脑苷脂对H2O2致PC12细胞氧化损伤的保护作用%Protective effects of cerebroside from sea cucumber on the oxidative damage of PC12 cells induced by H2O2

    Institute of Scientific and Technical Information of China (English)

    武风娟; 杜磊; 徐杰; 唐庆娟; 薛长湖; 王玉明

    2012-01-01

    Objective To study the protective effects of cerebroside from sea cucumber (SCC) on the oxidative damage of PC12 cells induced by H2O2. Methods An oxidative damage model of PC12 cells was induced by H2O2. The survival rate of the cells was determined by MTT method. The leakage of LDH into cell cultures was determined to evaluate the degree of cell damage. The generation of intracellular reactive oxygen species (ROS) was detected by using DCFH-DA fluorescent probe. The activity of total anti-oxidation capacity ( T-AOC) was determined by chemical colorimetric method and the SOD activity was measured by xanthine oxide method. Results The injury of PC12 cells induced by H2O2 could be reduced obviously by SCC Compared with the model group, SCC could improve the survival rate of injured cells, reduce the leakage of LDH into cell culture and reduce intracellular ROS ( P < 0. 01 ) , increase the intracellular T-AOC and SOD activity ( P < 0. 01 ). Conclusion SCC has significantly protective effect on the injured PC 12 cells induced by H2O2. The results would provide preliminary data on supporting the development of sea cucumber as a functional food, which might give benefits to our brain.%目的 研究海参脑苷脂(SCC)对H2O2诱导PC12细胞氧化损伤的保护作用.方法 建立H2O2致PC12细胞氧化损伤模型,MTT法检测细胞存活率;测定细胞培养液中乳酸脱氢酶( LDH)漏出量,评价细胞的损伤程度;利用荧光探针DCFH-DA对细胞内活性氧(ROS)进行荧光染色,检测荧光强度;化学比色法测定细胞内总抗氧化能力(T-AOC),黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性.结果 海参脑苷脂能明显改善H2O2诱导的PC12细胞的氧化损伤,与模型组相比,SCC处理组可使细胞存活率升高,细胞培养液中LDH的漏出量明显减少(P<0.01),细胞内ROS的积累量明显降低(P<0.01),同时细胞内T-AOC和SOD活性明显增加(P<0.01).结论 SCC对H2O2诱导的PC12细胞的氧化损伤具有一定的保护作用.

  20. ProNGF derived from rat sciatic nerves downregulates neurite elongation and axon specification in PC12 cells

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    Anna Sofia Trigos

    2015-09-01

    Full Text Available Several reports have shown that a sciatic nerve conditioned media (CM causes neuronal-like differentiation in PC12 cells. This differentiation is featured by neurite outgrowth, which are exclusively dendrites, without axon or sodium current induction. In previous studies, our group reported that the CM supplemented with a generic inhibitor for tyrosine kinase receptors (k252a enhanced the CM-induced morphological differentiation upregulating neurite outgrowth, axonal formation and sodium current elicitation. Sodium currents were also induced by depletion of endogenous proNGF from the CM (pNGFd-CM. Given that sodium currents, neurite outgrowth and axon specification are important features of neuronal differentiation, in the current manuscript, first we investigated if proNGF was hindering the full PC12 cell neuronal-like differentiation. Second, we studied the effects of exogenous wild type (pNGFwt and mutated (pNGFmut proNGF isoforms over sodium currents and, whether or not their addition to the pNGFd-CM would prevent sodium current elicitation. Third, we investigated if proNGF was exerting its negative regulation through the sortilin receptor, and for this, the proNGF action was blocked with neurotensin (NT, a factor known to compete with proNGF for sortilin. Thereby, here we show that pNGFd-CM enhanced cell differentiation, cell proportion with long neurites, total neurite length, induced axonal formation and sodium current elicitation. Interestingly, treatment of PC12 cells with wild type or mutated proNGF isoforms elicited sodium currents. Supplementing pNGFd-CM with pNGFmut reduced 35% the sodium currents. On the other hand, pNGFd-CM+pNGFwt induced larger sodium currents than pNGFd-CM. Finally, treatments with CM supplemented with NT showed that sortilin was mediating proNGF negative regulation, since its blocking induced similar effects than the pNGFd-CM treatment. Altogether, our results suggest that proNGF within the CM, is one of the

  1. Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells

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    Dimitriades-Schmutz Beatrice

    2009-02-01

    Full Text Available Abstract Background The major goal of the study was to compare the genetic programs utilized by the neuropoietic cytokine Interleukin-6 (IL-6 and the neurotrophin (NT Nerve Growth Factor (NGF for neuronal differentiation. Results The designer cytokine Hyper-IL-6 in which IL-6 is covalently linked to its soluble receptor s-IL-6R as well as NGF were used to stimulate PC12 cells for 24 hours. Changes in gene expression levels were monitored using Affymetrix GeneChip technology. We found different expression for 130 genes in IL-6- and 102 genes in NGF-treated PC12 cells as compared to unstimulated controls. The gene set shared by both stimuli comprises only 16 genes. A key step is upregulation of growth factors and functionally related external molecules known to play important roles in neuronal differentiation. In particular, IL-6 enhances gene expression of regenerating islet-derived 3 alpha (REG3A; 1084-fold, regenerating islet-derived 3 beta (REG3B/PAPI; 672-fold, growth differentiation factor 15 (GDF15; 80-fold, platelet-derived growth factor alpha (PDGFA; 69-fold, growth hormone releasing hormone (GHRH; 30-fold, adenylate cyclase activating polypeptide (PACAP; 20-fold and hepatocyte growth factor (HGF; 5-fold. NGF recruits GDF15 (131-fold, transforming growth factor beta 1 (TGFB1; 101-fold and brain-derived neurotrophic factor (BDNF; 89-fold. Both stimuli activate growth-associated protein 43 (GAP-43 indicating that PC12 cells undergo substantial neuronal differentiation. Moreover, IL-6 activates the transcription factors retinoic acid receptor alpha (RARA; 20-fold and early growth response 1 (Egr1/Zif268; 3-fold known to play key roles in neuronal differentiation. Ingenuity biological function analysis revealed that completely different repertoires of molecules are recruited to exert the same biological functions in neuronal differentiation. Major sub-categories include cellular growth and differentiation, cell migration, chemotaxis, cell

  2. A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells.

    Science.gov (United States)

    Um, Moonkyoung; Gross, Alec W; Lodish, Harvey F

    2007-03-01

    The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits

  3. Effect of 1-methyl, 4-phenyl pyridium on endogenous hydrogen sulfide production in PC12 cells%MPP+对PC12细胞内源性H2S生成的影响

    Institute of Scientific and Technical Information of China (English)

    唐小卿; 范黎黎; 杨春涛; 申新田; 赵静; 李玉娟; 郭瑞鲜; 冯鉴强

    2009-01-01

    目的:观察1-甲基4-苯基吡啶离子 (MPP+)对PC12细胞内源性硫化氢(H2S)生成的影响,以探讨MPP+损伤PC12细胞的新机制.方法:RT-PCR方法检测PC12细胞胱硫醚-β-合酶(CBS)mRNA 的表达;亚甲基蓝分光光度计法检测PC12细胞内源性H2S的含量及PC12细胞CBS活性;台盼蓝拒染色法观察PC12细胞的存活率.结果:MPP+ 可以抑制PC12细胞CBS的表达及其活性,减少内源性H2S的生成;MPP+可以明显地降低PC12细胞的存活率;H2S的供体硫氢化钠(NaHS)对MPP+诱导的PC12细胞损伤具有显著的拮抗作用.结论:MPP+能抑制CBS的表达和活性,减少内源性H2S生成,这可能与其损伤PC12细胞的机制有关.

  4. Trehalose Inhibits A53T Mutant α-Synuclein Overexpression and Neurotoxicity in Transduced PC12 Cells.

    Science.gov (United States)

    Zhao, Juan; Zhi, Xiuling; Pan, Luanfeng; Zhou, Ping

    2017-08-08

    Fibrillar accumulation of A53T mutant α-synuclein (A53T-AS) in Lewy bodies is a symptom of Parkinsonism. Inhibitions of the overexpression and fibrillar aggregation of α-synuclein (AS) in vivo could be a promising strategy for treating Parkinson's disease (PD). In this study, at concentrations lower than 1 mM, trehalose decreased the A53T-AS expression level in transduced PC12 cells. Although H₂O₂ and aluminum ions increased the expression level and neurotoxicity of A53T-AS in cells, proper trehalose concentrations inhibited the event. These studies adequately prove that trehalose at an appropriate dose would be potentially useful for PD treatment.

  5. Neurotropin promotes NGF signaling through interaction of GM1 ganglioside with Trk neurotrophin receptor in PC12 cells.

    Science.gov (United States)

    Fukuda, Yu; Fukui, Takao; Hikichi, Chika; Ishikawa, Tomomasa; Murate, Kenichiro; Adachi, Takeshi; Imai, Hideki; Fukuhara, Koki; Ueda, Akihiro; Kaplan, Allen P; Mutoh, Tatsuro

    2015-01-30

    Activation of the high-affinity nerve growth factor (NGF) receptor Trk occurs through multiple processes consisted of translocation and clustering within the plasma membrane lipid rafts, dimerization and autophosphorylation. Here we found that a nonprotein extract of inflamed rabbit skin inoculated with vaccinia virus (Neurotropin(®)) enhanced efficiency of NGF signaling. In rat pheochromocytoma PC12 cells overexpressing Trk (PCtrk cells), Neurotropin augmented insufficient neurite outgrowth observed at suboptimal concentration of NGF (2ng/mL) in a manner depending on Trk kinase activity. Cellular exposure to Neurotropin resulted in an accumulation of Trk-GM1 complexes without affecting dimerization or phosphorylation states of Trk. Following NGF stimulation, Neurotropin significantly facilitated the time course of NGF-induced Trk autophosphorylation. These observations provide a unique mechanism controlling efficiency of NGF signaling, and raise the therapeutic potential of Neurotropin for various neurological conditions associated with neurotrophin dysfunction.

  6. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces initiation factor 2 alpha phosphorylation and translation inhibition in PC12 cells.

    Science.gov (United States)

    Muñoz, F; Martín, M E; Salinas, M; Fando, J L

    2001-03-09

    We have investigated the effect of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on protein synthesis rate and initiation factor 2 (eIF2) phosphorylation in PC12 cells differentiated with nerve growth factor. FCCP treatment induced a very rapid 2-fold increase in intracellular Ca(2+) concentration that was accompanied by a strong protein synthesis rate inhibition (68%). The translation inhibition correlated with an increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) (25% vs. 7%, for FCCP-treated and control cells, respectively) and a 1.7-fold increase in the double-stranded RNA-dependent protein kinase activity. No changes in the PKR endoplasmic reticulum-related kinase or eIF2 alpha phosphatase were found. Translational regulation may play a significant role in the process triggered by mitochondrial calcium mobilization.

  7. Rat Nasal Respiratory Mucosa-Derived Ectomesenchymal Stem Cells Differentiate into Schwann-Like Cells Promoting the Differentiation of PC12 Cells and Forming Myelin In Vitro

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    2015-01-01

    Full Text Available Schwann cell (SC transplantation as a cell-based therapy can enhance peripheral and central nerve repair experimentally, but it is limited by the donor site morbidity for clinical application. We investigated weather respiratory mucosa stem cells (REMSCs, a kind of ectomesenchymal stem cells (EMSCs, isolated from rat nasal septum can differentiate into functional Schwann-like cells (SC-like cells. REMSCs proliferated quickly in vitro and expressed the neural crest markers (nestin, vimentin, SOX10, and CD44. Treated with a mixture of glial growth factors for 7 days, REMSCs differentiated into SC-like cells. The differentiated REMSCs (dREMSCs exhibited a spindle-like morphology similar to SC cells. Immunocytochemical staining and Western blotting indicated that SC-like cells expressed the glial markers (GFAP, S100β, Galc, and P75 and CNPase. When cocultured with dREMSCs for 5 days, PC12 cells differentiated into mature neuron-like cells with long neurites. More importantly, dREMSCs could form myelin structures with the neurites of PC12 cells at 21 days in vitro. Our data indicated that REMSCs, a kind of EMSCs, could differentiate into SC-like cells and have the ability to promote the differentiation of PC12 cells and form myelin in vitro.

  8. Hexabromocyclododecane inhibits depolarization-induced increase in intracellular calcium levels and neurotransmitter release in PC12 cells.

