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Sample records for activated hepatic stellate

  1. Epigenetic Changes during Hepatic Stellate Cell Activation.

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    Silke Götze

    Full Text Available Hepatic stellate cells (HSC, which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC.The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism.In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.

  2. Activated hepatic stellate cells in liver cirrhosis. A morphologic and morphometrical study.

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    Carpino, Guido; Franchitto, Antonio; Morini, Sergio; Corradini, Stefano Ginanni; Merli, Manuela; Gaudio, Eugenio

    2004-01-01

    Hepatic stellate cells have been considered the most important cell-type involved in hepatic fibrogenesis. Proliferation and differentiation of hepatic stellate cells into myofibroblast-like cells has been related to the development of liver fibrosis. The alpha-actin expressed by hepatic stellate cells was considered a marker of their activation to myofibroblast-like cell. The aim of this study is to evaluate the changes in morphology, distribution, percentage and alpha-smooth muscle actin expression of hepatic stellate cells in normal and cirrhotic livers, and to correlate activated hepatic stellate cells with the progression of fibrosis. Human liver biopsies (n=121) were divided in five groups: 1) normal livers (controls); 2) cirrhosis post-HCV hepatitis; 3) cirrhosis post-HBV hepatitis; 4) non viral related cirrhosis; 5) recurrent HCV hepatitis after orthotopic liver transplantation. Samples immunostained with anti alpha-smooth muscle actin antibody by immunoperoxidase method were semi-quantitatively evaluated. Liver fibrosis was quantified by computer image analysis on specimens stained with Masson's trichrome. In normal adult livers stellate cells were very rarely stained for alpha-smooth muscle actin. In cirrhotic livers, a strongly enhanced percentage of stellate cells expressing alpha-smooth muscle actin was detected in cirrhotic fragments with respect to the control group, with a significant correlation between alpha-smooth muscle actin positive stellate cells and the volume fraction of fibrosis. Moreover, liver biopsies of recurrent hepatitis revealed an increased number of activated stellate cells compared to normal livers, and intermediate volume fraction of fibrosis. These results confirmed that a direct correlation existed between activated stellate cells and the progression of fibrosis. Alpha-smooth muscle actin confirmed to be a reliable marker of hepatic stellate cells activation also in precocious stages of the disease.

  3. Roles of the Lipid Metabolism in Hepatic Stellate Cells Activation

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    Xin-yan Jing; Xue-feng Yang; Kai Qing; Yan Ou-Yang

    2013-01-01

    The lipids present in hepatic stellate cells (HSCs) lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Activation of HSCs is crucial to the development of fibrosis in liver disease. During activation, HSCs transform into myofibroblasts with concomitant loss of their lipid droplets and production of excessive extracellular matrix. Release of lipid droplets containing retinyl esters and triglyceride is a defining feature of activated HSCs. Accumulating evidence supports the proposal that recovering the accumulation of lipids would inhibit the activation of HSCs. In healthy liver, quiescent HSCs store 80%of total liver retinols and release them depending on the extracellular retinol status. However, in injured liver activated HSCs lose their retinols and produce a considerable amount of extracellular matrix, subsequently leading to liver fibrosis. Further findings prove that lipid metabolism of HSCs is closely associated with its activation, yet relationship between activated HSCs and the lipid metabolism has remained mysterious.

  4. Relationship between somatostatin receptors and activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 陆良勇; 尤汉宁; 徐芹芳

    2004-01-01

    Background Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs.Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation.SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining.Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc.Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  5. RELATIONSHIP BETWEEN SOMATOSTATIN RECEPTORS AND ACTIVATION OF HEPATIC STELLATE CELL

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    潘勤; 李定国; 陆汉明; 尤汉宁; 徐芹芳; 陆良勇

    2004-01-01

    Objective To investigate the relationship between expression of somatostatin receptors (SSTRs) and activation of rat hepatic stellate cell (HSC). Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation, and then SSTR1 ~5 mRNA levels in the differentiated first passage HSCs were detected by means of reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1 ~5 in normal as well as fibrotic liver was measured by immunohistochemical staining. Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs and normal rat liver. But SSTR1~3 mRNA appeared as HSCs became wholly activated, and SSTR1 ~3 could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc Conclusion The expression of SSTR1~3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  6. Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

    NARCIS (Netherlands)

    Testerink, N.; Ajat, M.A.; Houweling, M.; Brouwers, J.F.; Pully, V.V.; Manen, van H.J.; Otto, C.; Helms, J.B.; Vaandrager, A.B.

    2012-01-01

    Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed t

  7. Melatonin suppresses activation of hepatic stellate cells through ROR alpha-mediated inhibition of 5-lipoxygenase

    NARCIS (Netherlands)

    Shajari, Shiva; Laliena, Almudena; Heegsma, Janette; Jesus Tunon, Maria; Moshage, Han; Faber, Klaas Nico

    2015-01-01

    Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to prolifer

  8. Early hepatic stellate cell activation predicts severe hepatitis C recurrence after liver transplantation.

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    Gawrieh, Samer; Papouchado, Bettina G; Burgart, Lawrence J; Kobayashi, Shogo; Charlton, Michael R; Gores, Gregory J

    2005-10-01

    Only a subset of hepatitis C virus (HCV)-infected patients develop progressive hepatic fibrosis after liver transplantation (LT). Hepatic stellate cell (HSC) activation is a pivotal step in hepatic fibrosis and precedes clinically apparent fibrosis. We determined whether early HSC activation, measured in 4-month protocol post-LT biopsies, is predictive of subsequent development of more histologically severe recurrence of HCV. Early (4 month) post-LT HSC activation, as measured by alpha-smooth muscle actin (alpha-SMA) staining, was determined in liver biopsies from recipients with severe (fibrosis score > or = 2, n = 13) and with mild (fibrosis score of 0, n = 13) recurrence of HCV at one-year post-LT. Immunohistochemical staining for alpha-smooth muscle actin (alpha-SMA) was used to generate HSC activation scores (regional and total). Total HSC activation scores at 4 months were similar in patients with severe and mild HCV recurrence (3.9 +/- 2.0 vs. 2.7 +/- 2.2, P = 0.2). Regional HSC activation, assessed as parenchymal (zones 1, 2, and 3) or mesenchymal (portal tracts and fibrous septa), was different between the study groups, with higher mesenchymal scores predictive of progression. No patients in the mild recurrence group had detectable mesenchymal alpha-SMA staining vs. 46% (6/13) of patients with severe recurrence (P HCV or HSC-targeted therapy.

  9. Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells.

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    Feng, Yi; Ying, Hai-Yan; Qu, Ying; Cai, Xiao-Bo; Xu, Ming-Yi; Lu, Lun-Gen

    2016-09-01

    Matrine (MT), the effective component of Sophora flavescens Ait, has been shown to have anti-inflammation, immune-suppressive, anti-tumor, and anti-hepatic fibrosis activities. However, the pharmacological effects of MT still need to be strengthened due to its relatively low efficacy and short half-life. In the present study, we report a more effective thio derivative of MT, MD-1, and its inhibitory effects on the activation of hepatic stellate cells (HSCs) in both cell culture and animal models. Cytological experiments showed that MD-1 can inhibit the proliferation of HSC-T6 cells with a half-maximal inhibitory concentration (IC50) of 62 μmol/L. In addition, MD-1 more strongly inhibits the migration of HSC-T6 cells compared to MT and can more effectively induce G0/G1 arrest and apoptosis. Investigating the biological mechanisms underlying anti-hepatic fibrosis in the presence of MD-1, we found that MD-1 can bind the epidermal growth factor receptor (EGFR) on the surface of HSC-T6 cells, which can further inhibit the phosphorylation of EGFR and its downstream protein kinase B (Akt), resulting in decreased expression of cyclin D1 and eventual inhibition of the activation of HSC-T6 cells. Furthermore, in rats with dimethylnitrosamine (DMN)-induced hepatic fibrosis, MD-1 slowed the development and progression of hepatic fibrosis, protecting hepatic parenchymal cells and improving hepatic functions. Therefore, MD-1 is a potential drug for anti-hepatic fibrosis.

  10. Hepatitis B virus e antigen induces activation of rat hepatic stellate cells

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    Zan, Yanlu [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Zhang, Yuxia, E-mail: yzhang@wehi.edu.au [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Tien, Po, E-mail: tienpo@sun.im.ac.cn [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)

    2013-06-07

    Highlights: •HBeAg expression in HSCs induced production of ECM protein and liver fibrotic markers. •The activation and proliferation of HSCs were mediated by TGF-β. •HBeAg protein purified from cell medium directly activated HSCs. -- Abstract: Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs.

  11. RETARDING EFFECT OF SAL IANOLIIC ACID B ON ACTIVATION OF RAT HEPATIC STELLATE CELLS IN VITRO

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    王晓玲; 刘平; 王海南; 谭英姿

    2001-01-01

    To investigate the anti-fibrosis mechanism of salvianolic acid B in liver.Methods Hepatic stellate cells (HSCs) were isolated from liver of normal rats by in situ perfusion and density-gradient centrifugation with Nycodenz. Total RNA was extracted from cells to detect type Ⅰ collagen and smooth muscle α-actin mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR). Smooth muscle α-actin protein was assayed with immunoblotting analysis. Secretion of type Ⅰ collagen in the medium was determined by ELISA.Results Both 1 μmol/L and 10μmol/L SAB suppressed type Ⅰ collagen mRNA expression and its protein secretion. 10μmol/l SAB affected the expression of smooth muscle α-actin protein.Conclusion Retarding activation of stellate cells and inhibiting type Ⅰ collagen secretion were the main mechanism of SAB on anti-fibrosis of liver.

  12. Ionone Derivatives from the Mycelium of Phellinus linteus and the Inhibitory Effect on Activated Rat Hepatic Stellate Cells.

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    Huang, Shiow-Chyn; Kuo, Ping-Chung; Hung, Hsin-Yi; Pan, Tai-Long; Chen, Fu-An; Wu, Tian-Shung

    2016-05-06

    Three new γ-ionylideneacetic acid derivatives, phellinulins A-C (1-3), were characterized from the mycelium extract of Phellinus linteus. The chemical structures were established based on the spectroscopic analysis. In addition, phellinulin A (1) was subjected to the examination of effects on activated rat hepatic stellate cells and exhibited significant inhibition of hepatic fibrosis.

  13. Ionone Derivatives from the Mycelium of Phellinus linteus and the Inhibitory Effect on Activated Rat Hepatic Stellate Cells

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    Shiow-Chyn Huang

    2016-05-01

    Full Text Available Three new γ-ionylideneacetic acid derivatives, phellinulins A–C (1–3, were characterized from the mycelium extract of Phellinus linteus. The chemical structures were established based on the spectroscopic analysis. In addition, phellinulin A (1 was subjected to the examination of effects on activated rat hepatic stellate cells and exhibited significant inhibition of hepatic fibrosis.

  14. Ionone Derivatives from the Mycelium of Phellinus linteus and the Inhibitory Effect on Activated Rat Hepatic Stellate Cells

    OpenAIRE

    2016-01-01

    Three new γ-ionylideneacetic acid derivatives, phellinulins A–C (1–3), were characterized from the mycelium extract of Phellinus linteus. The chemical structures were established based on the spectroscopic analysis. In addition, phellinulin A (1) was subjected to the examination of effects on activated rat hepatic stellate cells and exhibited significant inhibition of hepatic fibrosis.

  15. Suppression of hepatic stellate cell activation by microRNA-29b

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    Sekiya, Yumiko; Ogawa, Tomohiro [Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka (Japan); Liver Research Center, Graduate School of Medicine, Osaka City University, Osaka (Japan); Yoshizato, Katsutoshi [Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka (Japan); Liver Research Center, Graduate School of Medicine, Osaka City University, Osaka (Japan); PhoenixBio Co. Ltd., Hiroshima (Japan); Ikeda, Kazuo [Department of Anatomy and Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Aichi (Japan); Kawada, Norifumi, E-mail: kawadanori@med.osaka-cu.ac.jp [Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka (Japan); Liver Research Center, Graduate School of Medicine, Osaka City University, Osaka (Japan)

    2011-08-19

    Highlights: {yields} Expression of miR-29b was found to be down-regulated during the activation of hepatic stellate cells in primary culture. {yields} Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs. {yields} It blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-b mRNAs essential for stellate cell activation. {yields} miR-29b overexpression led stellate cells to remain in a quiescent state, as evidenced by their star-like morphology. {yields} miR-29b overexpression suppressed the expression of c-fos mRNA. -- Abstract: MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has been previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3'UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-{beta}, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.

  16. Glial fibrillary acidic protein as an early marker of hepatic stellate cell activation in chronic and posttransplant recurrent hepatitis C.

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    Carotti, Simone; Morini, Sergio; Corradini, Stefano Ginanni; Burza, Maria Antonella; Molinaro, Antonio; Carpino, Guido; Merli, Manuela; De Santis, Adriano; Muda, Andrea Onetti; Rossi, Massimo; Attili, Adolfo Francesco; Gaudio, Eugenio

    2008-06-01

    Activated alpha-smooth muscle actin (alpha-SMA)-positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. In the rat, increased GFAP expression in the acute response to injury and down-regulation in the chronic response have been observed, whereas reports concerning GFAP expression in human liver are still conflicting. We investigated the utility of GFAP compared to alpha-SMA as an immunohistochemical marker of early activated HSCs in chronic and posttransplant recurrent hepatitis C and correlated GFAP expression with vascular remodeling and fibrosis progression. With immunohistochemistry and a semiquantitative scoring system, the expression of GFAP and alpha-SMA in HSCs and the microvessel density were analyzed in biopsies from normal livers obtained from cadaveric donors [donor liver (DL); n = 21] and from livers from posttransplant hepatitis C virus recurrent hepatitis (HCV-PTR) patients (n = 19), hepatitis C virus chronic hepatitis (HCV-CH) patients, (n = 12), and hepatitis C virus cirrhosis (HCV-C) patients (n = 16). The percentage of alpha-SMA-positive HSCs was significantly higher in the HCV-PTR, HCV-CH, and HCV-C groups compared to the DL group (P HCV-PTR group compared to the DL, HCV-C (P HCV-CH (P HCV-CH group compared to the DL group (P HCV-PTR group, the percentage of GFAP-positive HSCs correlated with fibrosis progression (P HCV-CH and seems to predict fibrosis progression in HCV-PTR.

  17. Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Hella Wobser; Christoph Dorn; Thomas S Weiss; Thomas Amann; Cornelius Bollheimer; Roland Büttner; Jürgen Sc(o)lmerich; Claus Hellerbrand

    2009-01-01

    Despite the initial belief that non-alcoholic fatty liver disease is a benign disorder, it is now recognized that fbrosis progression occurs in a significant number of patients. Furthermore, hepatic steatosis has been identified as a risk factor for the progression of hepatic fibrosis in a wide range of other liver diseases. Here, we established an in vitro model to study the effect of hepatic lipid accumulation on hepatic stellate cells (HSCs), the central mediators of liver fibrogenesis. Primary human hepatocytes were incubated with the saturated fatty acid palmitate to induce intracellular lipid accumulation. Subsequently, human HSCs were incubated with conditioned media (CM) from steatotic or control hepatocytes. Lipid accumulation in hepatocytes induced the release of factors that accelerated the activation and proliferation of HSC, and enhanced their resistance to apoptosis, largely mediated via activation of the PI-3-kinase pathway. Furthermore, CM from steatotic hepatocytes induced the expression of the profibrogenic genes TGF-β, tissue inhibitor of metallo-proteinase-1 (TIMP-1), TIMP-2 and matrix-metallo-proteinase-2, as well as nuclear-factor Κb-dependent MCP-1 expression in HSC. In summary, our in vitro data indicate a potential mechanism for the pathophysiological link between hepatic steatosis and fibrogenesis in vivo. Herewith, this study provides an attractive in vitro model to study the molecular mechanisms of steatosis-induced fibrogenesis, and to identify and test novel targets for antifibrotic therapies in fatty liver disease.

  18. Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death

    NARCIS (Netherlands)

    Dunning, Sandra; Rehman, Atta Ur; Tiebosch, Marjolein H.; Hannivoort, Rebekka A.; Haijer, Floris W.; Woudenberg, Jannes; van den Heuvel, Fiona A. J.; Buist-Homan, Manon; Faber, Klaas Nico; Moshage, Han

    2013-01-01

    Background: In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated i

  19. Inhibition of tyrosine kinase receptor Tie2 reverts HCV-induced hepatic stellate cell activation.

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    Samuel Martín-Vílchez

    Full Text Available BACKGROUND: Hepatitis C virus (HCV infection is a major cause of chronic liver disease (CLD and is frequently linked to intrahepatic microvascular disorders. Activation of hepatic stellate cells (HSC is a central event in liver damage, due to their contribution to hepatic renewal and to the development of fibrosis and hepatocarcinoma. During the progression of CLDs, HSC attempt to restore injured tissue by stimulating repair processes, such as fibrosis and angiogenesis. Because HSC express the key vascular receptor Tie2, among other angiogenic receptors and mediators, we analyzed its involvement in the development of CLD. METHODS: Tie2 expression was monitored in HSC cultures that were exposed to media from HCV-expressing cells (replicons. The effects of Tie2 blockade on HSC activation by either neutralizing antibody or specific signaling inhibitors were also examined. RESULTS: Media from HCV-replicons enhanced HSC activation and invasion and upregulated Tie2 expression. Notably, the blockade of Tie2 receptor (by a specific neutralizing antibody or signaling (by selective AKT and MAPK inhibitors significantly reduced alpha-smooth muscle actin (α-SMA expression and the invasive potential of HCV-conditioned HSC. CONCLUSIONS: These findings ascribe a novel profibrogenic function to Tie2 receptor in the progression of chronic hepatitis C, highlighting the significance of its dysregulation in the evolution of CLDs and its potential as a novel therapeutic target.

  20. Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation

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    Caliari, Steven R.; Perepelyuk, Maryna; Cosgrove, Brian D.; Tsai, Shannon J.; Lee, Gi Yun; Mauck, Robert L.; Wells, Rebecca G.; Burdick, Jason A.

    2016-02-01

    Tissue fibrosis contributes to nearly half of all deaths in the developed world and is characterized by progressive matrix stiffening. Despite this, nearly all in vitro disease models are mechanically static. Here, we used visible light-mediated stiffening hydrogels to investigate cell mechanotransduction in a disease-relevant system. Primary hepatic stellate cell-seeded hydrogels stiffened in situ at later time points (following a recovery phase post-isolation) displayed accelerated signaling kinetics of both early (Yes-associated protein/Transcriptional coactivator with PDZ-binding motif, YAP/TAZ) and late (alpha-smooth muscle actin, α-SMA) markers of myofibroblast differentiation, resulting in a time course similar to observed in vivo activation dynamics. We further validated this system by showing that α-SMA inhibition following substrate stiffening resulted in attenuated stellate cell activation, with reduced YAP/TAZ nuclear shuttling and traction force generation. Together, these data suggest that stiffening hydrogels may be more faithful models for studying myofibroblast activation than static substrates and could inform the development of disease therapeutics.

  1. Inhibitory effects of prostaglandin E1 on activation of hepatic stellate cells in rabbits with schistosomiasis

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    Wei-Long Zou; Zhen Yang; Yun-Jin Zang; Dong-Jian Li; Zhi-Peng Liang; Zhong-Yang Shen

    2007-01-01

    BACKGROUND:Liver ifbrosis is the result of an imbalance between synthesis and degradation of extracellular matrix proteins of the liver. At the cellular and molecular levels, this progressive process is mainly characterized by activation of hepatic stellate cells (HSCs). Schistosoma japonica is one of the most prevalent causes of liver ifbrosis in China. It is characterized by hepatocyte damage, inlfammation, and chronic parasite egg-induced granuloma formation leading to ifbrosis. This study aimed to investigate the inhibitory effects of prostaglandin E1 (PGE1) on activation of HSCs and the alteration of type Ⅰ and Ⅲ collagen in rabbits with schistosomiasis. The study may promote the clinical application of praziquantel and PGE1 as a combined therapy to reverse hepatic ifbrosis caused by schistosomiasis. METHODS: Rabbits were percutaneously infected with cercaria of S. japonicum. Seven rabbits were subjected to intravenous injections of PGE1 (2.5 μg/kg daily) from days 60 to 120 after infection. The ultrastructural changes in activated HSCs were observed under transmission electron microscopy. The expression ofα-smooth muscle actin (α-SMA) was detected by immunohistochemistry. Fibril-forming collagens were detected by picrosirius staining. RESULTS: Activation of HSCs was a characteristic alteration in schistosome-induced hepatic ifbrosis. The expression of contraction-related α-SMA and the content of collagens were increased. Exogenous PGE1 markedly inhibited the activation of HSCs and reduced the expression of α-SMA around the hepatic sinusoids (P CONCLUSIONS:Activation of HSCs may play a key role in the progress of schistosome-induced hepatic ifbrosis. PGE1 effectively protects rabbit liver from ifbrosis, at least in part by inhibiting the activation of HSCs.

  2. Bile acids induce hepatic stellate cell proliferation via activation of the epidermal growth factor receptor

    NARCIS (Netherlands)

    Svegliati-Baroni, G; Ridolfi, F; Hannivoort, R; Saccomanno, S; Homan, M; De Minicis, S; Jansen, PLM; Candelaresi, C; Benedetti, A; Moshage, H

    2005-01-01

    Background B Aims: Hepatic stellate cell (HSC) proliferation is a key event in the development of liver fibrosis. In many liver diseases, HSCs are exposed to inflammatory cytokines, reactive oxygen species, and bile acids. Although inflammatory cytokines and reactive oxygen species are known to prom

  3. Neferine inhibits cultured hepatic stellate cell activation and facilitates apoptosis: A possible molecular mechanism.

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    Ding, Hui; Shi, Jinghong; Wang, Ying; Guo, Jia; Zhao, Juhui; Dong, Lei

    2011-01-10

    Neferine is a major alkaloid component of "Lian Zi Xin", embryos of the seeds of Nelumbo nucifera Gaertner, Nymphaeaceae. Previous studies have shown that neferine has an inhibitory effect on pulmonary fibrosis through its anti-inflammatory and anti-oxidative activities and inhibition of cytokines and NF-κB. However, it is unknown whether neferine also has an inhibitory effect on liver fibrosis through inhibition of TGF-β1 and collagen I and facilitation of apoptosis of hepatic stellate cells. This study examined the effects of neferine on cultured hepatic stellate (HSC-T6) cells and explored its possible action mechanisms by means of MTT assay, enzyme-linked immunosorbent assay, flow-cytometric annexin V-PI assay and Hoechst 33258 staining, as well as real-time PCR and western blotting. The results showed that neferine administration (2, 4, 6, 8 and 10μmol/l) significantly decreased the TGF-β1 and collagen I produced in HSC-T6 cells, and increased the HSC-T6 cell apoptosis in a dose-dependent manner. Neferine treatment for 48h at concentrations of 6 and 10μmol/l significantly increased Bax and caspase 3 mRNAs and proteins, and reduced Bcl2 and alpha-smooth muscle actin (α-SMA) mRNAs and proteins. Our data indicate that neferine efficiently inhibits cultured HSC-T6 cell activation and induces apoptosis by increasing Bax and caspase 3 expression via the mitochondrial pathway.

  4. Tetramethylpyrazine Inhibits Activation of Hepatic Stellate Cells through Hedgehog Signaling Pathways In Vitro

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    Jue Hu

    2015-01-01

    Full Text Available Background and Aim. Tetramethylpyrazine (TMP, a major alkaloid isolated from Ligusticum chuanxiong, has been reported in hepatic fibrosis models. However, the action mechanism remains unclear. In the present study, effects of tetramethylpyrazine (TMP against hepatic stellate cell (HSC activation as well as the possible mechanisms were evaluated. Methods. Western blot assay was used to detect TMP effects on protein expression of Smo, Patched, Hhip, and Gli and to investigate the effects of TMP on Cyclin D1, Cyclin E1, CDK2, Bcl-2, Bax, and caspase expression with cyclopamine supplementation. Results. Our results showed that TMP significantly inhibits the expression of Cyclin D1, Cyclin E1, and Cyclin-dependent kinase CDK2 and changes the HSC cycle by inhibiting the proliferation of HSC. Moreover, TMP has also been shown to decrease the expression of Bcl-2 and increase the expression of Bax in HSC-T6 cells. Furthermore, TMP can inhibit the expression of connective tissue growth factor (CTGF, and the inhibitory effect was intensified after the application of joint treatment with TMP and cyclopamine. Conclusion. TMP may be an effective Hh signaling pathway inhibitor for hepatic fibrosis treatment.

  5. Bisdemethoxycurcumin Induces Apoptosis in Activated Hepatic Stellate Cells via Cannabinoid Receptor 2

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    Phil Jun Lee

    2015-01-01

    Full Text Available Activated Hepatic Stellate Cells (HSCs, major fibrogenic cells in the liver, undergo apoptosis when liver injuries cease, which may contribute to the resolution of fibrosis. Bisdemethoxycurcumin (BDMC is a natural derivative of curcumin with anti-inflammatory and anti-cancer activities. The therapeutic potential of BDMC in hepatic fibrosis has not been studied thus far in the context of the apoptosis in activated HSCs. In the current study, we compared the activities of BDMC and curcumin in the HSC-T6 cell line and demonstrated that BDMC relatively induced a potent apoptosis. BDMC-induced apoptosis was mediated by a combinatory inhibition of cytoprotective proteins, such as Bcl2 and heme oxygenase-1 and increased generation of reactive oxygen species. Intriguingly, BDMC-induced apoptosis was reversed with co-treatment of sr144528, a cannabinoid receptor (CBR 2 antagonist, which was confirmed with genetic downregulation of the receptor using siCBR2. Additionally, incubation with BDMC increased the formation of death-induced signaling complex in HSC-T6 cells. Treatment with BDMC significantly diminished total intracellular ATP levels and upregulated ATP inhibitory factor-1. Collectively, the results demonstrate that BDMC induces apoptosis in activated HSCs, but not in hepatocytes, by impairing cellular energetics and causing a downregulation of cytoprotective proteins, likely through a mechanism that involves CBR2.

  6. Inhibitory Effects of Ecklonia cava Extract on High Glucose-Induced Hepatic Stellate Cell Activation

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    Akiko Kojima-Yuasa

    2011-12-01

    Full Text Available Nonalcoholic steatohepatitis (NASH is a disease closely associated with obesity and diabetes. A prevalence of type 2 diabetes and a high body mass index in cryptogenic cirrhosis may imply that obesity leads to cirrhosis. Here, we examined the effects of an extract of Ecklonia cava, a brown algae, on the activation of high glucose-induced hepatic stellate cells (HSCs, key players in hepatic fibrosis. Isolated HSCs were incubated with or without a high glucose concentration. Ecklonia cava extract (ECE was added to the culture simultaneously with the high glucose. Treatment with high glucose stimulated expression of type I collagen and α-smooth muscle actin, which are markers of activation in HSCs, in a dose-dependent manner. The activation of high glucose-treated HSCs was suppressed by the ECE. An increase in the formation of intracellular reactive oxygen species (ROS and a decrease in intracellular glutathione levels were observed soon after treatment with high glucose, and these changes were suppressed by the simultaneous addition of ECE. High glucose levels stimulated the secretion of bioactive transforming growth factor-β (TGF-β from the cells, and the stimulation was also suppressed by treating the HSCs with ECE. These results suggest that the suppression of high glucose-induced HSC activation by ECE is mediated through the inhibition of ROS and/or GSH and the downregulation of TGF-β secretion. ECE is useful for preventing the development of diabetic liver fibrosis.

  7. Brivanib attenuates hepatic fibrosis in vivo and stellate cell activation in vitro by inhibition of FGF, VEGF and PDGF signaling.

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    Ikuo Nakamura

    Full Text Available Brivanib is a selective inhibitor of vascular endothelial growth factor receptor (VEGFR and fibroblast growth factor receptor (FGFR tyrosine kinases, which are both involved in mechanisms of liver fibrosis. We hypothesized that inhibition of VEGFR and FGFR by brivanib would inhibit liver fibrosis. We therefore examined the effect of brivanib on liver fibrosis in three mouse models of fibrosis.In vivo, we induced liver fibrosis by bile duct ligation (BDL, chronic carbon tetrachloride (CCl4, and chronic thioacetamide (TAA administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs to assess the effect of brivanib on stellate cell proliferation and activation.After in vivo induction with BDL, CCl4, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF, VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.Brivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.

  8. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

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    Iyer, Soumya C, E-mail: chidambaram.soumya@gmail.com [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Kannan, Anbarasu [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Gopal, Ashidha [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Devaraj, Niranjali [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Halagowder, Devaraj [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India)

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  9. Switching-on of serotonergic calcium signaling in activated hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Kyu-Sang Park; Pyo-Jin Sin; Dong Hyeon Lee; Seung-Kuy Cha; Min-Jeong Kim; Na-Hyun Kim; Soon-Koo Baik; Seong-Woo Jeong; In Deok Kong

    2011-01-01

    AIM: To investigate serotonergic Ca2+ signaling and the expression of 5-hydroxytryptamine (5-HT) receptors,as well as Ca2+ transporting proteins, in hepatic stellate cells (HSCs).METHODS: The intracellular Ca2+ concentration ([Ca2+]i)of isolated rat HSCs was measured with a fluorescence microscopic imaging system. Quantitative PCR was performed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum (ER) proteins involved in Ca2+ storage and release in cultured rat HSCs.RESULTS: Distinct from quiescent cells, activated HSCs exhibited [Ca2+]i transients following treatment with 5-HT, which was abolished by U-73122, a phospholipase C inhibitor. Upregulation of 5-HT2A and 5-HT2B receptors,but not 5-HT3, was prominent during trans-differentiation of HSCs. Pretreatment with ritanserin, a 5-HT2 antagonist, inhibited [Ca2+]i changes upon application of 5-HT. Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca2+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca2+ storage and release in activated HSCs. Ca2+ binding chaperone proteins of the ER, including calreticulin, calnexin and calsequestrin, were up-regulated following activation of HSCs.CONCLUSION: The appearance of 5-HT-induced [Ca2+]i response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca2+ transporting/chaperone ER proteins may participate in the activating process of HSCs.

  10. Paclitaxel ameliorates fibrosis in hepatic stellate cells via inhibition of TGF-β/Smad activity

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigated if paclitaxel can attenuate hepatic fi brosis in rat hepatic stellate cells (RHSCs). METHODS: RHSCs were cultured in vitro and randomly assigned to four groups: normal control group (treated only with Dulbecco's Modified Eagle's Medium), Taxol group (200 nmol/L paclitaxel was added to the cell culture), transforming growth factor (TGF)-β group (5 ng/mL recombinant human TGF-β1 was added to the cell culture), and TGF-β + Taxol group. TGF-β signaling cascade and status of various extracel...

  11. Explore the Molecular Mechanism of Apoptosis Induced by Tanshinone IIA on Activated Rat Hepatic Stellate Cells

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    Tai-Long Pan

    2012-01-01

    Full Text Available Since the activated hepatic stellate cell (HSC is the predominant event in the progression of liver fibrosis, selective clearance of HSC should be a potential strategy in therapy. Salvia miltiorrhiza roots ethanol extract (SMEE remarkably ameliorates liver fibrogenesis in DMN-administrated rat model. Next, tanshinone IIA (Tan IIA, the major compound of SMEE, significantly inhibited rat HSC viability and led to cell apoptosis. Proteome tools elucidated that increased prohibitin is involved in cell cycle arrest under Tan IIA is the treatment while knockdown of prohibitin could attenuate Tan IIA-induced apoptosis. In addition, Tan IIA mediated translocation of C-Raf which interacted with prohibitin activating MAPK and inhibiting AKT signaling in HSC. MAPK antagonist suppressed ERK phosphorylation which was necessary for Tan IIA-induced expression of Bax and cytochrome c. PD98059 also abolished Tan IIA-modulated cleavage of PARP. Our findings suggested that Tan IIA could contribute to apoptosis of HSC by promoting ERK-Bax-caspase pathways through C-Raf/prohibitin complex.

  12. Amphiregulin activates human hepatic stellate cells and is upregulated in non alcoholic steatohepatitis

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    McKee, Chad; Sigala, Barbara; Soeda, Junpei; Mouralidarane, Angelina; Morgan, Maelle; Mazzoccoli, Gianluigi; Rappa, Francesca; Cappello, Francesco; Cabibi, Daniela; Pazienza, Valerio; Selden, Claire; Roskams, Tania; Vinciguerra, Manlio; Oben, Jude A.

    2015-01-01

    Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways, and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD. PMID:25744849

  13. miRNA studies in in vitro and in vivo activated hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Gunter Maubach; Michelle Chin Chia Lim; Jinmiao Chen; Henry Yang; Lang Zhuo

    2011-01-01

    AIM: To understand which and how different miRNAs are implicated in the process of hepatic stellate cell (HSC) activation.METHODS: We used microarrays to examine the differential expression of miRNAs during in vitro activation of primary HSCs (pHSCs). The transcriptome changes upon stable transfection of rno-miR-146a into an HSC cell line were studied using cDNA microarrays. Selected differentially regulated miRNAs were investigated by quantitative real-time polymerase chain reaction during in vivo HSC activation. The effect of miRNA mimics and inhibitor on the in vitro activation of pHSCs was also evaluated.RESULTS: We found that 16 miRNAs were upregulated and 26 were downregulated significantly in 10-d in vitro activated pHSCs in comparison to quiescent pHSCs.Overexpression of rno-miR-146a was characterized by marked upregulation of tissue inhibitor of metalloproteinase-3, which is implicated in the regulation of tumor necrosis factor-α activity. Differences in the regulation of selected miRNAs were observed comparing in vitro and in vivo HSC activation. Treatment with miR-26a and 29a mimics, and miR-214 inhibitor during in vitro activation of pHSCs induced significant downregulation of collagen type Ⅰ transcription.CONCLUSION: Our results emphasize the different regulation of miRNAs in in vitro and in vivo activated pHSCs. We also showed that miR-26a, 29a and 214 are involved in the regulation of collagen type I mRNA.

  14. Hepatic stellate cell is activated by microRNA-181b via PTEN/Akt pathway.

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    Zheng, Jianjian; Wu, Cunzao; Xu, Ziqiang; Xia, Peng; Dong, Peihong; Chen, Bicheng; Yu, Fujun

    2015-01-01

    Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. MicroRNAs have been shown to play a pivotal role in regulating HSC functions such as cell proliferation, differentiation, and apoptosis. Recently, miR-181b has been reported to promote HSCs proliferation by targeting p27. But whether alpha-smooth muscle actin (α-SMA) or collagens could be promoted by miR-181b in activated HSCs is still not clear. Therefore, the understanding of the role of miR-181b in liver fibrosis remains limited. Our results showed that miR-181b expression was increased much higher than miR-181a expression in vitro in transforming growth factor-β1-induced HSC activation as well as in vivo in carbon tetrachloride-induced rat liver fibrosis. Of note, overexpression of miR-181b significantly increased the expressions level of α-SMA and type I collagen, and further promoted HSCs proliferation. Furthermore, phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of PI3K/Akt pathway, were confirmed as a direct target of miR-181b. We demonstrated that miR-181b could suppress PTEN expression and increase Akt phosphorylation in HSCs. Interestingly, the effects of miR-181b on the activation of HSCs were blocked down by Akt inhibitor LY294002. Our results revealed a profibrotic role of miR-181b in HSC activation and demonstrated that miR-181b could activate HSCs, at least in part, via PTEN/Akt pathway.

  15. Relevance of activated hepatic stellate cells in predicting the development of pediatric liver allograft fibrosis.

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    Venturi, Carla; Reding, Raymond; Quinones, Jorge Abarca; Sokal, Etienne; Rahier, Jacques; Bueno, Javier; Sempoux, Christine

    2016-06-01

    Activated hepatic stellate cells (HSCs) are the main collagen-producing cells in liver fibrogenesis. With the purpose of analyzing their presence and relevance in predicting liver allograft fibrosis development, 162 liver biopsies of 54 pediatric liver transplantation (LT) recipients were assessed at 6 months, 3 years, and 7 years after LT. The proportion of activated HSCs, identified by α-smooth muscle actin (ASMA) immunostaining, and the amount of fibrosis, identified by picrosirius red (PSR%) staining, were determined by computer-based morphometric analysis. Fibrosis was also staged by using the semiquantitative liver allograft fibrosis score (LAFSc), specifically designed to score fibrosis in the pediatric LT population. Liver allograft fibrosis displayed progression over time by PSR% (P ASMA expression decreased in the long term, with inverse evolution with respect to fibrosis (P ASMA-positive HSCs area ≥ 8% at 6 months (n = 20) developed a higher fibrosis proportion compared to those with ASMA-positive HSCs area ≤ 8% (n = 34) at the same period of time and in the long term (P = 0.03 and P ASMA expression ≥ 8% at 6 months was found to be an independent risk factor for 7-year fibrosis development by PSR% (r(2) = 0.5; P ASMA expression ≥ 8% at 3 years showed an association with the development of fibrosis at 7 years (P = 0.02). In conclusion, there is a high proportion of activated HSCs in pediatric LT recipients. ASMA ≥ 8% at 6 months seems to be a risk factor for early and longterm fibrosis development. In addition, activated HSCs showed inverse evolution with respect to fibrosis in the long term. Liver Transplantation 22 822-829 2016 AASLD.

  16. Gallic acid induces necroptosis via TNF-α signaling pathway in activated hepatic stellate cells.

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    Ya Ju Chang

    Full Text Available Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA, a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs, the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH. Elevated oxidative stress triggered the production of TNF-α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3, and the inactivation of caspase-8. Calmodulin and calpain-1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP. The TNF-α antagonist (SPD-304 and the RIP1 inhibitor (necrostatin-1, Nec-1 confirmed GA-induced TNFR1-mediated necroptosis. The inhibition of RIP1 by Nec-1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling-triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis.

  17. The role of dystroglycan in PDGF-BB-dependent migration of activated hepatic stellate cells/myofibroblasts

    Science.gov (United States)

    Kastanis, George John; Hernandez-Nazara, Zamira; Nieto, Natalia; Rincón-Sanchez, Ana Rosa; Popratiloff, Anastas; Dominguez-Rosales, Jose Alfredo; Lechuga, Carmen G.

    2011-01-01

    Hepatic stellate cells are embedded in the loose connective tissue matrix within the space of Disse. This extracellular matrix contains several basement membrane components including laminin, but its composition changes during liver injury because of the production of extracellular matrix components found in scar tissue. These changes in extracellular matrix composition and in cell-extracellular matrix interactions may play a key role in hepatic stellate cell transdifferentiation. In this communication we used early passages of mouse hepatic stellate cells (activated HSC/myofibroblasts) to study the platelet-derived growth factor BB (PDGF-BB)-dependent expression and regulation of β-dystroglycan and its role in activated HSC/myofibroblast migration. We used Northern and Western analysis to study dystroglycan expression and confocal microscopy to investigate changes in subcellular distribution of the protein. Activated HSC migration was investigated using an in vitro wound-healing assay. PDGF-BB induced significant changes in dystroglycan regulation and subcellular distribution of the protein. Whereas steady-state levels of dystroglycan mRNA remained constant, PDGF-BB increased dystroglycan transcription but shortened the t1/2 by 50%. Moreover, PDGF-BB changed dystroglycan and α5-integrin cellular distribution. Cell migration experiments revealed that PDGF-BB-dependent migration of activated HSC/myofibroblasts was completely blocked by neutralizing antibodies to fibronectin, α5-integrin, laminin, and β-dystroglycan. Overall, these findings suggest that both laminin and fibronectin and their receptors play a key role in PDGF-BB-induced activated HSC migration. PMID:21659621

  18. Activated effects of parathyroid hormone-related protein on human hepatic stellate cells.

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    Fen-Fen Liang

    Full Text Available BACKGROUND & AIMS: After years of experiments and clinical studies, parathyroid hormone-related protein(PTHrP has been shown to be a bone formation promoter that elicits rapid effects with limited adverse reaction. Recently, PTHrP was reported to promote fibrosis in rat kidney in conjunction with transforming growth factor-beta1 (TGF-β1, which is also a fibrosis promoter in liver. However, the effect of PTHrP in liver has not been determined. In this study, the promoting actions of PTHrP were first investigated in human normal hepatic stellate cells (HSC and LX-2 cell lines. METHODS: TGF-β1, alpha-smooth muscle actin (α-SMA, matrix metalloproteinase 2 (MMP-2, and collagen I mRNA were quantified by real-time polymerase chain reaction (PCR after HSCs or LX-2 cells were treated with PTHrP(1-36 or TGF-β1. Protein levels were also assessed by western-blot analysis. Alpha-SMA were also detected by immunofluorescence, and TGF-β1 secretion was measured with enzyme-linked immunosorbent assay (ELISA of HSC cell culture media. RESULTS: In cultured human HSCs, mRNA and protein levels of α-SMA, collagen I, MMP-2, and TGF-β1 were increased by PTHrP treatment. A similar increasing pattern was also observed in LX-2 cells. Moreover, PTHrP significantly increased TGF-β1 secretion in cultured media from HSCs. CONCLUSIONS: PTHrP activated HSCs and promoted the fibrosis process in LX-2 cells. These procedures were probably mediated via TGF-β1, highlighting the potential effects of PTHrP in the liver.

  19. Up-regulation of interleukin-22 mediates liver fibrosis via activating hepatic stellate cells in patients with hepatitis C.