    Science.gov (United States)

    Dingemans, Milou M L; Heusinkveld, Harm J; de Groot, Aart; Bergman, Ake; van den Berg, Martin; Westerink, Remco H S

    2009-02-01

    Environmental levels of the brominated flame retardant (BFR) hexabromocyclododecane (HBCD) have been increasing. HBCD has been shown to cause adverse effects on learning and behavior in mice, as well as on dopamine uptake in rat synaptosomes and synaptic vesicles. For other BFRs, alterations in the intracellular Ca(2+) homeostasis have been observed. Therefore, the aim of this study was to investigate whether the technical HBCD mixture and individual stereoisomers affect the intracellular Ca(2+) concentration ([Ca(2+)](i)) in a neuroendocrine in vitro model (PC12 cells). [Ca(2+)](i) and vesicular catecholamine release were measured using respectively single-cell Fura-2 imaging and amperometry. Exposure of PC12 cells to the technical HBCD mixture or individual stereoisomers did neither affect basal [Ca(2+)](i), nor the frequency of basal neurotransmitter release. However, exposure to HBCD (0-20 microM) did cause a dose-dependent reduction of a subsequent depolarization-evoked increase in [Ca(2+)](i). This effect was apparent only when HBCD was applied at least 5 min before depolarization (maximum effect after 20 min exposure). The effects of alpha- and beta-HBCD were comparable to that of the technical mixture, whereas the inhibitory effect of gamma-HBCD was larger. Using specific blockers of L-, N- or P/Q-type voltage-gated Ca(2+) channels (VGCCs) it was shown that the inhibitory effect of HBCD is not VGCC-specific. Additionally, the number of cells showing depolarization-evoked neurotransmitter release was markedly reduced following HBCD exposure. Summarizing, HBCD inhibits depolarization-evoked [Ca(2+)](i) and neurotransmitter release. As increasing HBCD levels should be anticipated, these findings justify additional efforts to establish an adequate exposure, hazard and risk assessment.

  9. Extracellular α-synuclein leads to microtubule destabilization via GSK-3β-dependent Tau phosphorylation in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Magdalena Gąssowska

    Full Text Available α-Synuclein (ASN plays an important role in pathogenesis of Parkinson's disease (PD and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12 cells and the involvement of glycogen synthase kinase-3β (GSK-3β and cyclin-dependent kinase 5 (CDK5 in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763 prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.

  10. The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Mai [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Kitaguchi, Tetsuya [Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABOIS), Waseda University, 11 Biopolis Way, 05-01/02 Helios, Singapore 138667 (Singapore); Numano, Rika [The Electronics-Inspired Interdisciplinary Research Institute (EIIRIS), Toyohashi University of Technology, 1-1 Hibarigaoka, Tennpaku-cho, Toyohashi, Aichi 441-8580 (Japan); Ikematsu, Kazuya [Forensic Pathology and Science, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 (Japan); Kakeyama, Masaki [Laboratory of Environmental Health Sciences, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Murata, Masayuki; Sato, Ken [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Tsuboi, Takashi, E-mail: takatsuboi@bio.c.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

  11. Actin and dynamin recruitment and the lack thereof at exo- and endocytotic sites in PC12 cells.

    Science.gov (United States)

    Felmy, Felix

    2009-06-01

    Protein recruitment during endocytosis is well characterized in fibroblasts. Since fibroblasts do not engage in regulated exocytosis, only information about protein recruitment during constitutive endocytosis is provided. Furthermore, the cortical actin of fibroblasts is characterized by stress fibers rather than a thick cortical meshwork. A cell model, which differs in these features, could provide insight into the heterogeneity of protein recruitment to constitutive and exocytosis coupled endocytotic areas. Therefore, this study investigates the sequence of protein recruitment in PC12 cells, a well documented exocytotic cell model with thick actin cortex. Using real time total-internal-reflection fluorescence microscopy it was found that at the plasma membrane steady, but not transient, dynamin-1-EGFP or -mCherry fluorescence spots that rapidly dimmed coincided with markers for constitutive endocytotic such as clathrin-LC-dsRed and transferrin-receptor-pHluorin. Clathrin-LC-dsRed and dynamin-1-EGFP were further used to determine the temporal sequence of protein recruitment to areas of constitutive endocytosis. mCherry- and EGFP-beta-actin, Arp-3-EGFP and EGFP-mAbp1 were slowly recruited before the dynamin-1-mCherry fluorescence dimmed, but their fluorescence peaked after the loss of clathrin-LC-dsRed commenced. Furthermore, mCherry-beta-actin fluorescence increased before exocytosis, indicating redistribution prior to release. Also, no average dynamin-1-mCherry recruitment was observed within 50 s to regions of exocytosis marked by NPY-mGFP. This indicates that the temporal-spatial coupling between regulated exo-and endocytosis is rather limited in PC12 cells. Furthermore, the time course of the protein recruitment to constitutive endocytotic sites might depend on the subcellular morphology such as the size of the actin cortex.

  12. PGN激活BV2内吞Aβ寡聚体后对PC12的影响%Influences of peptidoglycan on PC12 cells after endocytosis of amyioid protein β oligomers in activated BV2 cells

    Institute of Scientific and Technical Information of China (English)

    姜新; 谢明; 白丽娟; 陈晓虹; 马恩龙

    2011-01-01

    探讨前炎性介质肽聚糖(peptidoglycan,PGN)激活BV2细胞内吞β淀粉样蛋白(amyloid protein β,Aβ)1~42寡聚体后对PC12的影响.方法采用细胞株传代法培养PC12细胞及BV2细胞,分别替代神经元细胞和小胶质细胞;用转移筛网进行PC12、BV2细胞共育培养;制备Aβ1-42寡聚体;分别采用MTT方法检测PC12细胞抑制率、Western blotting方法检测各组PC12细胞tau( pS396)蛋白表达情况、流式细胞仪检测PC12细胞凋亡.结果Aβ寡聚体、Aβ寡聚体作用BV2细胞后及PGN作用BV2细胞后均能抑制PC12细胞增殖、增加PC12细胞tau( pS396)表达量、增加PC12细胞凋亡率;而PGN激活BV2细胞内吞Aβ寡聚体后对PC12细胞增殖抑制、tau (pS396)表达量、PC12细胞凋亡率与前面三种情况比较明显增加.结论Aβ寡聚体可引起PC12细胞的细胞抑制率,tau蛋白异常磷酸化水平,细胞凋亡的增多;PGN激活BV2细胞内吞Aβ后,加重Aβ寡聚体引起PC12细胞的细胞抑制率,tau蛋白异常磷酸化水平,细胞凋亡的增多.

  13. IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

    Science.gov (United States)

    Nakatani, Yoshihiko; Nagaoka, Takumi; Hotta, Sayako; Utsunomiya, Iku; Yoshino, Hiide; Miyatake, Tadashi; Hoshi, Keiko; Taguchi, Kyoji

    2007-03-01

    We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

  14. Effects of methylmercury on the differentiation and survival of PC12 cells%甲基汞对PC12细胞分化及活性的影响

    Institute of Scientific and Technical Information of China (English)

    李永进; 毕晓颖; 宋春梅; 马洪波; 王长文; 李志超

    2005-01-01

    目的研究氯化甲基汞对PC12细胞活性和分化的影响. 方法培养PC12细胞,用不同浓度的甲基汞作用于PC12细胞,用倒置显微镜观察细胞形态学变化,通过MTT检测PC12细胞活性. 结果氯化甲基汞能抑制PC12细胞突起生长,降低细胞活性. 结论氯化甲基汞可抑制PC12细胞分化和活性.

  15. Pycnogenol Protects Against Rotenone-Induced Neurotoxicity in PC12 Cells Through Regulating NF-κB-iNOS Signaling Pathway.

    Science.gov (United States)

    Gao, Bo; Chang, Chongwang; Zhou, Jie; Zhao, Tianzhi; Wang, Chao; Li, Chen; Gao, Guodong

    2015-10-01

    Parkinson's disease (PD) is a common neurodegenerative disorder characterized by dopaminergic neurons degeneration and oxidative damage may underlie this process. However, there are still no efficient drugs to cure the disease. Pycnogenol (PYC) isolated from the procyanidin-rich French maritime pine (Pinus maritime) bark has shown various antioxidant activities in previous studies. In this study, we explored its effect against rotenone (Rot)-induced neurotoxicity and the underlying mechanisms in PC12 cells. Using Rot-induced cell model of PD, we found that PYC treatment significantly increased cell viability and decreased cell apoptosis in Rot-treated PC12 cells in a dose-dependent manner. Furthermore, data showed that PYC markedly reduced inducible nitric oxide synthase (iNOS)-nitric oxide (NO) signaling in Rot-treated PC12 cells. Pretreatment with the iNOS-specific inhibitor significantly attenuated Rot-induced neurotoxicity. Moreover, PYC was found to be capable of reducing Rot-induced NF-κB activation. Blocking NF-κB signaling with its inhibitor mimicked the biological effect of PYC on Rot-induced iNOS and NO expression levels, as well as neurotoxicity in PC12 cells, suggesting that the NF-κB-iNOS signaling pathway was likely to participate in the PYC-mediated protective progress. Our results suggest that PYC protects against Rot-induced neurotoxicity in PC12 cells, and the mechanism may be associated with the downregulation of NF-κB-iNOS signaling pathway.

  16. Protective effects of fractions from Artemisia biennis hydro-ethanolic extract against doxorubicin-induced oxidative stress and apoptosis in PC12 cells

    Directory of Open Access Journals (Sweden)

    Mahdi Mojarrab

    2016-05-01

    Full Text Available Objective(s: This study was designed to indicate whether different fractions from Artemisia biennis hydroethanolic extract could provide cytoprotection against oxidative stress and apoptosis induced by doxorubicin (DOX in rat pheochromocytoma cell line (PC12. Material and Methods:Cell viability was determined by MTT assay. Also, activation of caspase-3 and superoxide dismutase were evaluated by spectrophotometry. Detection of reactive oxygen species (ROS and measurement of mitochondrial membrane potential (MMP were performed by flowcytometry. Results:  Treatment of PC12 cells with DOX reduced viability dose dependently. For evaluation of the effect of fractions (A-G on DOX-induced cytotoxicity, PC12 cells were pretreated for 24 hr with the A. biennis fractions and then cells were treated with DOX.  The fractions C and D increased PC12 cells viability significantly compared to DOX treated cells.  Moreover, pretreatment with fractions C and D for 24 hr attenuated DOX-mediated apoptosis and the anti-apoptotic action of A. biennis fractions was partially dependent on inhibition of caspase 3 activity and also increasing the  mitochondrial membrane potential (MMP. Selected A. biennis fractions also suppressed the generation of ROS and increased superoxide dismutase (SOD activity. Conclusion: Taken together our observation indicated that subtoxic concentration of aforementioned fractions of A. biennis hydroetanolic extract has protective effect against apoptosis induced by DOX in PC12 cell. The results highlighted that fractions C and D may exert cytoprotective effects through their antioxidant actions.

  17. Effects of Angelica Oil and the Isolated Butylphthalides on Glutamate-induced Neurotoxicity in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Lu-Si Liu

    2017-03-01

    Full Text Available Angelica sinensis contains a large amount of essential oil (angelica oil, which is rich in phthalide derivatives with a lot of bioactivities. In vitro activity screening of angelica oil from the roots of A. sinensis found that it had concentration-dependent effect on glutamate-induced injury in PC12 cells. Further phytochemical investigation on this angelica oil led to the isolation of nine butylphthalides (1 –9 including two new compounds (1 and 2. Their structures were elucidated by extensive spectroscopic analyses. It is noteworthy that most of the isolated butylphthalides also displayed protective activity at low concentrations and cytotoxicity at high concentrations. These results imply that angelica oil and its main chemical components have protective effect for injured neurons only in appropriate concentration range.

  18. 人Slitrk1基因对PC12细胞增殖和分化的影响%The effect of human Slitrk1 gene on proliferation and differentiation of PC12 cells

    Institute of Scientific and Technical Information of China (English)

    靳雁斌; 吴燕; 王晓文; 吴海涛; 景孝堂; 刘毅; 范文红; 范明

    2006-01-01

    目的 研究人Slitrk1基因过表达对PC12细胞增殖和分化的影响.方法 PCR扩增人Slitrk1 CDS,克隆到pcDNA4/myc-His A载体,构建重组质粒pcDNA4/St1,分别以空载体和重组质粒转染PC12细胞,RT-PCR法鉴定出阳性转染细胞,观察空载体转染的PC12细胞(pcDNA4/PC12)和过表达Slitrk1的PC12细胞(ST1/PC12)的表型并用MTT法检测细胞增殖情况.结果 与空载体转染的PC12细胞相比,过表达Slitrk1基因的细胞增殖速度明显减慢.pcDNA4/PC12与野生型PC12细胞表型一致,均为悬浮成团生长,细胞少有突起,而ST1/PC12细胞大部分贴壁生长,突起多见,呈分化状态.结论过表达人Slitrk1基因能够抑制PC12细胞增殖,促进细胞的贴壁及突起长出.人Slitrk1基因可能有促进PC12细胞神经分化的作用.