    Science.gov (United States)

    Wu, Li-Yuan; Liu, Shuhong; Liu, Yuan; Guo, Chaonan; Li, Hanwei; Li, Wenshu; Jin, Xueyuan; Zhang, Keming; Zhao, Ping; Wei, Lai; Zhao, Jingmin

    2015-05-01

    Interleukin-22 (IL-22) is known to play a critical role in liver immunity. However, the role of IL-22 in HCV-associated liver fibrosis is poorly understood. In this study, patients with HCV infection disclosed significant increases in peripheral numbers of IL-22-producing cells as well as in IL-22 plasma levels. In the liver, the increased intrahepatic IL-22(+) cells were positively correlated with fibrotic staging scores and clinical progression from CHC to cirrhosis. Moreover, the majority of IL-22(+) cells were located in fibrotic areas in the liver of patients with cirrhosis and co-localized with α-smooth muscle actin (α-SMA) positive hepatic stellate cells (HSCs). In vitro, administration of IL-22 was accompanied with inhibited LX-2 cell apoptosis, promoted LX-2 cell proliferation, increased expression of α-SMA, and up-regulated collagen production by LX-2 cells. Collectively, our data provide evidence that IL-22 may contribute to the fibrogenesis of HCV-associated liver fibrosis by activating HSCs.

  20. Curcumin inhibits srebp-2 expression in activated hepatic stellate cells in vitro by reducing the activity of specificity protein-1.

    Science.gov (United States)

    Kang, Qiaohua; Chen, Anping

    2009-12-01

    Elevated levels of cholesterol/low-density lipoprotein (LDL) are a risk factor for the development of nonalcoholic steatohepatitis and its associated hepatic fibrosis. However, underlying mechanisms remain elusive. We previously reported that curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma and stimulated its activity, leading to the inhibition of the activation of hepatic stellate cells (HSCs), the major effector cells during hepatic fibrogenesis. We recently showed that curcumin suppressed gene expression of LDL receptor in activated HSCs in vitro by repressing gene expression of the transcription factor sterol regulatory element binding protein-2 (SREBP-2), leading to the reduction in the level of intracellular cholesterol in HSCs and to the attenuation of the stimulatory effects of LDL on HSCs activation. The current study aimed at exploring molecular mechanisms by which curcumin inhibits srebp-2 expression in HSCs. Promoter deletion assays, mutagenesis assays, and EMSAs localize a specificity protein-1 (SP-1) binding GC-box in the srebp-2 promoter, which is responsible for enhancing the promoter activity and responding to curcumin in HSCs. Curcumin suppresses gene expression of SP-1 and reduces its trans-activation activity, which are mediated by the activation of PPARgamma. The inhibitory effect of curcumin on SP-1 binding to the GC-box is confirmed by chromatin immuno-precipitation. In summary, our results demonstrate that curcumin inhibits srebp-2 expression in cultured HSCs by activating PPARgamma and reducing the SP-1 activity, leading to the repression of ldlr expression. These results provide novel insights into molecular mechanisms by which curcumin inhibits LDL-induced HSC activation.

  1. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

    Institute of Scientific and Technical Information of China (English)

    Xiao-Jing Liu; Li Yang; Feng-Ming Luo; Hong-Bin Wu; Qu-Qiang

    2004-01-01

    AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis.METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and singlestep density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted,quantified and reversely transcripted into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4 000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription- polymerase chain reaction (RT-PCR).RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation,apoptosis, and DNA damage response.CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis.

  2. Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation

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    Chen, Chao [Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, No.415 Fengyang Road, Shanghai 200003 (China); Wu, Chao-Qun [Genetics Institute, Fudan University, No. 220 Handan Road, Shanghai 200433 (China); Zhang, Zong-Qi [Department of Cardiology, No. 3 Hospital, Shanghai Jiao Tong University Medical school, No.280 Mohe Road, Shanghai 201900 (China); Yao, Ding-Kang; Zhu, Liang, E-mail: 15900611429@163.com [Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, No.415 Fengyang Road, Shanghai 200003 (China)

    2011-07-15

    Activation and migration of resident stellate cells (HSCs) within the hepatic space of Disse play an important role in hepatic fibrosis, which accounts for the increased numbers of activated HSCs in areas of inflammation during hepatic fibrosis. Currently, microRNAs have been found to play essential roles in HSC differentiation, proliferation, apoptosis, fat accumulation and collagen production. However, little is known about microRNA mediated HSC activation and migration. In this study, the miRNA expression profiles of quiescent HSCs, partially activated HSCs and fully activated HSCs were compared in pairs. Gene ontology (GO) and GO-Map network analysis indicated that the activation of HSCs was regulated by microRNAs. Among them miR-335 was confirmed to be significantly reduced during HSC activation by qRT-PCR, and restoring expression of miR-335 inhibited HSC migration and reduced {alpha}-SMA and collagen type I. Previous study revealed that tenascin-C (TNC), an extracellular matrix glycoprotein involved in cell migration, might be a target of miR-335. Therefore, we further studied the TNC expression in miR-335 over-expressed HSCs. Our data showed that exogenous TNC could enhance HSC migration in vitro and miR-335 restoration resulted in a significant inhibition of TNC expression. These results demonstrated that miR-335 restoration inhibited HSC migration, at least in part, via downregulating the TNC expression.

  3. Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity.

    Science.gov (United States)

    El-Mansy, A A; Mazroa, S A; Hamed, W S; Yaseen, A H; El-Mohandes, E A

    2016-01-01

    The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.

  4. Arg-gly-asp-mannose-6-phosphate inhibits activation and proliferation of hepatic stellate cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Lian-Sheng Wang; Ying-Wei Chen; Ding-Guo Li; Han-Ming Lu

    2006-01-01

    AIM: To investigate the effect of arg-gly-asp-mannose-6phosphate (RGD-M6P) on the activation and proliferation of primary hepatic stellate cells in vitro.METHODS: Hepatic stellate cells (HSCs) were isolated from rats by in situ collagenase perfusion of liver and 18% Nycodenz gradient centrifugation and cultured on uncoated plastic plates for 24 h with DMEM containing 10% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, HSCs were cultured in 2% FBS/DMEM with transforming growth factor β1, M6P, RGD, or RGD-M6P, respectively. Cell morphology was observed under inverted microscope, smooth muscle α-actin (α-SMA)was detected by immunocytochemistry, type Ⅲprocollagen (PCⅢ) in supernatant was determined by radioimmunoassay, and the proliferation rate of HSCs was assessed by flow cytometry.RESULTS: RGD-M6P significantly inhibited the morphological transformation and the α-SMA and PC Ⅲ expressions of HSCs in vitro and also dramatically prevented the proliferation of HSCs in vitro. Such effects were remarkably different from those of RGD or M6P.CONCLUSION: The new compound, RGD-M6P, which has a dramatic effect on primary cultured HSCs in vitro, can inhibit the transformation of HSCs in culture caused by TGFβ1, suppresses the expression of PCⅢand decreases proliferation rate of HSC. RGD-M6P can be applied as a selective drug carrier targeting at HSCs,which may be a new approach to the prevention and treatment of liver fibrosis.

  5. Inhibitory effect of angiotensin TT receptor antagonist on hepatic stellate cell activation in non-alcoholic steatohepatitis

    Institute of Scientific and Technical Information of China (English)

    Shiro Yokohama; Naoyuki Miyokawa; Masakazu Haneda; Masashi Yoneda; Yoshihiko Tokusashi; Kimihide Nakamura; Yosui Tamaki; Satoshi Okamoto; Mituyoshi Okada; Kazunobu Aso; Takenao Hasegawa; Masaru Aoshima

    2006-01-01

    AIM: To investigate the efficacy of angiotensin Ⅱ receptor antagonist on hepatic stellate cells (HSCs) activation in the patients with non-alcoholic steatohepatitis (NASH).METHODS: Seven patients with NASH were prescribed losartan, a selective angiotensin Ⅱ type 1 receptor antagonist (50 mg/d) for 48 wk. Liver biopsies were performed both at the entry and end of the study in all patients. Quiescent and activated HSCs were identified by double immunostaining using anti-p75 and α-smooth muscle actin antibodies, and the number of each phenotype was counted. Similarly, the liver specimens obtained from the eight patients with non-alcoholic fatty liver (NAFL) were also examined as controls.RESULTS: In NASH hepatic tissues, activated HSCs were dominantly distributed as compared with those in NAFL.The 48-wk losartan treatment induced a remarkable decrease in activated HSCs and a mild increase in quiescent phenotypes.CONCLUSION: Our data suggest the crucial involvement of HSCs in anti-fibrotic effect of angiotensin Ⅱ receptor antagonist on patients with NASH.

  6. The hop constituent xanthohumol exhibits hepatoprotective effects and inhibits the activation of hepatic stellate cells at different levels.

    Science.gov (United States)

    Weiskirchen, Ralf; Mahli, Abdo; Weiskirchen, Sabine; Hellerbrand, Claus

    2015-01-01

    Xanthohumol is the principal prenylated flavonoid of the female inflorescences of the hop plant. In recent years, various beneficial xanthohumol effects including anti-inflammatory, antioxidant, hypoglycemic activities, and anticancer effects have been revealed. This review summarizes present studies indicating that xanthohumol also inhibits several critical pathophysiological steps during the development and course of chronic liver disease, including the activation and pro-fibrogenic genotype of hepatic stellate cells. Also the various mechanism of action and molecular targets of the beneficial xanthohumol effects will be described. Furthermore, the potential use of xanthohumol or a xanthohumol-enriched hop extract as therapeutic agent to combat the progression of chronic liver disease will be discussed. It is notable that in addition to its hepatoprotective effects, xanthohumol also holds promise as a therapeutic agent for treating obesity, dysregulation of glucose metabolism and other components of the metabolic syndrome including hepatic steatosis. Thus, therapeutic xanthohumol application appears as a promising strategy, particularly in obese patients, to inhibit the development as well as the progression of non-alcoholic fatty liver disease.

  7. The hop constituent xanthohumol exhibits hepatoprotective effects and inhibits the activation of hepatic stellate cells at different levels

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    Ralf eWeiskirchen

    2015-05-01

    Full Text Available Xanthohumol is the principal prenylated flavonoid of the female inflorescences of the hop plant. In recent years, various beneficial xanthohumol effects including anti-inflammatory, antioxidant, hypoglycemic activities, and anticancer effects have been revealed. This review summarizes present studies indicating that xanthohumol also inhibits several critical pathophysiological steps during the development and course of chronic liver disease, including the activation and pro-fibrogenic genotype of hepatic stellate cells. Also the various mechanism of action and molecular targets of the beneficial xanthohumol effects will be described. Furthermore, the potential use of xanthohumol or a xanthohumol-enriched hop extract as therapeutic agent to combat the progression of chronic liver disease will be discussed. It is notable that in addition to its hepatoprotective effects, xanthohumol also holds promise as a therapeutic agent for treating obesity, dysregulation of glucose metabolism and other components of the metabolic syndrome including hepatic steatosis. Thus, therapeutic xanthohumol application appears as a promising strategy, particularly in obese patients, to inhibit the development as well as the progression of non-alcoholic fatty liver disease.

  8. Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

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    Yannan Qin

    2014-11-01

    Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

  9. Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells, Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells.

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    Laura Corbett

    Full Text Available The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis.

  10. Antihepatic Fibrosis Effect of Active Components Isolated from Green Asparagus (Asparagus officinalis L.) Involves the Inactivation of Hepatic Stellate Cells.

    Science.gov (United States)

    Zhong, Chunge; Jiang, Chunyu; Xia, Xichun; Mu, Teng; Wei, Lige; Lou, Yuntian; Zhang, Xiaoshu; Zhao, Yuqing; Bi, Xiuli

    2015-07-01

    Green asparagus (Asparagus officinalis L.) is a vegetable with numerous nutritional properties. In the current study, a total of 23 compounds were isolated from green asparagus, and 9 of these compounds were obtained from this genus for the first time. Preliminary data showed that the ethyl acetate (EtOAc)-extracted fraction of green asparagus exerted a stronger inhibitory effect on the growth of t-HSC/Cl-6 cells, giving an IC50 value of 45.52 μg/mL. The biological activities of the different compounds isolated from the EtOAc-extracted fraction with respect to antihepatic fibrosis were investigated further. Four compounds, C3, C4, C10, and C12, exhibited profound inhibitory effect on the activation of t-HSC/Cl-6 cells induced by TNF-α. The activation t-HSC/Cl-6 cells, which led to the production of fibrotic matrix (TGF-β1, activin C) and accumulation of TNF-α, was dramatically decreased by these compounds. The mechanisms by which these compounds inhibited the activation of hepatic stellate cells appeared to be associated with the inactivation of TGF-β1/Smad signaling and c-Jun N-terminal kinases, as well as the ERK phosphorylation cascade.

  11. Long non-coding RNA APTR promotes the activation of hepatic stellate cells and the progression of liver fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Fujun [Department of Gastroenterology, Jinshan Hospital of Fudan University, Jinshan, Shanghai, 201508 (China); Zheng, Jianjian [Wenzhou Key Laboratory of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000 (China); Mao, Yuqing [Department of Gastroenterology, Jinshan Hospital of Fudan University, Jinshan, Shanghai, 201508 (China); Dong, Peihong [Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000 (China); Li, Guojun [Department of Hepatology, Ningbo Yinzhou Second Hospital, Ningbo, 315000 (China); Lu, Zhongqiu [Department of Emergency, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000 (China); Guo, Chuanyong; Liu, Zhanju [Department of Gastroenterology, Shanghai Tenth People' s Hospital, Tongji University School of Medicine, Shanghai, 200072 (China); Fan, Xiaoming, E-mail: ktsqdph@163.com [Department of Gastroenterology, Jinshan Hospital of Fudan University, Jinshan, Shanghai, 201508 (China)

    2015-08-07

    In this study, we aimed at assessing a role of Alu-mediated p21 transcriptional regulator (APTR) in hepatofibrogenesis. APTR was upregulated in fibrotic liver samples and activated hepatic stellate cells (HSCs). Knockdown of APTR inhibited the activation of HSCs in vitro and mitigated the accumulation of collagen in vivo. Importantly, APTR silencing could abrogate TGF-β{sub 1}-induced upregulation of α-SMA in HSCs. In addition, inhibition of cell cycle and cell proliferation by APTR knockdown was attenuated by p21 siRNA1 in primary HSCs. Finally, serum APTR levels were increased in patients with liver cirrhosis, indicating a potential biomarker for liver cirrhosis. Collectively, evidence is proposed for a new biological role of APTR in hepatofibrogenesis. - Highlights: • APTR is upregulated in fibrotic liver tissues and activated HSCs. • APTR silencing inhibits HSC activation and the progression of liver fibrosis. • Antifibrotic effect of APTR silencing is achieved by increasing p21.

  12. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases human hepatic stellate cell activation.

    Science.gov (United States)

    Harvey, Wendy A; Jurgensen, Kimberly; Pu, Xinzhu; Lamb, Cheri L; Cornell, Kenneth A; Clark, Reilly J; Klocke, Carolyn; Mitchell, Kristen A

    2016-02-17

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-β. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.

  13. Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2

    Science.gov (United States)

    Xu, Jianfeng; Li, Jie; Hong, Zaifa; Yin, Zhenyu; Wang, Xiaomin

    2016-01-01

    Hepatic stellate cells (HSCs) are critical mediators of immunosuppression and the pathogenesis of hepatocellular carcinoma (HCC). Our previous work indicates that HSCs promote HCC progression by enhancing immunosuppressive cell populations including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). MDSCs are induced by inflammatory cytokines (e.g., prostaglandins) and are important in immune suppression. However, how HSCs mediate expansion of MDSCs is uncertain. Thus, we studied activated HSCs that could induce MDSCs from bone marrow cells and noted that HSC-induced MDSCs up-regulated immunosuppressive activity via iNOS, Arg-1, and IL-4Rα. After treating cells with a COX-2 inhibitor or an EP4 antagonist, we established that HSC-induced MDSC accumulation was mediated by the COX2-PGE2-EP4 signaling. Furthermore, in vivo animal studies confirmed that inhibition of HSC-derived PGE2 could inhibit HSC-induced MDSC accumulation and HCC growth. Thus, our data show that HSCs are required for MDSC accumulation mediated by the COX2-PGE2-EP4 pathway, and these data are the first to link HSC and MDSC subsets in HCC immune microenvironment and provide a rationale for targeting PGE2 signaling for HCC therapy. PMID:26758420

  14. Mannan binding lectin-associated serine protease 1 is induced by hepatitis C virus infection and activates human hepatic stellate cells.

    Science.gov (United States)

    Saeed, A; Baloch, K; Brown, R J P; Wallis, R; Chen, L; Dexter, L; McClure, C P; Shakesheff, K; Thomson, B J

    2013-11-01

    Mannan binding lectin (MBL)-associated serine protease type 1 (MASP-1) has a central role in the lectin pathway of complement activation and is required for the formation of C3 convertase. The activity of MASP-1 in the peripheral blood has been identified previously as a highly significant predictor of the severity of liver fibrosis in hepatitis C virus (HCV) infection, but not in liver disease of other aetiologies. In this study we tested the hypotheses that expression of MASP-1 may promote disease progression in HCV disease by direct activation of hepatic stellate cells (HSCs) and may additionally be up-regulated by HCV. In order to do so, we utilized a model for the maintenance of primary human HSC in the quiescent state by culture on basement membrane substrate prior to stimulation. In comparison to controls, recombinant MASP-1 stimulated quiescent human HSCs to differentiate to the activated state as assessed by both morphology and up-regulation of HSC activation markers α-smooth muscle actin and tissue inhibitor of metalloproteinase 1. Further, the expression of MASP-1 was up-regulated significantly by HCV infection in hepatocyte cell lines. These observations suggest a new role for MASP-1 and provide a possible mechanistic link between high levels of MASP-1 and the severity of disease in HCV infection. Taken together with previous clinical observations, our new findings suggest that the balance of MASP-1 activity may be proinflammatory and act to accelerate fibrosis progression in HCV liver disease.

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

    Institute of Scientific and Technical Information of China (English)

    赵东强; 姜慧卿; 修贺明; 张晓岚

    2002-01-01

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

  16. Diallyl Trisulfide Suppresses Oxidative Stress-Induced Activation of Hepatic Stellate Cells through Production of Hydrogen Sulfide.

    Science.gov (United States)

    Zhang, Feng; Jin, Huanhuan; Wu, Li; Shao, Jiangjuan; Zhu, Xiaojing; Chen, Anping; Zheng, Shizhong

    2017-01-01

    Accumulating data reveal that garlic has beneficial effects against chronic liver disease. We previously reported that diallyl trisulfide (DATS), the primary organosulfur compound in garlic, reduced fibrosis and attenuated oxidative stress in rat fibrotic liver. The present study was aimed at elucidating the underlying mechanisms. The primary rat hepatic stellate cells (HSCs) were cultured and stimulated with hydrogen peroxide (H2O2) for inducing HSC activation under oxidative stress. We examined the effects of DATS on the profibrogenic properties and oxidative stress in H2O2-treated HSCs. The results showed that DATS suppressed and reduced fibrotic marker expression in HSCs. DATS arrested cell cycle at G2/M checkpoint associated with downregulating cyclin B1 and cyclin-dependent kinase 1, induced caspase-dependent apoptosis, and reduced migration in HSCs. Moreover, intracellular levels of reactive oxygen species and lipid peroxide were decreased by DATS, but intracellular levels of glutathione were increased in HSCs. Furthermore, DATS significantly elevated hydrogen sulfide (H2S) levels within HSCs, but iodoacetamide (IAM) reduced H2S levels and significantly abrogated DATS production of H2S within HSCs. IAM also abolished all the inhibitory effects of DATS on the profibrogenic properties and oxidative stress in HSCs. Altogether, we demonstrated an H2S-associated mechanism underlying DATS inhibition of profibrogenic properties and alleviation of oxidative stress in HSCs. Modulation of H2S production may represent a therapeutic remedy for liver fibrosis.

  17. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation.

    Science.gov (United States)

    Twu, Yuh-Ching; Lee, Tzong-Shyuan; Lin, Yun-Lian; Hsu, Shih-Ming; Wang, Yuan-Hsi; Liao, Chia-Yu; Wang, Chung-Kwe; Liang, Yu-Chih; Liao, Yi-Jen

    2016-07-13

    In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

  18. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation

    Directory of Open Access Journals (Sweden)

    Yuh-Ching Twu

    2016-07-01

    Full Text Available In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2 protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1-induced collagen type 1 α1 (Col1a1, α-smooth muscle actin (α-SMA expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

  19. Activation of TGF-β1-CD147 positive feedback loop in hepatic stellate cells promotes liver fibrosis.

    Science.gov (United States)

    Li, Hai-Yan; Ju, Di; Zhang, Da-Wei; Li, Hao; Kong, Ling-Min; Guo, Yanhai; Li, Can; Wang, Xi-Long; Chen, Zhi-Nan; Bian, Huijie

    2015-11-12

    Activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) initiates HBV-associated fibrogenesis. The mechanism of TGF-β1 modulating HSC activation is not fully uncovered. We hypothesized a positive feedback signaling loop of TGF-β1-CD147 promoting liver fibrogenesis by activation of HSCs. Human HSC cell line LX-2 and spontaneous liver fibrosis model derived from HBV transgenic mice were used to evaluate the activation of molecules in the signaling loop. Wound healing and cell contraction assay were performed to detect the CD147-overexpressed HSC migration and contraction. The transcriptional regulation of CD147 by TGF-β1/Smad4 was determined using dual-luciferase reporter assay and chromatin immunoprecipitation. We found that a positive reciprocal regulation between TGF-β1 and CD147 mediated HSC activation. CD147 over-expression promoted HSC migration and accelerated TGF-β1-induced cell contraction. Phosphorylation of Smad2 and Smad3 in cooperation with Smad4 mediated the TGF-β1-regulated CD147 expression. Smad4 activated the transcription by direct interaction with CD147 promoter. Meanwhile, CD147 modulated the activated phenotype of HSCs through the ERK1/2 and Sp1 which up-regulated α-SMA, collagen I, and TGF-β1 synthesis. These findings indicate that TGF-β1-CD147 loop plays a key role in regulating the HSC activation and combination of TGF-β receptor inhibitor and anti-CD147 antibody might be promised to reverse fibrogenesis.

  20. Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Yan-Tong Guo; Xi-Sheng Leng; Tao Li; Ji-Run Peng; Sheng-Han Song; Liang-Fa Xiong; Zhi-Zhong Qin

    2005-01-01

    AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), on the expression of PPARγ in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs.METHODS: The activated HSCs were divided into three groups: control group, 3 μmol/L rosiglitazone group, and 10 μmol/L rosiglitazone group. The expression of PPARγ,α-smooth muscle actin (α-SMA), and type Ⅰ and Ⅲ collagen was detected by RT-PCR, Western blot and immunocytochemical staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry.RESULTS: The expression of PPARγ at mRNA and protein level markedly increased in HSCs of 10 μmol/L rosiglitazone group (tvalue was 10.870 and 4.627 respectively, P<0.01in both). The proliferation of HSCs in 10 μmol/L rosiglitazone group decreased significantly (t = 5.542, P<0.01), α-SMA expression level and type Ⅰ collagen synthesis ability were also reduced vs controls (tvalue= 10.256 and 14.627respectively, P<0.01 in both). The apoptotic rate of HSCs significantly increased in 10 μmol/L rosiglitazone group vs control (x2= 16.682, P<0.01).CONCLUSION: By increasing expression of PPARγ in activated HSCs, rosiglitazone, an agonist of PPARγ,decreases α-SMA expression and type Ⅰ collagen synthesis,inhibits cell proliferation, and induces cell apoptosis.

  1. Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types.

    Science.gov (United States)

    Salloum, Shadi; Holmes, Jacinta A; Jindal, Rohit; Bale, Shyam S; Brisac, Cynthia; Alatrakchi, Nadia; Lidofsky, Anna; Kruger, Annie J; Fusco, Dahlene N; Luther, Jay; Schaefer, Esperance A; Lin, Wenyu; Yarmush, Martin L; Chung, Raymond T

    2016-12-01

    Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta-1 (TGFβ1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFβ1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells.

  2. Dietary Flavonoid Hyperoside Induces Apoptosis of Activated Human LX-2 Hepatic Stellate Cell by Suppressing Canonical NF-κB Signaling

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    Liwen Wang

    2016-01-01

    Full Text Available Hyperoside, an active compound found in plants of the genera Hypericum and Crataegus, is reported to exhibit antioxidant, anticancer, and anti-inflammatory activities. Induction of hepatic stellate cell (HSC apoptosis is recognized as a promising strategy for attenuation of hepatic fibrosis. In this study, we investigated whether hyperoside treatment can exert antifibrotic effects in human LX-2 hepatic stellate cells. We found that hyperoside induced apoptosis in LX-2 cells and decreased levels of α-smooth muscle actin (α-SMA, type I collagen, and intracellular reactive oxygen species (ROS. Remarkably, hyperoside also inhibited the DNA-binding activity of the transcription factor NF-κB and altered expression levels of NF-κB-regulated genes related to apoptosis, including proapoptotic genes Bcl-Xs, DR4, Fas, and FasL and anti-apoptotic genes A20, c-IAP1, Bcl-XL, and RIP1. Our results suggest that hyperoside may have potential as a therapeutic agent for the treatment of liver fibrosis.

  3. HIV-1 gp120 signaling through TLR4 modulates innate immune activation in human macrophages and the biology of hepatic stellate cells.

    Science.gov (United States)

    Del Cornò, Manuela; Cappon, Andrea; Donninelli, Gloria; Varano, Barbara; Marra, Fabio; Gessani, Sandra

    2016-09-01

    Highly active antiretroviral therapy has significantly improved the prognosis of HIV-infected subjects. However, patients treated long term still manifest increased mortality and, even with undetectable plasma viremia, often experience persistent immune activation. Furthermore, liver-related mortality is now the most common cause of non-AIDS-related death in HIV-infected individuals on highly active antiretroviral therapy through accelerated fibrosis progression. TLRs are the first line of the host response to pathogens and play an important role in human host defense against viruses through sensing of viral structural proteins. Growing evidence points to TLR4 as a key player in chronic immune activation, HIV recognition/replication, and liver fibrosis progression, suggesting that HIV triggering of TLR4 may dictate some aspects of the multifaceted AIDS pathogenesis. In this study, we provide evidence for an interplay between host TLR4 and HIV-1 gp120 in human monocyte-derived macrophages and hepatic stellate cells, leading to intracellular pathways and biologic activities that mediate proinflammatory and profibrogenic signals. Finally, we hypothesize that CCR5 and TLR4 are likely part of a common receptor cluster, as the blocking of CCR5 by specific antagonists impairs the macrophage capacity to produce chemokines in response to LPS. Chronic immune activation and liver fibrosis remain important obstacles for highly active antiretroviral therapy success. Thus, the identification of gp120-TLR4 axis as a novel determinant of immune system and hepatic stellate cell biology opens new perspectives to the management of HIV infection and disease.

  4. Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays

    Institute of Scientific and Technical Information of China (English)

    Wu-Jun Xiong; Li-Juan Hu; Yi-Cheng Jian; Li-Jing Wang; Ming Jiang; Wei Li; Yi He

    2012-01-01

    AIM:To identify differentially expressed genes in quiescent and activated hepatic stellate cells (HSCs) and explore their functions.METHODS:HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation.Total RNA and mRNA of quiescent HSCs,and cultureactivated HSCs were extracted,quantified and reversely transcripted into cDNA.The global gene expression profile was analyzed by microarray with Affymetrix rat genechip.Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation,Visualization and Integrated Discovery.Microarray data were validated by quantitative real-time polymerase chain reaction (qRTPCR).The function of Wnt5a on human HSCs line LX-2was assessed with lentivirus-mediated Wnt5a RNAi.The expression of Wnt5a in fibrotic liver of a carbon tetrachloride (CCl4)-induced fibrosis rat model was also analyzed with Western blotting.RESULTS:Of the 28 700 genes represented on this chip,2566 genes displayed at least a 2-fold increase or decrease in expression at a P < 0.01 level with a false discovery rate.Of these,1396 genes were upregulated,while 1170 genes were downregulated in culture-activated HSCs.These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms.The most enriched GO terms included response to wounding,wound healing,regulation of cell growth,vasculature development and actin cytoskeleton organization.KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs.Wnt5a was significantly increased in cultureactivated HSCs as compared with quiescent HSCs.qRTPCR validated the microarray data.Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation,downregulated expressions of type I collagen and transforming

  5. Targeted elimination of activated hepatic stellate cells by an anti-epidermal growth factor-receptor single chain fragment variable antibody-tumor necrosis factor-related apoptosis-inducing ligand (scFv425-sTRAIL)

    NARCIS (Netherlands)

    Arabpour, Mohammad; Poelstra, Klaas; Helfrich, Wijnand; Bremer, Edwin; Haisma, Hidde J.

    2014-01-01

    BackgroundProgressive liver fibrosis is the result of chronic liver injury and is characterized by the excessive accumulation of extracellular matrix that may result in liver failure. Activated hepatic stellate cells are known to play a central role in this process and their elimination is a crucial

  6. Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

    Institute of Scientific and Technical Information of China (English)

    Yuan-Wen Chen; Jian-Xin Wu; Ying-Wei Chen; Ding-Guo Li; Han-Ming Lu

    2005-01-01

    AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.METHODS: HSCs were isolated from rats by in situperfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 hwith DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscleα-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲprocollagen (PCⅢ) in supernatants were determined byradioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2%(control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P<0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%,75.7% or 80.7% respectively (P<0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 m

  7. Green tea polyphenol epigallocatechin-3-gallate suppresses rat hepatic stellate cell invasion by inhibition of MMP-2 expression and its activation

    Institute of Scientific and Technical Information of China (English)

    Mao-chuan ZHEN; Xiao-hui HUANG; Qian WANG; Kai SUN; Yun-jian LIU; Wen LI; Long-Juan ZHANG; Liang-qi CAO; Xi-ling CHEN

    2006-01-01

    Aim: Epigallocatechin-3-gallate (EGCG) is the major component of green tea polyphenols, whose wide range of biological properties includes anti-fibrogenic activity. Matrix metalloproteinases (MMP) that participate in extracellular matrix degradation are involved in the development of hepatic fibrosis. The present study investigates whether EGCG inhibits activation of the major gelatinase matrix metalloproteinase-2 (MMP-2) in rat hepatic stellate cells (HSC). Methods: The expression of MMP-2, tissue inhibitors of metalloproteinases-2 (TIMP-2), and membrane-type 1-MMP (MT1-MMP) was assessed by RT-PCR and Western blot analyses. MMP-2 activity was evaluated by zymography and MT1-MMP activity was assessed by an enzymatic assay. HSC migration was measured by a wound healing assay and cell invasion was performed using Transwell cell culture chambers. Results: The expression of MMP-2 mRNA and protein in HSC was substantially reduced by EGCG treatment. EGCG treatment also reduced con-canavalin A (ConA)-induced activation of secreted MMP-2 and reduced MT1-MMP activity in a dose-dependent manner. In addition, EGCG inhibited either HSC migration or invasion. Conclusion: The abilities of EGCG to suppress MMP-2 activation and HSC invasiveness suggest that EGCG may be useful in the treatment and prevention of hepatic fibrosis.

  8. Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation.

    Science.gov (United States)

    Jiang, Shuang; Zhang, Yu; Zheng, Jin-Hua; Li, Xia; Yao, You-Li; Wu, Yan-Ling; Song, Shun-Zong; Sun, Peng; Nan, Ji-Xing; Lian, Li-Hua

    2017-03-01

    Purinergic receptor P2x7 (P2x7R) is a key modulator of liver inflammation and fibrosis. The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation. Lipopolysaccharide (LPS) or the conditioned medium (CM) from LPS-stimulated RAW 264.7 mouse macrophages was supplemented to human hepatic stellate cells, LX-2 for 24h and P2x7R selective antagonist A438079 (10μM) was supplemented to LX-2 cells 1h before LPS or CM stimulation. In addition LX-2 cells were primed with LPS for 4h and subsequently stimulated for 30min with 3mM of adenosine 5'-triphosphate (ATP). A438079 was supplemented to LX-2 cells 10min prior to ATP. Directly treated with LPS on LX-2 cells, mRNA expressions of interleukin (IL)-1β, IL-18 and IL-6 were increased, as well as mRNA expressions of P2x7R, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) mRNA. LPS also increased α-smooth muscle actin (α-SMA) and type I collagen mRNA expressions, as well as collagen deposition. Interestingly treatment of LX-2 cells with LPS-activated CM exhibited the greater increase of above factors than those in LX-2 cells directly treated with LPS. Pretreatment of A438079 on LX-2 cells stimulated by LPS or LPS-activated CM both suppressed IL-1β mRNA expression. LPS combined with ATP dramatically increased protein synthesis and cleavage of IL-1β and its mRNA level than those in HSC treated with LPS or ATP alone. Additionally LX-2 cells primed with LPS and subsequently stimulated for 30min with ATP greatly increased mRNA and protein expression of caspase-1, NLRP3 and P2x7R, as well as liver fibrosis markers, α-SMA and type I collagen. These events were remarkably suppressed by A438079 pretreatment. siRNA against P2x7R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type I collagen. The involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1

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  11. Inhibitory effect of tanshinone IIA on rat hepatic stellate cells.

    Directory of Open Access Journals (Sweden)

    Ya-Wei Liu

    Full Text Available Anti-inflammation via inhibition of NF-κB pathways in hepatic stellate cells (HSCs is one therapeutic approach to hepatic fibrosis. Tanshinone IIA (C19H18O3, Tan IIA is a lipophilic diterpene isolated from Salvia miltiorrhiza Bunge, with reported anti-inflammatory activity. We tested whether Tan IIA could inhibit HSC activation.The cell line of rat hepatic stellate cells (HSC-T6 was stimulated with lipopolysaccharide (LPS (100 ng/ml. Cytotoxicity was assessed by MTT assay. HSC-T6 cells were pretreated with Tan IIA (1, 3 and 10 µM, then induced by LPS (100 ng/ml. NF-κB activity was evaluated by the luciferase reporter gene assay. Western blotting analysis was performed to measure NF-κB-p65, and phosphorylations of MAPKs (ERK, JNK, p38. Cell chemotaxis was assessed by both wound-healing assay and trans-well invasion assay. Quantitative real-time PCR was used to detect gene expression in HSC-T6 cells.All concentrations of drugs showed no cytotoxicity against HSC-T6 cells. LPS stimulated NF-κB luciferase activities, nuclear translocation of NF-κB-p65, and phosphorylations of ERK, JNK and p38, all of which were suppressed by Tan IIA. In addition, Tan IIA significantly inhibited LPS-induced HSCs chemotaxis, in both wound-healing and trans-well invasion assays. Moreover, Tan IIA attenuated LPS-induced mRNA expressions of CCL2, CCL3, CCL5, IL-1β, TNF-α, IL-6, ICAM-1, iNOS, and α-SMA in HSC-T6 cells.Our results demonstrated that Tan IIA decreased LPS-induced HSC activation.

  12. Expression of AT1amRNA in rat hepatic stellate cells and its effects on cell growth collagen production

    Institute of Scientific and Technical Information of China (English)

    张艺军; 杨希山; 吴平生; 廖贵清; 杨国平; 张晓峰; 陈晓清

    2004-01-01

    @@ Activated hepatic stellate cells (HSCs) play important roles in hepatic fibrosis. Studies on HSCs activation in vitro have shown that this process is regulated by a wide variety of growth factors and cytokines.1 Recent data indicate that AngⅡ is responsible for the mechanisms of myocardial fibrosis and kidney fibrosis; but there are only few reports on hepatic fibrosis.2-8

  13. Effects of curcumin on peroxisome proliferator-activated receptor γ expression and nuclear translocation/redistribution in culture-activated rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    CHENG Yang; PING Jian; XU Lie-ming

    2007-01-01

    Background The function of peroxisome proliferator-activated receptor γ (PPARγ) in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells (HSCs) through PPARγ signaling were investigated.Methods HSCs were isolated from the normal Sprague Dawley rats through in situ perfusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy Isulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARγ subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay.Results Curcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARγ nuclear expression level decreased. Curcumin up-regulated PPARγ expression and significantly inhibited the production of α-SMA and collagen I. PPARγ is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFκB p65 protein and TGFβR-I protein expression were down-regulated significantly by curcumin. The

  14. Quercetin attenuates the activation of hepatic stellate cells and liver fibrosis in mice through modulation of HMGB1-TLR2/4-NF-κB signaling pathways.

    Science.gov (United States)

    Li, Xi; Jin, Qianwen; Yao, Qunyan; Xu, Beili; Li, Zheng; Tu, Chuantao

    2016-11-02

    This study aimed to investigate the effects of quercetin on liver fibrogenesis in mice and to elucidate the underlying molecular mechanisms. Mice were administered with carbon tetrachloride (CCl4) for eight weeks to induce liver fibrosis and concomitantly orally treated with quercetin (50mgkg(-1)day(-1)). Here, we demonstrated that quercetin dramatically ameliorated liver injury, inflammation, and hepatic fibrogenesis induced by CCl4. Quercetin also inhibited the activation of hepatic stellate cells (HSC) in vivo and in vitro, as evaluated by α-smooth muscle actin (α-SMA) expression, which is a specific marker of HSC activation. Moreover, reduced fibrosis was associated with decreased high-mobility group box 1 (HMGB1), toll like receptor (TLR) 2 and TLR4 genes, and protein expression. Quercetin also inhibited the cytoplasmic translocation of HMGB1 in hepatocytes of fibrotic livers. Further investigation demonstrated that quercetin treatment significantly attenuated CCl4-induced nuclear translocation of the nuclear factor-κB (NF-κB) p65 and inhibited degradation of IκBα (an inhibitor of NF-κB) expression in the liver compared with vehicle-treated fibrotic mice. Considered together, our data indicate that quercetin has hepatoprotective and anti-fibrotic effects in animal models of liver fibrosis, the mechanism of which may be involved in modulating the HMGB1-TLR2/4-NF-κB signaling pathways.

  15. Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC).

    Science.gov (United States)

    Breitkopf, Katja; Roeyen, Claudia van; Sawitza, Iris; Wickert, Lucia; Floege, Jürgen; Gressner, Axel M

    2005-09-07

    The platelet-derived growth factor (PDGF) family, which regulates many physiological and pathophysiological processes has recently been enlarged by two new members, the isoforms PDGF-C and -D. Little is known about the expression levels of these new members in hepatic fibrosis. We therefore investigated by quantitative real time PCR (Taqman) the mRNA expression profiles of all four PDGF isoforms in transdifferentiating primary cultured hepatic stellate cells (HSC), an in vitro model system of hepatic fibrogenesis, either with or without stimulation of the cells with PDGF-BB or TGF-beta1. All four isoforms were expressed in HSC transdifferentiating to myofibroblast-like cells (MFB) albeit with different profiles: while PDGF-A mRNA exhibited minor fluctuations only, PDGF-B was rapidly down-regulated. In contrast, both PDGF-C and -D mRNA were strongly induced: PDGF-C up to 5 fold from day 2 to day 8 and PDGF-D up to 8 fold from day 2 to day 5 of culture. Presence of PDGF-DD in activated HSC was confirmed at the protein level by immunocytochemistry. Stimulation of HSC and MFB with PDGF-BB led to down-regulation of the new isoforms, whereas TGF-beta1 upregulated PDGF-A only. We further show that PDGF receptor-beta (PDGFR-beta) mRNA was rapidly upregulated within the first day of culture and was constantly expressed from day 2 on while the expression profile of PDGFR-alpha mRNA was very similar to that of PDGF-A during transdifferentiation. Given the dramatic changes in PDGF-C and -D expression, which may compensate for down-regulation of PDGF-B, we hypothesize that the new PDGF isoforms may fulfil specific functions in hepatic fibrogenesis.

  16. Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Ning-Li Chai; Qiang Fu; Hui Shi; Chang-Hao Cai; Jun Wan; Shi-Ping Xu; Ben-Yan Wu

    2012-01-01

    AIM:To investigate the potential mechanism of ArgGly-Asp (RGD) peptide-labeled liposome loading oxymatrine (OM) therapy in CCl4-induced hepatic fibrosis in METHODS:We constructed a rat model of CCl4-induced hepatic fibrosis and treated the rats with different formulations of OM.To evaluate the antifibrotic effect of OM,we detected levels of alkaline phosphatase,hepatic histopathology (hematoxylin and eosin stain and Masson staining) and fibrosis-related gene expression of matrix metallopeptidase (MMP)-2,tissue inhibitor of metalloproteinase (TIMP)-1 as well as type Ⅰ procollagen via quantitative real-time polymerase chain reaction.To detect cell viability and apoptosis of hepatic stellate cells (HSCs),we performed 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay and flow cytometry.To reinforce the combination of oxymatrine with HSCs,we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM,and its targeting of HSCs was examined by fluorescent microscopy.RESULTS:OM attenuated CCl4-induced hepatic fibrosis,as defined by reducing serum alkaline phosphatase (344.47 ± 27.52 U/L vs 550.69 ± 43.78 U/L,P < 0.05),attenuating liver injury and improving collagen deposits (2.36% ± 0.09% vs 7.70% ± 0.60%,P < 0.05) and downregulating fibrosis-related gene expression,that is,MMP-2,TIMP-1 and type Ⅰ procollagen (P < 0.05).OM inhibited cell viability and induced apoptosis of HSCs in vitro.RGD promoted OM targeting of HSCs and enhanced the therapeutic effect of OM in terms of serum alkaline phosphatase (272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L,P < 0.05),liver injury,collagen deposits (0.26% ± 0.09% vs 2.36% ± 0.09%,P < 0.05) and downregulating fibrosis-related gene expression,that is,MMP-2,TIMP-1 and type Ⅰ procollagen (P < 0.05).Moreover,in vitro assay demonstrated that RGD enhanced the effect of OM on HSC viability and apoptosis.CONCLUSION:OM attenuated hepatic fibrosis by

  17. A Novel Matrine Derivative WM130 Inhibits Activation of Hepatic Stellate Cells and Attenuates Dimethylnitrosamine-Induced Liver Fibrosis in Rats

    Directory of Open Access Journals (Sweden)

    Yang Xu

    2015-01-01

    Full Text Available Activation of hepatic stellate cells (HSCs is a critical event in process of hepatic fibrogenesis and cirrhosis. Matrine, the active ingredient of Sophora, had been used for clinical treatment of acute/chronic liver disease. However, its potency was low. We prepared a high potency and low toxicity matrine derivate, WM130 (C30N4H40SO5F, which exhibited better pharmacological activities on antihepatic fibrosis. This study demonstrated that WM130 results in a decreased proliferative activity of HSC-T6 cells, with the half inhibitory concentration (IC50 of 68 μM. WM130 can inhibit the migration and induce apoptosis in HSC-T6 cells at both concentrations of 68 μM (IC50 and 34 μM (half IC50. The expression of α-SMA, Collagen I, Collagen III, and TGF-β1 could be downregulated, and the protein phosphorylation levels of EGFR, AKT, ERK, Smad, and Raf (p-EGFR, p-AKT, p-ERK, p-Smad, and p-Raf were also decreased by WM130. On the DMN-induced rat liver fibrosis model, WM130 can effectively reduce the TGF-β1, AKT, α-SMA, and p-ERK levels, decrease the extracellular matrix (ECM formation, and inhibit rat liver fibrosis progression. In conclusion, this study demonstrated that WM130 can significantly inhibit the activation of HSC-T6 cells and block the rat liver fibrosis progression by inducing apoptosis, suppressing the deposition of ECM, and inhibiting TGF-β/Smad and Ras/ERK pathways.