  19. EFFECTS OF PLACENTA FACTOR ON THE GROWTH OF PC12 CELLS%胎盘免疫调节因子对PC12细胞生长的影响

    Institute of Scientific and Technical Information of China (English)

    徐阳曦; 杨铁峥; 张学荣; 陈缨; 覃柳燕; 张钦乐; 舒雨雁

    2010-01-01

    目的:观察胎盘免疫调节因子(placenta factor,PF)对PC12细胞生长的影响.方法:培养PC12细胞,用MTT法观察PF对体外无血清培养PC12细胞存活率的影响及PF对低血清培养PC12细胞增殖、分化的影响.结果:对无血清培养的PC12细胞加入不同浓度的PF培养44 h后, PF在浓度为50、25、12.5、6.25 mg/L时可以显著提高无血清培养PC12细胞的存活率(P<0.05).对低血清培养的PC12细胞加入不同浓度的PF培养68 h后,PF在浓度为12.5 mg/L和6.25 mg/L时可以显著提高PC12细胞的数量(P<0.01).对低血清培养的PC12细胞加入不同浓度的PF培养10 d,PF在浓度为3.13 mg/L和1.56 mg/L作用第6天时,细胞长出突起.结论:PF能提高无血清培养PC12细胞的存活率,促进PC12细胞增殖,诱导PC12细胞分化.

  20. 他莫昔芬介导小胶质细胞 LRRK-2表达对炎症刺激 PC12细胞的影响%Effect of Tamoxifen on Inflammation-Stimulated PC12 Cells Mediated by LRRK-2 Gene Expression of Microglia

    Institute of Scientific and Technical Information of China (English)

    卢奎; 吴文军; 周敏; 黎捷; 钟健强; 张成

    2014-01-01

    Objective To study the effect of tamoxifen on the expressions of leucine-rich repeat kinase 2(LRRK-2) gene of microglia and related inflammatory factors .Methods Primary microglia was divided into 4 groups including control group,lipopolysaccharide(LPS) group,LPS plus tamoxifen group and LPS plus LRRK-2 group.LPS-activated microglia was regulated by tamoxifen .PC12 cells of rats were co-cultured with microglia for 24 hours in each group .The expressions of LRRK-2 gene and iNOS were detected by Western Blot .The levels of tumor necrosis factor α( TNF-α) and interleukin 1β(IL-1β) were tested by enzyme-linked immunosorbent assay ( ELISA).The apoptosis/survival ratio of PC12 cells was determined by Hochest staining .Results Tamoxifen down-regulated the LRRK-2 gene expression of microglia by LPS stimulation,inhibited the release of iNOS ,TNF-αand IL-1βby suppressing LRRK-2 gene,and decreased the co-cultured inflammatory expression of microglia and PC12 cells as well as apoptosis/survival ratio of PC12 cells.Conclusion LRRK-2 might be an upstream-regulated target for the release of iNOS in microglia .Tamoxifen can inhibit the activation of microglia by regulation of LRRK-2 gene,which might alleviate the inflammation-associated dopamine neuron injury .%目的:探讨他莫昔芬对小胶质细胞的富含亮氨酸重复序列激酶2( LRRK2)基因及相关炎症因子表达的影响。方法取原代小胶质细胞分为CON组、脂多糖( LPS)组、LPS+他莫昔芬组、LPS+LRRK2组;以LPS刺激激活小胶质细胞,用他莫昔芬调控小胶质细胞激活;将各组小胶质细胞与大鼠肾上腺嗜铬瘤细胞( PC12)细胞共培养24 h;Western Blot法检测小胶质细胞的LRRK2、一氧化氮合成酶( iNOS )表达,酶联免疫吸附法( ELISA)测定TNF-α、IL-1β水平;Hochest染核法检测PC12细胞凋亡/存活比。结果他莫昔芬抑制LPS刺激后小胶质细胞的LRRK2表达,通过抑制LRRK2

  1. Hydrogen sulfide inhibits formaldehyde-induced endoplasmic reticulum stress in PC12 cells by upregulation of SIRT-1.

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    Xiang Li

    Full Text Available BACKGROUND: Formaldehyde (FA, a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S, the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1, a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action. OBJECTIVE: We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis. PRINCIPAL FINDINGS: We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1. CONCLUSION/SIGNIFICANCE: These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity.

  2. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca(2+)-Independent Phospholipase A₂ Pathways.

    Science.gov (United States)

    Seo, Jihui; Maeng, Jeehye; Kim, Hwa-Jung

    2016-10-24

    The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [³H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca(2+)]i), the rTCTP-driven effect on dopamine release was mediated by a Ca(2+)-independent pathway, as evidenced by the fact that Ca(2+)-modulating agents such as Ca(2+) chelators and a voltage-gated L-type Ca(2+)-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A₂ (PLA₂) in rTCTP-induced dopamine release, the inhibitor for Ca(2+)-independent PLA₂ (iPLA₂) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca(2+)-dependent cytosolic PLA₂ (cPLA₂) and secretory PLA₂ (sPLA₂) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca(2+)-independent mechanism that involved PLA₂ in the process, suggesting the regulatory role of TCTP in the neuronal functions.

  3. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A2 Pathways

    Directory of Open Access Journals (Sweden)

    Jihui Seo

    2016-10-01

    Full Text Available The translationally controlled tumor protein (TCTP, initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF. TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12 cells. Treatment with recombinant TCTP (rTCTP enhanced both basal and depolarization (50 mM KCl-evoked [3H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i, the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2 in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2 produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2 and secretory PLA2 (sPLA2 inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions.

  4. Neurite Outgrowth of PC12 Mutant Cells Induced by Orange Oil and d-Limonene via the p38 MAPK Pathway

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    Shinomiya,Misae

    2012-04-01

    Full Text Available We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF, where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.

  5. Multi-porous electroactive poly(L-lactic acid)/polypyrrole composite micro/nano fibrous scaffolds promote neurite outgrowth in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Qiaozhen Yu; Shuiling Xu; Kuihua Zhang; Yongming Shan

    2013-01-01

    In this study, poly(L-lactic acid)/ammonium persulfate doped-polypyrrole composite fibrous scaffolds with moderate conductivity were produced by combining electrospinning with in situ polymerization. PC12 cells were cultured on these fibrous scaffolds and their growth following electrical stimulation scanning electron microscopy coupled with the MTT cell viability test. The results demonstrated that the poly(L-lactic acid)/ammonium persulfate doped-polypyrrole fibrous scaffold was a dual 10.0 μA for about 2 days enhanced neuronal growth and neurite outgrowth, while a high current intensity (over 15.0 μA) suppressed them. These results indicate that electrical stimulation with a moderate current intensity for an optimum time frame can promote neuronal growth and neurite outgrowth in an intensity- and time-dependent manner.

  6. Effects of iPSC-MSCs on mitochondria of PC12 cells injured by CoCl2%iPSC-MSCs 对 CoCl2诱导的 PC12细胞损伤时线粒体功能的影响

    Institute of Scientific and Technical Information of China (English)

    杨焰; 李慧; 孙占朋; 胡春林; 荆小莉; 魏红艳; 廖晓星; 李欣

    2015-01-01

    目的:观察诱导性多能干细胞来源的间充质干细胞(iPSC-MSCs)对氯化钴(CoCl2)诱导的PC12细胞损伤的影响,并探讨其可能机制。方法:用CoCl2处理PC12细胞建立化学损伤模型,加入iPSC-MSCs共培养。用细胞计数试剂盒-8(CCK-8)比色法检测细胞存活率,Annexin V/PI双染流式细胞术检测细胞凋亡比率,JC-1染色流式细胞术检测细胞线粒体膜电位,免疫荧光观察iPSC-MSCs向PC12细胞转移线粒体的情况。结果:用CoCl2(400μmol/L)处理PC12细胞24 h可使其凋亡明显增多,线粒体膜电位明显下降。与iPSC-MSCs共培养能减轻PC12细胞的凋亡,使其膜电位恢复。 iPSC-MSCs可以与PC12细胞间形成隧道纳米管并向PC12细胞转移线粒体。结论:iPSC-MSCs可以减轻CoCl2诱导的PC12细胞损伤,机制可能与其向PC12细胞转移线粒体有关。%[ ABSTRACT] AIM:To investigate the effects of induced pluripotent stem cells-derived mesenchymal stem cells ( iPSC-MSCs) on cobalt chloride ( CoCl2 )-induced injuries of PC12 cells and its possible mechanism.METHODS:PC12 cells were exposed to CoCl2 to set up a chemical-induced cellular injury model and were cocultured with iPSC-MSCs.The cell viability was tested by CCK-8 assay.The apoptosis was measured by flow cytometry using Annexin V/PI staining.The mitochondrial membrane potential (MMP) was analyzed by flow cytometry using JC-1 staining.Immunofluorescence was employed to observe mitochondrial transfer from iPSC-MSCs to PC12 cells.RESULTS: Apoptosis of PC12 cells was in-creased and MMP of PC12 cells was decreased after exposed to CoCl2 at concentration of 400μmol/L for 24 h.Coculture of PC12 cells with iPSC-MSCs reduced the apoptosis and recovered the MMP of the PC12 cells.Tunneling nanotubes were formed between iPSC-MSCs and PC12 cells, through which the iPSC-MSCs transferred the mitochondria to the PC12 cells. CONCLUSION:iPSC-MSCs protect PC12

  7. SUB-ACUTE TREATMENT WITH METHYLMERCURY DURING DIFFERENTIATION OF PHEOCHROMOCYTOMA (PC12) CELLS DOES NOT ALTER BINDING OF ION CHANNEL LIGANDS OR CELL MORPHOLOGY.

    Science.gov (United States)

    We demonstrated recently that 6 days of exposure to nanomolar concentrations (3-10 nM) of methylmercury (MeHg) during nerve growth factor (NGF) induced PC12 cell differentiation reduced the amplitude and density of voltage-gated sodium and calcium currents. In the present study,...

  8. Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells.

    Science.gov (United States)

    Tseng, Wei-Ting; Hsu, Ya-Wen; Pan, Tzu-Ming

    2016-08-01

    Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson's disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC). Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated. Materials and methods DMA (0-60 μM) or DFC (0-10 μM) was co-treated with 6-OHDA (200 μM, 24 h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression. Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells. Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.

  9. Oxidative modification of the molecular chaperone family in a PC12 cell model of Parkinson's disease induced by Z-lle-Glu(OtBu)-Ala-Leucinal

    Institute of Scientific and Technical Information of China (English)

    Ying Zhang; Yimin Yang; Jing Bai; Ming Chang; Linsen Hu

    2011-01-01

    Previous studies have demonstrated that ubiquitin-proteasome system function is significantly decreased in the substantia nigra of Parkinson's disease patients.In the present study, proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was used to inhibit the function of the ubiquitin-proteasome system in PC12 cells to simulate Parkinson's disease.Oxidatively modified proteins were identified to determine pathogenesis of Parkinson's disease.Results demonstrated that 24 hours of 10 μmol/L PSI-treatment in PC12 cells simulated pathological characteristics of Parkinson's disease: neuronal degeneration and eosinophilic inclusion formation in neurons.In PSI-treated PC12 cells, three oxidative proteins and a molecular chaperone family member were detected: chaperonin containing t-complex polypeptide 1 subunit 3, glucose-regulated protein 58,and heat shock protein 70.This is the first study to demonstrate oxidative modification of a molecule family in a cell model of Parkinson's disease induced with PSI.

  10. Inhibition Effects of FLOS ROSAE RUGOSAE Extracts on the Cytotoxity of Aβ25-35-induced PC12 Cell%玫瑰花对β-淀粉样蛋白诱导PC12神经细胞毒性的抑制作用

    Institute of Scientific and Technical Information of China (English)

    杨庆雄; 王聪聪; 张万全; 余天华; 孙黔云

    2011-01-01

    [目的]研究玫瑰花提取物对β-淀粉样蛋白(Aβ25-35)诱导的损伤PC12细胞的保护作用.[方法]PC12细胞加入Aβ25-35共同孵育后,采用四甲基偶氮唑盐(MTT)法检测PC12细胞活力,比色法检测细胞内丙二醛(MDA)含量,酶联免疫法检测细胞内Nf-κb水平.[结果]玫瑰花提取物对PC12无显著细胞毒性,可以显著抑制Aβ25-35诱导的PC12细胞毒性.与模型组相比,添加玫瑰花乙酸乙酯萃取物的样品组能降低PC12细胞内MDA浓度和Nf-κb表达.[结论]玫瑰花提取物对β-淀粉样蛋白致PC12细胞损伤具有一定的抑制作用,其作用机理可能与降低Aβ25-35所致的PC12细胞内的氧化应激有关.%[Objective] To study the neuroprotection effect of FLOS ROSAE RUGOSAE on Aβ25,-induced PC12 cells. [ Method] PC12 cell was co-incubated with Aβ25_35, and then the cell viability was determined by MTT method, content of MDA was determined by colorimetry, and the expression level of Nf-kb was evaluated by ELISIA. [ Result] The extract of FLOS ROSAE RUGOSAE did not show apparent cytotoxity towards PC12 cell line, instead, it inhibited the cytotoxity induced by the Aβ25-35.Compared with the model group, the content of MDA and expression of Nf-Kb in PC12 cell decreased after being treated with the extract of FLOS ROSAE RUGOSAE. [Conclusion] The extracts of FLOS ROSAE RUGOSAE showed neuroprotection effect on the cytotoxity of PC12 induced by Aβ25-35, and the mechanism of the neuroprotec tion may be related to the decreasing content of oxidative stress in the PC 12 cell .