  18. DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells.

    Science.gov (United States)

    Olaso, E; Ikeda, K; Eng, F J; Xu, L; Wang, L H; Lin, H C; Friedman, S L

    2001-11-01

    Type I collagen provokes activation of hepatic stellate cells during liver injury through mechanisms that have been unclear. Here, we tested the role of the discoidin domain tyrosine kinase receptor 2 (DDR2), which signals in response to type I collagen, in this pathway. DDR2 mRNA and protein are induced in stellate cells activated by primary culture or in vivo during liver injury. The receptor becomes tyrosine phosphorylated in response to either endogenous or exogenous type I collagen, whereas its expression is downregulated during cellular quiescence induced by growth on Matrigel. We developed stellate cell lines stably overexpressing either wild-type DDR2, a constitutively active chimeric DDR2 receptor (Fc-DDR2), a truncated receptor expressing the extracellular domain, or a kinase-dead DDR2 Cells overexpressing DDR2 showed enhanced proliferation and invasion through Matrigel, activities that were directly related to increased expression of active matrix metalloproteinase 2 (MMP-2). These data show that DDR2 is induced during stellate cell activation and implicate the phosphorylated receptor as a mediator of MMP-2 release and growth stimulation in response to type I collagen. Moreover, type I collagen-dependent upregulation of DDR2 expression establishes a positive feedback loop in activated stellate cells, leading to further proliferation and enhanced invasive activity.

  19. AMP-activated protein kinase inhibits TGF-β-induced fibrogenic responses of hepatic stellate cells by targeting transcriptional coactivator p300.

    Science.gov (United States)

    Lim, Joong-Yeon; Oh, Min-A; Kim, Won Ho; Sohn, Hee-Young; Park, Sang Ick

    2012-03-01

    Liver fibrosis is a common consequence of various chronic liver injuries, including virus infection and ethanol. Activated hepatic stellate cells (HSCs) contribute to liver fibrosis through the accumulation of extracellular matrix proteins, including type I alpha collagen (COL1A). The activation of adenosine monophosphate-activated protein kinase (AMPK) modulates HSCs activation, but its underlying mechanism remains unclear. Here, we report that AMPK inhibits transforming growth factor (TGF)-β-induced fibrogenic property of HSCs by regulating transcriptional coactivator p300. We treated human (LX-2) and rat (CFSC-2G) HSC lines with TGF-β to induce fibrogenic activation of HSCs. Pharmacological activation of AMPK by treatment with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), metformin, or adiponectin lowered TGF-β-induced expression of COL1A and myofibroblast marker alpha-smooth muscle actin (α-SMA). Transient transduction of constitutively active AMPKα (caAMPKα) was sufficient to attenuate COL1A and α-SMA expression, whereas an AMPK inhibitor considerably abrogated the inhibitory effect of AICAR on fibrogenic gene expression. Although AMPK significantly suppressed Smad-dependent transcription, it did not affect TGF-β-stimulated phosphorylation, nuclear localization, or DNA-binding activity of Smad2/3. AICAR rather attenuated TGF-β-induced Smad3 interaction with transcriptional coactivator p300 accompanying with reduction of Smad3 acetylation. Moreover, AICAR induced not only physical interaction between AMPK and p300 but also proteasomal degradation of p300 protein. Our data provide substantial evidence that AMPK could be a novel therapeutic target for treatment of liver fibrosis, by demonstrating the underlying mechanism of AMPK-induced antifibrotic function in HSCs.

  20. Effects of octreotide on expression of L-type voltage-operated calcium channels and on intracellular Ca2+ in activated hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    丁惠国; 王宝恩; 贾继东; 夏华向; 王振宇; 赵春惠; 徐燕琳

    2004-01-01

    Background The contractility of hepatic stellate cells (HSCs) may play an important role in the pathogenesis of cirrhosis with portal hypertension. The aim of this study was to research the effects of octreotide, an analogue of somatostatin, on intracellular Ca2+ and on the expression of L-type voltage-operated calcium channels (L-VOCCs) in activated HSCs, and to try to survey the use of octreotide in treatment and prevention of cirrhosis with portal hypertension complications. Methods HSC-T6, an activated HSCs line, was plated on small glass coverslips in 35-mm culture dishes at a density of 1×105/ml, and incubated in DMEM media for 24 hours. After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured by Laser Scanning Confocal Microscopy (LSCM). The dynamic changes in activated HSCs of intracellular Ca2+, stimulated by octreotide, endothelin-1, and KCl, respectively, were also determined by LSCM. Each experiment was repeated six times. L-VOCC expression in HSCs was estimated by immunocytochemistry. Results After octreotide stimulation, a signifcant decrease in the intracellular Ca2+ of activated HSCs was observed. However, octreotide did not inhibit the increases in intracellular Ca2+ after stimulation by KCl and endothelin-1. Moreover, octreotide did not significantly affect L-VOCC expression. These results suggest that neither L-VOCC nor endothelin-1 receptors in activated HSCs are inhibited by octreotide. Conclusions Octreotide may decrease portal hypertension and intrahepatic vascular tension by inhibiting activated HSCs contractility through decreases in intracellular Ca2+. The somatostatin receptors in activated HSCs may be inhibited by octreotide.

  1. X4 Human immunodeficiency virus type 1 gp120 promotes human hepatic stellate cell activation and collagen I expression through interactions with CXCR4.

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    Feng Hong

    Full Text Available BACKGROUND & AIMS: Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the in vitro impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the in vivo expression of gp120 in HIV/HCV coinfected livers. METHODS: Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/- either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies. RESULTS: X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers. CONCLUSIONS: X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.

  2. Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells.

    Science.gov (United States)

    Devhare, Pradip B; Sasaki, Reina; Shrivastava, Shubham; Di Bisceglie, Adrian M; Ray, Ranjit; Ray, Ratna B

    2017-03-15

    Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor β (TGF-β) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3-TGF-β pathway in HSC. This study contributes to the

  3. Cytoglobin is expressed in hepatic stellate cells, but not in myofibroblasts, in normal and fibrotic human liver.

    Science.gov (United States)

    Motoyama, Hiroyuki; Komiya, Tohru; Thuy, Le Thi Thanh; Tamori, Akihiro; Enomoto, Masaru; Morikawa, Hiroyasu; Iwai, Shuji; Uchida-Kobayashi, Sawako; Fujii, Hideki; Hagihara, Atsushi; Kawamura, Etsushi; Murakami, Yoshiki; Yoshizato, Katsutoshi; Kawada, Norifumi

    2014-02-01

    Cytoglobin (CYGB) is ubiquitously expressed in the cytoplasm of fibroblastic cells in many organs, including hepatic stellate cells. As yet, there is no specific marker with which to distinguish stellate cells from myofibroblasts in the human liver. To investigate whether CYGB can be utilized to distinguish hepatic stellate cells from myofibroblasts in normal and fibrotic human liver, human liver tissues damaged by infection with hepatitis C virus (HCV) and at different stages of fibrosis were obtained by liver biopsy. Immunohistochemistry was performed on histological sections of liver tissues using antibodies against CYGB, cellular retinol-binding protein-1 (CRBP-1), α-smooth muscle actin (α-SMA), thymocyte differentiation antigen 1 (Thy-1), and fibulin-2 (FBLN2). CYGB- and CRBP-1-positive cells were counted around fibrotic portal tracts in histological sections of the samples. The expression of several of the proteins listed above was examined in cultured mouse stellate cells. Quiescent stellate cells, but not portal myofibroblasts, expressed both CYGB and CRBP-1 in normal livers. In fibrotic and cirrhotic livers, stellate cells expressed both CYGB and α-SMA, whereas myofibroblasts around the portal vein expressed α-SMA, Thy-1, and FBLN2, but not CYGB. Development of the fibrotic stage was positively correlated with increases in Sirius red-stained, α-SMA-positive, and Thy-1-positive areas, whereas the number of CYGB- and CRBP-1-positive cells decreased with fibrosis development. Primary cultured mouse stellate cells expressed cytoplasmic CYGB at day 1, whereas they began to express α-SMA at the cellular margins at day 4. Thy-1 was undetectable throughout the culture period. In human liver tissues, quiescent stellate cells are CYGB positive. When activated, they also become α-SMA positive; however, they are negative for Thy-1 and FBLN2. Thus, CYGB is a useful marker with which to distinguish stellate cells from portal myofibroblasts in the damaged human

  4. Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation.

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    Tian Lan

    Full Text Available Reactive oxygen species (ROS produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as pro-fibrotic genes in hepatic stellate cells (HSCs as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4 to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS, platelet-derived growth factor (PDGF, or sonic hedgehog (Shh in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days. Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

  5. 丙型肝炎病毒F蛋白诱导LX2肝星形细胞活化%Activation of hepatic stellate LX2 cells by F protein of hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    邵圣文; 徐伯赢; 卢添宝; 徐菊玲; 顾福萍; 段劲

    2011-01-01

    目的 探讨丙型肝炎病毒(HCV)F蛋白对肝星形细胞(HSC)功能的影响.方法 采用脂质体转染法将pcDNA3.1-f(实验组)或pcDNA3.1空质粒(对照组)转染HSC株LX2细胞,24、48 h后收集细胞,用Western blot法检测α-肌动蛋白(α-SMA)含量,RT-PCR法测定金属蛋白酶组织抑制因子1(TIMP-1)和基质金属蛋白酶2(MMP-2)基因mRNA表达水平.结果 转染后24、48 h,实验组LX2细胞α-SMA蛋白表达量以及TIMP-1基因mRNA水平均明显高于对照组,两组细胞MMP-2 基因mRNA水平接近.结论 HCV F蛋白能够活化HSC,上调其TIMP-1基因转录水平,但不影响MMP-2基因转录水平,使MMP-2蛋白的有效酶活性下降,造成肝组织细胞外基质(ECM)降解速率减慢,进而发生纤维化.%Objective To investigate the effect of hepatitis C virus( HCV) F protein on function of hepatic stellate cells.Methods 24 h and 48 h after transfection pcDNA3.1-f(test group)or pcDNA3.1(control group) plasmid into hepatic stellate LX2 cells by liposome , the protein of smooth muscle α-actin( α-SMA) was detected by Western blot, and the mRNA level of tissue inhibitor factor 1 of metalloproteinases(TIMP-1) and matrix metalloproteinase 2(MMP-2) gene were examined by RT-PCR.Results 24 h and 48 h after LX2 cells transfected pcDNA3.1-f(test group) or pcDNA3.1(control group) , the content of α-SMA protein and the mRNA level of TIMP-1 gene of cells in test group was high than that in control group,and the mRNA level of MMP-2 gene of cells showed no difference between test and control groups.Conclusion HCV F protein is able to activate hepatic stellate LX2 cells.enhance the mRNA level of TIMP-1 gene,but not affect MMP-2 gene transcription,which make the effective enzyme activity of MMP-2 protein be decreased,the degradation rate of ECM in hepatic tissue slowed down.and result in hepatic fibrosis.

  6. Standardized Salvia miltiorrhiza Extract Suppresses Hepatic Stellate Cell Activation and Attenuates Steatohepatitis Induced by a Methionine-Choline Deficient Diet in Mice

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    Hak Sung Lee

    2014-06-01

    Full Text Available The aim of this study was to examine the effect of standardized extract of Salvia miltiorrhiza (SME on gene and protein expression of non-alcoholic steatohepatitis (NASH-related factors in activated human hepatic stellate cells (HSC, and in mice with steatohepatitis induced by a methionine-choline deficient (MCD diet. Male C57BL/6J mice were placed on an MCD or control diet for 8 weeks and SME (0, 0.1, 0.5 and 1 mg/kg body weight was administered orally every other day for 4 or 6 weeks. HSCs from the LX-2 cell line were treated with transforming growth factor β-1 (TGF-β1 or TGF-β1 plus SME (0.1–10 μg/mL. To investigate the effect of SME on reactive oxygen species (ROS-induced condition, LX-2 cells were treated with hydrogen peroxide (H2O2 or H2O2 plus SME (0.1–100 μg/mL. MCD administration for 12 weeks increased mRNA expression of tumor necrosis factor (TNF-α, TGF-β1, interleukin-1β (IL-1β, C-reactive protein (CRP, α-smooth muscle actin (α-SMA, type I collagen, matrix metalloproteinase-2 (MMP-2 and MMP-9. TGF-β1-induced LX-2 cells exhibited similar gene expression patterns. SME treatment significantly reduced the mRNA and protein expression of NASH-related factors in the mouse model and HSCs. Histopathological liver analysis showed improved non-alcoholic fatty liver disease (NAFLD activity and fibrosis score in SME-treated mice. The in vivo studies showed that SME had a significant effect at low doses. These results suggest that SME might be a potential therapeutic candidate for NAFLD treatment.

  7. Inhibition on the production of collagen type Ⅰ, Ⅲ of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid

    Institute of Scientific and Technical Information of China (English)

    Wen-Bin Liu; Chang-Qing Yang; Wei Jiang; Yi-Qing Wang; Jing-Sheng Guo; Bo-Ming He; Ji-Yao Wang

    2003-01-01

    AIM: To investigate the inhibition effects on the productionof collagen type I, Ⅲ secreted by activated rat hepatic stellatecells (rHSCs) by antisense tissue inhibitors of metalloproteinase1 (TIMP-1) recombinant plasmid through elevating interstitialcollagenase activity.METHODS: rHSCs were extracted from normal rat liverby pronase and collagenase digestion and purified bycentrifugal elutriation, and were cultured on plastic dishesuntil they were activated to a myofibroblastic phenotypeafter 7-10 days. RT-Nest-PCR and gene recombinanttechniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryoticcells. The recombinant plasmid and the pcDNA3 emptyplasmid were transfected in rHSCs by Effectene (QIAGEN)separately. Cells were selected after growing in DMEMcontaining 400 μg/ml G418 for 2-3 weeks. Expression ofexogenous gene was assessed by Northern blot, andexpression oflIMP-1 in rHSCs was determined by Northernblot and Western blot. We tested the interstitial collagenaseactivity with FITC-labled type I collagen as substrate.Ultimately, we quantified the type Ⅰ, Ⅲ collagen byWestern blot.RESULTS: The exogenous antisense TIMP-1 recombinantplasmid could be expressed in rHSCs well, which couldblock the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNAlevel (P<0.05); the ratio of TIMP-1/β-actin was 0.31, 0.98and 1.32 separately at protein level (P<0.05); It mightelevate active and latent interstitial collagenase activity,the collagenase activity was 0.3049, 0.1411 and 0.1196respectively. (P<0.05), which led to promotion thedegradation of type Ⅰ, Ⅲ collagen, the ratio of collagen I/β-actin was 0.63, 1.78 and 1.92 separately (P<0.05); andthe ratio of collagen Ⅲ/β-actin was 0.59, 1.81 and 1.98separately (P<0.05).CONCLUSION: These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on theproduction of type Ⅰ, Ⅲ collagens

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  16. Hepatic stellate cells increase the immunosuppressive function of natural Foxp3+ regulatory T cells via IDO-induced AhR activation.

    Science.gov (United States)

    Kumar, Sudhir; Wang, Jiang; Thomson, Angus W; Gandhi, Chandrashekhar R

    2017-02-01

    Immunosuppressive, naturally occurring CD4(+)CD25(+)forkhead box p3(+) (Foxp3(+)) regulatory T cells (nTregs) offer potential for the treatment of immune-mediated inflammatory disorders. However, potential instability of ex vivo-expanded nTregs following their adoptive transfer may be a significant limitation. LPS-stimulated hepatic stellate cells (HSCs) induce expansion and enhance the suppressive function and stability of allogeneic nTregs We aimed to delineate mechanisms underlying HSC-induced expansion and increased potency of nTregs HSCs and nTregs were isolated from mouse livers and spleens, respectively. Following coculture with LPS-pretreated allogeneic HSCs (LPS/HSCs), proliferation of nTregs was measured by CFSE dilution, and Foxp3 expression and acetylation were determined by immunoprecipitation (IP) and Western blotting analysis. Expression of various genes associated with immunologic tolerance was determined by quantitative RT-PCR (qRT-PCR). LPS stimulation increased the expression and activity of the immunoregulatory enzyme IDO1 in HSCs, and LPS/HSCs stimulated aryl hydrocarbon receptor (AhR) signaling in cocultured nTregs Reciprocally, Tregs increased IDO1 expression in HSCs. IDO1(-/-) LPS/HSCs were inferior to WT LPS/HSCs in stimulating nTreg expansion. Pharmacologic inhibition of IDO1 in HSCs by 1-methyltryptophan (1MT) inhibited LPS/HSC-induced AhR signaling in nTregs, which was responsible for their expansion, Foxp3 expression, and stabilization of Foxp3 by increasing acetylation of lysine residues. Finally, HSCs cryopreserved, following 2-3 passages, were as potent as primary-cultured HSCs in expanding nTregs In conclusion, LPS/HSCs expand allogeneic nTregs through an IDO-dependent, AhR-mediated mechanism and increase their stability through lysine-acetylation of Foxp3. nTregs expanded by cryopreserved HSCs may have potential for clinical use.

  17. Daily genetic profiling indicates JAK/STAT signaling promotes early hepatic stellate cell transdifferentiation

    Institute of Scientific and Technical Information of China (English)

    Ashley; M; Lakner; Cathy; C; Moore; Alyssa; A; Gulledge; Laura; W; Schrum

    2010-01-01

    AIM: To identify signaling pathways and genes that initiate and commit hepatic stellate cells (HSCs) to transdifferentiation. METHODS: Primary HSCs were isolated from male Sprague-Dawley rats and cultured on plastic for 0-10 d. Gene expression was assessed daily (quiescent to day 10 culture-activation) by real time polymerase chain reaction and data clustered using AMADA software. The significance of JAK/STAT signaling to HSC transdifferentiation was determined by treating cells with a JAK2 inhibitor. RESUL...

  18. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

    OpenAIRE

    Deng, Xin; Liang, Jian; LIN, ZHI-XIU; Wu, Fa-Sheng; Zhang, Ya-ping; Zhang, Zhi-Wei

    2010-01-01

    AIM: To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.

  19. Activation of TGF-β1 promoter by hepatitis C virus-induced AP-1 and Sp1: role of TGF-β1 in hepatic stellate cell activation and invasion.

    Science.gov (United States)

    Presser, Lance D; McRae, Steven; Waris, Gulam

    2013-01-01

    Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-β1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-β1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-β1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-β1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-β1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-β1 is critical for the induction of alpha smooth muscle actin (α-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-β1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-β1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-β1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection.

  20. Activation of TGF-β1 promoter by hepatitis C virus-induced AP-1 and Sp1: role of TGF-β1 in hepatic stellate cell activation and invasion.

    Directory of Open Access Journals (Sweden)

    Lance D Presser

    Full Text Available Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-β1 in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-β1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-β1 gene expression. Using chromatin immunoprecipitation (ChIP assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-β1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-β1 in human hepatic stellate cells (HSCs activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-β1 is critical for the induction of alpha smooth muscle actin (α-SMA and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-β1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-β1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-β1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection.

  1. Suppressive effect of microRNA-29b on hepatic stellate cell activation and its crosstalk with TGF-β1/Smad3.

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    Liang, Chunli; Bu, Shurui; Fan, Xiaoming

    2016-07-01

    The microRNA (miR)-29 family is closely associated with fibrotic processes by virtue of its low expression in many tissues during organ fibrosis. The present study investigated whether miR-29b overexpression suppressed hepatic stellate cell (HSC) activation and its interactions with transforming growth factor (TGF)-β1/mothers against decapentaplegic homolog 3 (Smad3), a classical signal transduction pathway contributing to the activation of HSCs. The results showed that transfection of LX-2 (human HSC) cells with miR-29b mimic or pSUPER-Smad3 silencing (si)RNA resulted in significantly increased expression of miR-29b and decreased expression of Smad3. miR-29b overexpression inhibited proliferation of LX-2 cells 24 h after transfection. Both miR-29b overexpression and Smad3 silencing antagonized the effects of TGF-β1 on the expression of α-smooth muscle actin (α-SMA) and collagen type I (col-1). Furthermore, infection with miR-29b mimics suppressed Smad3 and TGF-β1 expression, suggesting that miR-29b inhibited LX-2 activation mediated by both Smad3 and TGF-β1. Nevertheless, primary miR-29a/b1, miR-29b2/c and mature miR-29b were downregulated by TGF-β1 and stimulated by Smad3 silencing, suggesting that TGF-β1/Smad3 signalling pathway regulate not just mature miR-29b but also its transcription. In summary, our results show overwhelming evidence corroborating the suppressive effect of miR-29b on TGF-β1-induced LX-2 cell activation. The results also revealed the existence of crosstalk between miR-29b and TGF-β1/Smad3 during LX-2 activation, suggesting a feedback loop between miR-29b and TGF-β1/Smad3 signalling that promotes liver fibrosis. Copyright © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.

  2. Effect of Kruppel-like factor 4 on Notch pathway in hepatic stellate cells.

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    Xue, Yin-Kai; Tan, Jun; Dou, Dong-Wei; Chen, Ding; Chen, Lu-Jia; Ren, Huan-Ping; Chen, Li-Bo; Xiong, Xin-Gao; Zheng, Hai

    2016-12-01

    The relationship between Kruppel-like factor 4 (KLF4) and the Notch pathway was determined to investigate the effect of KLF4 on the activation of hepatic stellate cells and underlying mechanisms. Fifty SPF BALB/c mice were randomly divided into two groups. A liver fibrosis model was established in 25 mice as the experimental group, and the remaining 25 mice served as controls. On the day 0, 7, 14, and 35, liver tissues were removed for immunofluorescent detection. The Notch pathway inhibitor DAPT was added to the primary original hepatic stellate cells, and KLF4 and Notch-associated factor expression was detected by qRT-PCR. Additionally, the hepatic stellate cell line LX-2 was used to establish control and experimental groups, and was cultured in vitro. LX-2 cells in the experimental groups were treated with DAPT and the Notch activator transforming growth factor-beta 1 separately, whereas those in the control group were given isotonic culture medium. After 48 h, KLF4 expression was examined by Western blotting. After transient transfection of LX-2 cells to increase KLF4, the expression of Notch factor was examined. Immunofluorescence analysis showed that, with the aggravation of liver fibrosis, the absorbance (A) values of KLF4 were decreased (day 0: 980.73±153.19; day 7: 1087.99±230.23; day 14: 390.95±93.56; day 35: 245.99±87.34). The expression of Notch pathway- related factors (Notch-1, Notch-2, and Jagged-1) in the hepatic stellate cell membrane was negatively correlated to KLF4 expression. With the increase of KLF4 expression, Notch-2 (0.73±0.13) and Jagged-1 (0.43±0.12) expression decreased, whereas Notch-1 level was not detectable. When the Notch pathway was inhibited, KLF4 levels generally increased (18.12±1.31). Our results indicate that KLF4 expression is negatively correlated to the Notch pathway in hepatic stellate cells, which may provide a reference for the treatment of hepatic fibrosis.

  3. Metformin-mediated Bambi expression in Hepatic Stellate Cells induces pro-survival Wnt/β-catenin signaling

    OpenAIRE

    Subramaniam, Nanthakumar; Sherman, Mara H.; Rao, Renuka; Wilson, Caroline; Coulter, Sally; Atkins, Annette R.; Evans, Ronald M.; Liddle, Christopher; Downes, Michael

    2012-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) regulates lipid, cholesterol and glucose metabolism in specialized metabolic tissues, such as muscle, liver and adipose tissue. Agents that activate AMPK, such as metformin and AICAR, have beneficial effects on liver glucose and lipid metabolism. Additionally, AMPK activation in proliferating hepatic stellate cells (HSCs) induces growth arrest and inhibits hepatic fibrosis. As metformin and AICAR act in different ways to achieve their ef...

  4. MicroRNA-130a and -130b enhance activation of hepatic stellate cells by suppressing PPARγ expression: A rat fibrosis model study

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Le; Wang, Jinlong; Lu, Hongwei [Department of General Surgery, The Second Affiliated Hospital of Xi' an Jiaotong University, No.157, West 5th Road, Xi' an, Shaanxi 710004 (China); Zhang, Guoyu [West Hospital Ward 1, Shaanxi Provincial People' s Hospital, No.256, Youyi Road(west), Xi' an, Shaanxi 710068 (China); Liu, Yang; Wang, Jiazhong; Zhang, Yafei; Shang, Hao; Ji, Hong; Chen, Xi; Duan, Yanxia [Department of General Surgery, The Second Affiliated Hospital of Xi' an Jiaotong University, No.157, West 5th Road, Xi' an, Shaanxi 710004 (China); Li, Yiming, E-mail: yiminngli@163.com [Department of General Surgery, The Second Affiliated Hospital of Xi' an Jiaotong University, No.157, West 5th Road, Xi' an, Shaanxi 710004 (China)

    2015-09-25

    Hepatic stellate cells (HSCs) are the primary sources of extracellular matrix (ECM) in normal and fibrotic liver. Peroxisome proliferator-activated receptor gamma (PPARγ) maintains HSCs in a quiescent state, and its downregulation induces HSC activation. MicroRNAs (miRNAs) can induce PPARγ mRNA degradation, but the mechanism by which miRNAs regulate PPARγ in rat HSCs is unclear. This study aimed to investigate some miRNAs which putatively bind to the 3′-untranslated region (3′-UTR) of PPARγ mRNA, and increase expression of ECM genes in rat HSCs. In carbon tetrachloride injection (CCl{sub 4}) and common bile duct ligation (CBDL) liver fibrosis models, miRNAs miR-130a, miR-130b, miR-301a, miR-27b and miR-340 levels were found to be increased and PPARγ expression decreased. Overexpression of miR-130a and miR-130b enhanced cell proliferation by involving Runx3. MiR-130a and miR-130b decreased PPARγ expression by targeting the 3′-UTR of PPARγ mRNA in rat HSC-T6 cells. Transforming growth factor-β1 (TGF-β1) may mediate miR-130a and miR-130b overexpression, PPARγ downregulation, and ECM genes overexpression in cell culture. These findings suggest that miR-130a and miR-130b are involved in downregulation of PPARγ in liver fibrosis. - Highlights: • MiR-130a and miR-130b are increased and PPARγ is decreased in liver fibrosis models. • MiR-130a and miR-130b decreased PPARγ by targeting the 3′-UTR of PPARγ mRNA. • MiR-130a and miR-130b enhanced HSC cell proliferation by involving Runx3. • TGF-β1 may mediate miR-130a and miR-130b overexpression.

  5. Hepatic Stellate Cells Support Hematopoiesis and are Liver-Resident Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Claus Kordes

    2013-02-01

    Full Text Available Background/Aims: Hematopoiesis can occur in the liver, when the bone marrow fails to provide an adequate environment for hematopoietic stem cells. Hepatic stellate cells possess characteristics of stem/progenitor cells, but their contribution to hematopoiesis is not known thus far. Methods: Isolated hepatic stellate cells from rats were characterized with respect to molecular markers of bone marrow mesenchymal stem cells (MSC and treated with adipocyte or osteocyte differentiation media. Stellate cells of rats were further co-cultured with murine stem cell antigen-1+ hematopoietic stem cells selected by magnetic cell sorting. The expression of murine hematopoietic stem cell markers was analyzed by mouse specific quantitative PCR during co-culture. Hepatic stellate cells from eGFP+ rats were transplanted into lethally irradiated wild type rats. Results: Desmin-expressing stellate cells were associated with hematopoietic sites in the fetal rat liver. Hepatic stellate cells expressed MSC markers and were able to differentiate into adipocytes and osteocytes in vitro. Stellate cells supported hematopoietic stem/progenitor cells during co-culture similar to bone marrow MSC, but failed to differentiate into blood cell lineages after transplantation. Conclusion: Hepatic stellate cells are liver-resident MSC and can fulfill typical functions of bone marrow MSC such as the differentiation into adipocytes or osteocytes and support of hematopoiesis.

  6. The hop constituent xanthohumol exhibits hepatoprotective effects and inhibits the activation of hepatic stellate cells at different levels

    OpenAIRE

    Ralf eWeiskirchen; Abdo eMahli; Sabine eWeiskirchen; Claus eHellerbrand

    2015-01-01

    Xanthohumol is the principal prenylated flavonoid of the female inflorescences of the hop plant. In recent years, various beneficial xanthohumol effects including anti-inflammatory, antioxidant, hypoglycemic activities, and anticancer effects have been revealed. This review summarizes present studies indicating that xanthohumol also inhibits several critical pathophysiological steps during the development and course of chronic liver disease, including the activation and pro-fibrogenic genotyp...

  7. Clinical associations of hepatic stellate cell (HSC) hyperplasia.

    Science.gov (United States)

    Mounajjed, Taofic; Graham, Rondell P; Sanderson, Schuyler O; Smyrk, Thomas C

    2014-07-01

    Hepatic stellate cell (HSC) hyperplasia has been principally attributed to hypervitaminosis A. There are sporadic reports of HSC hyperplasia in other conditions such as chronic biliary disease and hepatitis C, but clinical associations of this entity have not been studied in detail. We aimed to investigate the clinical associations of HSC hyperplasia aside from hypervitaminosis A. We identified 34 patients whose liver histology showed HSC hyperplasia. We reviewed the liver samples; additional histologic findings in addition to HSC hyperplasia were consolidated into a histologic diagnosis. We collected clinical, laboratory, and radiologic data; the histologic diagnosis was combined with this data to reach an "overall diagnosis." Four patients had hypervitaminosis A (all native livers). In native livers (n = 24), HSC hyperplasia also occurred in association with drug-induced hepatitis [n = 6, niacin was the most common inducing agent (n = 3)], reactive hepatitis (n = 4), chronic hepatitis C (n = 4), autoimmune hepatitis (n = 3), steatohepatitis (n = 1), chronic biliary disease (n = 1), and portal venopathy (n = 1). In liver allografts (n = 10), HSC hyperplasia was seen in protocol biopsies without other significant abnormalities (n = 5), chronic biliary disease (n = 4), and acute cellular rejection (n = 1). All patients used medications (total of 99) and 82 % were on multiple medications. HSC hyperplasia is an uncommon and relatively nonspecific finding that most commonly occurs in multimedicated patients, often in the absence of hypervitaminosis A. Associated conditions include drug toxicity (such as niacin), post-liver transplant setting, reactive hepatitis (due to systemic illness or inflammatory disorders of the gastrointestinal tract), and chronic liver disease.

  8. Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells

    NARCIS (Netherlands)

    Svegliati-Baroni, G; Saccomanno, S; van Goor, H; Jansen, P; Benedetti, A; Moshage, H

    2001-01-01

    Background/Aims: Reactive oxygen species (ROS) induce HSCs activation, proliferation and collagen gene expression in vitro. Nitric oxide (NO) represents a reactive molecule that reacts with ROS, yielding peroxynitrite. We thus verified the effect of NO on ROS-induced HSCs proliferation in vitro and

  9. The improving effects on hepatic fibrosis of interferon-γ liposomes targeted to hepatic stellate cells

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    Li, Qinghua; Yan, Zhiqiang; Li, Feng; Lu, Weiyue; Wang, Jiyao; Guo, Chuanyong

    2012-07-01

    No satisfactory anti-fibrotic therapies have yet been applied clinically. One of the main reasons is the inability to specifically target the responsible cells to produce an available drug concentration and the side-effects. Exploiting the key role of the activated hepatic stellate cells (HSCs) in both hepatic fibrogenesis and over-expression of platelet-derived growth factor receptor-β (PDGFR-β), we constructed targeted sterically stable liposomes (SSLs) modified by a cyclic peptide (pPB) with affinity for the PDGFR-β to deliver interferon (IFN)-γ to HSCs. The pPB-SSL-IFN-γ showed satisfactory size distribution. In vitro pPB-SSL could be taken up by activated HSCs. The study of tissue distribution via living-body animal imaging showed that the pPB-SSL-IFN-γ mostly accumulated in the liver until 24 h. Furthermore, the pPB-SSL-IFN-γ showed more significant remission of hepatic fibrosis. In vivo the histological Ishak stage, the semiquantitative score for collagen in fibrotic liver and the serum levels of collagen type IV-C in fibrotic rats treated with pPB-SSL-IFN-γ were less than those treated with SSL-IFN-γ, IFN-γ and the control group. In vitro pPB-SSL-IFN-γ was also more effective in suppressing activated HSC proliferation and inducing apoptosis of activated HSCs. Thus the data suggest that pPB-SSL-IFN-γ might be a more effective anti-fibrotic agent and a new opportunity for clinical therapy of hepatic fibrosis.

  10. Curcumin, the main active constituent of turmeric (Curcuma longa L.), induces apoptosis in hepatic stellate cells by modulating the abundance of apoptosis-related growth factors.

    Science.gov (United States)

    He, Ya-Jun; Kuchta, Kenny; Lv, Xia; Lin, Yu; Ye, Guo-Rong; Liu, Xu-You; Song, Hui-Dong; Wang, Le-Xin; Kobayashi, Yuta; Shu, Jian-Chang

    2015-11-01

    In order to elucidate the mechanism of action of curcumin against hepatic fibrosis, cultured rat hepatic stellate cells (HSC) (HSC-T6) were incubated with curcumin for 24 h, after which apoptosis was measured by flow-cytometry. The protein levels of the pro-apoptotic factors Fas and p53b as well as of the anti-apoptotic factor Bcl-2 were monitored by immunocytochemical ABC staining after incubation with curcumin for 24 h. In the case of 20 μM curcumin, not only was the respective apoptosis index increased, but also the abundance of the pro-apoptotic factors Fas and p53 were amplified, whereas that of the anti-apoptotic factor Bcl-2 decreased. All these effects were highly reproducible (P<0.05). Consequently, curcumin has an up-regulating effect on pro-apoptotic factors like Fas and p53 as well as a down-regulating effect of the anti-apoptotic factor Bcl-2, thus inducing apoptosis in HSC.

  11. Hepatic stellate cells and innate immunity in alcoholic liver disease

    Institute of Scientific and Technical Information of China (English)

    Yang-Gun Suh; Won-Il Jeong

    2011-01-01

    Constant alcohol consumption is a major cause of chronic liver disease, and there has been a growing concern regarding the increased mortality rates worldwide. Alcoholic liver diseases (ALDs) range from mild to more severe conditions, such as steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The liver is enriched with innate immune cells (e.g. natural killer cells and Kupffer cells) and hepatic stellate cells (HSCs), and interestingly, emerging evidence suggests that innate immunity contributes to the development of ALDs (e.g. steatohepatitis and liver fibrosis). Indeed, HSCs play a crucial role in alcoholic steatosis via production of endocannabinoid and retinol metabolites. This review describes the roles of the innate immunity and HSCs in the pathogenesis of ALDs, and suggests therapeutic targets and strategies to assist in the reduction of ALD.

  12. Influence of serum collected from rat perfused with compound Biejiaruangan drug on hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Shun-Gen Guo; Wei Zhang; Tao Jiang; Min Dai; Lu-Fen Zhang; Yi-Chun Meng; Li-Yun Zhao; Jian-Zhao Niu

    2004-01-01

    AIM: To observe the effect of compound Biejiaruangan decoction (CBJRGC) (composite prescription of Carapax trionycis for softening the liver) on proliferation, activation,excretion of collagen and cytokine of hepatic stellate cells (HSCs) and to find the mechanism of prevention and treatment of hepatic fibrosis by CBJRGC.METHODS: Using MTT, immunohistochemistry and image analysis technology, the related indexes for proliferation,activation, excretion of collagen and cytokine of hepatic stellate cells were detected in 24 h, 48 h, and 72 h after adminstration of different dosages of CBJRGC.RESULTS: Statistical analysis showed that serum collected from rat perfused with CBJRGC could restrain the proliferation of HSC in 48 h and 72 h especially in high and medium dosage groups, markedly decrease the expression of desmin, synapsin and platelet derived growth factor (PDGF) in HSC in 24 h, 48 h and 72 h, as well as the expression of α-SMA, collagen Ⅲ, TIMP and TGFβ1 in 48 h and 72 h, decrease the excretion of collagen Ⅰ in 72 h.CBJRGC serum had no significant effect on collagens Ⅰ, Ⅲ and TIMP in 24 h.CONCLUSION: CBJRGC serum has a good curative effect on hepatic fibrosis. Its main mechanism may be related to the following factors. The drug serum can restrain the proliferation and activation of HSC, decrease the number of activated HSC and the total number of HSC, the excretion of collagens Ⅰ, Ⅲ, enhance the degradation of collagen and restore the balance of synthesis and degradation of collagen,inhibit the expression of transforming growth factor β1 (TGFβ1) and platelet derived growth factor (PDGF) in HSC,block and delay the process of hepatic fibrosis. Synapsin is a new marker of activation of HSC, which provides a theoretical and testing basis for neural regulation in the developing process of hepatic fibrosis.

  13. The possible role of NS3 protease activity of hepatitis C virus on fibrogenesis and miR-122 expression in hepatic stellate cells.

    Science.gov (United States)

    Khanizadeh, S; Ravanshad, M; Hosseini, S Y; Davoodian, P; Zadeh, A N; Sabahi, F; Sarvari, J; Khanlari, Z; Hasani-Azad, M

    The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-β) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-β cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

  14. Inverse association between hepatic stellate cell apoptosis and fibrosis in chronic hepatitis C virus infection.

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    Gonzalez, S A; Fiel, M I; Sauk, J; Canchis, P W; Liu, R-C; Chiriboga, L; Yee, H T; Jacobson, I M; Talal, A H

    2009-02-01

    Perisinusoidal hepatic stellate cells (HSC) are the principal fibrogenic cells in the liver. In animal models, HSC apoptosis is the predominant clearance mechanism of activated HSC, although data evaluating whether the same processes occur in humans are limited. We conducted a cross-sectional study to evaluate the association between HSC apoptosis and fibrosis stage in subjects with chronic hepatitis C virus (HCV) infection (n = 44) and HCV-negative controls with normal liver histology (n = 9). We used immunohistochemical techniques to identify activated (alpha-smooth muscle actin+), proliferative (Ki-67+) and apoptotic (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end-labelling+) HSC in liver biopsy specimens from all subjects. The same pathologist enumerated positive cells per high-power field (HPF, x 200) in 20 periportal/lobular areas. HSC apoptosis was decreased in HCV-positive subjects compared with controls (median 0.4, range 0.0-3.1 vs 1.1, 0.2-3.5 cells/HPF, P = 0.02). Among HCV-positive subjects, HSC apoptosis was decreased in those with moderate to advanced fibrosis (P = 0.04) compared with those with mild fibrosis. By multivariate analysis, HSC apoptosis decreased by an average of 0.14 cells/HPF (95% confidence interval 0.01-0.28 cells/HPF) per increase in fibrosis stage (P = 0.04). While the number of activated and proliferative HSC was significantly increased in HCV-infected subjects compared with that in uninfected controls, the numbers of these cells did not differ between HCV-infected subjects with mild vs moderate/advanced fibrosis. In conclusion, the number of apoptotic HSC was significantly decreased in HCV-infected subjects with advanced fibrosis. In chronic HCV infection, inhibition of HSC apoptosis may be one mechanism by which fibrosis progresses.

  15. Regulation of Hepatic Stellate Cells and Fibrogenesis by Fibroblast Growth Factors

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    Justin D. Schumacher

    2016-01-01

    Full Text Available Fibroblast growth factors (FGFs are a family of growth factors critically involved in developmental, physiological, and pathological processes, including embryogenesis, angiogenesis, wound healing, and endocrine functions. In the liver, several FGFs are produced basally by hepatocytes and hepatic stellate cells (HSCs. Upon insult to the liver, expression of FGFs in HSCs is greatly upregulated, stimulating hepatocyte regeneration and growth. Various FGF isoforms have also been shown to directly induce HSC proliferation and activation thereby enabling autocrine and paracrine regulation of HSC function. Regulation of HSCs by the endocrine FGFs, namely, FGF15/19 and FGF21, has also recently been identified. With the ability to modulate HSC proliferation and transdifferentiation, targeting FGF signaling pathways constitutes a promising new therapeutic strategy to treat hepatic fibrosis.

  16. Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and trans-differentiation

    Institute of Scientific and Technical Information of China (English)

    Hong Shen; Guo-Jiang Huang; Yue-Wen Gong

    2003-01-01

    AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

  17. Variable expression of cystatin C in cultured trans-differentiating rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Axel M Gressner; Birgit Lahme; Steffen K Meurer; Olav Gressner; Ralf Weiskirchen

    2006-01-01

    AIM: To study the expression of cystatin C (CysC), its regulation by transforming growth factor-β1 (TGF-β1)and platelet-derived growth factor (PDGF) and the potential interference of CysC with TGF-β1 signaling in this special cell type.METHODS: We evaluated the CysC expression in cultured, profibrogenic hepatic stellate cells and transdifferentiated myofibroblasts by Northern and Western blotting and confocal laser scanning microscopy.RESULTS: CysC was increased significantly in the course of trans-differentiation. Both TGF-β1 and PDGFBB suppressed CysC expression. Furthermore, CysC secretion was induced by the treatment with TGF-β1.Although CysC induced an increased binding affinity of TGF-β receptor type Ⅲ (beta-glycan) as assessed by chemical cross-linking with [125I]-TGF-β1, it did not modulate TGF-β1 signal transduction as shown by evaluating the Smad2/3 phosphorylation status and [CAGA]-MLP-luciferase reporter gene assay. Interestingly,the shedding of type Ⅲ TGF-β receptor beta-glycan was reduced in CysC-treated cells. Our data indicated that CysC expression was upregulated during transdifferentiation.CONCLUSION: Increased CysC levels in the serum of patients suffering from liver diseases are at least partially due to a higher expression in activated hepatic stellate cells. Furthermore, TGF-β1 influences the secretion of CysC, highlighting a potentially important role of cysteine proteases in the progression of hepatic fibrogenesis.

  18. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells

    NARCIS (Netherlands)

    Tuohetahuntila, Maidina; Spee, Bart; Kruitwagen, Hedwig S; Wubbolts, Richard; Brouwers, Jos F; van de Lest, Chris H; Molenaar, Martijn R; Houweling, Martin; Helms, J Bernd; Vaandrager, Arie B

    2015-01-01

    Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. We previously observed that the levels of triacylglycerol (TAG) species containing long polyunsaturated fatty acids (PUFAs) are increased in in vitro activated HSCs. Here we investigated the cause

  19. Caffeine inhibits the activation of hepatic stellate cells induced by acetaldehyde via adenosine A2A receptor mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK signal pathway.

    Directory of Open Access Journals (Sweden)

    He Wang

    Full Text Available Hepatic stellate cell (HSC activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR. Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine's inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway.Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III.

  20. Hepatic stellate cell-targeted delivery of hepatocyte growth factor transgene via bile duct infusion enhances its expression at fibrotic foci to regress dimethylnitrosamine-induced liver fibrosis.

    Science.gov (United States)

    Narmada, Balakrishnan Chakrapani; Kang, Yuzhan; Venkatraman, Lakshmi; Peng, Qiwen; Sakban, Rashidah Binte; Nugraha, Bramasta; Jiang, Xuan; Bunte, Ralph M; So, Peter T C; Tucker-Kellogg, Lisa; Mao, Hai-Quan; Yu, Hanry

    2013-05-01

    Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A-coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A-coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-β1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

  1. Role of ethanol in the regulation of hepatic stellate cell function

    Institute of Scientific and Technical Information of China (English)

    Jian-Hua Wang; Robert G Batey; Jacob George

    2006-01-01

    Evidence has accumulated to suggest an important role of ethanol and/or its metabolites in the pathogenesis of alcohol-related liver disease. In this review, the fibrogenic effects of ethanol and its metabolites on hepatic stellate cells (HSCs) are discussed. In brief,ethanol interferes with retinoid metabolism and its signaling, induces the release of fibrogenic cytokines such as transforming growth factor β-1 (TGFβ-1) from HSCs, up-regulates the gene expression of collagen I and enhances type Ⅰ collagen protein production by HSCs.Ethanol further perpetuates an activated HSC phenotype through extracellular matrix remodeling. The underlying pathophysiologic mechanisms by which ethanol exerts these pro-fibrogenic effects on HSCs are reviewed.

  2. Immunoreactive hepatic stellate cells in biopsy material in children with chronic hepatitis B. The first report in pediatric patients.

    Science.gov (United States)

    Łotowska, Joanna M; Lebensztejn, Dariusz M

    2015-09-01

    The research objective was to identify and quantify the immunohistochemically (IHC) stained hepatic stellate cells (HSCs) in children with chronic hepatitis B (CHB), including staging (S), location in the hepatic lobule, and correlation with hepatocyte count. Retrospective morphological analysis was based on liver biopsies obtained from 70 CHB children before antiviral treatment. To determine fibrosis stage, the Batts and Ludwig scoring system was applied. Immunohistochemical examinations used monoclonal antibodies against - SMA. IHC observations in CHB children revealed a significant positive correlation between the mean number of SMA immunopositive HSCs within the hepatic lobule (r = 0.518; p biopsy specimens with intensive fibrosis, most HSCs had an elongated shape and demonstrated evidently strong immunoexpression of cytoskeletal protein - SMA. The mean counts of HSCs/100 hepatocytes (in high power field) in 4 study groups, i.e. with S-0, S-1, S-2, S-3, were 5.00; 5.98; 9.80; 12.19, respectively. Interestingly, in most groups the highest count of immunoreactive HSCs/100 hepatocytes was in the intermediate zone, indicating its high metabolic activity in liver fibrogenesis. Immunohistochemical and statistical investigations of HSCs in children with CHB showed a close positive correlation of cell count with fibrosis intensity, which may have prognostic implications in this pathology.

  3. Interaction of targeted liposomes with primary cultured hepatic stellate cells : Involvement of multiple receptor systems

    NARCIS (Netherlands)

    Adrian, Joanna Ewa; Poelstra, Klaas; Scherphof, Gerrit; Molema, Ingrid; Meijer, D.K F; Reker-Smit, Catharina; Morselt, Henriette; Kamps, Jan

    2006-01-01

    Background/Aims: In designing a versatile liposomal drug carrier to hepatic stellate cells (HSC), the interaction of mannose 6-phosphate human serum albumin (M6P-HSA) liposomes with cultured cells was studied. Methods: M6P-HSA was covalently coupled to the liposomal surface and the uptake and bindin

  4. Transcriptome-based repurposing of apigenin as a potential anti-fibrotic agent targeting hepatic stellate cells

    Science.gov (United States)

    Hicks, Daniel F.; Goossens, Nicolas; Blas-García, Ana; Tsuchida, Takuma; Wooden, Benjamin; Wallace, Michael C.; Nieto, Natalia; Lade, Abigale; Redhead, Benjamin; Cederbaum, Arthur I; Dudley, Joel T.; Fuchs, Bryan C.; Lee, Youngmin A.; Hoshida, Yujin; Friedman, Scott L.

    2017-01-01

    We have used a computational approach to identify anti-fibrotic therapies by querying a transcriptome. A transcriptome signature of activated hepatic stellate cells (HSCs), the primary collagen-secreting cell in liver, and queried against a transcriptomic database that quantifies changes in gene expression in response to 1,309 FDA-approved drugs and bioactives (CMap). The flavonoid apigenin was among 9 top-ranked compounds predicted to have anti-fibrotic activity; indeed, apigenin dose-dependently reduced collagen I in the human HSC line, TWNT-4. To identify proteins mediating apigenin’s effect, we next overlapped a 122-gene signature unique to HSCs with a list of 160 genes encoding proteins that are known to interact with apigenin, which identified C1QTNF2, encoding for Complement C1q tumor necrosis factor-related protein 2, a secreted adipocytokine with metabolic effects in liver. To validate its disease relevance, C1QTNF2 expression is reduced during hepatic stellate cell activation in culture and in a mouse model of alcoholic liver injury in vivo, and its expression correlates with better clinical outcomes in patients with hepatitis C cirrhosis (n = 216), suggesting it may have a protective role in cirrhosis progression.These findings reinforce the value of computational approaches to drug discovery for hepatic fibrosis, and identify C1QTNF2 as a potential mediator of apigenin’s anti-fibrotic activity. PMID:28256512

  5. Hop bitter acids exhibit anti-fibrogenic effects on hepatic stellate cells in vitro.

    Science.gov (United States)

    Saugspier, Michael; Dorn, Christoph; Thasler, Wolfgang E; Gehrig, Manfred; Heilmann, Jörg; Hellerbrand, Claus

    2012-04-01

    Female inflorescences of the hop plant Humulus lupulus L. contain a variety of secondary metabolites with bitter acids (BA) as quantitatively dominating secondary metabolites. The use of hops in beer brewing has a long history due to the antibacterial effects of the BA and their typical bitter taste. Furthermore, hop cones are used in traditional medicine and for pharmaceutical purposes. Recent studies indicate that BA may affect activity of the transcription factor NFκB. NFκB plays a key role in the activation process of hepatic stellate cells (HSC), which is the key event of hepatic fibrosis. The aim of this study was to investigate the effect of BA on HSC (activation) and their potential to inhibit molecular processes involved in the pathogenesis of hepatic fibrosis. HSC were isolated from murine and human liver tissue and incubated with a characterized fraction of bitter acids purified from a CO(2) hop extract. At a concentration of 25μg/ml BA started to induce LDH leakage. Already at lower concentrations BA lead to a dose dependent inhibition of HSC proliferation and inhibited IκB-α-phosphorylation, nuclear p65 translocation and binding activity in a dose dependent way (up to 10μg/ml). Accordingly, the same BA-doses inhibited the expression of pro-inflammatory and NFκB regulated genes as MCP-1 and RANTES, but did not affect expression of genes not related to NFκB signaling. In addition to the effect on activated HSC, BA inhibited the in vitro activation process of freshly isolated HSC as evidenced by delayed expression of collagen I and α-SMA mRNA and protein. Together, these findings indicate that BA inhibit NFκB activation, and herewith the activation and development of profibrogenic phenotype of HSC. Thus, bitter acids appear as potential functional nutrients for the prevention or treatment hepatic fibrosis in chronic liver disease.

  6. Albumin modified with mannose 6-phosphate : A potential carrier for selective delivery of antifibrotic drugs to rat and human hepatic stellate cells

    NARCIS (Netherlands)

    Beljaars, Leonie; Molema, Ingrid; Weert, B; Olinga, Peter; Groothuis, Geny; Meijer, D.K F; Poelstra, Klaas

    1999-01-01

    The hallmark of liver fibrosis is an increased extracellular matrix deposition, caused by an activation of hepatic stellate cells (HSC). Therefore, this cell type is an important target for pharmacotherapeutic intervention. Antifibrotic drugs are not efficiently taken up by HSC or may produce unwant

  7. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.METHODS: A proteomic strategy combining two-dimensional gel electrophoresis and ultraperform ance liquid chromatographyelectrospray ionizationtandem mass spectrometry (UPLCESIMS/MS) was used to study the differential expression of proteins and Western blotting was used to validate the re...

  8. Berberine Inhibition of Fibrogenesis in a Rat Model of Liver Fibrosis and in Hepatic Stellate Cells

    Directory of Open Access Journals (Sweden)

    Ning Wang

    2016-01-01

    Full Text Available Aim. To examine the effect of berberine (BBR on liver fibrosis and its possible mechanisms through direct effects on hepatic stellate cells (HSC. Methods. The antifibrotic effect of BBR was determined in a rat model of bile duct ligation- (BDL- induced liver fibrosis. Multiple cellular and molecular approaches were introduced to examine the effects of BBR on HSC. Results. BBR potently inhibited hepatic fibrosis induced by BDL in rats. It exhibited cytotoxicity to activated HSC at doses nontoxic to hepatocytes. High doses of BBR induced apoptosis of activated HSC, which was mediated by loss of mitochondrial membrane potential and Bcl-2/Bax imbalance. Low doses of BBR suppressed activation of HSC as evidenced by the inhibition of α-smooth muscle actin (α-SMA expression and cell motility. BBR did not affect Smad2/3 phosphorylation but significantly activated 5′ AMP-activated protein kinase (AMPK signalling, which was responsible for the transcriptional inhibition by BBR of profibrogenic factors α-SMA and collagen in HSC. Conclusion. BBR is a promising agent for treating liver fibrosis through multiple mechanisms, at least partially by directly targeting HSC and by inhibiting the AMPK pathway. Its value as an antifibrotic drug in patients with liver disease deserves further investigation.

  9. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

    Science.gov (United States)

    Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

    2015-01-15

    Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation.

  10. Modulation of Bcl-x Alternative Splicing Induces Apoptosis of Human Hepatic Stellate Cells

    Directory of Open Access Journals (Sweden)

    Lin Wu

    2016-01-01

    Full Text Available Liver fibrosis is a major cause of morbidity and mortality worldwide due to chronic viral hepatitis and, more recently, from fatty liver diseases. Activation and proliferation of hepatic stellate cells (HSCs represent a key aspect of fibrogenesis and are associated with progressive reduction of HSC apoptosis. Bcl-x, an antiapoptotic member of Bcl-2 gene family, plays a role in apoptosis regulation in mammalian cells. Through alternative splicing, the Bcl-x gene yields two major protein isoforms with opposing functions, antiapoptotic Bcl-xL and proapoptotic Bcl-xS. This study aimed to investigate the role of Bcl-x and its alternate splicing in HSC apoptosis. The results indicated that the expression of Bcl-xL was dramatically higher than Bcl-2 in activated human HSCs. The relative expression of Bcl-xL over Bcl-xS increased gradually when HSCs were activated in cell culture, which was consistent with the increase in apoptosis resistance of activated HSCs. Redirection of Bcl-x splicing by an antisense oligonucleotide from the antiapoptotic isoform to the proapoptotic isoform induced death of HSCs without other apoptosis stimuli. We conclude that Bcl-x plays a role in regulation of HSC apoptosis and modulation of Bcl-x alternative splicing may become a novel molecular therapy for liver fibrosis.

  11. miRNA参与肝星状细胞活化及慢性肝病发展的作用机制研究进展%Recent progress in the role of miRNA in hepatic stellate cells activation and the chronic hepatic diseases

    Institute of Scientific and Technical Information of China (English)

    宋立莹; 彭向东; 王春江; 郭韧; 刘世坤

    2014-01-01

    微小RNA(microRNA.miRNA)为内源性短小(18~25个核苷酸组成)单链非编码RNA,通过与靶基因mRNA序列3'末端非翻译区(3'-UTR)结合抑制蛋白翻译过程或干扰mRNA降低靶蛋白水平,miRNA异常表达影响多种器官重构中细胞增殖及分化.近期研究表明miRNA这一转录后水平的调控分子参与各种慢性肝病发生发展,肝星状细胞的激活是导致早期肝损伤的关键因素,本文将就miRNA在肝星状细胞激活及慢性肝病发展中的作用作一系统综述.%Small RNA (known as microRNA,miRNA) is single-stranded non-coding RNA (18-25 nucleotides length),it regulates various proteins by targeting the 3'-untranslated region (3-UTR) of various transcripts,thus dysregulation of miRNA affects a wide range of processes such as cell proliferation and differentiation involved in organ remodeling processes.Current studies demonstrated that post-transcriptional effect of miRNA is involved in the development of chronic liver diseases,while hepatic stellate cells (HSCs) play a vital role in the primary formation of ECM and undergo progressive activation to become myofibroblasts (MFB)-like cells.The aim of this paper is to discuss the role of miRNA in hepatic stellate cells activation and the chronic hepatic diseases.

  12. Effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with IL-2 or TNF-α

    Institute of Scientific and Technical Information of China (English)

    展玉涛; 王宇; 魏来; 陈红松

    2003-01-01

    Objective To study the effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with interleukin-2 (IL-2 ) or tumor necrosis factor-α (TNF-α).Methods Hepatic stellate cells were isolated from male Sprague-Dawley rats by using modified Friedman's method. Using the isolated cells cultured and treated with IL-2 or TNF-α, we studied the effects of vitamin E on their proliferation and collagen synthesis through an 3 H-thymidine and 3 H-proline incorporation assay, as well as through observation of these cells under a contrary phase microscope. Results Adding IL-2 increased the both proliferation and collagen synthesis of hepatic stellate cells. Their proliferation was also increased by the addition of TNF-α, although it decreased collagen synthesis. Vitamin E had marked inhibitory effects on the ability of cells treated with IL-2 or TNF-α to reproduce or synthesize collagen.Conclusion Vitamin E can inhibit the proliferation and collagen synthesis of hepatic stellate cells. It is possible that vitamin E affects liver fibrosis through these activities.

  13. Hepatic stellate cells secreted hepatocyte growth factor contributes to the chemoresistance of hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Guofeng Yu

    Full Text Available As the main source of extracellular matrix proteins in tumor stroma, hepatic stellate cells (HSCs have a great impact on biological behaviors of hepatocellular carcinoma (HCC. In the present study, we have investigated a mechanism whereby HSCs modulate the chemoresistance of hepatoma cells. We used human HSC line lx-2 and chemotherapeutic agent cisplatin to investigate their effects on human HCC cell line Hep3B. The results showed that cisplatin resistance in Hep3B cells was enhanced with LX-2 CM (cultured medium exposure in vitro as well as co-injection with LX-2 cells in null mice. Meanwhile, in presence of LX-2 CM, Hep3B cells underwent epithelial to mesenchymal transition (EMT and upregulation of cancer stem cell (CSC -like properties. Besides, LX-2 cells synthesized and secreted hepatic growth factor (HGF into the CM. HGF receptor tyrosine kinase mesenchymal-epithelial transition factor (Met was activated in Hep3B cells after LX-2 CM exposure. The HGF level of LX-2 CM could be effectively reduced by using HGF neutralizing antibody. Furthermore, depletion of HGF in LX-2 CM abolished its effects on activation of Met as well as promotion of the EMT, CSC-like features and cisplatin resistance in Hep3B cells. Collectively, secreting HGF into tumor milieu, HSCs may decrease hepatoma cells sensitization to chemotherapeutic agents by promoting EMT and CSC-like features via HGF/Met signaling.

  14. 1,25-(OH){sub 2}-vitamin D{sub 3} prevents activation of hepatic stellate cells in vitro and ameliorates inflammatory liver damage but not fibrosis in the Abcb4{sup −/−} model

    Energy Technology Data Exchange (ETDEWEB)

    Reiter, Florian P., E-mail: florian.reiter@med.uni-muenchen.de [Department of Medicine II, Liver Center Munich, University of Munich, Marchioninistr. 15, D-81377 Munich (Germany); Hohenester, Simon; Nagel, Jutta M.; Wimmer, Ralf; Artmann, Renate; Wottke, Lena [Department of Medicine II, Liver Center Munich, University of Munich, Marchioninistr. 15, D-81377 Munich (Germany); Makeschin, Marie-Christine; Mayr, Doris [Institute of Pathology, University of Munich, Thalkirchner Str. 36, D-80337 Munich (Germany); Rust, Christian [Department of Medicine I, Krankenhaus Barmherzige Brüder, Romanstr. 93, D-80639 Munich (Germany); Trauner, Michael [Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna (Austria); Denk, Gerald U. [Department of Medicine II, Liver Center Munich, University of Munich, Marchioninistr. 15, D-81377 Munich (Germany)

    2015-04-03

    Background/Purpose of the study: Vitamin D{sub 3}-deficiency is common in patients with chronic liver-disease and may promote disease progression. Vitamin D{sub 3}-administration has thus been proposed as a therapeutic approach. Vitamin D{sub 3} has immunomodulatory effects and may modulate autoimmune liver-disease such as primary sclerosing cholangitis. Although various mechanisms of action have been proposed, experimental evidence is limited. Here we test the hypothesis that active 1,25-(OH){sub 2}-vitamin D{sub 3} inhibits activation of hepatic stellate cells (HSC) in vitro and modulates liver-injury in vivo. Methods: Proliferation and activation of primary murine HSC were assessed by BrdU- and PicoGreen{sup ®}-assays, immunoblotting, immunofluorescence-microscopy, quantitative-PCR, and zymography following calcitriol-treatment. Wild-type and ATP-binding cassette transporter b4{sup −/−} (Abcb4{sup −/−})-mice received calcitriol for 4 weeks. Liver-damage, inflammation, and fibrosis were assessed by serum liver-tests, Sirius-red staining, quantitative-PCR, immunoblotting, immunohistochemistry and hydroxyproline quantification. Results: In vitro, calcitriol inhibited activation and proliferation of murine HSC as shown by reduced α-smooth muscle actin and platelet-derived growth factor-receptor-β-protein-levels, BrdU and PicoGreen®-assays. Furthermore, mRNA-levels and activity of matrix metalloproteinase 13 were profoundly increased. In vivo, calcitriol ameliorated inflammatory liver-injury reflected by reduced levels of alanine aminotransferase in Abcb4{sup −/−}-mice. In accordance, their livers had lower mRNA-levels of F4/80, tumor necrosis factor-receptor 1 and a lower count of portal CD11b positive cells. In contrast, no effect on overall fibrosis was observed. Conclusion: Calcitriol inhibits activation and proliferation of HSCs in vitro. In Abcb4{sup −/−}-mice, administration of calcitriol ameliorates inflammatory liver-damage but has

  15. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Roskams, Tania [Department of Morphology and Molecular Pathology, University of Leuven (Belgium); Oben, Jude A., E-mail: j.oben@ucl.ac.uk [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Department of Gastroenterology and Hepatology, Guy' s and St Thomas' Hospital, London SE1 7EH (United Kingdom)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  16. Murine junctional adhesion molecules JAM-B and JAM-C mediate endothelial and stellate cell interactions during hepatic fibrosis.

    Science.gov (United States)

    Hintermann, Edith; Bayer, Monika; Ehser, Janine; Aurrand-Lions, Michel; Pfeilschifter, Josef M; Imhof, Beat A; Christen, Urs

    2016-07-03

    Classical junctional adhesion molecules JAM-A, JAM-B and JAM-C influence vascular permeability, cell polarity as well as leukocyte recruitment and immigration into inflamed tissue. As the vasculature becomes remodelled in chronically injured, fibrotic livers we aimed to determine distribution and role of junctional adhesion molecules during this pathological process. Therefore, livers of naïve or carbon tetrachloride-treated mice were analyzed by immunohistochemistry to localize all 3 classical junctional adhesion molecules. Hepatic stellate cells and endothelial cells were isolated and subjected to immunocytochemistry and flow cytometry to determine localization and functionality of JAM-B and JAM-C. Cells were further used to perform contractility and migration assays and to study endothelial tubulogenesis and pericytic coverage by hepatic stellate cells. We found that in healthy tissue, JAM-A was ubiquitously expressed whereas JAM-B and JAM-C were restricted to the vasculature. During fibrosis, JAM-B and JAM-C levels increased in endothelial cells and JAM-C was de novo generated in myofibroblastic hepatic stellate cells. Soluble JAM-C blocked contractility but increased motility in hepatic stellate cells. Furthermore, soluble JAM-C reduced endothelial tubulogenesis and endothelial cell/stellate cell interaction. Thus, during liver fibrogenesis, JAM-B and JAM-C expression increase on the vascular endothelium. More importantly, JAM-C appears on myofibroblastic hepatic stellate cells linking them as pericytes to JAM-B positive endothelial cells. This JAM-B/JAM-C mediated interaction between endothelial cells and stellate cells stabilizes vessel walls and may control the sinusoidal diameter. Increased hepatic stellate cell contraction mediated by JAM-C/JAM-C interaction may cause intrahepatic vasoconstriction, which is a major complication in liver cirrhosis.

  17. Rat hepatic stellate cells alter the gene expression profile and promote the growth, migration and invasion of hepatocellular carcinoma cells.

    Science.gov (United States)

    Wang, Zhi-Ming; Zhou, Le-Yuan; Liu, Bin-Bin; Jia, Qin-An; Dong, Yin-Ying; Xia, Yun-Hong; Ye, Sheng-Long

    2014-10-01

    The aim of the present study was to examine the effects of activated hepatic stellate cells (HSCs) and their paracrine secretions, on hepatocellular cancer cell growth and gene expression in vitro and in vivo. Differentially expressed genes in McA-RH7777 hepatocellular carcinoma (HCC) cells following non-contact co-culture with activated stellate cells, were identified by a cDNA microarray. The effect of the co-injection of HCC cells and activated HSCs on tumor size in rats was also investigated. Non-contact co-culture altered the expression of 573 HCC genes by >2-fold of the control levels. Among the six selected genes, ELISA revealed increased protein levels of hepatic growth factor, matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9). Incubation of HCC cells with medium conditioned by activated HSCs significantly increased the proliferation rate (Pprofile of HCC cells and affected their growth, migration and invasiveness. The results from the present study indicate that the interaction between the activated HSCs and HCC has an important role in the development of HCC.

  18. 毛蕊异黄酮抗肝星状细胞活化的作用机制研究%Study on the mechanism of Calycosin in inhibition of hepatic stellate cell activation

    Institute of Scientific and Technical Information of China (English)

    任爽; 张杰

    2016-01-01

    目的:观察毛蕊异黄酮对TGF-β1/Smads信号通路的影响,探讨毛蕊异黄酮抗肝星状细胞活化的作用机制。方法以人肝星状细胞株(LX-2)为研究对象,采用高内涵系统观察细胞增殖( EdU)及细胞内F-actin骨架的聚合情况,RT-PCR法检测细胞I型胶原( Col-Ⅰ)、α-SMA、 TGF-β1 mRNA表达水平, Wstern-blot 法检测细胞内p-Smad2、Smad2及Smad7蛋白表达水平。结果 TGF-β1可显著促进LX-2细胞增殖、活化,EdU阳染细胞明显增加,α-SMA、TGF-β1、p-Smad2及Col-ⅠmRNA 或蛋白表达显著增加( P均<0.05),Smad7表达显著下降;毛蕊异黄酮可显著降低TGF-β1诱导的LX-2中EdU阳染细胞率以及α-SMA、p-Smad2与Col-Ⅰ的mRNA或蛋白表达(P均<0.05),显著提高Smad7蛋白表达,且作用与TβRI激酶抑制剂SB-431542相当。结论毛蕊异黄酮可显著抑制肝星状细胞活化,其机制与抑制TGF-β1、p-Smad2的表达,提高Smad7蛋白表达,进而抑制TGF-β1/Smads信号转导有关。%Objective It is to observe the influence of Calycosin (CA) in the signaling pathway of TGF -β1/Smads, and investigate its mechanism of action in inhibition of hepatic stellate cell activation .Methods Human hepatic stellate cell line LX-2 cells were taken as the research object , the high content system was used to observe the cell proliferation ( EdU) and the aggregation of F -actin skeleton in the cell , RT-PCR method was used to detect the mRNA expression levels of collagen type I ( Col -Ⅰ) , α-SMA and TGF -β1 , Wstern-blot method was used to detect the protein expression levels of p -Smad2, Smad2 and Smad7 in the cell.Results Compared to the control , the proliferation and activation of LX -2 was signifi-cantly promoted by TGF-β1 , and the expression of mRNA or protein of EdU , F-action,α-SMA, TGF-β1 , p-Smad2, Col-Ⅰwere significantly increased (all P<0.05), as well

  19. Metformin-mediated Bambi expression in hepatic stellate cells induces prosurvival Wnt/β-catenin signaling.

    Science.gov (United States)

    Subramaniam, Nanthakumar; Sherman, Mara H; Rao, Renuka; Wilson, Caroline; Coulter, Sally; Atkins, Annette R; Evans, Ronald M; Liddle, Christopher; Downes, Michael

    2012-04-01

    AMP-activated protein kinase (AMPK) regulates lipid, cholesterol, and glucose metabolism in specialized metabolic tissues, such as muscle, liver, and adipose tissue. Agents that activate AMPK, such as metformin and 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), have beneficial effects on liver glucose and lipid metabolism. In addition, AMPK activation in proliferating hepatic stellate cells (HSC) induces growth arrest and inhibits hepatic fibrosis. As metformin and AICAR act in different ways to achieve their effects, our aim was to examine the effects of AMPK activation in quiescent HSCs with these two agents on HSC function. We found that phospho-AMPK levels were markedly upregulated by both AICAR and metformin in quiescent HSCs. However, although AICAR treatment induced cell death, cells treated with metformin did not differ from untreated controls. AICAR-mediated HSC cell death was paralleled by loss of expression of the TGF-β decoy receptor Bambi, whereas metformin increased Bambi expression. Transfection of siRNA-Bambi into HSCs also induced cell death, mimicking the effects of AICAR, whereas overexpression of Bambi partially rescued AICAR-treated cells. As Bambi has previously been shown to promote cell survival through Wnt/β-catenin signaling, a reporter incorporating binding sites for a downstream target of this pathway was transfected into HSCs and was induced. We conclude that although AICAR and metformin both activate AMPK in quiescent HSCs, AICAR rapidly induced cell death, whereas metformin-treated cells remained viable. The finding that metformin increases Bambi expression and activates Wnt/β-catenin signaling provides a possible mechanistic explanation for this observation. These results suggest that AICAR and metformin may confer disease-specific therapeutic benefits.

  20. Intracellular Glutathione Depletion by Oridonin Leads to Apoptosis in Hepatic Stellate Cells

    Directory of Open Access Journals (Sweden)

    Liang-Mou Kuo

    2014-03-01

    Full Text Available Proliferation of hepatic stellate cells (HSCs plays a key role in the pathogenesis of liver fibrosis. Induction of HSC apoptosis by natural products is considered an effective strategy for treating liver fibrosis. Herein, the apoptotic effects of 7,20-epoxy-ent-kaurane (oridonin, a diterpenoid isolated from Rabdosia rubescens, and its underlying mechanisms were investigated in rat HSC cell line, HSC-T6. We found that oridonin inhibited cell viability of HSC-T6 in a concentration-dependent manner. Oridonin induced a reduction in mitochondrial membrane potential and increases in caspase 3 activation, subG1 phase, and DNA fragmentation. These apoptotic effects of oridonin were completely reversed by thiol antioxidants, N-acetylcysteine (NAC and glutathione monoethyl ester. Moreover, oridonin increased production of reactive oxygen species (ROS, which was also inhibited by NAC. Significantly, oridonin reduced intracellular glutathione (GSH level in a concentration- and time-dependent fashion. Additionally, oridonin induced phosphorylations of extracellular signal-regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38 mitogen-activated protein kinase (MAPK. NAC prevented the activation of MAPKs in oridonin-induced cells. However, selective inhibitors of MAPKs failed to alter oridonin-induced cell death. In summary, these results demonstrate that induction of apoptosis in HSC-T6 by oridonin is associated with a decrease in cellular GSH level and increase in ROS production.

  1. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Genz, Berit [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Thomas, Maria [Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart (Germany); Pützer, Brigitte M. [Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Rostock (Germany); Siatkowski, Marcin; Fuellen, Georg [Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock (Germany); Vollmar, Brigitte [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Abshagen, Kerstin, E-mail: kerstin.abshagen@uni-rostock.de [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany)

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  2. Inhibitory effects of capsaicin on hepatic stellate cells and liver fibrosis.

    Science.gov (United States)

    Yu, Fu-Xiang; Teng, Yin-Yan; Zhu, Qian-Dong; Zhang, Qi-Yu; Tang, Yin-He

    2014-10-01

    Hepatic stellate cells (HSCs) play an important role in the process of liver fibrosis. In this study, we investigated the inhibitory effects of capsaicin on HSCs and liver fibrosis. Cultured HSCs were incubated with various concentrations of capsaicin. Cell proliferation was examined using a cell counting kit. Production of hydrogen peroxide was determined using a 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay. The mRNA and protein expression of target genes was analyzed by reverse transcription PCR and Western blot analysis, respectively. Cell apoptosis was evaluated by annexin V-FITC and propidium iodide (PI) costaining followed by flow cytometric analysis. A CCl4 rat liver fibrosis model was used to assess in vivo effects of capsaicin by histological examination and measurement of liver fibrosis markers, including hydroxyproline content, serum type III collagen, and hyaluronic acid (HA) levels. Our results show that capsaicin dose-dependently inhibited cell proliferation, suppressed cell activation, and decreased hydrogen peroxide production in cultured HSCs. Capsaicin reduced the mRNA levels of tissue inhibitors of metalloproteinase 1 (TIMP-1) and transforming growth factor-β1 (TGF-β1) in HSCs. Moreover, capsaicin-induced cell apoptosis was associated with increased expression of Bax, cytochrome c (cyt c), and caspase-3, but reduced levels of Bcl-2. The animal studies further revealed that capsaicin efficiently reduced the extent of liver fibrosis, inhibited HSC proliferation, and promoted cell apoptosis. Our findings suggest that capsaicin might inhibit fibrogenesis by inhibiting the activities of HSCs.

  3. Glucocorticoids Have Opposing Effects on Liver Fibrosis in Hepatic Stellate and Immune Cells.

    Science.gov (United States)

    Kim, Kang Ho; Lee, Jae Man; Zhou, Ying; Harpavat, Sanjiv; Moore, David D

    2016-08-01

    Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid receptor (GR), acting in immune cells and HSCs, in liver fibrosis. In the carbon tetrachloride hepatotoxin-induced fibrosis model, both steroidal and nonsteroidal GR ligands suppressed expression of fibrotic genes and decreased extracellular matrix deposition but also inhibited immune cell infiltration and exacerbated liver injury. These counteracting effects of GR ligands were dissociated in mice with conditional GR knockout in immune cells (GR(LysM)) or HSC (GR(hGFAP)): the impacts of dexamethasone on immune cell infiltration and liver injury were totally blunted in GR(LysM) mice, whereas the suppression of fibrotic gene expression was diminished in GR(hGFAP) mice. The effect of GR activation in HSC was further confirmed in the LX-2 HSC cell line, in which antifibrotic effects were mediated by GR ligand inhibition of Sma and mad-related protein 3 (SMAD3) expression. We conclude that GR has differential roles in immune cells and HSCs to modulate liver injury and liver fibrosis. Specific activation of HSC-GR without alteration of GR activity in immune cells provides a potential therapeutic approach to treatment of hepatic fibrosis.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  5. Hepatitis C virus core protein induces fibrogenic actions of hepatic stellate cells via toll-like receptor 2.

    Science.gov (United States)

    Coenen, Martin; Nischalke, Hans Dieter; Krämer, Benjamin; Langhans, Bettina; Glässner, Andreas; Schulte, Daniela; Körner, Christian; Sauerbruch, Tilman; Nattermann, Jacob; Spengler, Ulrich

    2011-09-01

    Hepatic stellate cells (HSCs) represent the main fibrogenic cell type accumulating extracellular matrix in the liver. Recent data suggest that hepatitis C virus (HCV) core protein may directly activate HSCs. Therefore, we examined the influence of recombinant HCV core protein on human HSCs. Primary human HSCs and the human HSC line LX-2 were stimulated with recombinant HCV proteins core and envelope 2 protein. Expression of procollagen type I α-1, α-smooth muscle actin, cysteine- and glycine-rich protein 2, glial fibrillary acidic protein, tissue growth factor β1, matrix metalloproteinases 2 (MMP2) and 13, tissue inhibitor of metalloproteinases 1 and 2 was investigated by real-time PCR. Intracellular signaling pathways of ERK1/2, p38 and, jun-amino-terminal kinase (JNK) were analyzed by western blot analysis. Recombinant HCV core protein induced upregulation of procollagen type I α-1, α-smooth muscle actin, MMP 2 and 13, tissue inhibitor of metalloproteinases 1 and 2, tissue growth factor β1, cysteine- and glycine-rich protein 2, and glial fibrillary acidic protein mRNA expression, whereas HCV envelope 2 protein did not exert any significant effect. Blocking of toll-like receptor 2 (TLR2) with a neutralizing antibody prevented mRNA upregulation by HCV core protein confirming that the TLR2 pathway was involved. Furthermore, western blot analysis revealed HCV-induced phosphorylation of the TLR2-dependent signaling molecules ERK1/2, p38 and JNK mitogen-activated kinases. Our in vitro results demonstrate a direct effect of HCV core protein on activation of HSCs toward a profibrogenic state, which is mediated via the TLR2 pathway. Manipulating the TLR2 pathway may thus provide a new approach for antifibrotic therapies in HCV infection.

  6. Copper ions stimulate the proliferation of hepatic stellate cells via oxygen stress in vitro.

    Science.gov (United States)

    Xu, San-qing; Zhu, Hui-yun; Lin, Jian-guo; Su, Tang-feng; Liu, Yan; Luo, Xiao-ping

    2013-02-01

    This study examined the effect of copper ions on the proliferation of hepatic stellate cells (HSCs) and the role of oxidative stress in this process in order to gain insight into the mechanism of hepatic fibrosis in Wilson's disease. LX-2 cells, a cell line of human HSCs, were cultured in vitro and treated with different agents including copper sulfate, N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO) for different time. The proliferation of LX-2 cells was measured by non-radioactive cell proliferation assay. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of platelet-derived growth factor receptor β subunit (PDGFβR), ELISA to determine the level of glutathione (GSH) and oxidized glutathione (GSSG), dichlorofluorescein assay to measure the level of reactive oxygen species (ROS), and lipid hydroperoxide assay to quantify the level of lipid peroxide (LPO). The results showed that copper sulfate over a certain concentration range could promote the proliferation of LX-2 cells in a time- and dose-dependent manner. The effect was most manifest when LX-2 cells were treated with copper sulfate at a concentration of 100 μmol/L for 24 h. Additionally, copper sulfate could dose-dependently increase the levels of ROS and LPO, and decrease the ratio of GSH/GSSG in LX-2 cells. The copper-induced increase in mRNA and protein expression of PDGFβR was significantly inhibited in LX-2 cells pre-treated with NAC, a precursor of GSH, and this phenomenon could be reversed by the intervention of BSO, an inhibitor of NAC. It was concluded that copper ions may directly stimulate the proliferation of HSCs via oxidative stress. Anti-oxidative stress therapies may help suppress the copper-induced activation and proliferation of HSCs.

  7. Effects of dietary supplementation with vitamin E and selenium on rat hepatic stellate cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Xiu-Hua Shen; Wu-Feng Cheng; Xuan-Hai Li; Jian-Qin Sun; Feng Li; Ling Ma; Liang-Min Xie

    2005-01-01

    AIM: To evaluate the effects of dietary supplementation with vitamin E and selenium on proliferation and apoptosis of hepatic stellate cells (HSCs), in acute liver injury induced by CCl4, and to explore their role in the recovery from hepatic fibrosis phase.METHODS: An acute liver damage model of rats was established by intraperitoneal injection of carbon tetrachloride (0.3 mL/100 g body weight) twice a week,then the rats were killed at 6, 24, 48, and 72 h after the first and third injection, respectively. A liver fibrosis model was established by the same injection for 8 wk. Then three rats were killed at 3, 7, 14, and 28 d after the last injection,respectively. The rats from the intervention group were fed with chow supplemented with vitamin E (250 mg/kg)and selenium (0.2 mg/kg), and the rats in the normal control group and pathological group were given standard chow.Livers were harvested and stained with hematoxylin and eosin, Sirius red. Activated HSCs were determined by α-smooth muscle actin immunohistochemistry staining.Apoptotic HSCs were determined by dual staining with the terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) and α-smooth muscle actin immunohistochemistry. Serum alanine aminotransferase and aspartate aminotransferase were also analyzed.RESULTS: In the acute liver damage model, the degree of liver injury was more serious in the pathological group than in the intervention group. At each time point, the number of activated HSCs was less in the intervention group than in the pathological group, while the number of apoptotic HSCs was more in the intervention group than in the pathological group. In the liver fibrosis model,the degree of liver fibrosis was more serious in the pathological group than in the intervention group. At each time point, the number of activated HSCs was less in the intervention group than in the pathological group, and the number of apoptotic HSCs was more in the intervention group than in the

  8. Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells.

    Science.gov (United States)

    Coll, Mar; El Taghdouini, Adil; Perea, Luis; Mannaerts, Inge; Vila-Casadesús, Maria; Blaya, Delia; Rodrigo-Torres, Daniel; Affò, Silvia; Morales-Ibanez, Oriol; Graupera, Isabel; Lozano, Juan José; Najimi, Mustapha; Sokal, Etienne; Lambrecht, Joeri; Ginès, Pere; van Grunsven, Leo A; Sancho-Bru, Pau

    2015-06-22

    Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.

  9. The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells.

    Science.gov (United States)

    Arriola Benitez, Paula Constanza; Rey Serantes, Diego; Herrmann, Claudia Karina; Pesce Viglietti, Ayelén Ivana; Vanzulli, Silvia; Giambartolomei, Guillermo Hernán; Comerci, Diego José; Delpino, María Victoria

    2015-12-14

    The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway.