  11. 1,8-cineole (eucalyptol) mitigates inflammation in amyloid Beta toxicated PC12 cells: relevance to Alzheimer's disease.

    Science.gov (United States)

    Khan, Andleeb; Vaibhav, Kumar; Javed, Hayate; Tabassum, Rizwana; Ahmed, Md Ejaz; Khan, Mohd Moshahid; Khan, M Badruzzaman; Shrivastava, Pallavi; Islam, Farah; Siddiqui, M Saeed; Safhi, M M; Islam, Fakhrul

    2014-02-01

    Inflammatory process has a fundamental role in the pathogenesis of Alzheimer's disease and insoluble amyloid beta deposits and neurofibrillary tangles provide the obvious stimuli for inflammation. The present study demonstrate the effect of pretreatment of 1,8-cineole (Cin) on inflammation induced by Aβ(25-35) in differentiated PC12 cells. The cells were treated with Cin at different doses for 24 h and then replaced by media containing Aβ(25-35) for another 24 h. The cell viability was decreased in Aβ(25-35) treated cells which was significantly restored by Cin pretreatment. Cin successfully reduced the mitochondrial membrane potential, ROS and NO levels in Aβ(25-35) treated cells. Cin also lowered the levels of proinflammatory cytokines TNF-α, IL-1β and IL-6 in Aβ(25-35) treated cells. Moreover, Cin also succeeded in lowering the expression of NOS-2, COX-2 and NF-κB. This study suggests the protective effects of Cin on inflammation and provides additional evidence for its potential beneficial use in therapy as an anti-inflammatory agent in neurodegenerative disease.

  12. The ethanol response gene Cab45 can modulate the impairment elicited by ethanol and ultraviolet in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Yunfeng Zhu; Quanli Wang; Wangru Xu; Sha Li

    2008-01-01

    High consumption of ethanolic beverages facilitates neurodegeneration,but the mechanism of this process still remained elusive.Suppression subtractive hybridization (SSH) is a technique for detection of rare transcripts.With SSH approach,we identified one ethanol response gene Cab45,which was down-regulated by ethanol with time-dependent manner in B104 cells.The full-length sequence of Cab45 gene was obtained by 5'-RACE (5'Rapid Amplification of cDNA Ends) for the first time in rat.Based on the sequence of deduced amino acid of rat Cab45,the alignment was conducted with its counterparts in different species and displayed a high conservation.Using different tissues in rat and cell lines,Cab45 was characterized by a ubiquitous expression and differentiation dependent down-regulation.Given that ethanol facilitates some cell differentiation,we hypothesize that Cab45 is involved in ethanol-mediated differentiation.With transient transfection,the function of Cab45 was investigated by up-regulation and down-regulation in PC12 cells.Ethanol treatment and UV exposure were conducted subsequently and cell proliferations were detected by MTT (Methyl Thiazolyl Tetrazolium) approach.It revealed that the up-regulation of Cab45 modulated the impairment elicited by ethanol and UV in transfected cells.As a member of new calcium binding protein family,the exact role of Cab45 still remains unclear.

  13. Protective effect of serotonin derivatives on glucose-induced damage in PC12 rat pheochromocytoma cells.

    Science.gov (United States)

    Piga, Rosaria; Naito, Yuji; Kokura, Satoshi; Handa, Osamu; Yoshikawa, Toshikazu

    2010-01-01

    Oxidative damage is believed to be associated with ageing, cancer and several degenerative diseases. Previous reports have shown that safflower-seed extract and its major antioxidant constituents, serotonin hydroxycinnamic amides, possess a powerful free radical-scavenging and antioxidative activity, paying particular attention to atherosclerotic reactive oxygen species (ROS)-related dysfunctions. In the present report, we examined a still unknown cell-based mechanism of serotonin derivatives against ROS-related neuronal damage, phenomena that represent a crucial event in neurodegenerative diseases. Serotonin derivatives N-(p-coumaroyl)serotonin and N-feruloylserotonin exerted a protective effect on high glucose-induced cell death, inhibited the activation of caspase-3 which represents the last and crucial step within the cascade of events leading to apoptosis, and inhibited the overproduction of the mitochondrial superoxide, which represents the most dangerous radical produced by hyperglycaemia, by acting as scavengers of the superoxide radical. In addition, serotonin derivative concentration inside the cells and inside the mitochondria was increased in a time-dependent manner. Since recent studies support the assertion that mitochondrial dysfunctions related to oxidative damage are the major contributors to neurodegenerative diseases, these preliminary cell-based results identify a mitochondria-targeted antioxidant property of serotonin derivatives that could represent a novel therapeutic approach against the neuronal disorders and complications related to ROS.

  14. Identification of the alternative spliced form of the alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 cells.

    Science.gov (United States)

    Gilad, B; Shenkar, N; Halevi, S; Trus, M; Atlas, D

    1995-07-07

    The alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 has been cloned and partially sequenced. The message observed in Northern blot analysis displays a 7.5 kb transcript, identical in size to mRNA of rabbit skeletal muscle and rat brain. The nucleotide sequence of the cloned alpha 2 subunit of the PC12 specific cDNA is > 99% identical to rat brain sequence and 85% to skeletal muscle. Reverse-transcriptase-polymerase chain reaction (RT-PCR) of the alternative splicing region identifies two deleted regions of 57 bp and 21 bp in PC12 expressed alpha 2/delta transcript. The alternative variant alpha 2e of alpha 2/delta subunit which is expressed in PC12 cells was previously identified in human embryonic kidney (HEK293) cells. RT-PCR analysis show two different sized alternative PCR fragments in rat lung and none in rat spleen, kidney and intestine. Antibodies prepared against a 19 amino acid peptide within the alternative spliced region effectively inhibits [3H]dopamine release in PC12 cells. This implies that the alternatively spliced region is positioned extracellularly and is involved in regulation of the L-type Ca2+ channel-mediated transmitter release.

  15. Protective effect of cyclovirobuxine D derivatives on alcohol-induced inju-ry in PC12 cells%环维黄杨星D新衍生物对酒精诱导PC12细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    石贞玉; 厉永强

    2013-01-01

    Objective To explore the effect of the new cyclovirobuxine D derivative (CVB-D) on the model of alcohol-induced injury in PC12 cells .Methods The model of alcohol-induced injury in PC12 cells was established . The protective effect of the new cyclovirobuxine D derivative on PC12 cells was confirmed by MTT assay , Hoechst 33258 staining and lactate dehydrogenase ( LDH) activity . Results The new derivative inhibited the damage caused by alcohol in PC12 cells , and apoptosis rate and LDH release reduced . Conclusion The new derivatives of the cyclovirobuxine D had a protective effect of alcohol-induced injury in PC12 cells .%目的探讨环维黄杨星D(CVB-D)新衍生物对酒精诱导的PC12细胞损伤模型的影响。方法建立酒精诱导PC12细胞损伤模型,通过MTT染色、Hoechst 33258染色和乳酸脱氢酶(LDH)活性的测定研究环维黄杨星D新衍生物对PC12细胞的保护作用。结果环维黄杨星D新衍生物抑制酒精对PC12细胞的增殖抑制作用,凋亡率下降,同时LDH的漏出减少。结论环维黄杨星D新衍生物对酒精诱导的PC12细胞损伤具有保护作用。

  16. Structure and function of the ssDNA aptamer specifically recognizing differentiated PC12 cells%特异识别已分化PC12细胞的ssDNA适体的结构和功能研究

    Institute of Scientific and Technical Information of China (English)

    王成龙; 邵宁生; 张明; 杨光; 张达矜; 丁红梅; 王会信

    2004-01-01

    目的:研究特异识别已分化PC12细胞的ssDNA适体的二级结构及截短前后的适体与已分化PC12细胞的结合能力.方法:利用RNA structure 3.5软件对截短前后的适体的空间结构进行模建,体外合成这些截短适体,利用流式细胞术和荧光显微镜研究适体与已分化PC12细胞的结合能力.结果:单茎环结构是适体与已分化PC12细胞结合的二级结构;截短后的适体与已分化PC12细胞的结合能力明显大于与未分化PC12细胞结合能力;AP17-38与已分化PC12细胞结合的荧光强度明显大于与未分化PC12细胞结合的荧光强度;截短前后的适体均能够特异识别混合物中已分化PC12细胞.结论:截短后的适体能够保留适体的空间结构,而且与已分化PC12细胞的亲合力不会降低.

  17. Comparative Study on the Protective Effects of Salidroside and Hypoxic Preconditioning for Attenuating Anoxia-Induced Apoptosis in Pheochromocytoma (PC12) Cells.

    Science.gov (United States)

    Hu, Yao; Lv, Xiumei; Zhang, Jing; Meng, Xianli

    2016-10-30

    BACKGROUND Hypoxia is an important sign that can result from body injuries or a special condition such as being at a high altitude or deep water diving. In the current studies, hypoxic preconditioning (HPC) plays a key role in reducing hypoxia-induced apoptosis. We aimed to study the pharmacologic preconditioning effects of salidroside versus those of HPC in hypoxia-/anoxia-induced apoptosis in PC12 cells (pheochromocytoma). MATERIAL AND METHODS PC12 cells were treated by different experimental conditions: control condition, hypoxia condition, HPC condition, low-/middle-/high-dose condition of salidroside, cyclosporine A (CsA), and oratractyloside (ATR). The cell viability, lactate dehydrogenase (LDH) activity, apoptosis, mitochondrial membrane potential (MMP), intracellular Ca2+, caspase-3 activity, and expression of Bcl-2 were detected in PC12 cells after the hypoxia treatment. Salidroside, extracted from the traditional Chinese herb Rhodiola rosea L, plays an essential role in reducing hypoxia-induced apoptosis in PC12 cells by the mitochondrial pathway. RESULTS Salidroside decreased the apoptosis and increased the viability of hypoxia-induced PC12 cells more effectively than HPC Moreover, salidroside markedly stabilized MMP and intracellular Ca2+, reduced or inhibited LDH and caspase-3 activity, and up-regulated Bcl-2; CsA and ATR showed corresponding function. CONCLUSIONS Salidroside administration restrains apoptosis induced by hypoxia in PC12 cells. The protective effects are mediated by preservation of mitochondrial integrity and MMP to inhibit the excessive Ca2+ influx and caspase-3 activity and to promote the Bcl-2 expression, providing a potential clinical and effective therapeutic mechanism to reduce deaths from ischemic or hypoxic injury.

  18. Synthesis and Protective Effect of New Ligustrazine-Benzoic Acid Derivatives against CoCl2-Induced Neurotoxicity in Differentiated PC12 Cells

    Directory of Open Access Journals (Sweden)

    Haimin Lei

    2013-10-01

    Full Text Available A series of novel ligustrazine-benzoic acid derivatives were synthesized and evaluated for their protective effect against cobalt chloride-induced neurotoxicity in differentiated PC12 cells. Combining hematoxylin and eosin staining, we found compound that (3,5,6-trimethylpyrazin-2-ylmethyl 3-methoxy-4-[(3,5,6-trimethylpyrazin-2-ylmethoxy]benzoate (4a displayed promising protective effect on the proliferation of the injured PC12 cells (EC50 = 4.249 µM. Structure-activity relationships are briefly discussed.

  19. Endothelin-2/Vasoactive Intestinal Contractor: Regulation of Expression via Reactive Oxygen Species Induced by CoCl22, and Biological Activities Including Neurite Outgrowth in PC12 Cells

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    Eiichi Kotake-Nara

    2006-01-01

    Full Text Available This paper reviews the local hormone endothelin-2 (ET-2, or vasoactive intestinal contractor (VIC, a member of the vasoconstrictor ET peptide family, where ET-2 is the human orthologous peptide of the murine VIC. While ET-2/VIC gene expression has been observed in some normal tissues, ET-2 recently has been reported to act as a tumor marker and as a hypoxia-induced autocrine survival factor in tumor cells. A recently published study reported that the hypoxic mimetic agent CoCl2 at 200 µM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced intracellular reactive oxygen species (ROS increase and neurite outgrowth in neuronal model PC12 cells. The ROS was generated by addition of CoCl2 to the culture medium, and the CoCl2-induced effects were completely inhibited by the antioxidant N-acetyl cysteine. Furthermore, interleukin-6 (IL-6 gene expression was up-regulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by CoCl2-induced ROS may be associated with neuronal differentiation through the regulation of IL-6 expression. CoCl2 acts as a pro-oxidant, as do Fe(II, III and Cu(II. However, some biological activities have been reported for CoCl2 that have not been observed for other metal salts such as FeCl3, CuSO4, and NiCl2. The characteristic actions of CoCl2 may be associated with the differentiation of PC12 cells. Further elucidation of the mechanism of neurite outgrowth and regulation of ET-2/VIC expression by CoCl2 may lead to the development of treatments for neuronal disorders.