  10. 结缔组织生长因子对Kupffer细胞诱导的肝星状细胞激活的影响%Effects of CTGF on Kupffer cells induced activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    李涛; 宋盛晗; 冷希圣; 朱继业; 彭吉润; 魏玉华

    2009-01-01

    Objective To investigate the effect of RNA interference connective tissue growth factor (CTGF)expression on Kulclffer cells(KC)induced activation of hepatic stellate cells(HSC).Methods Rat CTGF RNA interference vector Psilencer 3.1H1-Neo-CTGF was constructed and identified.HSC cell line rHSC-99 cells were divided into three groups,group A served as control,group B transfected with vector without CTGF interference.group C was RNA interferenee CTGF expression of HSC.RT-PCR was used to measure the expression of CTGF in HSC.Rat Kur)ffer cells were isolated and identified.and cocultured with HSC in the 3 groups respectively.MTT assay was used to evaluate the proliferation of HSC.RT-PCR was used to measure the expression of TGF-β1 and precollagen type I in HSC.Western blot was used to measure the expression of TGF-β1 in HSC.ELISA was used to detect the production of precollagen type I protein.Immunofluorescence was used to detect the expression of ot-smooth muscle actin(α-SMA)in HSC.Resuits After CTGF RNA interference vector transfection.CTGF expression of HSC decreased by 22%(P<0.01).The yield rate of Kupffer cell was 5×107 and the cell viability exceeded 98%.In the HSC and KC co-culture system.the proliferation and activation of HSC were inhibited while RNA interferenee CTGF of HSC.As compared with control,HSC proliferation decreased by 29%(P<0.01).Precollagen type I and ot-SMA expression decreased by 38%(P<0.01).Production of precollagen type I protein in culture medium decreased bv 48%(P<0.01).Conclusions Blockade CTGF expression of HSC inhibits KC induced activation of HSC.%目的 探讨肝星状细胞(hepatic stellate cell,HSC)结缔组织生长因子(connective tissue growth factor,CTGF)表达对Kupffer细胞(Kc)诱导的HSC激活的影响.方法 构建含CTGF的RNA干扰载体.将肝星状细胞系rHSC-99分为3组:对照组、空载体组和RNA干扰组.采用RT-PCR方法 检测HSC的CTGF表达.分离培养KC,建立各组HSC与KC的共培养体

  11. Endotoxin-stimulated Rat Hepatic Stellate Cells Induce Autophagy in Hepatocytes as a Survival Mechanism.

    Science.gov (United States)

    Dangi, Anil; Huang, Chao; Tandon, Ashish; Stolz, Donna; Wu, Tong; Gandhi, Chandrashekhar R

    2016-01-01

    Bacterial lipopolysaccharide (LPS)-stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα, and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/mL) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS-stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6 h. In vitro, HSCs were treated with 100 ng/mL LPS till 24 h. The medium was transferred to hepatocytes, and determinations were made at 0-12 h. Controls were HSC-conditioned medium or medium-containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c-Jun NH2 terminal kinase (JNK) and apoptosis of ~4-5% of hepatocytes. In vitro, LPS-conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ~10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS-stimulated HSCs produced IFNβ, and LPS/HSC-induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti-IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS-stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia.

  12. Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model.

    Science.gov (United States)

    Karthikeyan, Swathi; Potter, James J; Geschwind, Jean-Francois; Sur, Surojit; Hamilton, James P; Vogelstein, Bert; Kinzler, Kenneth W; Mezey, Esteban; Ganapathy-Kanniappan, Shanmugasundaram

    2016-01-15

    Liver fibrosis and cirrhosis result from uncontrolled secretion and accumulation of extracellular matrix (ECM) proteins by hepatic stellate cells (HSCs) that are activated by liver injury and inflammation. Despite the progress in understanding the biology liver fibrogenesis and the identification of potential targets for treating fibrosis, development of an effective therapy remains elusive. Since an uninterrupted supply of intracellular energy is critical for the activated-HSCs to maintain constant synthesis and secretion of ECM, we hypothesized that interfering with energy metabolism could affect ECM secretion. Here we report that a sublethal dose of the energy blocker, 3-bromopyruvate (3-BrPA) facilitates phenotypic alteration of activated LX-2 (a human hepatic stellate cell line), into a less-active form. This treatment-dependent reversal of activated-LX2 cells was evidenced by a reduction in α-smooth muscle actin (α-SMA) and collagen secretion, and an increase in activity of matrix metalloproteases. Mechanistically, 3-BrPA-dependent antifibrotic effects involved down-regulation of the mitochondrial metabolic enzyme, ATP5E, and up-regulation of glycolysis, as evident by elevated levels of lactate dehydrogenase, lactate production and its transporter, MCT4. Finally, the antifibrotic effects of 3-BrPA were validated in vivo in a mouse model of carbon tetrachloride-induced liver fibrosis. Results from histopathology & histochemical staining for collagen and α-SMA substantiated that 3-BrPA promotes antifibrotic effects in vivo. Taken together, our data indicate that sublethal, metronomic treatment with 3-BrPA blocks the progression of liver fibrosis suggesting its potential as a novel therapeutic for treating liver fibrosis.

  13. Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells.

    Science.gov (United States)

    Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

    2016-04-11

    Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC's anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

  14. Verapamil inhibits the activation and function of hepatic stellate cells%钙通道阻滞剂维拉帕米抑制肝星状细胞活化和功能

    Institute of Scientific and Technical Information of China (English)

    戴夫; 李海文; 彭琼; 李元元; 林沪

    2011-01-01

    探讨维拉帕米(Ver)对人肝星状细胞系(HSC)-LX2的活化及分泌转化生长因子(TGF-β)的抑制作用.方法 体外用内皮素-1(endothelin,ET-1)刺激HSC-LX2活化建立培养体系,设对照、ET-1和ET-1+Ver 3组,对照组仅加入培养基,ET-1组加入ET-1和培养基,ET-1+Ver组加入ET-1、Ver和培养基,分别在(0、12、24、48、72 h)观察HSC分沁α-平滑肌肌动蛋白(α-SMA)和细胞因子的能力,采用细胞爬片和免疫组化技术鉴定活化HSC的细胞形态;流式细胞术分析HSC表达α-SMA水平,ELISA测定不同组HSC分泌TGF-β的浓度.结果 与对照组比较,ET-1组HSC活化和分泌TGF-β的能力较强(P<0.05);与ET-1组比较,ET-1+Ver组HSC活化和分泌TGF-β能力较弱(P<0.05).结论 Ver体外实验中能抑制HSC的增殖和活化,具有潜在的抗纤维化作用.%Objective To investigate the effect of Verapamil on the regulation of proliferation and function of human hepatic stellate cell lines ( HSC), and illuminate its further pharmacological role. Methods Activation of HSC culture system stimulated by ET-1 was established in vitro, which was divided into three groups: control group, ET-1 group and ET-1 + Verapamil group. The control group was added with medium only. The ET-1 group was added with medium and ET-1. The ET-1 + Verapamil group was added with medium, ET-1 and Verapamil. Slide of crawling cell was adopted to identify morphology of HSC. Flow cytometry was employed to detect the activation of HSC by analyzing the level of αSMA. ELISA was used to determine the concentration of TGF-β. Results Compared with control group, the expression of α-SMA and TGF-β of HSC in vitro significantly increased in ET-1group (P <0.05). However, the expression of αSMA and TGF-β of HSC significantly decreased in ET-1 + Verapamil group compared with ET-1 group (P < 0. 05).Conclusion Verapamil can inhibit the proliferation and activation of HSC. Verapamil can suppress the occurrence of liver fibrosis.

  15. RNA Interference Targeting Leptin Gene Effect on Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    XUE Xiulan; LIN Jusheng; SONG Yuhu; SUN Xuemei; ZHOU Hejun

    2005-01-01

    To construct the specific siRNA expression vectors and investigate their effect on leptin and collagen I in HSC, which provide a new approach to the prevent and treat hepatic fibrosis. The five siRNAs against leptin gene were transcript synthesized intracellularly by expression templates of plasmid vector psiRNA-hH1neo. The recombinant leptin siRNA plasmid vectors could express in eukaryocyte , and then to evaluate them by using enzyme cutting and sequencing. The recombinant plasmids were transfected into HSCs using Lipofectamine methods respectively. The cells were selected after growing in DMEM containing 300 μg/mL G418 for about 4 weeks. Gene expression of leptin and collagen I were showed by Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Identification by enzyme cutting and sequencing showed that the leptin siRNA expression vectors were constructed successfully, and leptin siRNA could inhibit the leptin and collagen I gene expression effectively. It was concluded that RNA interference-mediated silencing of leptin gene diminished leptin and collagen I gene expression in HSCs. Furthermore, attenuated the extracellular matrix over-deposition at the same time. Leptin gene is ideal targets of gene therapy for liver fibrosis.

  16. The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis

    NARCIS (Netherlands)

    Bansal, Ruchi; Van Baarlen, Joop; Storm, G|info:eu-repo/dai/nl/073356328; Prakash, Jai

    2015-01-01

    Hepatic stellate cells (HSCs) known as master producers and macrophages as master regulators, are the key cell types that strongly contribute to the progression of liver fibrosis. Since Notch signaling regulates multiple cellular processes, we aimed to study the role of Notch signaling in HSCs diffe

  17. The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis

    NARCIS (Netherlands)

    Bansal, Ruchi; Baarlen, van Joop; Storm, G.; Prakash, Jai

    2015-01-01

    Hepatic stellate cells (HSCs) known as “master producers” and macrophages as “master regulators”, are the key cell types that strongly contribute to the progression of liver fibrosis. Since Notch signaling regulates multiple cellular processes, we aimed to study the role of Notch signaling in HSCs d

  18. Clonorchis sinensis ferritin heavy chain triggers free radicals and mediates inflammation signaling in human hepatic stellate cells.

    Science.gov (United States)

    Mao, Qiang; Xie, Zhizhi; Wang, Xiaoyun; Chen, Wenjun; Ren, Mengyu; Shang, Mei; Lei, Huali; Tian, Yanli; Li, Shan; Liang, Pei; Chen, Tingjin; Liang, Chi; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing

    2015-02-01

    Clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis, is associated with hepatobiliary damage, inflammation, periductal fibrosis, and the development of cholangiocarcinoma. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators which drive fibrogenesis; however, their endogenous sources and pathophysiological roles in host cells were not determined. C. sinensis ferritin heavy chain (CsFHC) was previously confirmed as a component of excretory/secretory products and exhibited a number of extrahepatic immunomodulatory properties in various diseases. In this study, we investigated the expression pattern and biological role of CsFHC in C. sinensis. CsFHC was expressed throughout life stages of C. sinensis. More importantly, we found that treatment of human hepatic stellate cell line LX-2 with CsFHC triggered the production of free radicals via time-dependent activation of NADPH oxidase, xanthine oxidase, and inducible nitric oxide synthase. The increase in free radicals substantially promoted the degradation of cytosolic IκBα and nuclear translocation of NF-κB subunits (p65 and p50). CsFHC-induced NF-κB activation was markedly attenuated by preincubation with specific inhibitors of corresponding free radical-producing enzyme or the antioxidant. In addition, CsFHC induced an increased expression level of proinflammatory cytokines, IL-1β and IL-6, in NF-κB-dependent manner. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

  19. Aqueous Date Flesh or Pits Extract Attenuates Liver Fibrosis via Suppression of Hepatic Stellate Cell Activation and Reduction of Inflammatory Cytokines, Transforming Growth Factor-β1 and Angiogenic Markers in Carbon Tetrachloride-Intoxicated Rats

    Directory of Open Access Journals (Sweden)

    Nouf M. Al-Rasheed

    2015-01-01

    Full Text Available Previous data indicated the protective effect of date fruit extract on oxidative damage in rat liver. However, the hepatoprotective effects via other mechanisms have not been investigated. This study was performed to evaluate the antifibrotic effect of date flesh extract (DFE or date pits extract (DPE via inactivation of hepatic stellate cells (HSCs, reducing the levels of inflammatory, fibrotic and angiogenic markers. Coffee was used as reference hepatoprotective agent. Liver fibrosis was induced by injection of CCl4 (0.4 mL/kg three times weekly for 8 weeks. DFE, DPE (6 mL/kg, coffee (300 mg/kg, and combination of coffee + DFE and coffee + DPE were given to CCl4-intoxicated rats daily for 8 weeks. DFE, DPE, and their combination with coffee attenuated the elevated levels of inflammatory cytokines including tumor necrosis factor-α, interleukin-6, and interleukin-1β. The increased levels of transforming growth factor-β1 and collagen deposition in injured liver were alleviated by both extracts. CCl4-induced expression of α-smooth muscle actin was suppressed indicating HSCs inactivation. Increased angiogenesis was ameliorated as revealed by reduced levels and expression of vascular endothelial growth factor and CD31. We concluded that DFE or DPE could protect liver via different mechanisms. The combination of coffee with DFE or DPE may enhance its antifibrotic effects.

  20. Corona-directed nucleic acid delivery into hepatic stellate cells for liver fibrosis therapy.

    Science.gov (United States)

    Zhang, Zhengping; Wang, Chunming; Zha, Yinhe; Hu, Wei; Gao, Zhongfei; Zang, Yuhui; Chen, Jiangning; Zhang, Junfeng; Dong, Lei

    2015-03-24

    Strategies to modify nanoparticles with biological ligands for targeted drug delivery in vivo have been widely studied but met with limited clinical success. A possible reason is that, in the blood circulation, serum proteins could rapidly form a layer of protein "corona" on the vehicle surface, which might block the modified ligands and hamper their targeting functions. We speculate that strategies for drug delivery can be designed based upon elegant control of the corona formation on the vehicle surfaces. In this study, we demonstrate a retinol-conjugated polyetherimine (RcP) nanoparticle system that selectively recruited the retinol binding protein 4 (RBP) in its corona components. RBP was found to bind retinol, and direct the antisense oligonucleotide (ASO)-laden RcP carrier to hepatic stellate cells (HSC), which play essential roles in the progression of hepatic fibrosis. In both mouse fibrosis models, induced by carbon tetrachloride (CCl4) and bile duct ligation (BDL), respectively, the ASO-laden RcP particles effectively suppressed the expression of type I collagen (collagen I), and consequently ameliorated hepatic fibrosis. Such findings suggest that this delivery system, designed to exploit the power of corona proteins, can serve as a promising tool for targeted delivery of therapeutic agents for the treatment of hepatic fibrosis.

  1. Dihydroartemisinin counteracts fibrotic portal hypertension via farnesoid X receptor-dependent inhibition of hepatic stellate cell contraction.

    Science.gov (United States)

    Xu, Wenxuan; Lu, Chunfeng; Zhang, Feng; Shao, Jiangjuan; Yao, Shunyu; Zheng, Shizhong

    2017-01-01

    Portal hypertension is a frequent pathological symptom occurring especially in hepatic fibrosis and cirrhosis. Current paradigms indicate that inhibition of hepatic stellate cell (HSC) activation and contraction is anticipated to be an attractive therapeutic strategy, because activated HSC dominantly facilitates an increase in intrahepatic vein pressure through secreting extracellular matrix and contracting. Our previous in vitro study indicated that dihydroartemisinin (DHA) inhibited contractility of cultured HSC by activating intracellular farnesoid X receptor (FXR). However, the effect of DHA on fibrosis-related portal hypertension still requires clarification. In this study, gain- and loss-of-function models of FXR in HSC were established to investigate the mechanisms underlying DHA protection against chronic CCl4 -caused hepatic fibrosis and portal hypertension. Immunofluorescence staining visually showed a decrease in FXR expression in CCl4 -administrated rat HSC but an increase in that in DHA-treated rat HSC. Serum diagnostics and morphological analyses consistently indicated that DHA exhibited hepatoprotective effects on CCl4 -induced liver injury. DHA also reduced CCl4 -caused inflammatory mediator expression and inflammatory cell infiltration. These improvements were further enhanced by INT-747 but weakened by Z-guggulsterone. Noteworthily, DHA, analogous to INT-747, significantly lowered portal vein pressure and suppressed fibrogenesis. Experiments on mice using FXR shRNA lentivirus consolidated the results above. Mechanistically, inhibition of HSC activation and contraction was found as a cellular basis for DHA to relieve portal hypertension. These findings demonstrated that DHA attenuated portal hypertension in fibrotic rodents possibly by targeting HSC contraction via a FXR activation-dependent mechanism. FXR could be a target molecule for reducing portal hypertension during hepatic fibrosis.

  2. Graptopetalum paraguayense ameliorates chemical-induced rat hepatic fibrosis in vivo and inactivates stellate cells and Kupffer cells in vitro.

    Directory of Open Access Journals (Sweden)

    Li-Jen Su

    Full Text Available BACKGROUND: Graptopetalum paraguayense (GP is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN- and carbon tetrachloride (CCl(4-induced liver injury rats. METHODS: Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs and Kupffer cells, respectively, were evaluated. RESULTS: Oral administration of MGP significantly alleviated DMN- or CCl(4-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression. CONCLUSIONS: The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis.

  3. Loss of discoidin domain receptor 2 promotes hepatic fibrosis after chronic carbon tetrachloride through altered paracrine interactions between hepatic stellate cells and liver-associated macrophages.

    Science.gov (United States)

    Olaso, Elvira; Arteta, Beatriz; Benedicto, Aitor; Crende, Olatz; Friedman, Scott L

    2011-12-01

    Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl(4)) to DDR2(+/+) and DDR2(-/-) mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after chronic CCl(4) administration, DDR2(-/-) livers had increased collagen deposition, gelatinolytic activity, and HSC density. Increased basal gene expression of osteopontin, transforming growth factor-β1, monocyte chemoattractant protein-1, and IL-10 and reduced basal gene expression of matrix metalloproteinase-2, matrix metalloproteinase-13, and collagen type I in quiescent DDR2(-/-) HSCs were amplified further after chronic CCl(4). In concordance, DDR2(-/-) HSCs isolated from chronically injured livers had enhanced in vitro migration and proliferation, but less extracellular matrix degradative activity. Macrophages from chronic CCl(4)-treated DDR2(-/-) livers showed stronger chemoattractive activity toward DDR2(-/-) HSCs than DDR2(+/+) macrophages, increased extracellular matrix degradation, and higher cytokine mRNA expression. In conclusion, loss of DDR2 promotes chronic liver fibrosis after CCl(4) injury. The fibrogenic sinusoidal milieu generated in chronic DDR2(-/-) livers recruits more HSCs to injured regions, which enhances fibrosis. Together, these findings suggest that DDR2 normally orchestrates gene programs and paracrine interactions between HSCs and macrophages that together attenuate chronic hepatic fibrosis.

  4. Biological effects of extract from newborn porcine liver on hepatocytes, hepatic stellate cells, and hepatoma cell line

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective: Porcine liver extract has been shown to be effective in the clinical treatment of severe hepatitis. The aim of the present study was to study its antifibrotic as well as immune regulatory effect in vitro. Methods: Hepatocytes, hepatic stellate cells (HSCs), hepatoma cell line (HepG2) and human peripheral blood mononuclear cells (PMNCs) were studied with respect to proliferation, extracellular matrix production and apoptotic activities by proliferation assay, radioimmunoassay, gene transfection, reporter gene analysis and flow cytometry, respectively. Results: A strong stimulatory proliferation effect was observed in hepatocytes, and an inhibitory effect was found in HSCs. Hyaluronic acid (HA) production and reporter gene activities driven by various α1(Ⅰ) procollagen gene promoters in HSC-T6 were significantly decreased after treatment with the extract. Fluo-Anexin V binding apoptotic HepG2 cells were more prominent in the presence of 60 μg/ml extract. More CD4+/CD69+ positive T lymphocytes existed in the presence of the extract. Conclusion: Porcine liver extract is effective for antifibrogenesis via hepatocyte regeneration, HSC and hepatoma cell inhibition in vitro. The elevation of active T lymphocytes is helpful for immune surveillance. Fine mapping of the extract is necessary in order to get definite molecules which are essential in all described functions.

  5. Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury.

    Science.gov (United States)

    Antoine, Marianne; Wirz, Werner; Tag, Carmen G; Gressner, Axel M; Marvituna, Meltem; Wycislo, Mathias; Hellerbrand, Claus; Kiefer, Paul

    2007-09-21

    Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.

  6. Wnt3a对肝星状细胞增殖活化以及转化生长因子β/Smad表达的影响%Effects of Wnt3a on proliferation,activation and the expression of TGFβ/Smad in rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    王燕平; 贺琪; 吴飞; 朱兰兰; 刘卫; 张雅楠; 贺永文

    2013-01-01

    Objective To observe the effects of Wnt3a on proliferation and,activation of hepatic stellate cells(HSCs)and their the expression of the transforming growth factor beta(TGFβ)and/Smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line.Methods Sychronized HSC-T6 cells were stimulated with various concentrations of recombinant Wnt3a(50,100,200,250 and 300 ng/mL).Unstimulated cells served as controls.Edu Effects on proliferation were determined by EdU(5-ethynyl-2’-deoxyuridine)incorporation assay and fluorescence microscopy.analysis was used to observe the proliferation of the hepatic stellate cells stimulated by different concentration of recombinant Wnt3a,and theEffects on the protein expression of TGFβ/Smad signaling factors was assessed by western blot detection(gray-value analysis)of alpha-smooth muscle actin(α-SMA),α-SMA,TGFβ1,Smad3,and and Smad7;glyceraldehyde 3-phosphate dehydrogenase(GAPDH)was detected as the normalization control in the hepatic stellate cells was observed by Western blot analysis.The correlation was also observed.The significance of inter-group differences was assessed by one-way ANOVA,and correlations were determined using bivariate statistical modeling.Results In general,HSCThe proliferation of hepatic stellate cells increased after the addition ofin response to Wnt3a stimulation for 24 h.,reaching its peak atThe maximum proliferation rate was observed with the 200 ng/mL Wnt3a concentration(63.00 ± 2.30%),and it increased dramatically compared with those inwhich was significantly higher than the proliferation rates of the unstimulated control cells,and the cells stimulated with 50,100 and 150 ng/mL1 group(P < 0.05),but the increase was not significantly different from that in the compared cells stimulated with 250 and 300 ng/mLl group,it had no obvious increase(P > 0.05).;The Wnt3a stimulation also led to time-dependent increases in the protein expressions of α-SMA,TGFβ1,and Smad3 increased

  7. Loss of Discoidin Domain Receptor 2 Promotes Hepatic Fibrosis after Chronic Carbon Tetrachloride through Altered Paracrine Interactions between Hepatic Stellate Cells and Liver-Associated Macrophages

    OpenAIRE

    Olaso, Elvira; ARTETA, BEATRIZ; BENEDICTO, AITOR; Crende, Olatz; Friedman, Scott L.

    2011-01-01

    Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl4) to DDR2+/+ and DDR2−/− mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after...

  8. TRPM7 channel regulates PDGF-BB-induced proliferation of hepatic stellate cells via PI3K and ERK pathways

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Ling, E-mail: fangling_1984@126.com; Zhan, Shuxiang; Huang, Cheng; Cheng, Xi; Lv, Xiongwen; Si, Hongfang; Li, Jun, E-mail: lj@ahmu.edu.cn

    2013-11-01

    TRPM7, a non-selective cation channel of the TRP channel superfamily, is implicated in diverse physiological and pathological processes including cell proliferation. Recently, TRPM7 has been reported in hepatic stellate cells (HSCs). Here, we investigated the contribution role of TRPM7 in activated HSC-T6 cell (a rat hepatic stellate cell line) proliferation. TRPM7 mRNA and protein were measured by RT-PCR and Western blot in rat model of liver fibrosis in vivo and PDGF-BB-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPM7 were dramatically increased in CCl{sub 4}-treated rat livers. Stimulation of HSC-T6 cells with PDGF-BB resulted in a time-dependent increase of TRPM7 mRNA and protein. However, PDGF-BB-induced HSC-T6 cell proliferation was inhibited by non-specific TRPM7 blocker 2-aminoethoxydiphenyl borate (2-APB) or synthetic siRNA targeting TRPM7, and this was accompanied by downregulation of cell cycle proteins, cyclin D1, PCNA and CDK4. Blockade of TRPM7 channels also attenuated PDGF-BB induced expression of myofibroblast markers as measured by the induction of α-SMA and Col1α1. Furthermore, the phosphorylation of ERK and AKT, associated with cell proliferation, decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TRPM7 channels contribute to perpetuated fibroblast activation and proliferation of PDGF-BB induced HSC-T6 cells via the activation of ERK and PI3K pathways. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 mRNA and protein in the fibrotic livers from CCl{sub 4}-treated rats. • Increasing expression of TRPM7 mRNA and protein during HSC activation. • Blockade of TRPM7 inhibited the PDGF-BB induced proliferation of HSC-T6 cells. • Blockade of TRPM7 decreased α-SMA and Col1α1 expressions in activated HSC-T6 cells. • TRPM7 up-regulation contributes to the activation of ERK and AKT pathways.

  9. 自噬抑制剂对乙醇诱导的肝星状细胞活化作用的影响%The Effects of Autophagy Inhibitor on Activation of Alcohol induced Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    何月; 贾宝辉; 刘曼; 罗文; 张吉翔

    2014-01-01

    Objective To observe the effect of autophagy inhibitor on the activation of alcohol induced hepatic stel-late cells, and the mechanisms thereof. Methods HSC-T6 cells were cultured in vitro and divided into four groups, includ-ing blank control group, alcohol group, 5 mmol/L 3-MA+alcohol group (low alcohol group) and 10 mmol/L 3-MA+alcohol group (high alcohol group). RT-PCR was used to detect the expression levels ofα-smooth muscle actin (α-SMA) and typeⅠcollagen. The levels of LC3Ⅱ,α-SMA and typeⅠcollagen were detected by Western blot assay. The cell viability of HSC-T6 was detected by MTT assay. Results The mRNA expressions ofα-SMA, typeⅠcollagen and the protein of expressionsα-SMA, typeⅠcollagen and LC3Ⅱwere significantly up-regulated in alcohol group compared with those of control group (P<0.05), while the expressions of those parameters were significantly down-regulated in 10 mmol/L 3-MA+alcohol group (P<0.01). The mRNA and protein levels ofα-SMA and typeⅠcollagen were significantly decreased in two 3-MA-treated groups compared with those in alcohol group (P<0.05). Meanwhile, compared with the 5 mmol/L 3-MA+alcohol group,the protein expressions ofα-SMA, typeⅠcollagen and LC3Ⅱwere significantly decreased in10 mmol/L 3-MA+alcohol group (P < 0.05 ). Compared with the alcohol group,there was significantly lower proliferation activity in all two 3-MA-treated groups (P<0.05). Conclusion 3-MA can inhibit the protein expression of LC3Ⅱ,α-SMA and typeⅠcollagen induced by alcohol in HSC-T6 cells, and inhibit the proliferation of HSC cells.%目的:观察自噬抑制剂3-甲基腺嘌呤(3-MA)对乙醇诱导的肝星状细胞(HSC)活化作用,并探讨其作用机制。方法体外培养大鼠肝星状细胞株HSC-T6,设立空白对照组、阳性对照组(乙醇刺激组)、低剂量组(5 mmol/L 3-MA+100 mmol/L乙醇)、高剂量组(10 mmol/L 3-MA+100 mmol/L乙醇)。RT-PCR分析各组HSC活化标志蛋白α-平滑

  10. Hedgehog信号通路对肝星状细胞激活和增殖的影响%Regulation of hepatic stellate cell activation and proliferation by Hedgehog signal pathway

    Institute of Scientific and Technical Information of China (English)

    王刚; 李涛; 封益飞; 冷希圣

    2009-01-01

    Objective To study the expression of Hedgehog signal pathway in rat hepatic stellate cell (HSC) line rHSC-99. The method of RNAi was adopted to inhibit Hedgehog signal pathway,and estimate the regulation role of Hedgehog signal pathway in activation and proliferation of HSC. Methods RTPCR was used to detect the expression of Hedgehog signal pathway in rat HSC line rHSC-99. Transcripts of siRNA sequence of the genes Ihh,Smo,and Gli2 were designed,and transfected into HSC respectively. Then the expression of these mRNAs were detected by SYBR green flurogenic quantitative PCR. The expression of α-SMA was detected by Western blot. The variation of type I collagen in culture supernatant of HSC was detected by ELISA. The proliferation of HSC was measured by MTT assay. Results HSC expressed mRNAs of Ihh,Smo,Ptc,Gli2,Gli3. The expression of these mRNAs could be reduced by trans-fecting plasmids encoded siRNA of Ihh,Smo or Gli2 (0. 254 ±0.130,0.221 ±0. 150,0. 235 ±0. 110 vs 1 ,P<0.01). Transfection experiment demonstrated the reduction of the expression of α-SMA (0. 191 ± 0.014,0. 357 ± 0. 021,0. 086 ± 0. 016 vs 1. 143 ± 0. 017, P<0. 01) and secretion of collagen I (22.9±2.0,16.4±1.4,17.6±1.8 vs 40.7 ±4.3,P<0.01) in HSC,and HSC proliferation was decreased (0.204 ±0.019,0. 226 ±0. 014,0. 228 ±0.015 vs 0. 412 ±0. 016,P<0.05). Conclusion This study showed the expression of Hedgehog signal pathway in HSC. Down-regulation of Hedgehog signal pathway may inhibit HSC activation and proliferation.%目的 观察Hedgehog信号通路在肝星状细胞(HSC)中的表达情况及Hedgehog信号通路对HSC激活和增殖的调控作用.方法 采用逆转录-聚合酶链反应(RT-PCR)的方法检测大鼠HSC细胞株rHSC-99中Hedgehog信号通路各成分的表达.构建含Ihh、Smo、Gli2的干扰片段的质粒,分别转染HSC,用SYBR Green荧光定量PCR的方法检测转染后Ihh、Smo、Gli2的表达,Western blot方法检测HSC中α-SMA表达,酶联免疫吸附试

  11. Hepcidin inhibits Smad3 phosphorylation in hepatic stellate cells by impeding ferroportin-mediated regulation of Akt.

    Science.gov (United States)

    Han, Chang Yeob; Koo, Ja Hyun; Kim, Sung Hoon; Gardenghi, Sara; Rivella, Stefano; Strnad, Pavel; Hwang, Se Jin; Kim, Sang Geon

    2016-12-22

    Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl4 treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGFβ1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGFβ1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGFβ1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin.

  12. Hepatitis C virus E2 protein induce reactive oxygen species (ROS)-related fibrogenesis in the HSC-T6 hepatic stellate cell line.

    Science.gov (United States)

    Ming-Ju, Hsieh; Yih-Shou, Hsieh; Tzy-Yen, Chen; Hui-Ling, Chiou

    2011-01-01

    Chronic infection of hepatitis C virus (HCV) leads to hepatic fibrosis and subsequently cirrhosis, although the underlying mechanisms have not been established. Previous studies have indicated that the binding of HCV E2 protein and CD81 on the surface of hepatic stellate cells (HSCs) lead to the increased protein level and activity of matrix metallopeptidase (MMP) 2, indicating that E2 may involve in the HCV-induced fibrosis. This study was designed to investigate the involvement of HCV E2 protein in the hepatic fibrogenesis. Results showed that E2 protein may promote the expression levels of α-smooth muscle actin (α-SMA) and collagen α(I). Furthermore, several pro-fibrosis or pro-inflammatory cytokines, including transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF), interleukin (IL)-6 and IL-1β, were significantly increased in E2 transfected-HSC cell lines, while the expression of MMP-2 are also considerably increased. Moreover, the significant increases of CTGF and TGF-β1 in a stable E2-expressing Huh7 cell line were also observed the same results. Further molecular studies indicated that the impact of E2 protein on collagen production related to higher production of ROS and activated Janus kinase (JAK)1, JAK2 and also enhance the activation of ERK1/2 and p38, while catalase and inhibitors specific for JAK, ERK1/2, and p38 abolish E2-enhanced expression of collagen α(I). Taken together, this study indicated that E2 protein involve in the pathogenesis of HCV-mediated fibrosis via an up-regulation of collagen α(I) and oxidative stress, which is JAK pathway related.

  13. Effects of sinusoidal endothelial cell conditioned medium on the expressionof connective tissue growth factor in rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Xiao Jing Liu; Fang Liu; Wen Jun Xiao; Ming Hui Huang; Song Min Huang; Yi Ping Wang

    2000-01-01

    AIM To investigate the effects of sinusoidal endothelial cell (SEC) conditioned medium on the expression ofconnective tissue growth factor (CTGF) in rat hepatic stellate cells (HSC).METHODS By in situ collagenase perfusion and two-step Percoll gradient centrifugation, SECs wereisolated and cultured from normally and CCl4-treated Wistar rats, and the SEC conditioned media werecollected. HSCs were prepared from Wistar rats by in situ perfusion and single-step Nycodenz gradient, andwere cultured with SEC conditioned media. Expression of CTGF in HSC was assessed using reversetranscription-polymerase chain reaction (RT-PCR).RESULTS Expression of CTGF was not found in freshly isolated HSC and in primary culture of HSC onday 4 with SEC conditioned media from normal rats, but was present in primary culture of HSC on day 4 withSEC conditioned media from CCl4-induced liver fibrosis rats. Expression of CTGF was observed in culture-activated HSCs, and the effect of SEC conditioned media from CCl4-induced liver fibrosis rats on theexpression of CTGF gene in activated HSCs was not significant.CONCLUSION Expression of CTGF might be relative to the activation of HSC and the liver fibrogenesis,and damaged SECs play a very important role in the early stage of activation of HSC.

  14. Periostin down-regulation attenuates the pro-fibrogenic response of hepatic stellate cells induced by TGF-β1.

    Science.gov (United States)

    Hong, Li; Shejiao, Dai; Fenrong, Chen; Gang, Zhao; Lei, Dong

    2015-10-01

    Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)-β1-induced HSC activation remains unclear. We used RT-PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α-smooth muscle actin (α-SMA), collagen I, TGF-β1, p-Smad2 and p-Smad3 were determined by western blot. Our study found that periostin was up-regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA-periostin suppressed TGF-β1-induced HSC proliferation. The HSC transfected with siRNA-periostin significantly inhibited TGF-β1-induced expression levels of α-SMA and collagen I. Furthermore, TGF-β1 stimulated the expression of periostin, and siRNA-periostin attenuated TGF-β1-induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF-β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.

  15. Transcriptomic and proteomic analysis of human hepatic stellate cells treated with natural taurine.

    Science.gov (United States)

    Liang, Jian; Deng, Xin; Wu, Fa-Sheng; Tang, Yan-Fang

    2013-05-01

    The aim of this study was to investigate the differential expression of genes and proteins between natural taurine (NTau)‑treated hepatic stellate cells (HSCs) and control cells as well as the underlying mechanism of NTau in inhibiting hepatic fibrosis. A microculture tetrazolium (MTT) assay was used to analyze the proliferation of NTau‑treated HSCs. Flow cytometry was performed to compare the apoptosis rate between NTau-treated and non‑treated HSCs. Proteomic analysis using a combination of 2-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) was conducted to identify the differentially expressed proteins. Microarray analysis was performed to investigate the differential expression of genes and real-time polymerase chain reaction (PCR) was used to validate the results. The experimental findings obtained demonstrated that NTau decreased HSC proliferation, resulting in an increased number of cells in the G0/G1 phase and a reduced number of cells in the S phase. Flow cytometric analysis showed that NTau-treated HSCs had a significantly increased rate of apoptosis when compared with the non‑treated control group. A total of 15 differentially expressed proteins and 658 differentially expressed genes were identified by 2DE and MS, and microarray analysis, respectively. Gene ontology (GO) functional analysis indicated that these genes and proteins were enriched in the function clusters and pathways related to cell proliferation, cellular apoptosis and oxidation. The transcriptome and proteome analyses of NTau-treated HSCs demonstrated that NTau is able to significantly inhibit cell proliferation and promote cell apoptosis, highlighting its potential therapeutic benefits in the treatment of hepatic fibrosis.

  16. Effects of endothelin- 1 on hepatic stellate cell proliferation, collagen synthesis and secretion, intracellular free calcium concentration

    Institute of Scientific and Technical Information of China (English)

    Chuan-Yong Guo; Jian-Ye Wu; Yun-Bin Wu; Min-Zhang Zhong; Han-Ming Lu

    2004-01-01

    AIM: To explore the effects of endothelin-1(ET-1) on hepatic stellate cells (HSCs) DNA uptake, DNA synthesis, collagen synthesis and secretion, inward whole-cell calcium concentration ([Ca2+]i) as well as the blocking effect of verapamil on ET-1-stimulated release of inward calcium (Ca2+) of HSC in vitro.METHODS: Rat hepatic stellate cells (HSCs) were isolated and cultivated. 3H-TdR and 3H-proline incorporation used for testing DNA uptake and synthesis, collagen synthesis and secretion of HSCs cultured in vitro; Fluorescent calciumindicator Fura-2/AM was used to measure [Ca2+]i inward HSCs.RESULTS: ET-1 at the concentration of 5×10-8 mol/L,caused significant increase both in HSC DNA synthesis(2 247±344 cpm, P<0.05) and DNA uptake (P<0.05) whencompared with the control group. ET-1 could also increase collagen synthesis (P<0.05 vs control group) and collagen secretion (P<0.05 vs control group). Besides, inward HSC [Ca2+]i reached a peak concentration (422±98 mol/L, P<0.001)at 2 min and then went down slowly to165±51 mol/L(P<0.01) at 25 min from resting state (39±4 mol/L)aftertreated with ET-1. Verapamil (5 mol/L) blocked ET-1activated [Ca2+]i inward HSCs compared with control group(P<0.05). Fura-2/AM loaded HSC was suspended in no Ca2+ buffer containing 1 mol/L EGTA, 5 min later, 10-8 mol/Lof ET-1 was added, [Ca2+]i inward HSCs rose from restingstate to peak 399±123 mol/L, then began to come downby the time of 20 min. It could also raise [Ca2+]i inwardHSCs even without Ca2+ in extracellular fluid, and had a remarkable dose-effect relationship(P<0.05). Meanwhile, verapamil could restrain the action of ET-1(P<0.05). CONCLUSION: Actions of ET-1 on collagen metabolism of HSCs may depend on the transportation of inward wholecell calcium.

  17. The Role of Lipin-1 in the Regulation of Fibrogenesis and TGF-β Signaling in Hepatic Stellate Cells.

    Science.gov (United States)

    Jang, Chang Ho; Kim, Kyu Min; Yang, Ji Hye; Cho, Sam Seok; Kim, Seung Jung; Shin, Sang Mi; Cho, Il Je; Ki, Sung Hwan

    2016-09-01

    The adipogenic transcriptional regulation was reported to inhibit transdifferentiation of hepatic stellate cells (HSCs), which constitute the main fibrogenic cell type in the liver. Lipin-1 exhibits a dual function: an enzyme that catalyzes the conversion of phosphatidate to diacylglycerol and a transcriptional regulator. However, the involvement of Lipin-1 in the regulation of transforming growth factor-β (TGF-β) signaling and fibrogenesis in HSCs is not fully understood. Here, we showed that Lipin-1 was downregulated in activated primary HSCs and TGF-β-treated LX-2 cells, immortalized human HSC cell lines. The downregulation of Lipin-1 by TGF-β was not dependent on altered mRNA stability but rather on protein stability. Treatment of LX-2 cells with the proteasome inhibitor led to the accumulation of Lipin-1. Moreover, we observed a significant increase in Lipin-1 polyubiquitination. Overexpression of Lipin-1 attenuated TGF-β-induced fibrogenic gene expression. In addition, Lipin-1 inhibited TGF-β-mediated activation of Sma and Mad-related family (SMAD), a major transcription factor that transduces intracellular signals from TGF-β. Resveratrol, a well-known natural polyphenolic antioxidant, is known to inhibit liver fibrosis, although its mechanism of action remains unknown. Our data showed that resveratrol significantly increased the levels of Lipin-1 protein and mRNA in HSCs. Further investigation revealed that resveratrol blocked the polyubiquitination of Lipin-1. Resveratrol inhibited TGF-β-induced fibrogenic gene expression. TGF-β-induced SMAD binding element-luciferase reporter activity was significantly diminished by resveratrol with a simultaneous decrease in SMAD3 phosphorylation. Consistently, knockdown of the Lipin-1 gene using siRNA abolished the inhibitory effect of resveratrol. We conclude that Lipin-1 can antagonize HSC activation through the inhibition of TGF-β/SMAD signaling and that resveratrol may affect Lipin-1 gene induction and

  18. Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage.

    Directory of Open Access Journals (Sweden)

    Diana N D'Ambrosio

    Full Text Available Hepatic stellate cell (HSC lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i increased expression of typical markers of HSC activation; (ii decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT; (iii decreased triglyceride levels; (iv increased expression of genes associated with lipid catabolism; and (v an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1.Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.

  19. Construction of recombinant adenoviruses carrying human urokinase-type plasminogen activator and its expression in hepatic stellate cells in vitro%人尿激酶型纤溶酶原激活剂重组腺病毒的构建及其在肝星形细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    谢渭芬; 林勇; 张新; 张忠兵; 陈伟忠; 程志红; 陈岳祥; 张兴荣

    2002-01-01

    @@ 我们利用AdEasy系统在细菌内构建携带非分泌型尿激酶型纤溶酶原激活剂(urokinase-type plasminogen acti-vator,uPA)和绿色荧光蛋白(green fluorescent protein, GFP)cDNA的复制缺陷型腺病毒,并观察其在肝星形细胞(hepatic stellate cell, HSC)中的表达.

  20. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage

    Science.gov (United States)

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-01-01

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (ENKO) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in ENKO mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings. PMID:25680861

  1. Betaine treatment decreased oxidative stress, inflammation, and stellate cell activation in rats with alcoholic liver fibrosis.

    Science.gov (United States)

    Bingül, İlknur; Başaran-Küçükgergin, Canan; Aydın, A Fatih; Çoban, Jale; Doğan-Ekici, Işın; Doğru-Abbasoğlu, Semra; Uysal, Müjdat

    2016-07-01

    The aim of this study was to investigate the effect of betaine (BET) on alcoholic liver fibrosis in rats. Fibrosis was experimentally generated with ethanol plus carbon tetrachloride (ETH+CCl4) treatment. Rats were treated with ETH (5% v/v in drinking water) for 14 weeks. CCl4 was administered intraperitoneally (i.p.) 0.2mL/kg twice a week to rats in the last 6 weeks with/without commercial food containing BET (2% w/w). Serum hepatic damage markers, tumor necrosis factor-α, hepatic triglyceride (TG) and hydroxyproline (HYP) levels, and oxidative stress parameters were measured together with histopathologic observations. In addition, α-smooth muscle-actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type I collagen (COL1A1) protein expressions were assayed immunohistochemically to evaluate stellate cell (HSC) activation. mRNA expressions of matrix metalloproteinase-2 (MMP-2) and its inhibitors (TIMP-1 and TIMP-2) were also determined. BET treatment diminished TG and HYP levels; prooxidant status and fibrotic changes; α-SMA, COL1A1 and TGF-β protein expressions; MMP-2, TIMP-1 and TIMP-2 mRNA expressions in the liver of fibrotic rats. In conclusion, these results indicate that the antifibrotic effect of BET may be related to its suppressive effects on oxidant and inflammatory processes together with HSC activation in alcoholic liver fibrosis.