  20. Novel fermented chickpea milk with enhanced level of γ-aminobutyric acid and neuroprotective effect on PC12 cells

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    Wen Li

    2016-08-01

    Full Text Available In this study, novel fermented chickpea milk with high γ -aminobutyric acid (GABA content and potential neuroprotective activity was developed. Fermentation starter that can produce GABA was selected from 377 strains of lactic acid bacteria isolated from traditional Chinese fermented foods. Among the screened strains, strain M-6 showed the highest GABA-producing capacity in De Man–Rogosa and Sharp (MRS broth and chickpea milk. M-6 was identified as Lactobacillus plantarum based on Gram staining, API carbohydrate fermentation pattern testing, and 16s rDNA sequencing. The complete gene encoding glutamate decarboxylase was cloned to confirm the presence of the gene in L. plantarum M-6. The fermentation condition was optimized by response surface methodology. Results demonstrated that L. plantarum M-6 produced the highest GABA content of 537.23 mg/L. The optimal condition included an inoculum concentration of 7%, presence of 0.2% (m/v monosodium glutamate and 55 µ M pyridoxal-5-phosphate, incubation temperature of 39 °C and fermentation time of 48 h . GABA-enriched chickpea milk exerted protective effects on PC12 cells against MnCl2 -induced injury. GABA-enriched chickpea milk improved cell viability and markedly attenuated the release of lactate dehydrogenase compared with the impaired cells.

  1. Early Decrease in Respiration and Uncoupling Event Independent of Cytochrome c Release in PC12 Cells Undergoing Apoptosis

    Science.gov (United States)

    Berghella, Libera; Ferraro, Elisabetta

    2012-01-01

    Cytochrome c is a key molecule in mitochondria-mediated apoptosis. It also plays a pivotal role in cell respiration. The switch between these two functions occurs at the moment of its release from mitochondria. This process is therefore extremely relevant for the fate of the cell. Since cytochrome c mediates respiration, we studied the changes in respiratory chain activity during the early stages of apoptosis in order to contribute to unravel the mechanisms of cytochrome c release. We found that, during staurosporine (STS)- induced apoptosis in PC12 cells, respiration is affected before the release of cytochrome c, as shown by a decrease in the endogenous uncoupled respiration and an uncoupling event, both occurring independently of cytochrome c release. The decline in the uncoupled respiration occurs also upon Bcl-2 overexpression (which inhibits cytochrome c release), while the uncoupling event is inhibited by Bcl-2. We also observed that the first stage of nuclear condensation during STS-induced apoptosis does not depend on the release of cytochrome c into the cytosol and is a reversibile event. These findings may contribute to understand the mechanisms affecting mitochondria during the early stages of apoptosis and priming them for the release of apoptogenic factors. PMID:22666257

  2. Maslinic Acid Protected PC12 Cells Differentiated by Nerve Growth Factor against β-Amyloid-Induced Apoptosis.

    Science.gov (United States)

    Yang, Yu-wan; Tsai, Chia-wen; Mong, Mei-chin; Yin, Mei-chin

    2015-12-01

    β-Amyloid peptide (Abeta) was used to induce apoptosis in PC12 cells differentiated by nerve growth factor, and the protective activities of maslinic acid (MA) at 2-16 μM were examined. Abeta treatment lowered Bcl-2 expression, raised Bax expression, and decreased cell viability. MA pretreatments decreased Bax expression, raised the Bcl-2/Bax ratio, and increased cell viability. MA pretreatments retained glutathione content and decreased subsequent Abeta-induced release of reactive oxygen species, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Abeta treatment up-regulated protein expression of p47(phox), gp91(phox), mitogen-activated protein kinase, advanced glycation end product receptor (RAGE), and nuclear factor-κ B (NF-κB). MA pretreatments at 2-16 μM suppressed the expression of proteins including gp91(phox), p47(phox), p-p38, and NF-κB p65, at 4-16 μM down-regulated RAGE and NF-κB p50 expression, and at 8 and 16 μM reduced p-ERK1/2 expression. These novel findings suggest that maslinic acid is a potent compound against Abeta-induced cytotoxicity.

  3. Antagonism of astragalus on PC12 cells apoptosis induced by manganese%黄芪对锰致PC12细胞凋亡的拮抗作用

    Institute of Scientific and Technical Information of China (English)

    陈斯琦; 黄国香; 李莉; 宋世震

    2008-01-01

    目的:研究黄芪对锰致多巴胺能神经元PC12细胞凋亡的拮抗作用.方法:体外染锰培养多巴胺能神经元PC12细胞,MTT比色法观察染锰PC12细胞的增殖情况;流式细胞仪(FMC)和原位缺口,末端标记法(TUNEL)检测细胞凋亡状况及黄芪的拮抗作用.结果:锰对PC12细胞的生长有明显的抑制作用,并呈剂量-反应关系;FMC和TUNEL检测结果均显示锰能诱导PC12细胞凋亡,并呈浓度依赖性,而加入黄芪后,各组凋亡率明显降低.结论:锰能够诱导PC12细胞凋亡,黄芪对锰诱导PC12细胞的凋亡有一定的拮抗作用.

  4. P38信号通路在Aβ介导的PC12细胞损伤中的作用%P38 signaling pathway participates in PC12 cell injury induced by Aβ

    Institute of Scientific and Technical Information of China (English)

    徐芳; 魏桂荣

    2010-01-01

    目的 探讨P38信号通路在Aβ介导的PC12细胞损伤中的作用.方法 体外培养PC12细胞,不同浓度的Aβ处理PC12细胞.用MTT法检测Aβ对PC12细胞活力的影响,免疫印迹法检测P38通路活化水平,并观察P38通路抑制剂SB203580对细胞活力的影响.结果 Aβ处理后,PC12细胞活力随Aβ浓度的增高而逐渐下降(P<0.05);Aβ处理后P38表达水平从4 h开始升高,12 h 达到高峰,并一直持续到24 h;SB203580预处理能够明显抑制P38信号通路活化,并对PC12细胞起到保护作用.结论 P38信号通路活化参与Aβ介导的PC12细胞损伤.

  5. NAC1, a POZ/BTB protein present in the adult mammalian brain, triggers apoptosis after adenovirus-mediated overexpression in PC-12 cells.

    Science.gov (United States)

    Korutla, Laxminarayana; Neustadter, Jason H; Fournier, Keith M; Mackler, Scott A

    2003-05-01

    POZ/BTB proteins influence cellular development and in some examples act as oncoproteins. However, several POZ/BTB transcription factors have been found in terminally differentiated neurons, where their functions remain unknown. One example is NAC1, a constitutively-expressed protein that can regulate behaviors associated with cocaine use. The present study represents an initial attempt to understand the actions of NAC1 within neurons by using adenoviral-mediated gene transfer into differentiated PC-12 cells. Cell survival in PC-12 cells overexpressing NAC1 was greatly reduced compared with cells infected by a control Ad-GFP. The morphological appearance of the dying cells was consistent with programmed cell death. Fragmentation of genomic DNA occurred in PC-12 cells infected with adenoviruses encoding NAC1 but not control viruses. NAC1 over expression was followed by the down regulation of the anti-apoptotic proteins Bcl-2 and Bcl-2-xl. Concurrently, levels of the pro-apoptotic proteins Bax and p53 increased following NAC1 overexpression. These observations suggest that NAC1expression in PC-12 cells induces apoptosis by altering the expression of these upstream mediators of the execution phase of programmed cell death. These findings raise the possibility that aberrantly regulated NAC1 expression in the mammalian brain may contribute to programmed cell death.

  6. Modulatory effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells.

    Science.gov (United States)

    Zhang, Min; Lee, Hak Ju; Park, Keun Hong; Park, Hyun Jin; Choi, Hyun Sook; Lim, Sung Cil; Lee, Myung Koo

    2012-06-01

    The effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Sesamin at concentration ranges of 20-75 μM exhibited a significant increase in intracellular dopamine levels at 24 h: 50 μM sesamin increased dopamine levels to 133% and tyrosine hydroxylase (TH) activity to 128.2% of control levels. Sesamin at 20-100 μM rapidly increased the intracellular levels of cyclic AMP (cAMP) to 158.3%-270.3% of control levels at 30 min. At 50 μM, sesamin combined with L-DOPA (50, 100 and 200 μM) further increased the intracellular dopamine levels for 24 h compared to L-DOPA alone. In the absence or presence of L-DOPA (100 and 200 μM), sesamin (50 μM) increased the phosphorylation of TH, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), as well as the mRNA levels of TH and CREB for 24 h, an effect which was reduced by L-DOPA (100 and 200 μM). In addition, 50 μM sesamin exhibited a protective effect against L-DOPA (100 and 200 μM)-induced cytotoxicity via the inhibition of reactive oxygen species (ROS) production and superoxide dismutase reduction, induction of extracellular signal-regulated kinase (ERK)1/2 and BadSer112 phosphorylation and Bcl-2 expression, and inhibition of cleaved-caspase-3 formation. These results suggested that sesamin enhanced dopamine biosynthesis and L-DOPA-induced increase in dopamine levels by inducing TH activity and TH gene expression, which was mediated by cAMP-PKA-CREB systems. Sesamin also protected against L-DOPA (100-200 μM)-induced cytotoxicity through the suppression of ROS activity via the modulation of ERK1/2, BadSer112, Bcl-2, and caspase-3 pathways in PC12 cells. Therefore, sesamin might serve as an adjuvant phytonutrient for neurodegenerative diseases.

  7. Hypoxia-induced cell death and changes in hypoxia-inducible factor-1 activity in PC12 cells upon exposure to nerve growth factor.

    Science.gov (United States)

    Charlier, Nico; Leclere, Norbert; Felderhoff, Ursula; Heldt, Julia; Kietzmann, Thomas; Obladen, Michael; Gross, Johann

    2002-07-15

    The transcription factor hypoxia-inducible factor-1 (HIF-1) strongly contributes to the expression of adaptive genes under hypoxic conditions. In addition, HIF-1 has been implicated in the regulation of delayed neuronal cell death. Suspension-grown and adherent PC12 cells treated with NGF were used as an experimental model for studying the relationship between hypoxia-induced cell death and activation of HIF-1. Cell damage was assessed by flow cytometry of double-stained (Annexin V and propidiumiodide) cells, and by analysis of the overall death parameters LDH and mitochondrial dehydrogenase. In parallel, cells were transfected with a control and a three-hypoxia-responsive-elements (HRE)-containing vector and HIF-1-driven luciferase activity was determined. Exposure of NGF-treated PC12 cells to hypoxia resulted in a higher cell death rate when compared to untreated controls. PC12 cells exposed for 2 days to NGF exhibited a decrease of HIF-1 activity up to a factor of ten. This decrease may contribute to the enhanced hypoxia-induced cell death via reduced expression of HIF-1alpha-regulated genes responsible for adaptation to hypoxia, like those for glucose transport proteins and enzymes of the glycolytic chain. The decrease in HIF-1 activity and the increase in hypoxia sensitivity may suggest that NGF act as an hierarchically organized signaling molecule.

  8. Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells.

    Science.gov (United States)

    Yen, Jui-Hung; Wu, Pei-Shan; Chen, Shu-Fen; Wu, Ming-Jiuan

    2017-04-17

    Fisetin (3,7,3',4'-tetrahydroxyflavone) is a dietary flavonol and exhibits antioxidant, anti-inflammatory, and neuroprotective activities. However, high concentration of fisetin is reported to produce reactive oxygen species (ROS), induce endoplasmic reticulum (ER) stress and cause cytotoxicity in cancer cells. The aim of this study is to investigate the cytoprotective effects of low concentration of fisetin against tunicamycin (Tm)-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptotic and autophagic markers were analyzed by Western blot. Gene expression of unfolded protein response (UPR) and Phase II enzymes was further investigated using RT-Q-PCR or Western blotting. Intracellular ROS level was measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) by a fluorometer. The effects of fisetin on mitogen activated protein kinases (MAPKs) and SIRT1 (Sirtuin 1) signaling pathways were examined using Western blotting and specific inhibitors. Fisetin (Fisetin attenuated Tm-mediated expression of ER stress genes, such as glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP also known as GADD153) and Tribbles homolog 3 (TRB3), but induced the expression of nuclear E2 related factor (Nrf)2-targeted heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) and cystine/glutamate transporter (xCT/SLC7A11), in both the presence and absence of Tm. Moreover, fisetin enhanced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-JUN NH₂-terminal protein kinase), and p38 MAPK. Addition of JNK and p38 MAPK inhibitor significantly antagonized its cytoprotective activity and modulatory effects on UPR. Fisetin also restored Tm-inhibited SIRT1 expression and addition of sirtinol (SIRT1 activation inhibitor) significantly blocked fisetin-mediated cytoprotection. In conclusion, this result shows that fisetin activates

  9. Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia-reoxygenation injury in pheochromocytoma (PC12) cells

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Can [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Zhang, Li-Yang [Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Cancer Research Institute, Central South University, 110 Xiang Ya Road, Changsha 410078 (China); Chen, Hong [Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Xiao, Ling [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Liu, Xian-Peng, E-mail: xliu@lsuhsc.edu [Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932 (United States); Zhang, Jian-Xiang, E-mail: jianxiangzhang@yahoo.cn [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Overexpression of human CUL4A (hCUL4A) in PC12 cells. Black-Right-Pointing-Pointer The effects of hCUL4A on hypoxia-reoxygenation injury were investigated. Black-Right-Pointing-Pointer hCUL4A suppresses apoptosis and DNA damage and thus promotes cell survival. Black-Right-Pointing-Pointer hCUL4A regulates apoptosis-related proteins and cell cycle regulators. -- Abstract: The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.