  2. In vivo effects of Chinese herbal recipe, Danshaohuaxian, on apoptosis and proliferation of hepatic stellate cells in hepatic fibrotic rats

    Institute of Scientific and Technical Information of China (English)

    Xiao-Xia Geng; Qin Yang; Ru-Jia Xie; Xin-Hua Luo; Bing Han; Li Ma; Cheng-Xiu Li; Ming-Liang Cheng

    2005-01-01

    AIM: To investigate the effects of Danshaohuaxian (DSHX),a Chinese herbal recipe, on the apoptosis and cell cycles of hepatic stellate cells (HSCs) in rat hepatic fibrosis and its possible mechanisms. METHODS: Seventy-six male Wistar rats were randomly divided into normal control group, hepatic fibrosis group,non-DSHX-treated group and DSHX-treated group. Except for the normal control group, rat hepatic fibrotic models were induced by subcutaneous injection of carbon tetrachloride (CCl4), drinking alcohol, giving diet of hyperlipid and hypoprotein for 8 wk. When the hepatic fibrotic models were produced, 12 rats of hepatic fibrosis group (15 rats survived, others died during the 8 wk) were sacrificed to collect blood and livers. HSCs were isolated from the other 3 rats to detect the apoptotic index (AI) and cell cycles by flow cytometry. DSHX was then given to the DSHX-treated group (1.0 g/kg, PO daily) for 8 wk. At the same time, normal control group and non-DSHX-treated group were given normal saline for 8 wk. At end of the experiment, some rats in these three groups were sacrificed to collect blood and livers, the other rats were used for HSC isolation to detect the apoptotic index (AI) and cell cycles. Then the liver index, serum hyaluronic acid (HA) and alanine aminotransferase (ALT),degree of hepatic fibrosis, urinary excretion of hydroxyproline (Hyp) and expression of collagen types Ⅰ and Ⅲ (COL Ⅰ and Ⅲ) in these four groups were detected respectively.RESULTS: Compared with the indexes of the hepatic fibrosis group and non-DSHX-treated group, the DSHX-treated group revealed a liver index of (0.0267±0.0017 vs 0.0423±0.0044, 0.0295±0.0019, P<0.05), levels of serum HA (200.78±31.71 vs316.17±78.48, 300.86±72.73, P<0.05)and ALT(93.13±5.79 vs 174.5±6.02, 104.75±6.54, P<0.01),and stage of hepatic fibrosis (1.30 vs 4.25, 2.60, P<0.01)all reduced. The urinary excretion of Hyp increased (541.09±73.39 vs 62.00±6.40, 182.44±30.83, P<0

  3. Characterization and sub-cellular localization of GalNAc-binding proteins isolated from human hepatic stellate cells.

    Science.gov (United States)

    Zhong, Yaogang; Zhang, Jing; Yu, Hanjie; Zhang, Jiaxu; Sun, Xiu-Xuan; Chen, Wentian; Bian, Huijie; Li, Zheng

    2015-12-25

    Although the expression levels of total GalNAc-binding proteins (GNBPs) were up-regulated significantly in human hepatic stellate cells (HSCs) activated with transforming growth factor-β1(TGF-β1), yet little is known about the precise types, distribution and sub-cellular localization of the GNBPs in HSCs. Here, 264 GNBPs from the activated HSCs and 257 GNBPs from the quiescent HSCs were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated HSCs. For example, the GNBPs (i.e. BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The western blotting showed the expression levels of COX17, and PRMT1 were significantly up-regulated, while, the expression level of CLIC1(B5) was down-regulated in the activated HSCs and liver cirrhosis tissues. Moreover, the GNBPs were sub-localized in the Golgi apparatus of HSCs. In conclusion, the precision alteration of the GNBPs referred to pathological changes in liver fibrosis/cirrhosis may provide useful information to find new molecular mechanism of HSC activation and discover the biomarkers for diagnosis of liver fibrosis/cirrhosis as well as development of new anti-fibrotic strategies.

  4. Hepatitis B virus infects hepatic stellate cells and affects their proliferation and expression of collagen type Ⅰ

    Institute of Scientific and Technical Information of China (English)

    LIU Xuan; ZHU Sheng-tao; YOU Hong; CONG Min; LIU Tian-hui; WANG Bao-en; JIA Ji-dong

    2009-01-01

    Background Hepatitis B is at particularly high risk of fibrosis progression. Unfortunately, the mechanism of hepatic fibrogenesis induced by hepatitis B virus (HBV) has not been fully understood to date. The aim of this study was to observe whether HBV can infect hepatic stellate cells (HSCs), and to examine the effects of HBV or HBV S protein (HBs) on the proliferation and collagen type Ⅰ expression of HSCs.Methods The supernatants of HepG2.2.15 cells which contained HBV-DNA or HBs were added to LX-2 cells for 72 hours. Cell survival was determined by MTT assay. HBV particles in LX-2 cells were detected by transmission electron microscopy. The expression of HBs and HBV C protein (HBc) was determined by confocal fluorescence microscopy. The expression levels of HBV-DNA were measured by real-time PCR. The cellular collagen type Ⅰ mRNA and protein levels were quantified by reverse transcription-PCR and ELISA, respectively.Results High concentrations of HBV (1.2x105-5.0x105 copies/ml) or HBs (1.25-20 μg/ml) inhibited the proliferation of LX-2 cells, while low concentrations of HBV (1.0x103-6.2x104 copies/ml) or HBs (0.04-0.62 μg/ml) promoted the proliferation. After treating LX-2 cells with HBV for 72 hours, about 42 nm HBV-sized particles and strong expression of HBs and HBc were found in the cytoplasm of LX-2 cells. HBV-DNA in the culture medium of LX-2 cells decreased at 24 hours, rose at 48 hours and thereafter, decreased again at 72 hours. The mRNA and protein expression of cellular collagen type Ⅰ in LX-2 cells were significantly increased by HBV infection but not by recombinant HBs. Conclusions HBV and HBs affect the proliferation of HSCs; HBV can transiently infect and replicate in cultured HSCs and express HBs and HBc in vitro. Furthermore, HBV can significantly increase the expression of collagen type Ⅰ mRNA and protein in HSCs.

  5. KN-93, a specific inhibitor of CaMK Ⅱ inhibits human hepatic stellate cell proliferation in vitro

    Institute of Scientific and Technical Information of China (English)

    Ping An; Jun-Yong Zhu; Yan Yang; Peng Lv; Yi-Hao Tian; Ming-Kai Chen; He-Sheng Luo

    2007-01-01

    AIM: To investigate the effects of KN-93, a CaMKⅡ selective inhibitor on cell proliferation and the expression of p53 or p21 protein in human hepatic stellate ceils.METHODS: Human hepatic stellate cells (LX-2) were incubated with various concentrations (0-50 μmol/L) of KN-93 or its inactive derivative, KN-92. Cell proliferation was measured by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was determined by SDS-PAGE and Western blotting.RESULTS: KN-93 (5-50 μmol/L) decreased the proliferation of human hepatic stellate cells in a dosedependent manner from 81.76% (81.76% + 2.58% vs 96.63% + 2.69%, P < 0.05) to 27.15% (27.15% + 2.86% vs 96.59% + 2.44%, P < 0.01) after 24 h treatment.Incubation of 10 μmol/L KN-93 induced the cell growth reduction in a time-dependent manner from 78.27% at 8 h to 11.48% at 48 h. However, KN-92, an inactive derivative of KN-93, did not inhibit cell proliferation effectively. Moreover, analysis of cell cycle regulator expression revealed that KN-93 rather than KN-92 reduced the expression of p53 and p21.CONCLUSION: KN-93 has potent inhibitory effect on proliferation of LX-2 cells by modulating the expression of two special cell cycle regulators, p53 and p21.

  6. Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model.

    Science.gov (United States)

    Dusabineza, Ange-Clarisse; Najimi, Mustapha; van Hul, Noémi; Legry, Vanessa; Khuu, Dung Ngoc; van Grunsven, Leo A; Sokal, Etienne; Leclercq, Isabelle A

    2015-01-01

    Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.

  7. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

    Science.gov (United States)

    Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M; Verma, Vikas K; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C; Shah, Vijay H

    2015-12-25

    Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

  8. Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

    Institute of Scientific and Technical Information of China (English)

    Run-Ping Gao; David R Brigstock

    2009-01-01

    AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-b1 to the culture medium. Semiquantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen Ⅰ, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen Ⅰ, and an increase in produced and secreted CCN2 or extracellular collagen Ⅰ protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen Ⅰ protein. Furthermore, the TGF-b1-induced increase in mRNA or protein for CCN2 or collagen Ⅰ was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-b1-induced collagen Ⅰ production in human HSCs and regulates entry of the cells into Sphase.

  9. Liver cirrhosis and hepatic stellate cells Cirrose hepática e células estreladas do figado

    Directory of Open Access Journals (Sweden)

    Daniel Ferracioli Brandão

    2006-01-01

    Full Text Available The cirrhosis represents the final stage of several chronic hepatic diseases and it is characterized by the presence of fibrosis and morphologic conversion from the normal hepatic architecture into structurally abnormal nodules. In the evolution of the disease there is loss of the normal vascular relationship and portal hypertension. There are also regenerative hepatocelular alterations that become more prominent with the progression of the disease. The liver transplantation continues to be the only therapeutic option in cases of disease in terminal phase. The hepatic stellate cells (HSC are perisinusoidal cells that store vitamin A and produce growth factors, citocins, prostaglandins and other bioactive substances. They can suffer an activation process that convert them to cells with a phenotype similar to myofibroblasts. When activated, they present increased capacity of proliferation, mobility, contractility and synthesis of collagen and other components of extracelular matrix. They possess cytoplasmic processes adhered to sinusoids and can affect the sinusoidal blood flow. HSC are important in pathogenesis of fibrosis and portal hypertension.A cirrose representa o estágio final de diversas doenças hepáticas crônicas e é caracterizada pela presença de fibrose e conversão da arquitetura hepática normal em nódulos estruturalmente anormais. Na evolução da doença ocorre perda da relação vascular normal e hipertensão portal. Há também alterações regenerativas hepatocelulares que se tornam mais proeminentes com a progressão da doença. O transplante hepático permanece como a única opção terapêutica nos casos de doença em fase terminal. As células estreladas hepáticas (CEH são células perisinusoidais que armazenam vitamina A e produzem fatores de crescimento, citocinas, prostaglandinas e outras substâncias bioativas. Podem sofrer um processo de ativação para um fenótipo semelhante a miofibroblastos. Quando ativadas

  10. HIV and HCV Co-Culture Promotes Profibrogenic Gene Expression through an Epimorphin-Mediated ERK Signaling Pathway in Hepatic Stellate Cells

    Science.gov (United States)

    Shi, Lei; Qin, Enqiang; Zhou, Junnian; Zhao, Juanjuan; Nie, Weimin; Jiang, Tianjun; Chen, Weiwei; Wu, Dan; Huang, Lei; Liu, Liying; Lv, Liping; Zhao, Min; Zhang, Zheng; Wang, Fusheng

    2016-01-01

    Accelerated fibrosis in patients co-infected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has been a major cause of mortality in the highly active anti-retroviral therapy (HAART) era. However, the role of co-infection in accelerating the progression of liver fibrosis, particularly with regard to the effects of co-infection on hepatic stellate cells (HSCs), remains unclear. We hypothesized that HIV and HCV induce liver fibrosis synergistically by altering the regulation of epimorphin production, and thereby indirectly alter HSC function. Here, we examined the effects of epimorphin on HSC proliferation and invasion, and the changes in fibrogenesis-related gene activity in HSCs (LX2) in the presence of inactivated CXCR4-tropic HIV and HCV (JFH1). The combination of HIV and HCV significantly increased epimorphin expression, which increased the proliferation and invasion capabilities of HSCs. Epimorphin also induced the expression of profibrogenic tissue inhibitor of metalloproteinase 1 (TIMP1) in an extracellular signal-regulated kinase (ERK)-dependent manner. These data indicated that the effects of HIV/HCV co-infection on hepatic fibrosis might be mediated in part by EPM. Strategies to limit the expression of EPM might represent a novel therapeutic approach to prevent the progression of hepatic fibrosis during HIV/HCV co-infection. PMID:27362846

  11. HIV and HCV Co-Culture Promotes Profibrogenic Gene Expression through an Epimorphin-Mediated ERK Signaling Pathway in Hepatic Stellate Cells.

    Directory of Open Access Journals (Sweden)

    Lei Shi

    Full Text Available Accelerated fibrosis in patients co-infected with hepatitis C virus (HCV and human immunodeficiency virus (HIV has been a major cause of mortality in the highly active anti-retroviral therapy (HAART era. However, the role of co-infection in accelerating the progression of liver fibrosis, particularly with regard to the effects of co-infection on hepatic stellate cells (HSCs, remains unclear. We hypothesized that HIV and HCV induce liver fibrosis synergistically by altering the regulation of epimorphin production, and thereby indirectly alter HSC function. Here, we examined the effects of epimorphin on HSC proliferation and invasion, and the changes in fibrogenesis-related gene activity in HSCs (LX2 in the presence of inactivated CXCR4-tropic HIV and HCV (JFH1. The combination of HIV and HCV significantly increased epimorphin expression, which increased the proliferation and invasion capabilities of HSCs. Epimorphin also induced the expression of profibrogenic tissue inhibitor of metalloproteinase 1 (TIMP1 in an extracellular signal-regulated kinase (ERK-dependent manner. These data indicated that the effects of HIV/HCV co-infection on hepatic fibrosis might be mediated in part by EPM. Strategies to limit the expression of EPM might represent a novel therapeutic approach to prevent the progression of hepatic fibrosis during HIV/HCV co-infection.

  12. Hepatic Stellate Cell-Derived Microvesicles Prevent Hepatocytes from Injury Induced by APAP/H2O2

    Directory of Open Access Journals (Sweden)

    Renwei Huang

    2016-01-01

    Full Text Available Hepatic stellate cells (HSCs, previously described for liver-specific mesenchymal stem cells (MSCs, appear to contribute to liver regeneration. Microvesicles (MVs are nanoscale membrane fragments, which can regulate target cell function by transferring contents from their parent cells. The aim of this study was to investigate the effect of HSC-derived MVs on xenobiotic-induced liver injury. Rat and human hepatocytes, BRL-3A and HL-7702, were used to build hepatocytes injury models by n-acetyl-p-aminophenol n-(APAP or H2O2 treatment. MVs were prepared from human and rat HSCs, LX-2, and HST-T6 and, respectively, added to injured BRL-3A and HL-7702 hepatocytes. MTT assay was utilized to determine cell proliferation. Cell apoptosis was analyzed by flow cytometry and hoechst33258 staining. Western blot was used for analyzing the expression of activated caspase-3. Liver injury indicators, alanine aminotransferase (ALT, aspartate aminotransferase (AST, and lactate dehydrogenase (LDH in culture medium were also assessed. Results showed that (1 HSC-MVs derived from LX-2 and HST-T6 were positive to CD90 and annexin V surface markers; (2 HSC-MVs dose-dependently improved the viability of hepatocytes in both injury models; (3 HSC-MVs dose-dependently inhibited the APAP/H2O2 induced hepatocytes apoptosis and activated caspase-3 expression and leakage of LDH, ALT, and AST. Our results demonstrate that HSC-derived MVs protect hepatocytes from toxicant-induced injury.

  13. Role of RhoA in platelet-derived growth factor-BB-induced migration of rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    LI Lei; LI Jing; WANG Ji-yao; YANG Chang-qing; JIA Ming-lei; JIANG Wei

    2010-01-01

    Background Although the migration of hepatic stellate cells (HSCs) is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases (especially RhoA) in platelet-derived growth factor (PDGF)-BB-induced migration of HSCs.Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases (RhoA, Rac1 and Cdc42) within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active (CA, Q63L) or dominant negative (DN, T19N) RhoA mutants. Data were analyzed using SPSS 16.0 software. Student's t test was used to analyze differences between two groups and one-way analysis of variance (ANOVA) was used among multiple groups.Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic (direct) and chemotactic (indirect) stimulation by PDGF-BB. PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Rac1 and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls.Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of

  14. Effects of adrenomedullin gene overexpression on biological behavior of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Yi Wang; Jin-Sheng Zhang; Guang-Cun Huang; Qi Cheng; Zhong-Hua Zhao

    2005-01-01

    AIM: To investigate the effects of adrenomedullin (AM) gene overexpression on the biological characteristics of human hepatic stellate cells (hHSCs) by stable transfection.METHODS: hHSCs which express low basal levels of AM were stably transfected with an expression construct containing rat AM gene or with an empty expression vector. Expression of AM in hHSCs was determined by reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). Cell proliferation was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and immunocytochemistry. RT-PCR and Western blot were used to test the expression of procollagen types Ⅰ and Ⅲ. Protein expressions of interstitial collagenase (MMP-1), gelatinase (MMP-2) and tissue inhibitors of matrix metalloproteinases-2 (TIMP-2) were assessed by Western blot.RESULTS: Two cell clones (A-2, A-8) transfected withthe AM gene expressed higher levels of AM mRNA (nontransfected group: 0.86±0.11, empty vector group: 1.01±0.11, A-2 clone group: 1.44±0.08 and A-8 clone group: 1.36±0.05) and protein (12.31±0.17, 12.35±0.12,12.56±0.06 and 12.62±0.07) (P<0.05). AM geneoverexpression had inhibitory effects on cell proliferation of hHSCs (29.6%, 30.9%, 18.9% and 21.8%, respectively. P<0.05) and expression of procollagen type Ⅰ (0.58±0.1,0.48±0.11, 0.3±0.06 and 0.31±0.07 at mRNA level)(0.27±0.07, 0.3±0.06, 0.14±0.05 and 0.13±0.05 at protein level) (P<0.05) and procollagen type Ⅲ (0.17±0.04, 0.15±0.03, 0.1±0.02 and 0.09±0.02 at mRNA level) (0.22±0.04, 0.2±0.03, 0.11±0.04 and 0.13±0.03 at protein level) (P<0.05). Compared with cells non-transfected (TIMp2: 2.77±0.03, MMP-2: 0.5±0.04, MMP-1: 0.49±0.07) and transfected with empty vector (TIMP2: 2.79±0.04,MMP-2: 0.48±0.03, MMP-1: 0.45±0.09), these two clones had lower expression levels of TIMP2(A-2 clone group: 2.7±0.02 and A-8 clone group: 2.71±0.02) (P<0.05) and MMP-2(A-2 clone group: 0.15±0.05 and A-8 clone group: 0.13±0

  15. Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Fowell, Andrew J., E-mail: ajf2@soton.ac.uk [Liver and Pancreas Group, University of Southampton, Division of Infection, Inflammation and Immunity, Southampton General Hospital, Southampton (United Kingdom); Collins, Jane E.; Duncombe, Dale R.; Pickering, Judith A. [Liver and Pancreas Group, University of Southampton, Division of Infection, Inflammation and Immunity, Southampton General Hospital, Southampton (United Kingdom); Rosenberg, William M.C. [Centre for Hepatology, Division of Medicine, University College London, London (United Kingdom); Benyon, R. Christopher [Liver and Pancreas Group, University of Southampton, Division of Infection, Inflammation and Immunity, Southampton General Hospital, Southampton (United Kingdom)

    2011-04-08

    Research highlights: {yields} Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis. {yields} We used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. {yields} Specific silencing of TIMP-1, but not TIMP-2, significantly reduces HSC proliferation and is associated with reduced Akt phosphorylation. {yields} TIMP-1 is localised in part to the HSC nucleus. {yields} TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. -- Abstract: Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover

  16. TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Ling, E-mail: fangling_1984@126.com [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); The First Affiliated Hospital of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Huang, Cheng; Meng, Xiaoming; Wu, Baoming; Ma, Taotao; Liu, Xuejiao; Zhu, Qian [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); Zhan, Shuxiang [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); The First Affiliated Hospital of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Li, Jun, E-mail: lj@ahmu.edu.cn [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China)

    2014-10-15

    Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 protein in human fibrotic livers • Upregulation of TRPM7 by TGF-β1 elicited Smad signaling in HSC-T6 cells

  17. 辛伐他汀抑制肝星状细胞活化及其对腺苷酸活化蛋白激酶活性的影响%Simvastatin inhibits activation of hepatic stellate cells and promotes activation of adenosine monophosphate activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    曹伟; 闫蕾; 王玮; 赵彩彦

    2012-01-01

    the underlying molecular mechanism ofthe cholesterol-blocking drug,simvastatin,in treating nonalcoholic fatty liver fibrosis.Method A rat model of nonalcoholic fatty liver fibrosis was established by feeding Wistar rats a fat-rich diet.After treatment with simvastatin (4 mg/kg/day),liver histological specimens were stained with hematoxylin-eosin and Masson's trichrome for microscopic analysis.Expression of adenosine monophosphate-activated protein kinase-alpha (AMPKα) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR; for mRNA) and Western blotting (protein).The levels of serum total cholesterol (TC),triglycerides (TG),alanine aminotransferase (ALT),aspartate aminotransferase (AST),and tumor necrosis factor-alpha (TNFa) were measured by standard biochemical assays.The human hepatic stellate cell line,LX-2 (quiescent or activated),was treated with transforming growth factor-beta l (TGF-β1) alone,simvastatin alone,or TGF-β1 +simvastatin.RT-PCR and Western blotting were used to determine changes in AMPKα mRNA and protein expression,respectively.Results In the rat model of nonalcoholic fatty liver fibrosis,the extent of pathological changes in hepatic tissues correlated with severity of disease progression.The levels of serum TC,TG,ALT,AST and TNFα were increased significantly in model rats (vs.healthy controls; all,P< 0.01).AMPKα mRNA expression and activity was significantly decreased in model rats (vs.healthy controls; P< 0.01 and P< 0.05,respectively).Simvastatin,treatment significantly improved all of these parameters in model rats (vs.untreated model rats; all,P< 0.05).In vitro simvastatin treatment of human HSCs significantly increased AMPKα activity (quiescent LX-2:0.93 -0.10 vs.0.72±0.09,activated LX-2:0.72±0.10 vs.0.54±0.10,q=7.00,6.00; all,P<0.01),decreased a-smooth muscle actin expression (mRNA:0.30±0.02 vs.0.36±0.02,protein:0.30±0.03 vs.0.38±0.02,q=11.245,11.216; all,P<0.01),and decreased collagen I expression

  18. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica*

    OpenAIRE

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Ampli...

  19. Involvement of the serine/threonine p70S6 kinase in TGF-beta1-induced ADAM12 expression in cultured human hepatic stellate cells

    DEFF Research Database (Denmark)

    Le Pabic, Hélène; L'Helgoualc'h, Annie; Coutant, Alexandre;

    2005-01-01

    In chronic liver injury, quiescent hepatic stellate cells change into proliferative myofibroblast-like cells, which are a main source of fibrosis. We have recently reported that these cells synthesize ADAM12, a disintegrin and metalloprotease whose expression is up-regulated by TGF-beta1 in liver...... cancers. Here, we studied the role of the serine/threonine p70S6 kinase (p70S6K) in regulating TGF-beta1-induced ADAM12 expression....

  20. Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Wen-Song Ge; Jian-Xin Wu; Jian-Gao Fan; Yao-Jun Wang; Ying-Wei Chen

    2011-01-01

    AIM: To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA. METHODS: Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine, and expression of HMGB1 was detected by immunohistochemistry. HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis. Expression of α-smooth muscle actin (α-SMA) and collagen types Ⅰ and Ⅲ was evaluated by real-time PCR. Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method. Finally, collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay. RESULTS: The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen. siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner. Moreover, HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs. CONCLUSION: This study suggests a significant functional role for HMGB1 in the development of liver fibrosis. It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

  1. Characterization of intracellular pathways leading to coinduction of thrombospondin-1 and TGF-beta1 expression in rat hepatic stellate cells.

    Science.gov (United States)

    Breitkopf, Katja; Sawitza, Iris; Gressner, Axel M

    2005-06-01

    Accumulating evidence has identified Thrombospondin (TSP)-1 as important activator of latent TGF-beta. Since little is known about signal transduction pathways regulating TSP expression in liver, we investigated cytokine-mediated upregulation of TSP-1 and TGF-beta1 in primary rat hepatic stellate cells (HSC). PDGF-BB and TNF-a rapidly coinduce mRNA levels of TSP-1 and TGF-beta1. Interestingly, blockade of basal Erk activity by synthetic Erk-binding peptides also leads to strong induction of both mRNA transcripts in non-stimulated cells. We show that PDGF-BB induces TSP-1 and TGF-beta1 via the src kinase pathway whereas TNF-a utilizes the MAPK/Erk pathway. However, especially TSP-1 induction by both cytokines involves a pathway, which depends to a certain extent on PI3 kinase activity. In summary the data illustrate specific pathways activated by PDGF-BB and TNF-a in HSC giving new insights into the tightly controlled mechanisms regulating TSP-1 and TGF-beta1 expression in these cells.

  2. Fuzheng Huayu Recipe Ameliorates Liver Fibrosis by Restoring Balance between Epithelial-to-Mesenchymal Transition and Mesenchymal-to-Epithelial Transition in Hepatic Stellate Cells.

    Science.gov (United States)

    Pan, Qin; Wang, Yu-Qin; Li, Guang-Ming; Duan, Xiao-Yan; Fan, Jian-Gao

    2015-01-01

    Activation of hepatic stellate cells (HSCs) depending on epithelial-to-mesenchymal transition (EMT) reflects the key event of liver fibrosis. Contrastively, mesenchymal-to-epithelial transition (MET) of HSCs facilitates the fibrosis resolution. Here we investigated the effect of Fuzheng Huayu (FZHY) recipe, a Chinese herbal decoction made of Radix Salviae Miltiorrhizae, Semen Persicae, Cordyceps sinensis, Pollen Pini, and Gynostemma pentaphyllum, on liver fibrosis concerning the balance of EMT and MET in HSCs. In contrast to the increased TGF-β 1/BMP-7 ratio in activated HSCs, FZHY administration induced significant upregulation of BMP-7 and downregulation of TGF-β 1 at both transcription and translation levels. Restoration of TGF-β 1/BMP-7 ratio inhibited the expression of p38 MAPK and phosphorylated p38 MAPK, resulting in the reversal of epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) as characterized by the abolishment of EMT markers (α-SMA and desmin) and reoccurrence of MET marker (E-cadherin). In vivo treatment of FZHY recipe also demonstrated the statistical reduction of activated HSCs with EMT phenotype, which attenuated the carbon tetrachloride- (CCl4-) induced liver fibrosis in a dose-dependent manner. These findings may highlight a novel antifibrotic role of FZHY recipe on the basis of rebalancing EMT and MET in HSCs.

  3. Regulator of G-protein signaling-5 is a marker of hepatic stellate cells and expression mediates response to liver injury.

    Directory of Open Access Journals (Sweden)

    Arya J Bahrami

    Full Text Available Liver fibrosis is mediated by hepatic stellate cells (HSCs, which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR-mediated signaling, via endothelin-1 (ET-1 and angiotensin II (AngII, increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5, an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs. Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.

  4. Study on Effects of Extracts from Salvia Miltiorrhiza and Curcuma Longa in Inhibiting Phosphorylated Extracellular Signal Regulated Kinase Expression in Rat's Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    CHENG Yang; PING Jian; LIU Cheng; TAN Ying-zi; CHEN Gao-feng

    2006-01-01

    Objective: To study the effect of salvianolic acid B (SAB) and curcumin, the extracts of Salvia Miltiorrhiza and Curcuma Longa, on the proliferation and activation of hepatic stellate cell (HSC), and the extracellular signal regulated kinase (ERK) expression in it. Methods: Rat's HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-(4,5-dimthyl-2-2thiazoly)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and the expression levels of α smooth actin (α-SMA), collagen type Ⅰ , and ERK were determined by Western blot. Results: SAB and curcumin inhibited the proliferation and activation of rat's HSC-T6 in dose-dependent fashion and significantly reduced the expression level of α-SMA ( P<0.01 ). Curcumin significantly reduced the expression of collagen type Ⅰ( P<0.05). Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group ( P<0.01 and P<0.05 respectively). Conclusion: SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type Ⅰ collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.

  5. Fuzheng Huayu Recipe Ameliorates Liver Fibrosis by Restoring Balance between Epithelial-to-Mesenchymal Transition and Mesenchymal-to-Epithelial Transition in Hepatic Stellate Cells

    Directory of Open Access Journals (Sweden)

    Qin Pan

    2015-01-01

    Full Text Available Activation of hepatic stellate cells (HSCs depending on epithelial-to-mesenchymal transition (EMT reflects the key event of liver fibrosis. Contrastively, mesenchymal-to-epithelial transition (MET of HSCs facilitates the fibrosis resolution. Here we investigated the effect of Fuzheng Huayu (FZHY recipe, a Chinese herbal decoction made of Radix Salviae Miltiorrhizae, Semen Persicae, Cordyceps sinensis, Pollen Pini, and Gynostemma pentaphyllum, on liver fibrosis concerning the balance of EMT and MET in HSCs. In contrast to the increased TGF-β1/BMP-7 ratio in activated HSCs, FZHY administration induced significant upregulation of BMP-7 and downregulation of TGF-β1 at both transcription and translation levels. Restoration of TGF-β1/BMP-7 ratio inhibited the expression of p38 MAPK and phosphorylated p38 MAPK, resulting in the reversal of epithelial-to-mesenchymal transition (EMT to mesenchymal-to-epithelial transition (MET as characterized by the abolishment of EMT markers (α-SMA and desmin and reoccurrence of MET marker (E-cadherin. In vivo treatment of FZHY recipe also demonstrated the statistical reduction of activated HSCs with EMT phenotype, which attenuated the carbon tetrachloride- (CCl4- induced liver fibrosis in a dose-dependent manner. These findings may highlight a novel antifibrotic role of FZHY recipe on the basis of rebalancing EMT and MET in HSCs.

  6. Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition%碧萝芷通过下调 ERK 磷酸化及自噬水平抑制TGF-β1诱导的肝星状细胞活化

    Institute of Scientific and Technical Information of China (English)

    杨淑娟; 何英利; 马晓华; 姜娜

    2016-01-01

    AIM:To explore the effect of Pycnogenol on transforming growth factor-β1 ( TGF-β1)-induced he-patic stellate cell activation .METHODS:Cultured LX-2 cells were treated with 5μg/L TGF-β1 and different concentra-tions (0, 10, 25 and 50 mg/L) of Pycnogenol.The viability of the LX-2 cells under the conditions with or without autoph-agy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay .The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot .RESULTS:Compared with con-trol group, 5μg/L TGF-β1 treatment elevated the cell viability , and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05).However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most sig-nificant in the LX-2 cells (P<0.05).Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnog-enol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells ( P<0.05 ) .CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition .%目的:探究碧萝芷对转化生长因子β1( TGF-β1)诱导的肝星状细胞活化的影响。方法:5μg/L TGF-β1和不同浓度碧萝芷(0、10、25、50 mg/L)分别作用于LX-2细胞,在有或无自噬抑制剂3-MA和ERK抑制剂PD98059的情况下,用MTT法检测细胞活力的变化,Western blot实验检测α-SMA、ColⅠ、TIMP-1、LC3-Ⅱ/Ⅰ、beclin 1、p-ERK1/2和ERK1/2蛋白水平的变化。结果:与对照组相比,5μg/L TGF-β1组的LX-2细胞活力以及α-SMA、ColⅠ、TIMP-1、LC3-Ⅱ/Ⅰ、beclin 1、p-ERK1/2和ERK1/2蛋白水平明显增加(P<0.05)。而碧萝芷预处理能逆转上述效应,并呈

  7. Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis.

    Science.gov (United States)

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run; Guo, Cheng

    2015-01-01

    Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis.

  8. 转录因子c-myb及肝脏激活蛋白对α1(Ⅰ)胶原基因在肝星状细胞中表达的调控作用%Regulation of transcription factors c-myb and liver activator protein on expression ofα1(I) collagen gene in activated hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    刘小菁

    2000-01-01

    目的:了解激活的HSC中c-myb及肝脏组织特异的转录因子—肝脏激活蛋白(liver activator protein,LAP)在α1(Ⅰ)基因表达调控中的作用。 方法:采用链霉蛋白酶,胶原酶原位灌注、Nycodenz密度梯度离心分离大鼠HSC,并进行体外培养使之激活。构建含人α1(Ⅰ)胶原基因启动子片段(-804~+1452或-804~+222碱基)的荧光素酶报告基因质粒。用构建的报告基因质粒与c-myb和(或)LAP表达质粒一起,用阳离子脂质体介导的方法,瞬时共转染激活的HSC。 结果:瞬时共转染LAP表达质粒可明显增强含α1(Ⅰ)基因第一内含子片段的荧光素酶报告基因(PGL3-col)及不含α1(Ⅰ)基因第一内含子的报告基因[PGL3-col(△intron)]报告基因在HSC中的表达[荧光素酶活性分别为每毫克蛋白(587±62)U对(315±45)U及(326±52)U对(220±70)U,t值分别为10.4和3.6,两者P值均小于0.05]。C-myb对这两个报告基因均无反式激活作用。但共转染c-myb及LAP表达质粒,却使PGL3-col报告基因在HSC中的表达增强近3倍[每毫克蛋白(1261±130)U对(315±45)U,t=20.6,P<0.01=,而共转染反义c-myb及LAP表达质粒,却抑制了LAP对α1(Ⅰ)基因启动子的反式激活作用[每毫克蛋白(334±29)U对(315± 45)U,t=1.06,P>0.05]。C-myb的这种作用只在 PGL3-col质粒中观察到。 结论:C-myb在α1(Ⅰ)基因转录调控中起重要作用,而此作用由转录因子LAP及α1(Ⅰ)基因第一内含子中的调控元件介导。%Objective: To elucidate the role of two transcription factors, c-myb and liver activator protein (LAP, a member of the C/EBP family) in the expression of α1(Ⅰ) collagen gene in activated hepatic stellate cells (HSCs). Methods: Rat HSCs were prepared from SD rats by in situ perfusion and single-step density Nycodenz gradient. Two chimeric luciferase reporter gene plasmids containing

  9. Pancreatic Stellate Cells and Chronic Alcoholic Pancreatitis

    Directory of Open Access Journals (Sweden)

    Raffaele Pezzilli

    2007-03-01

    fibrillary acidic protein, neural cell adhesion molecule and neurotrophin nerve growth factor just as hepatic stellate cells do. Pancreatic stellate cells contain the enzyme alcohol dehydrogenase [11] and, when activated, they assume a myofibroblastlike phenotype [12]. Activated pancreatic stellate cells are characterized by the disappearance of fat globules and the expression of alpha-smooth muscle actin. These cells have proliferative and migratory [13, 14, 15] functions and they also synthesize and secrete extracellular fibrous tissue matrix proteins, matrix metalloproteinases and their inhibitors [16]; it has also been demonstrated that pancreatic stellate cells have phagocytic activity [17]. Thus, the ability of pancreatic stellate cells to synthesize as well as to degrade extracellular matrix proteins suggests their role in maintaining a normal pancreatic architecture which can shift towards fibrogenesis if the balance is altered. Ethanol, acetaldehyde and oxidant stress are capable of activating activate pancreatic stellate cells via three mitogen-activated protein kinase pathways [18], namely extracellular signal kinase, p38 kinase and c-jun amino terminal kinase [19, 20, 21], and ethanol and acetaldehyde are also capable of activating phosphatidylinositol 3-kinase and protein kinase C [22]. On the other hand, extracellular signal kinase activation occurs via a signal transduction pathway which involves Gprotein Ras and serine threonine protein kinase Raf-1 [23, 24]. The Ras superfamily G proteins undergo post-translational modification involving isoprenylation, a process which requires intermediate substrates of cholesterol biosynthesis [25, 26] which is regulated by HMG CoA reductase [27]. The paracrine pro-fibrogenic effect of TGF-beta on pancreatic stellate cells is mediated via smad while the autocrine effect is mediated through the extracellular signal kinase pathway [28]; furthermore, the role of the peroxisome proliferator-activated receptor-gamma seems to be

  10. Apoptotic and survival signals in hepatic stellate cells%肝星状细胞中细胞凋亡和存活的信号调控

    Institute of Scientific and Technical Information of China (English)

    Hong Shen; Jianghong Fan; Geraid Minuk; Yuewen Gong

    2007-01-01

    肝星状细胞(hepatic stellate cells,HSCs)在肝脏纤维化发生过程中起着关键作用.当正常肝脏受到损伤时,HSCs由静息状态转分化为类肌成纤维细胞,并保持这种处于激活状态的表型,它们接收到的凋亡和存活的生物信号将决定激活态HSCs的最终细胞寿命.HSCs凋亡的发生与一系列复杂而又相互关联的生物信号传导和调控有关,HSCs凋亡信号来自于细胞膜受体,如死亡受体、神经生长因子受体和外周型苯甲二氮卓受体(peripheral-type benzodiazepine receptor);以及胞浆蛋白,如Bcl-2家族蛋白和细胞周期蛋白等.HSCs存活信号受到多种激酶和细胞因子的诱导,如金属蛋白酶组织抑制剂-1(tissue jnhibitors of metalloproteinase-1)、Rho/Rho激酶、血小板源生长因子(platelet-derived growth factor)、转化生长因子-β1(transforming growth factor-β1)和胰岛素样生长因子(insulin-like growth factor-1)等.特异性地诱导HSCs发生凋亡是治疗肝脏纤维化的直接和有效手段,虽然目前对HSCs由激活态到静息状态的转归尚需进一步研究,但诱导HSCs凋亡将是治疗肝脏纤维化和肝硬化的研究热点和主要发展方向.%Hepatic stellate cells (HSCs) play an important role in hepatic fibrogenesis.In response to liver injury, HSCs undergo a process called activation, which involves 2 stepsinitiation from quiescent phenotype to myofibroblast-like phenotype, and perpetuation that maintains the activated phenotype of HSCs. The fate of the activated HSCs depends on the apoptotic and survival signals that they receive. The apoptosis of HSCs results from a series of complex and interrelated signaling events. Apoptotic signals for the activated HSCs include proteins from membrane receptors, such as death receptors, nerve growth factor receptor and peripheral-type benzodiazepine receptor, as well as proteins from cytoplasm such as Bcl-2 family members. The survival signals for the activated HSCs are

  11. Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Lin Wang; Bao-En Wang; Jian Wang; Pei-Gen Xiao; Xue-Hai Tan

    2008-01-01

    AIM: To identify the role of herbal compound 861 (Cpd 861) in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells (HSCs).METHODS: mRNA levels of collagen types I and III, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), membrane type-1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and transforming growth factor β1 (TGF-βi) in cultured-activated HSCs treated with Cpd 861 or interferon-γ (IFN-γ) were determined by real-time PCR.RESULTS: Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-βl. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to (6.3 + 0.3)-fold compared to the control group.CONCLUSION: The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type in and TGF-pi and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

  12. Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in rat hepatic stellate cells.

    Science.gov (United States)

    Koide, Shigeki; Kobayashi, Yoshimasa; Oki, Yutaka; Nakamura, Hirotoshi

    2004-09-01

    Prostaglandin (PG) E2 inhibits hepatic stellate cell (HSC) mitogenesis. PGE-specific receptors are divided into four subtypes that are coupled either to Ca2+ mobilization (EP1 and EP3) or to the stimulation of adenyl cyclase (EP2 and EP4). The aims of the current study were to identify PGE receptor subtypes in cultured rat HSC and to examine which PGE receptor subtype(s) mediates the inhibitory effect of PGE2 on platelet-derived growth factor (PDGF)-stimulated proliferation. Reverse transcription-polymerase chain reaction analysis was performed to detect PGE receptor subtype mRNA expression. Cell proliferation was determined by measuring [3H]thymidine incorporation, and intracellular cyclic AMP was measured by radioimmunoassay. Cultured rat HSC expressed mRNAs for all four subtypes of PGE receptor. PGE2- and EP2-selective agonist produced dose-dependent inhibitory effects on PDGF-stimulated proliferation. Neither EP1-, EP3-, nor EP4-selective agonists showed any inhibitory effect. An adenylate cyclase inhibitor strongly blunted the inhibition of DNA synthesis elicited by PGE2 and the EP2 agonist. The EP2 agonist generated higher and more prolonged increases in intracellular cyclic AMP than the EP4 agonist. Activation of the PGE EP2 receptor has an antiproliferative effect in HSC that may be mediated by cyclic AMP-related signal transduction pathways.

  13. The endocannabinoid N-arachidonoyl dopamine (NADA) selectively induces oxidative stress-mediated cell death in hepatic stellate cells but not in hepatocytes.

    Science.gov (United States)

    Wojtalla, Alexandra; Herweck, Frank; Granzow, Michaela; Klein, Sabine; Trebicka, Jonel; Huss, Sebastian; Lerner, Raissa; Lutz, Beat; Schildberg, Frank Alexander; Knolle, Percy Alexander; Sauerbruch, Tilman; Singer, Manfred Vincenz; Zimmer, Andreas; Siegmund, Sören Volker

    2012-04-15

    The endocannabinoid system is a crucial regulator of hepatic fibrogenesis. We have previously shown that the endocannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces death in hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, but not in hepatocytes. However, the effects of other endocannabinoids such as N-arachidonoyl dopamine (NADA) have not yet been investigated. The NADA-synthesizing enzyme tyrosine hydroxylase was mainly expressed in sympathetic neurons in portal tracts. Its expression pattern stayed unchanged in normal or fibrotic liver. NADA dose dependently induced cell death in culture-activated primary murine or human HSCs after 2-4 h, starting from 5 μM. Despite caspase 3 cleavage, NADA-mediated cell death showed typical features of necrosis, including ATP depletion. Although the cannabinoid receptors CB1, CB2, or transient receptor potential cation channel subfamily V, member 1 were expressed in HSCs, their pharmacological or genetic blockade failed to inhibit NADA-mediated death, indicating a cannabinoid-receptor-independent mechanism. Interestingly, membrane cholesterol depletion with methyl-β-cyclodextrin inhibited AEA- but not NADA-induced death. NADA significantly induced reactive oxygen species formation in HSCs. The antioxidant glutathione (GSH) significantly decreased NADA-induced cell death. Similar to AEA, primary hepatocytes were highly resistant against NADA-induced death. Resistance to NADA in hepatocytes was due to high levels of GSH, since GSH depletion significantly increased NADA-induced death. Moreover, high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in hepatocytes also conferred resistance towards NADA-induced death, since pharmacological or genetic FAAH inhibition significantly augmented hepatocyte death. Thus the selective induction of cell death in HSCs proposes NADA as a novel antifibrogenic mediator.

  14. Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-β signaling in hepatic stellate cells

    Science.gov (United States)

    Cao, Sheng; Yaqoob, Usman; Das, Amitava; Shergill, Uday; Jagavelu, Kumaravelu; Huebert, Robert C.; Routray, Chittaranjan; Abdelmoneim, Soha; Vasdev, Meher; Leof, Edward; Charlton, Michael; Watts, Ryan J.; Mukhopadhyay, Debabrata; Shah, Vijay H.

    2010-01-01

    PDGF-dependent hepatic stellate cell (HSC) recruitment is an essential step in liver fibrosis and the sinusoidal vascular changes that accompany this process. However, the mechanisms that regulate PDGF signaling remain incompletely defined. Here, we found that in two rat models of liver fibrosis, the axonal guidance molecule neuropilin-1 (NRP-1) was upregulated in activated HSCs, which exhibit the highly motile myofibroblast phenotype. Additionally, NRP-1 colocalized with PDGF-receptor β (PDGFRβ) in HSCs both in the injury models and in human and rat HSC cell lines. In human HSCs, siRNA-mediated knockdown of NRP-1 attenuated PDGF-induced chemotaxis, while NRP-1 overexpression increased cell motility and TGF-β–dependent collagen production. Similarly, mouse HSCs genetically modified to lack NRP-1 displayed reduced motility in response to PDGF treatment. Immunoprecipitation and biochemical binding studies revealed that NRP-1 increased PDGF binding affinity for PDGFRβ-expressing cells and promoted downstream signaling. An NRP-1 neutralizing Ab ameliorated recruitment of HSCs, blocked liver fibrosis in a rat model of liver injury, and also attenuated VEGF responses in cultured liver endothelial cells. In addition, NRP-1 overexpression was observed in human specimens of liver cirrhosis caused by both hepatitis C and steatohepatitis. These studies reveal a role for NRP-1 as a modulator of multiple growth factor targets that regulate liver fibrosis and the vascular changes that accompany it and may have broad implications for liver cirrhosis and myofibroblast biology in a variety of other organ systems and disease conditions. PMID:20577048

  15. Adenoviral transduction of PTEN induces apoptosis of cultured hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    HAO Li-sen; ZHANG Xiao-lan; AN Jun-yan; YAO Dong-mei; Justin Karlin; FANG Shu-ming; JIANG Hui-qing; BAI Wen-yuan; CHEN Shuang

    2009-01-01

    @@ Hepatic fibrosis is the liver's wound healing response to virtually all forms of chronic liver injury: toxic insult, viral infection, immunological conditions and metabolic diseases. Uncontrolled liver fibrosis eventually results in cirrhosis and associated complications, such as cancer and liver failure.

  16. The hepatic stellate cell in sight : targeting antiproliferative drugs to the fibrotic liver

    NARCIS (Netherlands)

    Greupink, Albert Hendrikus

    2006-01-01

    Liver fibrosis is characterized by the accumulation of excessive amounts of scar tissue in response to chronic liver injury. Important causes of chronic liver injury are viral hepatitis, metabolic disorders such as Wilson’s disease, autoimmune diseases and chronic exposure to certain chemicals, alco

  17. Oxidative stress plays a role in high glucose-induced activation of pancreatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Gyeong Ryul; Lee, Esder; Chun, Hyun-Ji; Yoon, Kun-Ho; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho, E-mail: kihos@catholic.ac.kr

    2013-09-20

    Highlights: •High glucose increased production of reactive oxygen species in cultured pancreatic stellate cells. •High glucose facilitated the activation of these cells. •Antioxidant treatment attenuated high glucose-induced activation of these cells. -- Abstract: The activation of pancreatic stellate cells (PSCs) is thought to be a potential mechanism underlying islet fibrosis, which may contribute to progressive β-cell failure in type 2 diabetes. Recently, we demonstrated that antioxidants reduced islet fibrosis in an animal model of type 2 diabetes. However, there is no in vitro study demonstrating that high glucose itself can induce oxidative stress in PSCs. Thus, PSCs were isolated and cultured from Sprague Dawley rats, and treated with high glucose for 72 h. High glucose increased the production of reactive oxygen species. When treated with high glucose, freshly isolated PSCs exhibited myofibroblastic transformation. During early culture (passage 1), PSCs treated with high glucose contained an increased number of α-smooth muscle actin-positive cells. During late culture (passages 2–5), PSCs treated with high glucose exhibited increases in cell proliferation, the expression of fibronectin and connective tissue growth factor, release of interleukin-6, transforming growth factor-β and collagen, and cell migration. Finally, the treatment of PSCs with high glucose and antioxidants attenuated these changes. In conclusion, we demonstrated that high glucose increased oxidative stress in primary rat PSCs, thereby facilitating the activation of these cells, while antioxidant treatment attenuated high glucose-induced PSC activation.

  18. Effects of drug serum of anti-fibrosis I herbal compound on calcium in hepatic stellate cell and its molecular mechanism

    Institute of Scientific and Technical Information of China (English)

    Yong-Hong Xiao; Dian-Wu Liu; Qing Li

    2005-01-01

    AIM: To investigate the effects of anti-fibrosis I herbal compound on intracellular Ca2+ in activated hepatic stellate cell (HSC) and to try to survey its molecular mechanism in treatment and prevention of hepatic fibrosis and portal hypertension.METHODS: The activated HSC line was plated on small glass cover slips in 24 wells culture dishes at a density of 5x106/mL, and incubated in RPMI-1640 media for 24 h.After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured with laser scanning confocal microscopy (LSCM). The dynamic changes of intracellular Ca2+,stimulated by carbon tetrachloride, TGF-β1 antibody and the drug serum of anti-fibrosis I herbal compound and under orthogonal design were determined by LSCM. The effect of anti-fibrosis I herbal compound on intracellular Ca2+ was observed before and after the addition of TGF-β1antibody.RESULTS: The intracellular Ca2+ were significantly different in different dosage of carbon tetrachloride anti-fibrosis Iformula drug serum, TGF-β1 antibody and different turn of these substance, but their interval time between CCl4and TGF-β1 antibody, CCl4 and anti-fibrosis I drug serum had no influence on intracellular Ca2+. The result showed intracellular Ca2+ wasn't significantly different between rat serum without anti-fibrosis I and untreated group.After carbon tetrachloride stimulation, intracellular Ca2+ of activated HSC increased significantly when the dosage of CCl4 from 5 to 15 mmol/L, however, decreased significantly after stimulation by 5-20 μg/mL TGF-β1antibody or 5-20 mL/L drug serum. Moreover, before and after the addition of TGF-β1 antibody, intracellular Ca2+was significantly different. These results suggested that the molecular mechanism was independent of blocking TGF-β1 effects.CONCLUSION: Anti-fibrosis I herbal compound may treat hepatic fibrosis and decrease portal hypertension by inhibiting activated HSC contractility through decrease of intracellular Ca2+.

  19. Transforming growth factor-beta induces nerve growth factor expression in pancreatic stellate cells by activation of the ALK-5 pathway.

    Science.gov (United States)

    Haas, Stephan L; Fitzner, Brit; Jaster, Robert; Wiercinska, Eliza; Gaitantzi, Haristi; Jesnowski, Ralf; Jesenowski, Ralf; Löhr, J-Matthias; Singer, Manfred V; Dooley, Steven; Breitkopf, Katja

    2009-10-01

    Nerve growth factor (NGF), a survival factor for neurons enforces pain by sensitizing nociceptors. Also in the pancreas, NGF was associated with pain and it can stimulate the proliferation of pancreatic cancer cells. Hepatic stellate cells (HSC) respond to NGF with apoptosis. Transforming growth factor (TGF)-beta, one of the strongest pro-fibrogenic activators of pancreatic stellate cells (PSC) induced NGF and its two receptors in an immortalized human cell line (ihPSC) and primary rat PSC (prPSC) as determined by RT-PCR, western blot, and immunofluorescence. In contrast to HSC, PSC expressed both NGF receptors, although p75(NTR) expression was weak in prPSC. In contrast to ihPSC TGF-beta activated both Smad signaling cascades in prPSC. NGF secretion was diminished by the activin-like kinase (ALK)-5 inhibitor SB431542, indicating the predominant role of ALK5 in activating the NGF system in PSC. While NGF did not affect proliferation or survival of PSC it induced expression of Inhibitor of Differentiation-1. We conclude that under conditions of upregulated TGF-beta, like fibrosis, NGF levels will also increase in PSC which might contribute to pancreatic wound healing responses.

  20. Genetic characteristics of the human hepatic stellate cell line LX-2.

    Directory of Open Access Journals (Sweden)

    Ralf Weiskirchen

    Full Text Available The human hepatic cell line LX-2 has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds. It was originally generated by immortalisation with the Simian Vacuolating Virus 40 (SV40 transforming (T antigen and subsequent propagation in low serum conditions. Although this immortalized line is used in an increasing number of studies, detailed genetic characterisation has been lacking. We here have performed genetic characterisation of the LX-2 cell line and established a single-locus short tandem repeat (STR profile for the cell line and characterized the LX-2 karyotype by several cytogenetic and molecular cytogenetic techniques. Spectral karyotyping (SKY revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers. In addition, various subclonal and single cell aberrations were detected. Our study provides criteria for genetic authentication of LX-2 and offers insights into the genotype changes which might underlie part of its phenotypic features.

  1. Gene profile of chemokines on hepatic stellate cells of schistosome-infected mice and antifibrotic roles of CXCL9/10 on liver non-parenchymal cells.

    Science.gov (United States)

    Liang, Yue-jin; Luo, Jie; Lu, Qiao; Zhou, Ying; Wu, Hai-wei; Zheng, Dan; Ren, Yong-ya; Sun, Ke-yi; Wang, Yong; Zhang, Zhao-song

    2012-01-01

    Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis caused by schistosomiasis. Chemokines were widely expressed and involved in cellular activation, proliferation and migration in inflammatory and infectious diseases. However, little is known about the expressions of chemokines on HSCs in the schistosoma infection. In addition, the roles of chemokines in pathogenesis of liver fibrosis are not totally clear. In our study, we used microarray to analyze the temporal gene expressions of primary HSCs isolated from mice with both acute and chronic schistosomiasis. Our microarray data showed that most of the chemokines expressed on HSCs were upregulated at 3 weeks post-infection (p.i) when the egg granulomatous response was not obviously evoked in the liver. However, some of them like CXCL9, CXCL10 and CXCL11 were subsequently decreased at 6 weeks p.i when the granulomatous response reached the peak. In the chronic stage, most of the differentially expressed chemokines maintained persistent high-abundances. Furthermore, several chemokines including CCR2, CCR5, CCR7, CXCR3, CXCR4, CCL2, CCL5, CCL21, CXCL9 and CXCL10 were expressed by HCSs and the abundances of them were changed following the praziquantel treatment in the chronic stage, indicating that chemokines were possibly necessary for the persistence of the chronic stage. In vitro experiments, hepatic non-parenchymal cells, primary HSCs and human HSCs line LX-2 were stimulated by chemokines. The results showed that CXCL9 and CXCL10, but not CXCL11 or CXCL4, significantly inhibited the gene expressions of Col1α1, Col3α1 and α-SMA, indicating the potential anti-fibrosis effect of CXCL9 and CXCL10 in schistosomiasis. More interestingly, soluble egg antigen (SEA) of Schistosoma japonicum was able to inhibit transcriptional expressions of some chemokines by LX-2 cells, suggesting that SEA was capable of regulating the expression pattern of chemokine family and modulating the hepatic immune

  2. Salvianolic Acid B Inhibits ERK and p38 MAPK Signaling in TGF-β1-Stimulated Human Hepatic Stellate Cell Line (LX-2 via Distinct Pathways

    Directory of Open Access Journals (Sweden)

    Zhigang Lv

    2012-01-01

    Full Text Available Salvianolic acid B (SA-B is water-soluble component of Radix Salvia miltiorrhiza. The previous work indicated that SA-B can inhibit MAPK and Smad signaling in activated hepatic stellate cells (HSCs to perform anti-fibrotic activity Lv et al. 2010. However, some studies have shown that there is cross-talk between MAPK and Smad in certain cell types. Thus, the anti-fibrotic action of SA-B may be through the cross-talk. In order to clarify the mechanism of SA-B further, we knocked down Smad in LX-2 cells (SRV4 via RNAi, and then added TGF-β1, and PD98059 or SB203580 and SA-B. The levels of p-MEK and p-p38 were inhibited by SA-B in SRV4 independent of TGF-β1. The expression of Col I and α-SMA in SRV4 could be reduced by SA-B independent TGF-β1. SB203580 had not significant effect on p-MEK in SRV4 stimulated by TGF-β1. The levels of p-MEK in SRV4 were not increased significantly after TGF-β1 stimulation. PD98059 had no effect on the levels of p-p38 in SRV4 irrespective of TGF-β1. In conclusion, SA-B inhibits the synthesis of Col I in LX-2 cells independent of TGF-β1 stimulation, and the anti-fibrotic effect of SA-B is due to direct inhibition of p38 signaling and inhibition the cross-talk of Smad to ERK signaling.

  3. Salvianolic Acid B Inhibits ERK and p38 MAPK Signaling in TGF-β1-Stimulated Human Hepatic Stellate Cell Line (LX-2) via Distinct Pathways

    Science.gov (United States)

    Lv, Zhigang; Xu, Lieming

    2012-01-01

    Salvianolic acid B (SA-B) is water-soluble component of Radix Salvia miltiorrhiza. The previous work indicated that SA-B can inhibit MAPK and Smad signaling in activated hepatic stellate cells (HSCs) to perform anti-fibrotic activity Lv et al. 2010. However, some studies have shown that there is cross-talk between MAPK and Smad in certain cell types. Thus, the anti-fibrotic action of SA-B may be through the cross-talk. In order to clarify the mechanism of SA-B further, we knocked down Smad in LX-2 cells (SRV4) via RNAi, and then added TGF-β1, and PD98059 or SB203580 and SA-B. The levels of p-MEK and p-p38 were inhibited by SA-B in SRV4 independent of TGF-β1. The expression of Col I and α-SMA in SRV4 could be reduced by SA-B independent TGF-β1. SB203580 had not significant effect on p-MEK in SRV4 stimulated by TGF-β1. The levels of p-MEK in SRV4 were not increased significantly after TGF-β1 stimulation. PD98059 had no effect on the levels of p-p38 in SRV4 irrespective of TGF-β1. In conclusion, SA-B inhibits the synthesis of Col I in LX-2 cells independent of TGF-β1 stimulation, and the anti-fibrotic effect of SA-B is due to direct inhibition of p38 signaling and inhibition the cross-talk of Smad to ERK signaling. PMID:21860657

  4. Carvedilol Improves Inflammatory Response, Oxidative Stress and Fibrosis in the Alcohol-Induced Liver Injury in Rats by Regulating Kuppfer Cells and Hepatic Stellate Cells.

    Directory of Open Access Journals (Sweden)

    Raimundo Fernandes de Araújo Júnior

    Full Text Available To evaluate the anti-inflammatory, anti-oxidant and antifibrotic effects of carvedilol (CARV in rats with ethanol-induced liver injury.Liver injury was induced by gavage administration of alcohol (7 g/kg for 28 consecutive days. Eighty Wistar rats were pretreated with oral CARV at 1, 3, or 5 mg/kg or with saline 1 h before exposure to alcohol. Liver homogenates were assayed for interleukin (IL-1β, IL-10, and tumor necrosis factor (TNF-α level as well as for myeloperoxidase (MPO activity and malonyldialdehyde (MDA and glutathione (GSH levels. Serum aspartate aminotransferase (AST activity and liver triglyceride (TG levels were also assayed. Immunohistochemical analyses of cyclooxygenase 2 (COX-2, receptor activator of nuclear factor kappa-B/ligand (RANK/RANKL, suppressor of cytokine signalling (SOCS1, the Kupffer cell marker IBA-1 (ionized calcium-binding adaptor molecule 1, intercellular adhesion molecule 1 (ICAM-1, superoxide dismutase (SOD-1, and glutathione peroxidase (GPx-1 expression were performed. Confocal microscopy analysis of IL-1β and NF-κB expression and real-time quantitative PCR analysis for TNFα, PCI, PCIII, and NF-κB were performed.CARV treatment (5 mg/kg during the alcohol exposure protocol was associated with reduced steatosis, hepatic cord degeneration, fibrosis and necrosis, as well as reduced levels of AST (p < 0.01, ALT (p < 0.01, TG (p < 0.001, MPO (p < 0.001, MDA (p < 0.05, and proinflammatory cytokines (IL-1β and TNF-α, both p < 0.05, and increased levels of the anti-inflammatory cytokine IL-10 (p < 0.001 and GSH (p < 0.05, compared to the alcohol-only group. Treatment with CARV 5 mg/kg also reduced expression levels of COX-2, RANK, RANKL, IBA-1, and ICAM-1 (all p < 0.05, while increasing expression of SOCS1, SOD-1, and GPx-1 (all p < 0.05 and decreasing expression of IL-1β and NF-κB (both, p < 0.05. Real-time quantitative PCR analysis showed that mRNA production of TNF-α, procollagen type I (PCI, procollagen

  5. Effects of c-myb antisense RNA on TGF-β1 and α1-I collagen expression in cultured hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Hui-Hui Ma; Ji-Lu Yao; Gang Li; Chun-Lan Yao; Xue-Juan Chen; Shao-Ji Yang

    2004-01-01

    AIM: To investigate the effects of c-myb antisense RNA on cell proliferation and the expression of c-myb, TGF-β1 and α1-I collagen in cultured hepatic stellate cells (HSC) from rats.METHODS: Recombinant retroviral vector of c-myb antisense gene (pDOR-myb) was constructed, and then transfected into retroviral package cell line PA317 by means of DOTAP.The pseudoviruses produced from the resistant PA317 cells were selected with G418 to infect HSCs isolated from rat livers. The cell proliferation was measured by 3-[4, 5-Dimethylthiazolzyl]-2, 5-diphenyl tetrazo-dium bromide (MTT) method.The expression of c-myb, α1-I collagen and TGF-β1 mRNA, and c-myb protein in HSCs was detected with semi-quantitive reverse transeription-polymerase chain reaction (RT-PCR) and Western-blot respectively.RESULTS: HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, the cmyb protein expression, cell proliferation,and α1-I collagen and TGF-β1 mRNA expression were repressed significantly compared with their corresponding control groups (P<0.01).CONCLUSION: c-myb plays a key role in activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation, α1-I collagen and TGF-β1 mRNA expression,suggesting that inhibition of c-myb gene expression might be a potential way for the treatment of liver fibrosis.

  6. Effects of anandamide on the activation and proliferation of hepatic stellate cells through cannabinoid-2 receptors%大麻素受体2介导的N-花生四烯酸氨基乙醇对肝星状细胞增殖和活化的影响

    Institute of Scientific and Technical Information of China (English)

    刘红艳; 阳乔; 段瑞娴; 张曜文; 唐望先

    2008-01-01

    Objectives To study the effects of endogenous cannanbinoid anandamide (AEA) and its putative endocannabinoid receptors (CBR) on the activation and proliferation of hepatic stellate cells (HSC) and to study the role played by AEA during liver fibrosis. Methods By using immunofluorescence and cell culture, the expression of CBR 1 and 2 in the PDGF-stimulated HSCs was investigated. By using PCR and Western-blot, the effects of 10, 20 μmol/L AEA and CBR2 antagonist AM630 on the cultured and activated HSC were observed. Methyl thiazolyl tetrazolium and flow cytometry were used to investigate whether AEA induces growth inhibition or apoptsis in the activated HSCs. Results Both CBRI and CBR2 receptors were detectable in cultured HSCs with a higher level of CBR2 than CBRI (F=116.797, P<0.01). When HSCs were stimulated by PDGF, the expression of CBR2 receptors was significantly enhanced (F=7.878, P<0.05). HSC proliferation was dose-dependently inhibited by 10, 20, and 50 μmol/L AEA, with the rates of 7.12%±0.34%, 12.52%±0.78%, 80.13%±1.57% respectively (F=533.41, P<0.01). However, it did not induce apoptosis, but necrosis. The expressions of alpha-SMA, TGF β1, α1 (Ⅰ), α1 (Ⅲ) and TIMP-1 were significantly suppressed by 20 μmol/L AEA, but CBR2 antagonist AM630 reversed this suppressor action of AEA. Conclusions AEA may inhibit activation and proliferation of HSCs; CBR2 receptors mediate AEA-induced inhibitory action on the activation of HSCs. This CBR2 receptor-mediated action and AEA on HSCs could be used as a therapeutic target against liver fibrosis.%目的 观察内源性大麻素N-花生四烯酸氨基乙醇(AEA)及大麻素受体(CBR)2对肝星状细胞(HSC)增殖活化的影响,以探讨内源性大麻素及其受体系统在肝纤维化发展中的作用.方法 采用免疫荧光观察血小板衍生生长因子(PDGF)刺激前后HSC中CBR1和CBR2的表达.Western blot、PCR法观察不同浓度AEA及CBR2拮抗剂AM630对PDGF刺激下HSC增殖及活化的

  7. Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells.

    Science.gov (United States)

    Liu, Yu; Savtchouk, Iaroslav; Acharjee, Shoana; Liu, Siqiong June

    2011-07-01

    Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca(2+) and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the duration of action potentials can suppress the expression of GluR2-containing AMPARs at stellate cell synapses. We find that large-conductance Ca(2+)-activated potassium (BK) channels mediate a large proportion of the depolarization-evoked noninactivating potassium current in stellate cells. Pharmacological blockade of BK channels prolonged the action potential duration in postsynaptic stellate cells and altered synaptic AMPAR subtype from GluR2-lacking to GluR2-containing Ca(2+)-impermeable AMPARs. An L-type channel blocker abolished an increase in Ca(2+) entry that was associated with spike broadening and also prevented the BK channel blocker-induced switch in AMPAR phenotype. Thus blocking BK potassium channels prolongs the action potential duration and increases the expression of GluR2-containing receptors at the synapse by enhancing Ca(2+) entry in cerebellar stellate cells.

  8. Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment

    Science.gov (United States)

    Das, Amitava; Shergill, Uday; Thakur, Lokendra; Sinha, Sutapa; Urrutia, Raul; Mukhopadhyay, Debabrata

    2010-01-01

    Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 μg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase (P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 μM), an inhibitor of MEK, which is a kinase upstream from Erk (P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity (P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation (P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These

  9. Effects of ursolic acid on NADPH oxidase subunit p47Phox expression and ERK1/2 pathway activation in rat hepatic stellate cells%熊果酸对活化型肝星状细胞NADPH氧化酶亚基p47Phox表达及ERK1/2信号通路活化的影响

    Institute of Scientific and Technical Information of China (English)

    张新华; 何文华; 朱萱; 李弼民; 张焜和; 陈璐; 施凤

    2012-01-01

    目的 研究熊果酸(ursolic acid,UA)对瘦素诱导的大鼠肝星状细胞(HSC-T6) NADPH氧化酶(NOX)亚基p47Phox表达及ERK1/2信号通路活化的影响,并观察I 型胶原合成及细胞增殖情况.方法 将培养激活的HSC-T6细胞株分为6组:正常对照组,不加任何药物;瘦素组,给予重组大鼠瘦素(100 ng/ml)刺激细胞;各干预组分别给予UA (50 μmol/L)、JAK抑制剂AG490 (50 μmol/L)、NOX抑制剂DPI (20μmol/L)、ERK抑制剂PD98059(30 μmol/L)预处理30 min,再加入瘦素刺激不同时间.采用蛋白质印迹分析检测细胞膜移位的p47Phox蛋白、细胞总p47Phox蛋白和磷酸化的ERK1/2(p-ERK1/2)蛋白表达;采用RT-PCR法检测 I型胶原mRNA的表达;采用MTT法检测细胞增殖.结果 瘦素刺激HSC-T6细胞30 min后细胞膜p47Phox蛋白表达较正常对照组增高(P<0.01),细胞内p-ERK1/2蛋白表达也随之增高(P<0.05);UA、AG490、DPI、PD98059干预后抑制了p47Phox蛋白向细胞膜移位以及细胞内ERK1/2蛋白磷酸化.瘦素刺激HSO-T6细胞12h后I 型胶原的mRNA表达较正常对照组升高(P<0.01),UA、AG490、DPI及PD98059干预组I 型胶原mRNA的表达均低于瘦素组(P均<0.01).瘦素刺激HSC-T6细胞12、24、48 h后细胞增殖率高于正常对照组(P均<0.01);UA、AG490、DPI及PD98059干预不同时间点的细胞增殖率均低于瘦素组(P均<0.01),UA的抑制细胞增殖作用弱于DPI(P<0.01).结论 UA能抑制瘦素诱导的HSC-T6细胞增殖及I 型胶原表达,机制可能与抑制NOX亚基p47Phox向细胞膜移位及下游信号通路ERK1/2的激活有关.%Objective To investigate the effects of ursolic acid CUA) on leptin-induced NADPH oxidase (NOX) subunits p47phox expression and ERKi/2 pathway activation of rat hepatic stellate cells (HSOT6), and to observe the cells proliferation and collagen I synthesis. Methods Culture-activated HSC-T6 cells were divided into six groups: normal control group received no treatment; leptin

  10. TGF-beta1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k.

    Science.gov (United States)

    Lechuga, Carmen G; Hernández-Nazara, Zamira H; Domínguez Rosales, José-Alfredo; Morris, Elena R; Rincón, Ana Rosa; Rivas-Estilla, Ana María; Esteban-Gamboa, Andrés; Rojkind, Marcos

    2004-11-01

    Transforming growth factor-beta1 (TGF-beta1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-beta1 induces a transient elevation of alpha1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the alpha1(I) collagen mRNA. In the present study, we report that TGF-beta1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of alpha1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-beta1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70(S6k). Altogether, our data suggest that regulation of MMP-13 by TGF-beta1 is a complex process involving transcriptional and posttranscriptional mechanisms.

  11. In vitro structure-toxicity relationship of chalcones in human hepatic stellate cells

    KAUST Repository

    Zenger, Katharina

    2015-07-19

    Xanthohumol (XN), the major prenylated chalcone from hops (Humulus lupulus L.), has received much attention within the last years, due to its multiple pharmacological activities including anti-proliferative, anti-inflammatory, antioxidant, pro-apoptotic, anti-bacterial and anti-adhesive effects. However, there exists a huge number of metabolites and structurally-related chalcones, which can be expected, or are already known, to exhibit various effects on cells. We have therefore analyzed the effects of XN and 18 other chalcones in a panel, consisting of multiple cell-based assays. Readouts of these assays addressed distinct aspects of cell-toxicity, like proliferation, mitochondrial health, cell cycle and other cellular features. Besides known active structural elements of chalcones, like the Michael system, we have identified several moieties that seem to have an impact on specific effects and toxicity in human liver cells in vitro. Based on these observations, we present a structure-toxicity model, which will be crucial to understand the molecular mechanisms of wanted effects and unwanted side-effects of chalcones.

  12. In vitro structure-toxicity relationship of chalcones in human hepatic stellate cells.

    Science.gov (United States)

    Zenger, Katharina; Dutta, Subhajit; Wolff, Horst; Genton, Marc G; Kraus, Birgit

    2015-10-02

    Xanthohumol (XN), the major prenylated chalcone from hops (Humulus lupulus L.), has received much attention within the last years, due to its multiple pharmacological activities including anti-proliferative, anti-inflammatory, antioxidant, pro-apoptotic, anti-bacterial and anti-adhesive effects. However, there exists a huge number of metabolites and structurally-related chalcones, which can be expected, or are already known, to exhibit various effects on cells. We have therefore analyzed the effects of XN and 18 other chalcones in a panel, consisting of multiple cell-based assays. Readouts of these assays addressed distinct aspects of cell-toxicity, like proliferation, mitochondrial health, cell cycle and other cellular features. Besides known active structural elements of chalcones, like the Michael system, we have identified several moieties that seem to have an impact on specific effects and toxicity in human liver cells in vitro. Based on these observations, we present a structure-toxicity model, which will be crucial to understand the molecular mechanisms of wanted effects and unwanted side-effects of chalcones.

  13. Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1β or TNF-α Release from Human Hepatic Stellate Cells.

    Science.gov (United States)

    Robert, Sacha; Gicquel, Thomas; Bodin, Aude; Lagente, Vincent; Boichot, Elisabeth

    2016-01-01

    Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable

  14. Expression of cytosolic and membrane associated tissue transglutaminase in rat hepatic stellate cells and its upregulation during transdifferentiation to myofibroblasts in culture.

    Science.gov (United States)

    Schnabel, Claudia; Sawitza, Iris; Tag, Carmen G.; Lahme, Birgit; Gressner, Axel M.; Breitkopf, Katja

    2004-03-01

    Transdifferentiation of hepatic stellate cells (HSC) to collagen producing myofibroblasts (MFB) is a principal event in liver fibrogenesis. In our studies we investigated if tissue transglutaminase (tTG) from these cell types may play a role in liver fibrosis. Separation of cytosol and membrane components showed membrane associated tTG and during transdifferentiation an upregulation of total tTG on mRNA and protein level was found, but no modulation during stimulation with TGF-beta1. In HSC and fully differentiated MFB a significant amount of the total tTG synthesised during transdifferentiation is found to be membrane-associated whereas the remaining portion is cytosol-associated and only very little is found within the extracellular matrix (ECM). The data implicate that tTG in this cell type seems to play an important role in liver fibrogenesis.

  15. MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Sun, Ming; Stolz, Donna B. [Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (United States); He, Fengtian [Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038 (China); Fan, Jie [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Xie, Wen [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Li, Song, E-mail: sol4@pitt.edu [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States)

    2014-04-18

    Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

  16. Matrix metalloproteinase-1 induction by diethyldithiocarbamate is regulated via Akt and ERK/miR222/ETS-1 pathways in hepatic stellate cells.

    Science.gov (United States)

    Liu, Tianhui; Wang, Ping; Cong, Min; Zhang, Dong; Liu, Lin; Li, Hongyi; Zhai, Qingling; Li, Zhuo; Jia, Jidong; You, Hong

    2016-08-01

    Matrix metalloproteinase-1 (MMP-1) plays an important role in fibrolysis by degrading excessively deposited collagen I and III. We previously demonstrated that diethyldithiocarbamate (DDC) up-regulates MMP-1 in hepatic stellate cells via the ERK1/2 and Akt signalling pathways. In the current study, we attempted to further explore the molecular mechanisms involved in the regulation of MMP-1. We treated a co-cultured system that included hepatocytes (C3A) and hepatic stellate cells (LX-2) with DDC. The data revealed that the transcriptional factor ETS-1, which is an important regulator of MMP-1, was up-regulated in LX-2 cells following DDC treatment. Furthermore, the up-regulation of MMP-1 by DDC has been abrogated through employing si-ETS-1 to block expression of ETS-1. We found that DDC significantly inhibited the expression of miR-222 in LX-2 cells. We transfected miR-222 mimic into LX-2 cells and then co-cultured the cells with C3A. The up-regulation of ETS-1 and MMP-1 in LX-2 cells treated with DDC were inhibited after miR-222 mimic transfection. These data indicate that DDC up-regulated MMP-1 in LX-2 cells through the miR-222/ETS-1 pathway. Finally, we treated the co-cultured system with an Akt inhibitor (T3830) and an ERK1/2 inhibitor (U0126). Both T3830 and U0126 blocked the suppression of miR-222 by DDC in LX-2. Collectively, these data indicate that DDC up-regulated MMP-1 in LX-2 cells through the Akt and ERK/miR-222/ETS-1 pathways. Our study provides experimental data that will aid the control of the process of fibrolysis in liver fibrosis prevention and treatment.

  17. miR-181b Promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Baocan [Department of Gastroenterology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092 (China); Li, Wenxi [China Novartis Institutes for BioMedical Research Co. Ltd., Shanghai 201203 (China); Guo, Kun [Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200102 (China); Xiao, Yongtao [Shanghai Key Laboratory of Pediatric Gastroenterology and Nutrition, Shanghai 200092 (China); Wang, Yuqin, E-mail: wangvuqin00@sina.com [Department of Gastroenterology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092 (China); Fan, Jiangao, E-mail: fanjiangao@gmail.com [Department of Gastroenterology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092 (China)

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer miR-181a and miR-181b, especially, miR-181b could be induced by transforming growth factor-beta 1 (TGF-{beta}1) in hepatic stellate cells. Black-Right-Pointing-Pointer miR-181b could promote HSC-T6 cell proliferation by directly targeting the negative cell regulator-p27 in HSC-T6 cell. Black-Right-Pointing-Pointer miR-181b was identified as potential serum diagnostic marker for liver cirrhosis patients. -- Abstract: MicroRNAs, as a kind of negative gene regulators, were demonstrated to be involved in many types of diseases. In this study, we found that transforming growth factor-beta 1 could induce the expression of miR-181a and miR-181b, and miR-181b increased in the much higher folds than miR-181a. Because of the important role of transforming growth factor-beta 1 in HSC activation and liver cirrhosis, we investigate the effect of miR-181a and miR-181b on HSC proliferation. The results showed that miR-181b could promote HSC-T6 cell proliferation by regulating cell cycle. Further study showed p27, the cell cycle regulator, was the direct target of miR-181b in HSC-T6 cell. But miR-181a had no effects on HSC-T6 cell proliferation and cell cycle, and did not target p27. Interestingly, miR-181b is elevated significantly in serum of liver cirrhosis cases comparing to that of normal persons, whereas miR-181a expression was in the similar level with that of normal persons. These results suggested that miR-181b could be induced by TGF-{beta}1 and promote the growth of HSCs by directly targeting p27. The elevation of miR-181b in serum suggested that it may be potential diagnostic biomarkers for cirrhosis. As for miR-181a, it may work in TGF-{beta}1 pathway by a currently unknown mechanism.

  18. Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells

    OpenAIRE

    Yu LIU; Savtchouk, Iaroslav; Acharjee, Shoana; Liu, Siqiong June

    2011-01-01

    Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca2+ and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the durati...

  19. Pancreatic stellate cells contribute pancreatic cancer pain via activation of sHH signaling pathway.

    Science.gov (United States)

    Han, Liang; Ma, Jiguang; Duan, Wanxing; Zhang, Lun; Yu, Shuo; Xu, Qinhong; Lei, Jianjun; Li, Xuqi; Wang, Zheng; Wu, Zheng; Huang, Jason H; Wu, Erxi; Ma, Qingyong; Ma, Zhenhua

    2016-04-05

    Abdominal pain is a critical clinical symptom in pancreatic cancer (PC) that affects the quality of life for PC patients. However, the pathogenesis of PC pain is largely unknown. In this study, we show that PC pain is initiated by the sonic hedgehog (sHH) signaling pathway in pancreatic stellate cells (PSCs), which is activated by sHH secreted from PC cells, and then, neurotrophic factors derived from PSCs mediate the pain. The different culture systems were established in vitro, and the expression of sHH pathway molecules, neurotrophic factors, TRPV1, and pain factors were examined. Capsaicin-evoked TRPV1 currents in dorsal root ganglion (DRG) neurons were examined by the patch-clamp technique. Pain-related behavior was observed in an orthotopic tumor model. sHH and PSCs increased the expression and secretion of TRPV1, SP, and CGRP by inducing NGF and BDNF in a co-culture system, also increasing TRPV1 current. But, suppressing sHH pathway or NGF reduced the expression of TRPV1, SP, and CGRP. In vivo, PSCs and PC cells that expressed high levels of sHH could enhance pain behavior. Furthermore, the blockade of NGF or TRPV1 significantly attenuated the pain response to mechanical stimulation compared with the control. Our results demonstrate that sHH signaling pathway is involved in PC pain, and PSCs play an essential role in the process greatly by inducing NGF.

  20. 4-hydroxy-2, 3-nonenal activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Kazuhiro Kikuta; Atsushi Masamune; Masahiro Satoh; Noriaki Suzuki; Tooru Shimosegawa

    2004-01-01

    AIM: Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis,where oxidative stress is thought to play a key role. 4-hydroxy2,3-nonenal (HNE) is generated endogenously during the process of lipid peroxidation, and has been accepted as a mediator of oxidative stress. The aim of this study was to clarify the effects of HNE on the activation of signal transduction pathways and cellular functions in PSCs.METHODS: PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase P, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. PSCs were treated with physiologically relevant and non-cytotoxic concentrations (up to 5 μmol/L)of HNE. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay.Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Production of type Ⅰ collagen and monocyte chemoattractant protein-1was determined by enzyme-linked immunosorbent assay.The effect of HNE on the transformation of freshly isolated PSCs in culture was also assessed.RESULTS: HNE activated activator protein-1, but not nuclear factor κB. In addition, HNE activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. HNE increased type Ⅰ collagen production through the activation of p38 MAP kinase and c-Jun N-terminal kinase. HNE did not alter the proliferation,or monocyte chemoattractant protein-1 production. HNE did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype.CONCLUSION: Specific activation of these signal transduction pathways and altered cell functions such as collagen production by HNE may play a role in the pathogenesis of pancreatic

  1. Effect of angiotensin Ⅱ and angiotensin Ⅱ type 1 receptor antagonist on the proliferation,contraction and collagen synthesis in rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    LIU Jun; GONG Hao; ZHANG Zhong-tao; WANG Yu

    2008-01-01

    Background Angiotensin Ⅱ(Ang Ⅱ)is a very important vasoactive peptide that acts upon hepatic stellate cells(HSCs),which are major effector cells in hepatic cirrhosis and portal hypertension.The present study was aimed to investigate the effects of Ang Ⅱ and angiotensin Ⅱ type 1 receptor antagonist(AT1RA)on the proliferation,contraction and collagen synthesis in HSCs.Methods HSC-T6 rat hepatic stellate cell Iine was studied.The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by3 H-thymidine incorporation.The effects of angiotensin Ⅱ and AT1 RA on HSCs contraction were studied by analVSIs of the contraction of the collagen Iattice.CelI culture media were analyzed by RT-PCR to detect secretion of collagen Ⅰ(Col Ⅰ),collagen Ⅲ(Col Ⅲ)and transforming growth factor β1 (TGF-β1)by enzyme Iinked Immunosorbent assay.HSC was harvested to measure collagen Ⅰ,collagen Ⅲ and tissue inhibitor of metalloproteinase-1(TIMP-1)mRNA expression.Results Ang Ⅱ((1 x10-10-1×10-4)mol/L)stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells.AT1 RA inhibited angiotensin Ⅱ induced proliferation of HSCs.A Iinear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin Ⅱ(1×10-9-1×10-5mol/L)and with time over 48 hours.ATlRA blocks angiotensin Ⅱ induced contraction of collagen Iattice.Coll,Col Ⅲ and TGF-β1 levels of the Ang Ⅱ group were higher than those of control group and this increase was downregulated by AT1RA.The mRNA expressions of ColⅠ,CoI Ⅲ and TIMP-1 were higher in HSCs from the Ang Ⅱ group than the control group and downregulated by AT1RA.Conclusions Angiotensin Ⅱ increased DNA synthesis and proliferation of HSCs in a dose-dependent manner,stimulated the contraction of HSCs dose-and time-dependently.Angiotensin also promoted excretion of Col Ⅰ,ColⅢand TGF-β1 Ievels and stimulated Col Ⅰ,Col Ⅲ and

  2. Cthrc1抑制小鼠肝星状细胞的增殖%Cthrc1 Inhibits Proliferation of Hepatic Stellate Cells in Mice

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    钟巍; 卞兆连; 马雄

    2016-01-01

    背景:研究发现胶原三螺旋重复蛋白-1(Cthrc1)可能在肝脏疾病尤其是肝纤维化中发挥重要作用,但对肝星状细胞增殖的影响尚未完全阐明。目的:研究 Cthrc1对肝星状细胞增殖的影响。方法:构建 Cthrc1重组腺病毒载体,通过尾静脉注射小鼠体内,以实时 PCR 和蛋白质印迹法分别检测 Cthrc1 mRNA 和蛋白表达。通过胆总管结扎和3,5-二乙氧基羰基-1,4-二氢-2,4,6-三甲基吡啶(DDC)饲料喂养分别构建小鼠肝纤维化模型,分别尾静脉注射 Ad-Cthrc1或 Ad-GFP,以免疫荧光法检测肝星状细胞数量。结果:成功构建 Cthrc1重组腺病毒载体;实时 PCR 和蛋白质印迹法证实尾静脉注射 Ad-Cthrc1后小鼠肝内 Cthrc1 mRNA 和蛋白表达均较对照组增高。HE 和 Masson 染色示成功建立了小鼠肝纤维化模型。免疫荧光法显示 Cthrc1过表达能明显抑制肝纤维化模型小鼠肝星状细胞的增殖。结论:成功构建了重组腺病毒载体 Ad-Cthrc1并可在体内高效稳定表达,能抑制肝星状细胞的增殖,有望成为肝纤维化的治疗靶点。%Background:Collagen triple helix repeat containing protein-1(Cthrc1)has been reported playing an important role in liver diseases,especially in liver fibrosis,however,its effect on hepatic stellate cells proliferation is not fully clear. Aims:To investigate the effect of Cthrc1 on proliferation of hepatic stellate cells. Methods:Recombinant adenovirus vector of Cthrc1 was constructed. After injecting Ad-Cthrc1 through tail vein,mRNA and protein expressions of Cthrc1 were determined by real-time PCR and Western blotting,respectively. Liver fibrosis model was established by bile duct ligation and fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine(DDC)in mice,respectively. The liver fibrosis mice were injected with Ad-Cthrc1 or Ad-GFP through tail vein. Immunofluorescence was used to determine number of hepatic stellate cells

  3. Stellate Cell Activation in Tropical Calcific Pancreatitis Compared to Alcoholic Pancreatitis, Adenocarcinoma of Pancreas and Normal Pancreas

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    Johny Cyriac

    2012-07-01

    Full Text Available ContextPancreatic stellate cell (PSC is known to be the source of fibrosis in pancreatic pathology of various etiologies. However, there is no published data on activation of PSCs in tropical calcific pancreatitis. ObjectivesThe present study was undertaken to estimate the proportion of activated stellate cells, in a semi-quantitative manner, in normal pancreas and pancreatic fibrosis due to, tropical calcific pancreatitis, alcoholic chronic pancreatitis and pancreatic adenocarcinoma. PatientsSurgically resected specimen from patients with tropical calcific pancreatitis (n=22, alcoholic chronic pancreatitis(n=16, adenocarcinoma of pancreas (n=20 and normal pancreas (n=20 were included. Main outcome measuresExpression of CD34, and alpha-smooth muscle actin (α-SMA was assessed by immunohistochemistry. Morphometry was performed by a pointcounting procedure and CD34 positive areas were excluded from α-SMA positive areas for estimating activated PSCs. StatisticsThe one-way ANOVA and the Tukey multiple comparison test were used to compare the proportion ofactivated stellate cells among the four categories. ResultsIn all the disease conditions studied, namely, tropical calcific pancreatitis (16.7±14.5%, mean±SD, alcoholic chronic pancreatitis (13.6±12.4% and pancreatic adenocarcinoma (22.8±14.4%, there was highly significant (P<0.001 increased percentage of activated PSCs compared to normal pancreas (-0.9±6.4%. Proportion of activated PSCs in tropical calcific pancreatitis was similar to that in cases of alcoholic chronic pancreatitis and pancreatic adenocarcinoma. Such activation is documented for the first time in tropical calcific pancreatitis while it is known for the other causes. ConclusionsThe present study suggests that a final common pathway of PSC activation leads to fibrogenesis in tropical calcific pancreatitis just as in other pancreatic pathologies.