  10. Microarray and synchronization of neuronal differentiation with pathway changes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databank in nerve growth factor-treated PC12 cells.

    Science.gov (United States)

    Lin, Chih-Ming; Feng, Wayne

    2012-08-01

    The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a systems level. We performed microarray experiments and used the KEGG database, systems biology analysis, and annotation of pathway function to study nerve growth factor (NGF)-induced differentiation of PC12 cells. Cells were cultured to 70%-80% confluence, treated with NGF for 1 or 3 hours (h), and RNA was extracted. Stage 1 data analysis involved analysis of variance (ANOVA), and stage 2 involved cluster analysis and heat map generation. We identified 2020 NGF-induced PC12 genes (1038 at 1 h and 1554 at 3 h). Results showed changes in gene expression over time. We compared these genes with 6035 genes from the KEGG database. Cross-matching resulted in 830 genes. Among these, we identified 395 altered genes (155 at 1 h and 301 at 3 h; 2-fold increase from 1 h to 3 h). We identified 191 biologic pathways in the KEGG database; the top 15 showed correlations with neuronal differentiation (mitogen-activated protein kinase [MAPK] pathway: 35 genes at 1 h, 54 genes at 3 h; genes associated with axonal guidance: 12 at 1 h, 26 at 3 h; Wnt pathway: 16 at 1 h, 25 at 3 h; neurotrophin pathway: 4 at 1 h, 14 at 3 h). Thus, we identified changes in neuronal differentiation pathways with the KEGG database, which were synchronized with NGF-induced differentiation.

  11. (-)Schisandrin B ameliorates paraquat-induced oxidative stress by suppressing glutathione depletion and enhancing glutathione recovery in differentiated PC12 cells.

    Science.gov (United States)

    Lam, Philip Y; Ko, Kam Ming

    2011-01-01

    Exposure to paraquat (PQ; N,N'-dimethyl-4-4'-bipyridium), a potent herbicide, can lead to neuronal cell death and increased risk of Parkinson's disease because of oxidative stress. In this study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on PQ-induced cell injury in differentiated pheochromocytoma cells (PC12). PQ treatment caused cell injury in PC12 cells, as indicated by the significant increase in lactate dehydrogenase (LDH) leakage. Pretreatment with (-)Sch B (5 μM) protected against PQ-induced toxicity in PC12 cells, as evidenced by the significant decrease in LDH leakage. (-)Sch B induced the cytochrome P-450-mediated reactive oxygen species generation in differentiated PC12 cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with an increase in cellular reduced glutathione (GSH) level as well as the enhancement of γ-glutamylcysteine ligase (GCL) and glutathione reductase (GR) activity in PQ-challenged cells. Both GCL and GR inhibitors abrogated the cytoprotective effect of (-)Sch B in PQ-challenged cells. The biochemical mechanism underlying the GSH-enhancing effect of (-)Sch B was further investigated in PC12 cells subjected to an acute peroxide challenge. Although the initial GSH depletion induced by peroxide was reduced through GR-catalyzed regeneration of GSH in (-)Sch B-pretreated cells, the later enhanced GSH recovery was mainly mediated by GCL-catalyzed GSH synthesis. The results suggest that (-)Sch B treatment may increase the resistance of dopaminergic cells against PQ-induced oxidative stress through reducing the extent of oxidant-induced GSH depletion and enhancing the subsequent GSH recovery.

  12. The Study of the Transfection Efficiency of PC12 Cells Transfeced by Cationic Liposomes Lipofectamine2000%阳离子脂质体Lipofectamine2000对PC12细胞转染效率的研究

    Institute of Scientific and Technical Information of China (English)

    杨云廷; 付崇罗

    2011-01-01

    通过改变转染试剂及DNA用量和转染时间等影响转染效率的重要因素来实现阳离子脂质体lipofectamineTM2000(lipo2000)对PC12细胞的高效转染.结果表明,lipo2000转染PC12细胞的最佳转染条件:lipo2000用量为5μL,DNA 2μg,转染时间为6 h,这种条件下的PC12细胞转染率高达40%,且未影响细胞的正常分泌,这为应用PC12细胞进行神经生物学研究提供了基础资料.

  13. Propofol prevents autophagic cell death following oxygen and glucose deprivation in PC12 cells and cerebral ischemia-reperfusion injury in rats.

    Directory of Open Access Journals (Sweden)

    Derong Cui

    Full Text Available BACKGROUND: Propofol exerts protective effects on neuronal cells, in part through the inhibition of programmed cell death. Autophagic cell death is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. We therefore studied whether propofol could attenuate the formation of autophagosomes, and if so, whether the inhibition of autophagic cell death mediates the neuroprotective effects observed with propofol. METHODOLOGY/PRINCIPAL FINDINGS: The cell model was established by depriving the cells of oxygen and glucose (OGD for 6 hours, and the rat model of ischemia was introduced by a transient two-vessel occlusion for 10 minutes. Transmission electron microscopy (TEM revealed that the formation of autophagosomes and autolysosomes in both neuronal PC12 cells and pyramidal rat hippocampal neurons after respective OGD and ischemia/reperfusion (I/R insults. A western blot analysis revealed that the autophagy-related proteins, such as microtubule-associated protein 1 light chain 3 (LC3-II, Beclin-1 and class III PI3K, were also increased accordingly, but cytoprotective Bcl-2 protein was decreased. The negative effects of OGD and I/R, including the formation of autophagosomes and autolysosomes, the increase in LC3-II, Beclin-1 and class III PI3K expression and the decline in Bcl-2 production were all inhibited by propofol and specific inhibitors of autophagy, such as 3-methyladenine (3-MA, LY294002 and Bafilomycin A1 (Baf,. Furthermore, in vitro OGD cultures and in vivo I/R rats showed an increase in cell survival following the administration of propofol, as assessed by an MTT assay or histochemical analyses. CONCLUSIONS/SIGNIFICANCE: Our data suggest that propofol can markedly attenuate autophagic processes via the decreased expression of autophagy-related proteins in vitro and in vivo. This inhibition improves cell survival, which provides a novel explanation for the pleiotropic effects of

  14. Neferine attenuates the protein level and toxicity of mutant huntingtin in PC-12 cells via induction of autophagy.

    Science.gov (United States)

    Wong, Vincent Kam Wai; Wu, An Guo; Wang, Jing Rong; Liu, Liang; Law, Betty Yuen-Kwan

    2015-02-18

    Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington's disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74) in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7)-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

  15. Exposure of PC12 cells to NGF/ethanol results in accelerated differentiation and altered gene expression

    Energy Technology Data Exchange (ETDEWEB)

    White, K.R.; Wooten, M.W. (Auburn Univ., AL (United States))

    1991-03-11

    The role of alcohols in affecting neuromorphogenesis was investigated in the pheochromocytoma cell line, PC12. The effect of ethanol at physiological concentrations in this system leads to accelerated neurite extension in the presence of suboptimal concentrations of NGF. Accelerated morphological differentiation was dependent upon the side chain length of the alcohol and not inhibited by pyrazole. Ethanol/NGF induced neurite extension can be blocked with 50nM of K252a, but not sphingozine, H7, H89, genistein or okadaic acid. Changes in the expression of 17 NGF-induced and/or neuronal transcripts were examined in relationship to time of NGF/ethanol exposure; dose of NGF/ethanol; and side-chain length of NFG/alcohol. The authors studies indicate that ethanol potentiates the effects of NFG and subsequent neurogenesis through both protein kinase C and cAMP-independent pathways. In addition, these data show that ethanol is capable of altering gene expression in a specific manner.

  16. Neferine Attenuates the Protein Level and Toxicity of Mutant Huntingtin in PC-12 Cells via Induction of Autophagy

    Directory of Open Access Journals (Sweden)

    Vincent Kam Wai Wong

    2015-02-01

    Full Text Available Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington’s disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74 in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

  17. Cdk5及p35在NGF撤退诱导的已分化PC12细胞凋亡中的作用研究%The roles of Cdk5 and p35 in apoptosis of differentiated PC12 cells induced by NGF withdrawal

    Institute of Scientific and Technical Information of China (English)

    沈晗; 吴少波; 张百芳; 彭芳芳; 武栋成

    2012-01-01

    目的:探讨Cdk5及p35在神经生长因子(NGF)撤退诱导的PC12细胞凋亡中的作用机制.方法:建立NGF撤退诱导的已分化PC12细胞凋亡模型,Western blotting检测Cdk5及p35在凋亡过程中表达变化情况,利用Cdk5特异性抑制剂Roscovitine预处理已分化PC12细胞,检测其对NGF撤退诱导的凋亡作用影响,向已分化PC12细胞转染真核表达质粒pCMV-p35-IRES-Cdk5,检测过表达CdkS/p35对PC12细胞凋亡的影响.结果:NGF撤退36h会引起已分化PC12细胞出现典型的DNA Ladder凋亡特征,MTT检测结果也显示,NGF撤退对PC12细胞的损伤呈时间依赖性;Roscovitine预处理已分化PC12细胞可以抑制NGF撤退诱导的细胞凋亡率,但不影响Cdk5/p35蛋白表达水平;向已分化PC12细胞中转染真核表达质粒后,能检测到Cdk5/p35蛋白的过表达,并引起PC12细胞出现凋亡样改变.结论:Cdk5及p35的活化与NGF撤退诱导的已分化PC12细胞凋亡过程密切相关,抑制Cdk5的活化有抑制细胞凋亡保护神经元的作用.%Aim;To investigate the roles of Cdk5/p35 in apoptosis of induced by NGF withdrawal. Methods: The models of differentiated PC12 cells apoptosis were established with NGF withdrawal and Western blotting were carried out to detect the expressions of Cdk5 and p35 during the apoptotic process. Roscovitine is the specific inhibitor of Cdk5, which was used for pretreatment of differentiated PC12 cells to determine the impact on apoptosis. The Eukaryotic expression plasmids pCMV-p35-IRES-Cdk5 were transfected into differentiated PC 12 cells and the effects on apoptosis of PC 12 cells were observed. Results : The typical feature of apoptosis, DNA Ladder could be detected at 36 h after NGF withdrawal. The results of MTT indicated that the damages to PC12 cells showed a time-dependent. After preincubation with Roscovitine, the rates of apoptosis were decreased, but the protein expression levels of Cdk5 and p35 did not change significantly. The

  18. The PERK-eIF2α signaling pathway is involved in TCDD-induced ER stress in PC12 cells.

    Science.gov (United States)

    Duan, Zhiqing; Zhao, Jianya; Fan, Xikang; Tang, Cuiying; Liang, Lingwei; Nie, Xiaoke; Liu, Jiao; Wu, Qiyun; Xu, Guangfei

    2014-09-01

    Studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptotic cell death in neuronal cells. However, whether this is the result of endoplasmic reticulum (ER) stress-mediated apoptosis remains unknown. In this study, we determined whether ER stress plays a role in the TCDD-induced apoptosis of pheochromocytoma (PC12) cells and primary neurons. PC12 cells were exposed to different TCDD concentrations (1, 10, 100, 200, or 500nM) for varying lengths of time (1, 3, 6, 12, or 24h). TCDD concentrations much higher than 10nM (100, 200, or 500nM) markedly increased glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) levels, which are hallmarks of ER stress. We also evaluated the effects of TCDD on ER morphology in PC12 cells and primary neurons that were treated with different TCDD concentrations (1, 10, 50, or 200nM) for 24h. Ultrastructural ER alterations were observed with transmission electron microscopy in PC12 cells and primary neurons treated with high concentrations of TCDD. Furthermore, TCDD-induced ER stress significantly promoted the activation of the PKR-like ER kinase (PERK), a sensor for the unfolded protein response (UPR), and its downstream target eukaryotic translation initiation factor 2 α (eIF2α); in contrast, TCDD did not appear to affect inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), two other UPR sensors. Importantly, TCDD significantly inhibited eIF2α phosphorylation and triggered apoptosis in PC12 cells after 6-24h of treatment. Salubrinal, which activates the PERK-eIF2α pathway, significantly enhanced eIF2α phosphorylation in PC12 cells and attenuated the TCDD-induced cell death. In contrast, knocking down eIF2α using small interfering RNA markedly enhanced TCDD-induced cell death. Together, these results indicate that the PERK-eIF2α pathway plays an important role in TCDD-induced ER stress and apoptosis in PC12 cells.

  19. Protective Effect of Cordycepin on PC12 Cells Injury Induced by β-amyloid Protein%虫草素对β-淀粉样蛋白诱导PC12细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    赵国华; 韩笑

    2012-01-01

    目的 应用Aβ25-35孵育PC12细胞制作细胞损伤模型,探讨虫草素对PC12细胞损伤的保护作用.方法 将不同组别PC12细胞用Aβ25-35诱导48 h后,采用MTT法测定细胞活力,LDH法测定细胞膜通透性,化学比色法测定细胞中SOD活性和MDA含量,Western blot法检测PC12细胞中Caspase-3的表达.结果 与模型组相比,两种剂量的虫草素均使细胞存活率显著增加,细胞培养上清中LDH含量显著减少,细胞中SOD活性显著升高,mDA含量显著下降,并明显降低PC12细胞Caspase-3的表达.结论 虫草素对Aβ325-35诱导的PC12细胞死亡、氧化损伤和细胞凋亡有明显保护作用.

  20. 硫化氢对H2O2损伤PC12细胞的保护作用%Protective Effect of Hydrogen Sulfide on H2O2-Induced Damage in PC12 Cells

    Institute of Scientific and Technical Information of China (English)

    杨丝丝; 姜志胜; 唐小卿

    2007-01-01

    目的 探讨硫化氢对氧化应激损伤PC12细胞的保护作用.方法 以H2O2损伤PC12细胞为氧化应激损伤的模型,甲氮甲唑蓝法检测细胞增殖状况;Hoechst荧光染色观察细胞形态和核形态;碘化丙啶染色、流式细胞术检测细胞凋亡.结果 硫化氢可明显减少H2O2诱导的PC12细胞核呈浓染致密的固缩形态或颗粒状荧光的细胞数;200和400 μmol/L硫化氢均可降低200和400 μmol/L H2 O2作用24 h后对PC12细胞生长的抑制率,明显抑制200和400 μmol/L H2O2作用24 h后对PC12细胞凋亡的诱导作用.结论 硫化氢对氧化应激损伤PC12细胞具有保护作用.

  1. CART (cocaine- and amphetamine-regulated transcript) peptide specific binding sites in PC12 cells have characteristics of CART peptide receptors.

    Science.gov (United States)

    Nagelová, Veronika; Pirník, Zdeno; Železná, Blanka; Maletínská, Lenka

    2014-02-14

    CART (cocaine- and amphetamine-regulated transcript) peptide is a neuropeptide with a powerful central anorexigenic effect. Specific CART peptide binding sites, most likely CART peptide receptors, have been found in PC12 cells. This study further characterizes the CART peptide binding sites in PC12 cells. After differentiation to a neuronal phenotype with nerve growth factor, the number of CART peptide binding sites in PC12 cells tripled. Following dexamethasone treatment, which transforms PC12 cells into chromaffin-like cells, the number of CART peptide binding sites substantially decreased. CART peptide did not affect the differentiation or acetylcholinesterase activity of PC12 cells, indicating that CART peptide does not participate in differentiation or neuronal activity. CART peptide increased the phosphorylation of SAPK/JNK (stress-activated protein kinase/c-Jun-amino-terminal kinase) and subsequent c-Jun protein expression. These effects were reversed by SP600125, a specific JNK-kinase inhibitor. CART peptide did not significantly affect ERK (extracellular signal-regulated kinase), CREB (cAMP responsive element binding protein), or p38 phosphorylation and c-Fos protein expression. Central administration of CART peptide into mice also resulted in increased c-Jun positive cells in dorsomedial hypothalamic nucleus and nucleus of the solitary tract, areas involved in food intake regulation. Activation of c-Jun by CART peptide might indicate a possible role of CART peptide in managing stress conditions rather than a role in cell proliferation or differentiation as well as the more complex and/or specific regulation ways by transcription factors in some nuclei involved in food intake regulation. The characteristics of stress that CART peptide potentially mediates should be further studied.

  2. NFAT1 and NFAT3 cooperate with HDAC4 during regulation of alternative splicing of PMCA isoforms in PC12 cells.

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    Michalina Kosiorek

    Full Text Available The bulk of human genes undergo alternative splicing (AS upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs, abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells. PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4, whose transcript products undergo alternative splicing giving almost 30 variants.In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT and histone deacetylases (HDACs. Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation.We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

  3. Proteases inhibition assessment on PC12 and NGF treated cells after oxygen and glucose deprivation reveals a distinct role for aspartyl proteases.

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    Aristidis Kritis

    Full Text Available Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD or ischemic preconditioning (IPS. Nerve Growth Factor (NGF treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N(2/CO(2 (93:5% in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.

  4. Upregulation of the SERCA-type Ca2+ pump activity in response to endoplasmic reticulum stress in PC12 cells

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    Frandsen Aase

    2001-04-01

    Full Text Available Abstract Background Ca2+-ATPases of endoplasmic reticulum (SERCAs are responsible for maintenance of the micro- to millimolar Ca2+ ion concentrations within the endoplasmic reticulum (ER of eukaryotic cells. This intralumenal Ca2+ storage is important for the generation of Ca2+ signals as well as for the correct folding and posttranslational processing of proteins entering ER after synthesis. ER perturbations such as depletion of Ca2+ or abolishing the oxidative potential, inhibition of glycosylation, or block of secretory pathway, activate the Unfolded Protein Response, consisting of an upregulation of a number of ER-resident chaperones/stress proteins in an effort to boost the impaired folding capacity. Results We show here that in PC12 cells, depletion of ER Ca2+ by EGTA, as well as inhibition of disulphide bridge formation within the ER by dithiotreitol or inhibition of N-glycosylation by tunicamycin, led to a 2- to 3-fold increase of the SERCA-mediated 45Ca2+ transport to microsomes isolated from cells exposed to these stress agents. The time course of this response corresponded to that for transcriptional upregulation of ER stress proteins, as well as to the increase in the SERCA2b mRNA, as we recently observed in an independent study. Conclusions These findings provide the first functional evidence for the increase of SERCA pumping capacity in cells subjected to the ER stress. Since at least three different and unrelated mechanisms of eliciting the ER stress response were found to cause this functional upregulation of Ca2+ transport into the ER, these results support the existence of a coupling between the induction of the UPR pathway in general, and the regulation of expression of at least one of the SERCA pump isoforms.

  5. Differential effects of classical and atypical antipsychotic drugs on rotenone-induced neurotoxicity in PC12 cells.

    Science.gov (United States)

    Tan, Qing-Rong; Wang, Xin-Zhao; Wang, Chuan-Yue; Liu, Xiao-Jun; Chen, Yun-Chun; Wang, Huai-Hai; Zhang, Rui-Guo; Zhen, Xue-Chu; Tong, Yao; Zhang, Zhang-Jin

    2007-12-01

    Although classical and atypical antipsychotics may have different effects against neurotoxicity, the underlying mechanisms remain to be elucidated. In the present study, we compared the atypical agents, risperidone (RIP), olanzapine (OLZ), and quetiapine (QTP), with the classical agent haloperidol (HAL) in reducing cytotoxicity induced by rotenone, a mitochondrial complex I inhibitor, in PC12 cells. We also determined whether there were differential effects of RIP and HAL on the expression of brain-derived neurotrophic factor (BDNF), signal transducers and activators of transcription-3 (STAT-3), and the immediate early gene c-fos, as well as intracellular levels of calcium. Exposure to 6 muM rotenone for 24 h resulted in a significant decrease in cell viability and apoptotic alteration. The rotenone-induced cytotoxicity was dose-dependently worsened by pretreatment with HAL, but significantly improved by the aforementioned atypical agents at low doses. Real-time PCR analysis revealed that HAL pretreatment significantly increased BDNF mRNA expression but did not alter c-fos and STAT-3 expression compared to rotenone-exposed cells. Unlike HAL, RIP pretreatment produced a significant elevation of all the three substance mRNA expression and the expression intensity was 2.6- to 4.6-fold greater than HAL. Pretreatment with RIP, but not HAL, also effectively prevented an elevation of intracellular levels of calcium provoked by rotenone. These results suggest that the protective effects of atypical antipsychotics are associated with a greater capacity to enhance pro-cell survival factors, therapeutic biomarker expression, and blockade of calcium influx. This may provide an alternative for explaining therapeutic advantages of atypical agents observed in clinical use.

  6. Detection of the interaction between SNAP25 and rabphilin in neuroendocrine PC12 cells using the FLIM/FRET technique.

    Science.gov (United States)

    Lee, Jiung-De; Chang, Yu-Fen; Kao, Fu-Jen; Kao, Lung-Sen; Lin, Chung-Chih; Lu, Ai-Chu; Shyu, Bai-Chuang; Chiou, Shih-Hwa; Yang, De-Ming

    2008-01-01

    Exocytosis has been proposed to contain four sequential steps, namely docking, priming, fusion, and recycling, and to be regulated by various proteins-protein interactions. Synaptosomal-associated protein of 25 kDa (SNAP25) has recently been found to bind rabphilin, the Rab3A specific binding protein, in vitro. However, it is still unclear whether SNAP25 and rabphilin interact during exocytosis within cells in vivo. This problem was addressed by the integration of fluorescence resonance energy transfer (FRET) with high sensitivity fluorescence lifetime imaging microscopy (FLIM) to observe this protein-protein interaction. Enhanced green fluorescence protein-labeled SNAP25 (donor) and red fluorescence protein-labeled rabphilin (acceptor) were expressed in neuroendocrine PC12 cells as a FRET pair and ATP stimulation was carried out for various durations. With 10 s stimulation, a 0.17-ns left shift of the lifetime peak was found when compared with donor only. Analysis of the lifetime image further suggested that the lifetime recovered to a similar level as the donor only in a time dependent manner. Four-dimensional (4D) images by FLIM provided useful information indicating that the interaction of SNAP25 and rabphilin occurred particularly within optical sections near cell membrane. Together the results suggest that SNAP25 bound rabphilin loosely at docking step before exocytosis and the binding became tighter at the very start of exocytosis. Finally, these two proteins dissociated after stimulation. To our knowledge, this is the first report to demonstrate the interaction of SNAP25 and rabphilin in situ using the FLIM-FRET technique within neuroendocrine cells.

  7. Vitamin B(12), a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells.

    Science.gov (United States)

    Ina, Atsutoshi; Kamei, Yuto

    2006-11-01

    We previously isolated an analog to chlorophyll-related compounds, pheophytin a, from the marine brown alga Sargassum fulvellum and demonstrated that it is a neurodifferentiation compound. In the current study, we investigated the effects of the pheophytin a analog vitamin B(12) on PC12 cell differentiation. In the presence of a low level of nerve growth factor (10 ng ml(-1)), vitamin B(12 )demonstrated neurite outgrowth-promoting activity in PC12 cells. The effect was dose-dependent in the range of 6-100 muM. In the absence of nerve growth factor, vitamin B(12) did not promote differentiation. To investigate the mechanism for this effect, we conducted differentiation assays and western blot analysis with signal transduction inhibitors and found that vitamin B(12) did not promote PC12 cell differentiation in the presence of K252a or U0126 inhibitors. These results suggest that vitamin B(12 )stimulates PC12 cell differentiation through enhancement of the mitogen-activated protein kinase signal transduction pathway, which is also induced by nerve growth factor. Thus, vitamin B(12) may be a good candidate for treatment of neurodegenerative diseases such as Alzheimer's disease.

  8. Adenosine triphosphate-sensitive potassium channel opener protects PC12 cells against hypoxia-induced apoptosis through PI3K/Akt and Bcl-2 signaling pathways

    Institute of Scientific and Technical Information of China (English)

    Hong Zhang; Chunhong Jia; Danyang Zhao; Yang Lu; Runling Wang; Jia Li

    2010-01-01

    Although previous studies have shown the neuroprotective effects of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel opener against ischemic neuronal damage, little is known about the mechanisms involved. Phosphatidylinositol-3 kinase (PI3K)/v-akt murine thy-moma viral oncogene homolog (Akt) and Bcl-2 are thought to be important factors that mediate neuroprotection. The present study investigated the effects of KATP openers on hypoxia-induced PC12 cell apoptosis, as well as mRNA and protein expression of Akt and Bcl-2. Results demon-strated that pretreatment of PC12 cells with pinacidil, a KATP opener, resulted in decreased PC12 cell apoptosis following hypoxia, as detected by Annexin-V fluorescein isothiocyanate/ propidium iodide double staining flow cytometry. In addition, mRNA and protein expression of phosphorylated Akt (p-Akt) and Bcl-2 increased, as detected by immunofluorescence, Western blot analysis, and reverse-transcription polymerase chain reaction. The protective effect of this preconditioning was attenuated by glipizide, a selective KATP blocker. These results demonstrate for the first time that the protective mechanisms of KATP openers on PC12 cell apoptosis following hypoxia could result from activation of the PI3K/Akt signaling pathway, which further activates expression of the downstream Bcl-2 gene.

  9. Effects of Nogo-A Silencing on TNF-α and IL-6 Secretion and TH Downregulation in Lipopolysaccharide-Stimulated PC12 Cells

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    Jianbin Zhong

    2015-01-01

    Full Text Available Parkinson’s disease (PD is a common degenerative disease that lacks efficient treatment. Myelin-associated neurite outgrowth inhibitor A (Nogo-A is relevant with inhibition of nerve regeneration and may play vital role in pathogenesis of PD. The study aimed to establish the shRNA expression plasmids of Nogo-A gene and explore the regulatory effects of Nogo-A silencing on the expression of inflammation factor tumor necrosis factor-alpha (TNF-alpha and interleukin-6 (IL-6 as well as tyrosine hydroxylase (TH in lipopolysaccharide- (LPS- stimulated rat PC12 cells. The results showed that both mRNA and protein levels of Nogo-A in pGenesil-nogoA-shRNA group were downregulated. The viabilities of PC12 cells decreased with increase of LPS concentrations. LPS significantly increased the supernatant TNF-alpha and IL-6 concentrations and reduced TH protein expression in PC12 cells, while silencing Nogo-A could block these effects. These results suggested that LPS can activate PC12 cells to secrete inflammatory cytokines and lower the TH expression, which can be regulated by Nogo-A gene silencing. Nogo-A silencing might provide new ideas for PD treatment in the future.

  10. Dopamine as a potent inducer of cellular glutathione and NAD(P)H:quinone oxidoreductase 1 in PC12 neuronal cells: a potential adaptive mechanism for dopaminergic neuroprotection.

    Science.gov (United States)

    Jia, Zhenquan; Zhu, Hong; Misra, Bhaba R; Li, Yunbo; Misra, Hara P

    2008-11-01

    Dopamine auto-oxidation and the consequent formation of reactive oxygen species and electrophilic quinone molecules have been implicated in dopaminergic neuronal cell death in Parkinson's disease. We reported here that in PC12 dopaminergic neuronal cells dopamine at noncytotoxic concentrations (50-150 muM) potently induced cellular glutathione (GSH) and the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), two critical cellular defenses in detoxification of ROS and electrophilic quinone molecules. Incubation of PC12 cells with dopamine also led to a marked increase in the mRNA levels for gamma-glutamylcysteine ligase catalytic subunit (GCLC) and NQO1. In addition, treatment of PC12 cells with dopamine resulted in a significant elevation of GSH content in the mitochondrial compartment. To determine whether treatment with dopamine at noncytotoxic concentrations, which upregulated the cellular defenses could protect the neuronal cells against subsequent lethal oxidative and electrophilic injury, PC12 cells were pretreated with dopamine (150 muM) for 24 h and then exposed to various cytotoxic concentrations of dopamine or 6-hydroxydopamine (6-OHDA). We found that pretreatment of PC12 cells with dopamine at a noncytotoxic concentration led to a remarkable protection against cytotoxicity caused by dopamine or 6-OHDA at lethal concentrations, as detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. In view of the critical roles of GSH and NQO1 in protecting against dopaminergic neuron degeneration, the above findings implicate that upregulation of both GSH and NQO1 by dopamine at noncytotoxic concentrations may serve as an important adaptive mechanism for dopaminergic neuroprotection.

  11. Hydrogen peroxide induces the activation of the phospholipase C-γ1 survival pathway in PC12 cells: protective role in apoptosis

    Institute of Scientific and Technical Information of China (English)

    Wenli Yuan; Jiazhi Guo; Xingguo Li; Zhirong Zou; Guangxue Chen; Jun Sun; Tinghua Wang; Di Lu

    2009-01-01

    It has been reported that phospholipase C-γ1 (PLC-γ1) plays an important protective role in hydrogen peroxide (H2O2)-induced pheochromocytoma (PC) 12 cells death. However, most studies have used high doses of H2O2 and the downstream targets of PLC-γ1 activation remain to be identified. The present study was designed to examine the roles of PLC-γ1 signaling pathway in the apoptosis of PC12 cells induced by low dose of H2O2, as well as the downstream factors involved in this pathway. Low-dose treatment of H2O2 resulted in PLC-γ1 tyrosine phosphorylation in a time-dependent manner and H2O2 killed the PC12 cells by inducing necrosis. In contrast, pretreatment of PC12 cells with U73122, a specific inhibitor of PLC, markedly increased the percentage of dead cells. The mode of cell death was converted to apoptosis as determined by Hoechst/PI nuclear staining and fluorescence microscopy. Western blot analysis demonstrated that the expression of Bcl-2 protein and the activation of pro-caspase-3 were not significantly affected by low dose of H2O2 alone. However, after pretreatment with U73122, Bcl-2 protein expression was dramatically decreased and the activation of pro-caspase-3 was sig-nificantly increased. We concluded that PLC-γ1 plays an important protective role in H2O2-induced PC12 cells death. Bcl-2 and caspase-3 probably participate in the signaling pathway as downstream factors.

  12. The role of heat shock protein 70 in the protective effect of YC-1 on β-amyloid-induced toxicity in differentiated PC12 cells.

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    Yung-Chieh Tsai

    Full Text Available Neurodegenerative brain disorders such as Alzheimer's disease (AD have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70 plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC, on Aβ25-35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aβ25-35 (10 µM significantly increased p25 protein production in a pattern that was consistent with the increase in μ-calpain expression. Moreover, Aβ25-35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5-10 µM prevented the cell death induced by Aβ25-35. In addition, YC-1 (1, 10 µM significantly blocked Aβ25-35-induced μ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5-20 µM alone or combined with Aβ25-35 (10 µM significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM did not affect the neuroprotective effect of YC-1 against Aβ25-35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aβ25-35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.

  13. Antidepressant-like Effect of Cell-free Filtrate of Sodium Ferulate-induced and Differentioned PC12 Cell Lysates%阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液的抗抑郁样效果

    Institute of Scientific and Technical Information of China (English)

    廖铭能; 于立坚; 张永平; 马润娣; 张霄瑜; 于廷曦

    2011-01-01

    阿魏酸(ferulic acid,FA)是一种广泛存在的低毒酚酸,阿魏酸钠(sodium ferulate,SF)则是其钠盐.先前的研究已经证实,阿魏酸钠具有显著的神经保护和神经发生增强作用及抗抑郁效果.该研究的目的在于探讨阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液可能的抗抑郁效果.PC12细胞在含80 μmol/L阿魏酸钠的DMEM培养基中孵育6 d,无菌条件下制备阿魏酸钠诱导分化的PC12细胞液的无细胞滤液,测定PC12细胞裂解液无细胞滤液中残留的阿魏酸钠量.以慢性不可预期的多种刺激制造大鼠抑郁模型,用行为学、形态学、免疫组织化学和BrdU掺入等方法观察并检测阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液对慢性应激大鼠抑郁模型行为学、海马的组织病理学、海马和大脑皮质的神经生长因子(nerve growth factor,NGF)及脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)的表达及神经发生的影响.实验证实,阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液能改善抑郁症样模型大鼠的行为学障碍,上调其海马和大脑皮质NGF和BNDF的表达,增加海马神经干细胞/神经前体细胞的增殖.由此可见,阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液有明显的抗抑郁效果,而其抗抑郁效果可能源自它的上调NGF和BNDF以及其增强神经发生作用.%Ferulic acid (FA), 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid, is one of the most common phenolic acids with low toxicity, and sodium ferulate (SF) is its sodium salt.Our previous work demonstrates that SF has significant neuroprotective and neurogenesis-enhancing actions and antidepressant-like effects.The aim of this study was to investigate a potential antidepressant-like effect of cell-free filtrate of sodium ferulate-induced and differentioned PC 12 cell lysates (SFIDPC 12CL) in the chronic mild stress (CMS)-induced depression-like model rats.PC

  14. Cross talk among PMCA, calcineurin and NFAT transcription factors in control of calmodulin gene expression in differentiating PC12 cells.

    Science.gov (United States)

    Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena; Zylinska, Ludmila

    2017-04-01

    Brain aging is characterized by progressive loss of plasma membrane calcium pump (PMCA) and its activator - calmodulin (CaM), but the mechanism of this phenomenon remains unresolved. CaM encoded by three genes Calm1, Calm2, Calm3, works to translate Ca(2+) signal into changes in frequently opposite cellular activities. This unique function allows CaM to affect gene expression via stimulation of calcineurin (CaN) and its downstream target - nuclear factor of activated T-cells (NFAT) and to terminate Ca(2+) signal by stimulation of its extrusion. PMCA, which exists in four isoforms PMCA1-4, may in turn shape the pattern of Ca(2+) transients and control CaN activity by its direct binding. Therefore, the interplay between PMCA, CaM and CaN/NFAT is highly plausible. To verify that, we used differentiated PC12 cells with reduced expression of PMCA2 or PMCA3 to mimic the potential changes in aged brain. Manipulation in PMCAs level decreased CaM protein in PMCA2 or PMCA3-reduced lines that was accompanied by down-regulation of Calm1 and Calm2 in both lines, but Calm3 only in PMCA2-reduced cells. Further studies showed substantially higher NFATc2 nuclear accumulation and increased NFAT transcriptional activity. Blocking of CaN/NFAT signalling resulted in almost full CaM recovery, mainly due to up-regulation of Calm2 and Calm3 genes. Moreover, higher occupancy of Calm2 and Calm3 promoters by NFATc2 and increased expression of these genes in response to NFATc2 silencing were demonstrated in PMCA2 and PMCA3-reduced lines. Our results indicate that decrease in CaM level in response to PMCAs downregulation can be driven by CaN/NFAT pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Apoptosis of PC12 cell Induced by Amyloid Beta-peptide Fragment 25-35%β-淀粉样蛋白25-35片段诱导PC12细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    罗蔓; 谢瑞满

    2004-01-01

    目的探讨β-淀粉样蛋白25-35片段(Aβ25-35)对体外培养的PC12细胞的毒性作用机制.方法用四甲基偶氮唑蓝(MTT)代谢率检测,光镜吖啶橙荧光染色术,透射电镜以及流式细胞仪技术研究Aβ25-35损伤PC12细胞的途径.结果用Aβ25-35处理PC12细胞24 h,Aβ25-35剂量依赖性地引起PC12细胞的MTT代谢率减少,荧光染色及电镜观察发现经Aβ25-35处理的PC12细胞表现出凋亡细胞的特征,流式细胞仪检测发现,对照组20μmol/L及50 μmol/L的Aβ25-35组PC12细胞的凋亡率分别为0.08%±0.01%,14.8%±1.13%,25.9%±2.34%.结论Aβ对PC12细胞的损伤主要通过细胞凋亡的途径.

  16. H2S protects PC12 cells against toxicity of corticosterone by modulation of BDNF-TrkB pathway.

    Science.gov (United States)

    Gao, Shenglan; Li, Wenting; Zou, Wei; Zhang, Ping; Tian, Ying; Xiao, Fan; Gu, Hongfeng; Tang, Xiaoqing

    2015-11-01

    Corticosterone, one of the glucocorticoids, is toxic to neurons and plays an important role in depressive-like behavior and depression. We previously showed that hydrogen sulfide (H2S), a novel physiological mediator, plays an inhibitory role in depression. However, the mechanism underlying H2S-triggered antidepressant-like role is not clearly known. Brain-derived neurotrophic factor (BDNF), a neurotrophic factor, plays a neuroprotective role that is mediated by its high-affinity tropomysin-related kinase B (TrkB) receptor. In this study, to investigate the underlying mechanism of H2S-induced antidepressant-like role, we explored whether H2S could protect neurons against corticosterone-mediated cyctotoxicity and whether this protective role of H2S was involved in the regulation of BDNF-TrkB pathway. Our data demonstrated that sodium hydrosulfide (NaHS), the donor of H2S, could prevent corticosterone-induced cytotoxicity, apoptosis, accumulation of intracellular reactive oxygen species (ROS) and loss of mitochondrial membrane potential (MMP) in PC12 cells. NaHS not only induced the up-regulation of BDNF but also prevented the down-regulation of BDNF by corticosterone. It was also found that blocking BDNF-TrkB pathway by K252a, an inhibitor of TrkB, abolished the protection of H2S against corticosterone-induced cytotoxicity, apoptosis, accumulation of ROS, and loss of MMP. These results suggest that H2S protects against the neurotoxicity of corticosterone by modulation of the BDNF-TrkB pathway. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  17. Neuritogenic Monoglyceride Derived from the Constituent of a Marine Fish for Activating the PI3K/ERK/CREB Signalling Pathways in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Wei Yang

    2013-12-01