  4. Inhibition of pancreatic stellate cell activity by adipose-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    Fu-Xiang Yu; Long-Feng Su; Chun-Lei Dai; Yang Wang; Yin-Yan Teng; Jun-Hui Fu; Qi-Yu Zhang; Yin-He Tang

    2015-01-01

    BACKGROUND: Pancreatic stellate cells (PSCs) play a critical role in the development of pancreatic ifbrosis. In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells (ADSCs) on activation and proliferation of PSCs. METHODS: Pancreatic tissue was obtained from Sprague-Dawley rats for PSCs isolation. Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs prolifera-tion and apoptosis were determined using CCK-8 and lfow cytometry, respectively.α-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor (NGF), interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1)] in conditioned medium were detected by ELISA. Gene expression (MMP-2, MMP-9 and TIMP-1) was analyzed using qRT-PCR. RESULTS: This method produced 17.6±6.5×103 cells per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs signiifcantly inhib-ited PSCs proliferation and induced PSCs apoptosis. Moreover,α-SMA expression was signiifcantly reduced in PSCs+ADSCs compared with that in PSC-only cultures, while expression of ifbrinolytic proteins (e.g., MMP-2 and MMP-9) was up-regulated and anti-ifbrinolytic protein (TIMP-1) was down-regulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-β1 expressions were down-regulated in the co-culture conditioned medium compared with those in the PSC-only culture medium. CONCLUSIONS: This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibi-tory effects of ADSCs on the activation and proliferation of PSCsin vitro.

  5. Molecular regulation of pancreatic stellate cell function

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    Jaster Robert

    2004-10-01

    Full Text Available Abstract Until now, no specific therapies are available to inhibit pancreatic fibrosis, a constant pathological feature of chronic pancreatitis and pancreatic cancer. One major reason is the incomplete knowledge of the molecular principles underlying fibrogenesis in the pancreas. In the past few years, evidence has been accumulated that activated pancreatic stellate cells (PSCs are the predominant source of extracellular matrix (ECM proteins in the diseased organ. PSCs are vitamin A-storing, fibroblast-like cells with close morphological and biochemical similarities to hepatic stellate cells (also known as Ito-cells. In response to profibrogenic mediators such as various cytokines, PSCs undergo an activation process that involves proliferation, exhibition of a myofibroblastic phenotype and enhanced production of ECM proteins. The intracellular mediators of activation signals, and their antagonists, are only partially known so far. Recent data suggest an important role of enzymes of the mitogen-activated protein kinase family in PSC activation. On the other hand, ligands of the nuclear receptor PPARγ (peroxisome proliferator-activated receptor γ stimulate maintenance of a quiescent PSC phenotype. In the future, targeting regulators of the PSC activation process might become a promising approach for the treatment of pancreatic fibrosis.

  6. Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4-/- mouse model

    Institute of Scientific and Technical Information of China (English)

    Florian; P; Reiter; Ralf; Wimmer; Lena; Wottke; Renate; Artmann; Jutta; M; Nagel; Manuel; O; Carranza; Doris; Mayr; Christian; Rust; Peter; Fickert; Michael; Trauner; Alexander; L; Gerbes; Simon; Hohenester; Gerald; U; Denk

    2016-01-01

    AIM: To study the interleukin-1(IL-1) pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4-/-(Abcb4-/-) mouse model.METHODS: Female and male Abcb4-/- mice from 6 to 13 mo of age were analysed for the degree of cholestasis(liver serum tests), extent of liver fibrosis(hydroxyproline content and Sirius red staining) and tissue-specific activation of signalling pathways such as the IL-1 pathway [quantitative polymerase chain reaction(q PCR)]. For in vivo experiments, murine hepatic stellate cells(HSCs) were isolated via pronasecollagenase perfusion followed by density gradient centrifugation using female mice. Murine HSCs were stimulated with up to 1 ng/m L IL-1β with or without 2.5 μg/m L Anakinra, an IL-1 receptor antagonist, respectively. The proliferation of murine HSCs was assessed via the Brd U assay. The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis(FDH) assay. In vivo 8-wk-old Abcb4-/- mice with an already fully established hepatic phenotype were treated with Anakinra(1 mg/kg body-weight daily intraperitoneally) or vehicle and liver injury and liver fibrosis were evaluated via serum tests, q PCR, hydroxyproline content and Sirius red staining. RESULTS: Liver fibrosis was less pronounced in males than in female Abcb4-/- animals as defined by a lower hydroxyproline content(274 ± 64 μg/g vs 436 ± 80 μg/g liver, respectively; n = 13-15; P < 0.001; MannWhitney U-test) and lower m RNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1(TIMP)(1 ± 0.41 vs 0.66 ± 0.33 fold, respectively; n = 13-15; P < 0.05; Mann-Whitney U-test). Reduced liver fibrosis was associated with significantly lower levels of F4/80 m RNA expression(1 ± 0.28 vs 0.71 ± 0.41 fold, respectively; n = 12-15; P < 0.05; Mann-Whitney U-test) and significantly lower IL-1β m RNA expression levels(1 ± 0.38 vs 0.44 ± 0.26 fold

  7. Effect of quercetin on expression of matrix metallo-proteinases and tissue inhibitor of matalloproteinase-1 in cultured rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    康鲁平; 齐荔红; 张俊平; 周斌

    2003-01-01

    Objective: To study the effects of quercetin (QU) on matrix metallo-proteinases (MMPs), the tissue inhibitor of matalloproteinase-1 (TIMP-1), procollagen I and 2 proteoglycans (decorin and biglycan) mRNA expression in cultured rat hepatic stellate cell line HSC-T6 cells.Methods: Cells were treated with different concentrations of QU (12.5, 25, 50 μmol/L) or drug solvent (0.1 % Me2SO) for 24 h.mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR).Results: QU (12.5 - 50 μmol/L) enhanced collagenase (rat MMP-13) and membrane type1-MMP (MMP-14) mRNA expression, decreased procollagen I mRNA expression in a concentration-dependent manner, but did not affect gelatinase-A (MMP-2), TIMP-1, decorin and biglycan expression.Conclusion: QU may decrease matrix deposition and increase matrix degradation, which might be beneficial to liver fibrosis.

  8. Contact-dependent depletion of hydrogen peroxide by catalase is a novel mechanism of myeloid-derived suppressor cell induction operating in human hepatic stellate cells.

    Science.gov (United States)

    Resheq, Yazid J; Li, Ka-Kit; Ward, Stephen T; Wilhelm, Annika; Garg, Abhilok; Curbishley, Stuart M; Blahova, Miroslava; Zimmermann, Henning W; Jitschin, Regina; Mougiakakos, Dimitrios; Mackensen, Andreas; Weston, Chris J; Adams, David H

    2015-03-15

    Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14(+) monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14(+)HLADR(low/-) suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14(+) monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = -0.6555, p Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

  9. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica

    Institute of Scientific and Technical Information of China (English)

    Zheng Min; Wu Yi-jun; Cai Wei-min; Weng Hong-lei; Liu Rong-hua

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosomajaponicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes.Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library.Amplification of the library was carried out with transformation of DH5α. The amplified library contained more than 400 positive bacterial clones, which were then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. The subtracted cDNA library of differentially expressed genes of HSCs was constructed successfully,the library is efficient and lays foundation for screening and cloning new and specific genes of schistosomiasis.

  10. Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

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    Silvia Berardis

    Full Text Available Adult-derived human liver stem/progenitor cells (ADHLSC are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

  11. An experimental study on the effect of capsaicin on hepatic stellate cells and liver fibrogenesis%辣椒素调节肝星状细胞活性延缓肝纤维化的实验研究

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    俞富祥; 吴志伟; 朱千东; 符竣惠; 张启瑜

    2014-01-01

    tested.Results In dose dependent manner capsaicin inhibited the generation of the reactive oxygen species.Proliferation and activation of HSCs was inhibited by capsaicin (respectively F =13.267,57.392,all P < 0.05) and the apoptosis of HSCs was promoted by capsaicin (F =235.571,P < 0.05).Bax,cyt-c and caspase-3 was increased obviously (respectively F =29.334,38.274,138.329,all P < 0.05).Capsaicin changed the expression of fibrosis-related genes (TGF-β1,TIMP-1) in HSCs (respectively F =376.534,253.751,all P <0.05).Capsaicin downregulated the level of hydroxyproline,collagen Ⅲ and hyaluronic acid in the rat model (respectively F =153.397,27.149,38.392,all P < 0.05).Conclusions Capsaicin inhibits the proliferation and activation of hepatic stellate cells.Capsaicin promotes the apoptosis of hepatic stellate cells,and inhibits liver fibrogenesis.

  12. Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells

    Science.gov (United States)

    Shi, Hong-Bo; Lou, Jin-Li; Shi, Hong-Lin; Ren, Feng; Chen, Yu; Duan, Zhong-Ping

    2017-01-01

    AIM To prepare a Gpm6a/ReelinGFPCreERT2 construct with a rapid and reliable strategy using a bacterial artificial chromosome (BAC). METHODS Gpm6a and Reelin BACs were purified and transformed into SW102 E. coli by electroporation. The GFPCreERT2 fragment was prepared from a shuttle vector and transformed into SW102 E. coli carrying a BAC. Homologous recombination was induced in SW102 E. coli. Recombinant clones were screened and confirmed by PCR and restriction enzyme digestion. Recombinant clones were transformed into SW102 E. coli to remove the kanamycin unit. RESULTS A complete BAC was successfully transformed into SW102 E. coli by electroporation because BAC purified from SW102 E. coli showed the same pattern as the original BAC with BamHI digestion. The GFPCreERT2 fragment was deemed to have been prepared successfully because we obtained the same size fragment as expected. Homologous recombination was induced, and GFPCreERT2 was deemed to have been inserted into the correct site of the BAC because we found the band change was the same as the expected pattern after restriction enzyme digestion. The kanamycin unit was deemed to have been removed successfully because we obtained different sizes of bands that were consistent with the results expected by PCR with different primers. CONCLUSION The construct of Gpm6aGFPCreERT2 or ReelinGFPCreERT2 was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function. PMID:28127196

  13. Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib through the laminin-332/α3 integrin axis recovery of focal adhesion kinase ubiquitination.

    Science.gov (United States)

    Azzariti, Amalia; Mancarella, Serena; Porcelli, Letizia; Quatrale, Anna Elisa; Caligiuri, Alessandra; Lupo, Luigi; Dituri, Francesco; Giannelli, Gianluigi

    2016-12-01

    In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of α3β1 and α6β4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both α3β1 and α6β4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing α6β4 but lacking the α3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B α3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization.

  14. Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin Bound-Paclitaxel in Pancreatic Ductal Adenocarcinoma

    Science.gov (United States)

    Gonzalez-Villasana, Vianey; Rodriguez-Aguayo, Cristian; Arumugam, Thiruvengadam; Cruz-Monserrate, Zobeida; Fuentes-Mattei, Enrique; Deng, Defeng; Hwang, Rosa F.; Wang, Huamin; Ivan, Cristina; Garza, Raul Joshua; Cohen, Evan; Gao, Hui; Armaiz-Pena, Guillermo N.; Monroig-Bosque, Paloma del C.; Philip, Bincy; Rashed, Mohammed H.; Aslan, Burcu; Erdogan, Mumin Alper; Gutierrez-Puente, Yolanda; Ozpolat, Bulent; Reuben, James M.; Sood, Anil K.; Logsdon, Craig; Lopez-Berestein, Gabriel

    2014-01-01

    Pancreatic stellate cells (PSCs) have been recognized as the principal cells responsible for the production of fibrosis in PDAC. Recently PSCs have been noted to share characteristics with cells of monocyte-macrophage lineage (MML cells). Thus, we tested whether PSCs could be targeted with the nitrogen-containing bisphosphonates (NBPs) [pamidronate (Pam) or zoledronic acid (ZA)], which are potent MML cell inhibitors. In addition, we tested NBPs treatment combination with nanoparticle albumin-bound paclitaxel (nab-paclitaxel) to enhance antitumor activity. In vitro we observed that PSCs possess α-naphthyl butyrate esterase (ANBE) enzyme activity, a specific marker of MML cells. Moreover NBPs inhibited PSCs proliferation, activation, release of macrophage chemoattractant protein-1 (MCP-1) and type I collagen expression. NBPs also induced PSC apoptosis and cell cycle arrest in the G1 phase. In vivo, NBPs inactivated PSCs; reduced fibrosis; inhibited tumor volume, tumor weight, peritoneal dissemination, angiogenesis, and cell proliferation; and increased apoptosis in an orthotopic murine model of PDAC. These in vivo antitumor effects were enhanced when NBPs were combined with nab-paclitaxel but not gemcitabine (Gem). Our study suggests that targeting PSCs and tumor cells with NBPs in combination with nab-paclitaxel may be a novel therapeutic approach to PDAC. PMID:25193509

  15. Inhibition of pancreatic stellate cell activation by the vitamin A and vitamin E as a therapy for prevention fibrogenesis in experimental chronic alcoholic pancreatitis

    Directory of Open Access Journals (Sweden)

    Nichitaylo M. E.

    2012-01-01

    Full Text Available The aim of the study was to investigate the effects of Vitamin A and Vitamin E on activity of pancreatic stellate cells and fibrosis changes in pancreas after distal pancreatectomy in rats with experimental alcohol-induced chronic pancreatitis. Simultaneously Vitamin A and Vitamin E were administered after distal pancreatectomy in rats with experimental alcohol-induced chronic pancreatitis. The animals were treated withVitamin A at the dose of 33000 IU/kg body weight per day and Vitamin E at the dose of 100 mg/kg body weight per day for three weeks (21 days after operation. To estimate the efficacy of the treatment on activity and numbersof pancreatic stellate cells the immunohistochemicalinvestigation was made with alpha-smooth muscle actin, desmin, vimentin, glial fibrillary acidic protein (GFAP, matrix metalloproteinase 1 (MMP1, tissue inhibitor of metalloproteinase 2 (TIMP2 using. The treatment of rats after operation with vitamin A and vitamin E inhibited activity of pancreatic stellate cells and characterized by significant decreasing of the alpha-smooth muscle actin, Desmin, Vimentin, MMP1 and TIMP2 expression. The ratio of MMP1/TIMP2 was greater in the group with treatment then in the control group. This therapy had a trend to decrease the expression of GFAPand alleviate the fibrotic changes in pancreas.

  16. 血红铆钉菇子实体对人肝星状细胞LX-2和人肝癌细胞Hep G2增殖的影响%Effects of Chemical Fractions from Chroogomphus rutilus Fruiting Body on the Proliferation of Human Hepatic Stellate Cells LX-2 and Human Liver Carcinoma Hep G2 Cells

    Institute of Scientific and Technical Information of China (English)

    祝鹤鸣; 吴艳玲; 李雪; 姚大雷; 张昌浩; 崔炯谟; 李镐

    2013-01-01

    [Objective] To study the effects of chemical fractions from Chroogomphus rutilus fruiting body on the proliferation of human hepatic stellate cells LX-2 and human liver carcinoma Hep G2 cells,and to screen the active fractions with anti-hepatic fibrosis and anti-tumor activity from C.rutilus fruiting body.[Method] The polysaccharide,petroleum ether,dichloromethane,ethyl acetate,n-butanol and aqueous fractions from C.rutilus fruiting body were obtained by the methods of nature products chemistry.MTT assay was used to determine the effects of above fractions on the proliferation of human hepatic stellate cells LX-2 and human liver carcinoma Hep G2 cells.Morphological changes were observed through invert microscope.[Result] The petroleum ether fraction,dichloromethane fraction and ethyl acetate fraction showed inhibitory activities on human hepatic stellate cells LX-2 with IC50 values at 519.6,526,675.7 μg/ml,respectively,and human liver carcinoma Hep G2 cells with IC50 values at 727.6,610.1,618.1 μg/ml,respectively.Morphological change of apoptosis body formation was observed by invert microscope.[Conclusion] Among polysaccharide and solvent fractions from C.rutilus fruiting body,the petroleum ether fraction,dichloromethane fraction and ethyl acetate fraction have anti-hepatic fibrosis and anti-tumor activities as the main active parts.%[目的]探讨血红铆钉菇子实体对人肝癌细胞HepG2和人肝星状细胞LX-2增殖的影响,筛选出血红铆钉菇子实体抗肝纤维化和抗肿瘤作用的活性部位.[方法]利用天然药物化学方法提取得到血红铆钉菇子实体的石油醚、二氯甲烷、乙酸乙酯、正丁醇、水和粗多糖提取物,应用MTT法观察血红铆钉菇子实体不同溶剂提取物对体外培养的人肝星状细胞LX-2和人肝癌细胞Hep G2增殖的抑制作用,通过倒置显微镜观察细胞的形态学变化.[结果]血红铆钉菇子实体石油醚、二氯甲烷和乙酸乙酯提

  17. 脂筏在CB2受体介导的内源性大麻素AEA抑制大鼠肝星状细胞增殖活性中的作用%Lipid Rafts and Cannabinoid 2 Receptors-mediated Inhibitory Effects of Endogenous AEA on Proliferation of Hepatic Stellate Cells in Rats

    Institute of Scientific and Technical Information of China (English)

    吴文杰; 王密; 刘萍; 阳乔; 唐望先

    2012-01-01

    -treated hepatic stellate cells. Subcellular localization of lipid rafts and CB2 were examined by using laser confocal microscopy. The amount of CB2 in lipid-raft microdomains was detected after isolating lipid-rafts from hepatic stellate cells by Western blot. Results MTT assay showed either Cnr2-shRNA-trans-fected cells or MCD-treated cells had a noticeably higher ratio of cell proliferation than controls. No obvious difference in the ratio of cell proliferation was observed between Cnr2-shRNA-transfected cells and controls after MCD treatment. Meanwhile, the levels of P38 mitogen-activated protein kinases(p-P38MAPK)and c-Jun N-terminal kinases/stress-activated protein kinases(p-JNK)were significantly decreased after the hepatic stellate cells were treated with MCD. Laser confocal microscopy revealed the presence of lipid rafts and CB2 on the membranes of hepatic stellate cells. The lipid rafts isolated from hepatic stellate cells before AEA stimulation had little CB2. Conclusion Lipid rafts are required for the process of inhibition in the proliferation of HSC caused by AEA through CB2. This process was associated with the p-P38MAPK and p-JNK signaling pathway,suggesting that it is possible to be an effective treatment for liver fibrosis by controlling lipid rafts , CB2, and related signal.

  18. Autophagy in Hepatic Fibrosis

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    Yang Song

    2014-01-01

    Full Text Available Hepatic fibrosis is a leading cause of morbidity and mortality worldwide. Hepatic fibrosis is usually associated with chronic liver diseases caused by infection, drugs, metabolic disorders, or autoimmune imbalances. Effective clinical therapies are still lacking. Autophagy is a cellular process that degrades damaged organelles or protein aggregation, which participates in many pathological processes including liver diseases. Autophagy participates in hepatic fibrosis by activating hepatic stellate cells and may participate as well through influencing other fibrogenic cells. Besides that, autophagy can induce some liver diseases to develop while it may play a protective role in hepatocellular abnormal aggregates related liver diseases and reduces fibrosis. With a better understanding of the potential effects of autophagy on hepatic fibrosis, targeting autophagy might be a novel therapeutic strategy for hepatic fibrosis in the near future.

  19. 小RNA干扰DDR2基因表达对肝星状细胞的影响%Effects of siRNA targeting DDR2 on hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    张广林; 罗蒙; 孙勇伟; 徐庆; 陈炜

    2009-01-01

    Objective To explore the effects of inhibiting DDR2 expression by siRNA on hepatic stellate cells and evaluate the role of DDR2 gene in hepatic fibrogenesis. Methods (1) Three pairs of chemically synthesized siRNAs targeting DDR2 were respectively transfected into HSC-T6 cells for evaluation of silence efficacy, and the most effective siRNA was used. (2) HSC-T6 cells were divided into three groups, group A served as normal controls, group B served as negative control and group C was RNA interference DDR2 (siRNA-DDR2) expression of HSC. The most effective RNA interference sequences targeting DDR2 gene was chosen to transfect HSC-T6 cells by plasmid transfection. The tendency of DDR2, α-smooth muscle actin(α-SMA) and collagen-Ⅰ mRNA expression were estimated using RT-PCR, and the protein expression of DDR2 was evaluated by Western blot. Meanwhile, MTT assay was employed to analyze the proliferation of HSC. Results (1) DDR2 siRNA, which began at nt 868, inhibited DDR2 gone expression stronger than the other two siRNAs. (2) After transfection of siRNA-DDR2, the mRNA expression of DDR2 (P<0.01) and α-SMA (P<0.01) significantly decreased compared with the normal group, and the protein expression of DDR2 also significantly decreased (P<0.01). In addition, the proliferation of HSC was also markedly suppressed as compared with the normal group (P<0.01). However, compared with the negative control group, none of them was markedly suppressed. Conclusion SiRNA targeting DDR2 significantly suppresses the activation, proliferation of HSC, and thus attenuates hepatic fibrogonesis in vitro.%目的 探讨siRNA干扰大鼠盘状结构域受体2(discoidin domain receptor2,DDB2)基因对肝星状细胞(hepatic stellate cell,HSC)的影响,评估DDR2在肝纤维化发生中的作用.方法 (1)化学合成3对针对DDR2基因的siRNAs,转染肝星状细胞株(HSC-T6),筛选抑制效率最高的siRNA用于干扰实验;(2)将肝星状细胞株HSC-T6分为3组:正常

  20. PD98059 Inhibited the Activation of Pancreatic Stellate Cells Mediated by Platelet-Derived Growth Factor BB in Rats

    Institute of Scientific and Technical Information of China (English)

    WAN Yuantai; WANG Tiancai; ZHAO Qiu

    2005-01-01

    Summary: To determine the biological effects of extracelluar signal regulated kinase (ERK) specific inhibitor PD98059 on pancreatic stellate cells (PSCs) activated by platelet-derived factor-BB (PDGF-BB), cultured rat PSCs were co-incubated at 37 ℃ for 24 h with 25 ng/ml PDGF-BB and different doses of PD98059 (ranging from 5 ng/ml to 40 ng/ml). Expression of pERK1 protein was detected by Western blot and collagen α1 (Ⅰ) mRNA was tested by reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that there were statistical differences in the expression of pERK1 protein in all groups. Expression of pERK1 protein was up-regulated in the group treated by PDGF-BB, and gradually down-regulated in the other groups treated by PD98059 of different doses. An excellent positive correlation was revealed between the inhibitory effect and PD98059 concentrations. It was also observed that the expression of collagen α1 (Ⅰ) mRNA had similar response to pERK1. The level of collagen α1 (Ⅰ) mRNA was the highest in the PDGF-BB group, and gradually reduced in the other groups treated by PD98059 of different doses. It is concluded that expression of pERK1 protein and collagen α1 (Ⅰ) mRNA was up-regulated in rat PSCs activated by PDGF-BB. Meanwhile, PD98059 could inhibit PSCs activation mediated by PDGF. It is suggested that ERK1 protein plays an important role on PSCs activation mediated by PDGF signal pathway.

  1. Reelin expression in human liver of patients with chronic hepatitis C infection

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    Simone Carotti

    2017-03-01

    Full Text Available Reelin is a secreted extracellular glycoprotein that plays a critical role during brain development. Several studies have described Reelin expression in hepatic stellate cells of the human liver. In order to investigate the possible role of Reelin in the process of hepatic fibrogenesis, in this study we investigated Reelin expression in the liver tissue of patients infected with the Hepatitis C Virus (HCV. On this basis, Reelin expression was analysed by immunohistochemistry during liver biopsies of 81 patients with HCV-related chronic hepatitis. A Knodell score was used to stage liver fibrosis. Hepatic stellate cells/myofibroblast immunohistochemical markers (CRBP-1, alpha-SMA were also evaluated. As further confirmed by co-localization experiments (Reelin +CRBP-1, Reelin protein was expressed by hepatic stellate cells/myofibroblasts, and a significant positive correlation was found between Reelin expression and the stage of liver fibrosis (P=0.002. Moreover, Reelin correlated with CRBP-1 positive cells (P=0.002, but not with alpha-SMA, suggesting that Reelin should not be regarded as a marker of hepatic stellate cells/myofibroblasts differentiation but rather as a functional protein expressed during some phases of liver fibrosis. Furthermore, Disabled-1 (Dab1, a Reelin adaptor protein, was expressed in cells of ductular reaction suggesting a paracrine role for Reelin with regards these elements. In conclusion, Reelin was expressed by human hepatic stellate cells/myofibroblasts and the number of these cells increased significantly in the lobule as the liver fibrosis progressed, suggesting a role for Reelin in the activation of hepatic stellate cells/myofibroblasts during liver injury. Reelin may potentially be incorporated into liver injury evaluations in combination with other histological data.

  2. Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis.

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    Ursula Manuelpillai

    Full Text Available Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl(4 twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2 × 10(6 were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively. Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl(4 treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl(4 administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl(4 demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl(4 treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl(4 alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl(4 treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established

  3. 索拉非尼抑制人肝星状细胞胶原合成%Inhibitory effect of sorafenib on collagen synthesis in human hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    高俊茶; 王妍; 姜慧卿

    2012-01-01

    目的:研究索拉非尼(sorafenib)对人肝星状细胞胶原合成的影响.方法:应用人肝星状细胞株LX-2进行体外研究,采用[3H]-脯氨酸掺入法测定胶原的合成,采用免疫细胞化学法检测I型胶原蛋白表达,采用real-time PCR法测定I型胶原α1 mRNA表达.结果:免疫细胞化学研究显示血小板源性生长因子(PDGF)刺激可引起LX-2细胞胶原合成增加,10.0 μmol·L-1索拉非尼作用于LX-2细胞 24 h能明显抑制I型胶原蛋白的合成.无论有无PDGF的刺激,索拉非尼均呈剂量与时间依赖性地抑制LX-2细胞胶原合成(P<0.01);在10.0 μmol·L-1浓度下,索拉非尼作用于LX-2细胞 12 h、24 h和48 h对胶原合成的抑制率为22.69%、37.52%和71.74%.索拉非尼剂量依赖性地抑制PDGF诱导的I型胶原α1 mRNA表达上调;在2.5 μmol·L-1、5.0 μmol·L-1和10.0 μmol·L-1 索拉非尼作用下,I型胶原α1 mRNA表达较PDGF刺激组分别下调58.66%、67.06%和81.64%.结论:索拉非尼在体外能抑制人肝星状细胞胶原的合成,抑制I型胶原的表达,有可能成为一种新型的治疗肝纤维化药物.%AIM:To investigate the effects of sorafenib on collagen synthesis in human hepatic stellate cells ( HSCs ). METHODS : HSC cell line LX - 2 was used in vitro in this study. [3 H ] - proline incorporation assay was performed to measure the collagen synthesis. Immunocytochemistry was applied to detect type I collagen and real -time PCR was used to determine the mRNA expression of collagen al ( I). RESULTS: Stimulation with platelet - derived growth factor ( PDGF ) induced the increase in type I collagen synthesis, while treatment with sorafenib ( 10. 0μmol/L) for 24 h markedly decreased the collagen synthesis. Sorafenib resulted in dose - dependent and time - dependent decrease in collagen synthesis in LX -2 cells in the absence or presence of PDGF by [3 H ] - proline incorporation assay. The inhibition rates were 22. 69% , 37. 52% and 71.74% , respectively

  4. L-cysteine administration attenuates pancreatic fibrosis induced by TNBS in rats by inhibiting the activation of pancreatic stellate cell.

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    LiJuan Yang

    Full Text Available BACKGROUND AND AIMS: Recent studies have shown that activated pancreatic stellate cells (PSCs play a major role in pancreatic fibrogenesis. We aimed to study the effect of L-cysteine administration on fibrosis in chronic pancreatitis (CP induced by trinitrobenzene sulfonic acid (TNBS in rats and on the function of cultured PSCs. METHODS: CP was induced by TNBS infusion into rat pancreatic ducts. L-cysteine was administrated for the duration of the experiment. Histological analysis and the contents of hydroxyproline were used to evaluate pancreatic damage and fibrosis. Immunohistochemical analysis of α-SMA in the pancreas was performed to detect the activation of PSCs in vivo. The collagen deposition related proteins and cytokines were determined by western blot analysis. DNA synthesis of cultured PSCs was evaluated by BrdU incorporation. We also evaluated the effect of L-cysteine on the cell cycle and cell activation by flow cytometry and immunocytochemistry. The expression of PDGFRβ, TGFβRII, collagen 1α1 and α-SMA of PSCs treated with different concentrations of L-cysteine was determined by western blot. Parameters of oxidant stress were evaluated in vitro and in vivo. Nrf2, NQO1, HO-1, IL-1β expression were evaluated in pancreas tissues by qRT-PCR. RESULTS: The inhibition of pancreatic fibrosis by L-cysteine was confirmed by histological observation and hydroxyproline assay. α-SMA, TIMP1, IL-1β and TGF-β1 production decreased compared with the untreated group along with an increase in MMP2 production. L-cysteine suppressed the proliferation and extracellular matrix production of PSCs through down-regulating of PDGFRβ and TGFβRII. Concentrations of MDA+4-HNE were decreased by L-cysteine administration along with an increase in GSH levels both in tissues and cells. In addition, L-cysteine increased the mRNA expression of Nrf2, NQO1 and HO-1 and reduced the expression of IL-1β in L-cysteine treated group when compared with control

  5. Stellate Ganglion Block Reduces the Radicular Pain and Salivary Alpha-Amylase Activity in Patients with Cervical Spondylosis

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    Takashi Egashira

    2015-03-01

    Full Text Available Background The effects of stellate ganglion block (SGB on radicular pain associated with cervical spondylosis remain to be clarified. So we measured salivary alpha-amylase which reflects sympathetic nerve activity under psychological stress after SGB block or trigger points injection (TPI. Study Design A randomized, prospective, controlled trial Setting After institutional approval and informed consent, 40 patients who was suffered from neck-shoulder pain associated with cervical radiculopathy were randomly divided into two groups according to nerve block treatment. Group A (n=20, male 10 patients, female 10 patients, 50±8yr, mean±SD received SGB and group B (n=20, male 10 patients, female 10 patients, 52±6yr received TPI. SGB or TPI was produced by 6 ml of 1% mepivacaine a total of 5 times (twice per week. Visual analogue scale (VAS and the concentration of salivary alpha-amylase were measured before (T0 each nerve block and 3 days (T1, 6 days (T2, 9 days (T3, 12 days (T4 and 15days (T5 after each nerve block. The consumption of non-steroidal anti-inflammatory drug (NSAID was measured at T0 and T5 in each group. Results In group A, VAS was median 74 (range 60, 78 at T0 and showed a significant decrease at T3 [53 (48, 65, p<0.05], T4 [50 (42, 66, p<0.05] and T5 [48 (26,57, p<0.05]. The concentration of salivary alpha-amylase was median 116 (range 96, 144 KU/ml at T0 and showed a significant decrease at T3 [86 (79, 105, p<0.05], T4 [79 (68, 88] and T5 [70 (55, 84, p<0.05]. In group B, VAS and the concentration of salivary alpha-amylase showed no change throughout the time course. VAS in group A was significant lower than that in group B at T3, T4 and T5. The concentration of salivary alpha-amylase was significant lower than that in group B at T4 and T5. The consumption of NSAID in group A was significantly lower than that in group B at T5. Limitations Subjects are out patients. Patients include radicular pain due to different pathogenesis, e

  6. 天然生物碱川芎嗪通过阻断PDGF-β受体介导的ERK和/或p38信号通路抑制高糖高胰岛素和过氧化氢诱导的肝星状细胞的活化%Natural alkaloid tetramethylpyrazine inhibits glucose and insulin-and hydrogen peroxide-induced activation of hepatic stellate cell by disrupting PDGF-βR-mediated ERK and/or p38 pathways

    Institute of Scientific and Technical Information of China (English)

    张峰; 张自力; 张雪娇; 倪春艳; 陆茵; 郑仕中

    2013-01-01

    目的:分别以高糖高胰岛素(Glu/Ins)和过氧化氢(H2O2)处理肝星状细胞(HSC)模拟代谢综合征状态下高血糖高胰岛素血症和氧化应激,研究天然生物碱川芎嗪(TMP)体外对Glu/Ins和H2O2所诱导的HSC活化的影响及机制.方法:应用MTS实验检测细胞活力,免疫荧光染色检测α-SMA的表达,Real-time PCR和Western Blot分别检测关键因子在mRNA与蛋白水平的表达.结果:Glu/Ins和H2O2体外均可显著促进HSC的增殖与活化.在两种细胞模型中,TMP均能抑制HSC增殖并且在mRNA和蛋白水平减少α-SMA、α1(Ⅰ)procollagen和fibronectin的表达,并上调MMP-2,下调TIMP-1.此外,TMP抑制Glu/Ins和H2O2诱导的血小板源性生长因子-β受体(PDGF-βR)磷酸化,并在Glu/Ins激活的HSC中抑制细胞外信号调节激酶(ERK)磷酸化,在H2O2激活的HSC中抑制ERK和p38的磷酸化.运用受体酪氨酸激酶抑制剂依玛替尼发现,TMP抑制这两种HSC活化模型中α-SMA的表达均与对PDGF-βR的阻断作用相关.结论:TMP可以有效抑制Glu/Ins和H2O2诱导的HSC活化.这一作用可能与抑制PDGF-β R介导的ERK和/或p38信号通路相关.TMP可作为防治代谢综合征相关肝纤维化的潜在药物进一步开发.%Objective:To investigate the in vitro effects of tetramethylpyrazine (TMP) on hepatic stellate cell (HSC)activation under the conditions of high levels of glucose (glu) and insulin (Ins),and hydrogen peroxide (H2O2)-driven oxidative stress.Methods:HSCs were divided into control cells,model cells treated with Glu/Ins or H2O2,and TMP-treated cells.3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay was used to examine cell viability.Immunofluorescence staining was used to determine the expression of α-SMA.Real-time polymerase chain reaction (PCR)and Western blot analysis were applied to detect the expression of genes at mRNA and protein levels,respectively.Results:Glu/Ins or H2O2 significantly

  7. Hepatic stellate cell through SDF-1/CXCR4 axis induces epithelial-mesenchymal transition in hepatocellular carcinoma invasion%肝星状细胞通过SDF-1/CXCR4轴诱导肝癌细胞上皮间质转分化并促进其侵袭

    Institute of Scientific and Technical Information of China (English)

    李四光; 常远鸿; 刘凯歌

    2013-01-01

    Objective The aim of this study is to explore the impact of hepatic stellate cell in hepatocellular carcinoma invasion through SDF-1/CXCR4 axis.Methods The expression of SDF-1 and CXCR4 were examined in hepatic stellate cell LX02,four hepatocellular carcinoma cell lines by Western blot at protein levels and real-time RTPCR at mRNA level respectively.In addition,Transwell invasion assay was carried out to analyze the influence of hepatic stellate cell LX02 and SDF-1 on invasion of hepatocellular carcinoma cell HepG2 under normal condition or CXCR4 gene silence condition.Western blot was performed to evaluate the expression of epithelial marker E-cadherin and mesenchymal marker vimentin.Results The expression of SDF-1 was high in hepatic stellate cell LX02,and increased levels of expression of CXCR4 were found in all hepatocellular carcinoma cells.Co-culture with hepatic stellate cell LX02 or treatment with SDF-l both induced the epithelial-mesenchymal transition and increased the invasion of hepatocellular carcinoma cell HepG2.Furthermore,inhibition of CXCR4 by gene silence in HepG2 suppressed the enhanced invasion and epithelial-mesenchymal transition of HepG2 cells which induced by stellate cells or SDF-1.Conclusions Hepatic stellate cells promote hepatocellular carcinoma cell invasion through chemokine SDF-1/CXCR4 axis,the mechanism may involve the epithehal-mesenchymal transition of carcinoma cell.%目的 探讨肝星状细胞是否通过SDF-1/CXCR4轴促进肝癌细胞侵袭的作用和可能机制.方法 通过Westernblot和real time RT-PCR,检测肝星状细胞LX02和肝癌细胞系SDF-1、CXCR4表达.Transwell实验检测星状细胞LX02或外源性SDF-1干预对肝癌细胞HepG2以及CXCR4基因沉默后的HepG2侵袭的影响,Westem blot检测上皮标志E-cadherin和间质标志vimentin的表达变化.结果 肝星状细胞LX02中趋化因子SDF-1高表达,4株人肝细胞癌细胞系均有CXCR4高表达,其中HepG2细胞表达最强.星状细胞或SDF-1

  8. Transdifferentiation-dependent expression of alpha-SMA in hepatic stellate cells does not involve TGF-beta pathways leading to coinduction of collagen type I and thrombospondin-2.

    Science.gov (United States)

    Lindert, Susanne; Wickert, Lucia; Sawitza, Iris; Wiercinska, Eliza; Gressner, Axel M; Dooley, Steven; Breitkopf, Katja

    2005-05-01

    Hepatic stellate cells (HSC) cultured on plastic spontaneously transdifferentiate to a myofibroblast-like cell type (MFB). This model system of hepatic fibrogenesis is characterized by phenotypic changes of the cells and increased matrix synthesis. Here, we analyzed if transdifferentiation-dependent induction of ECM components, e.g., collagen type I and thrombospondin-2 (TSP-2), and phenotypic changes are coregulated events and if both processes are mediated via TGF-beta pathway(s). Blocking the TGF-beta-dependent p38 MAPK pathway in HSC with the specific inhibitor SB203580 strongly reduces collagen I and TSP-2 mRNA expression without inhibiting upregulation of the typical MFB-marker, alpha-smooth-muscle actin (alpha-SMA). Similarly, interference with the Smad2/3/4 pathway using dexamethasone also heavily decreased expression of collagen type I and TSP-2 whereas transdifferentiation of HSC to the typical morphology of MFB with loss of fat droplets and increasing alpha-SMA was unchanged. Further, p38 MAPK mediated induction of collagen I and TSP-2 expression by TGF-beta1 was still achieved in the presence of dexamethasone, showing that dexamethasone does not block p38 while it delays Smad2 phosphorylation and antagonizes stimulation of a Smad3/Smad4 dependent TGF-beta reporter construct. Interestingly, in contrast to SB203580 and dexamethasone, overexpression of the TGF-beta antagonist Smad7 reduced ECM expression and simultaneously inhibited morphologic transdifferentiation, indicating that Smad7 fulfills additional features in HSC. In conclusion, our data show that phenotypic changes of transdifferentiating HSC and induction of matrix synthesis are independent processes, the latter being stimulated by both, Smad dependent and MAPK dependent TGF-beta signaling.

  9. Recruitment and activation of pancreatic stellate cells from the bone marrow in pancreatic cancer: a model of tumor-host interaction.

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    Christopher J Scarlett

    Full Text Available BACKGROUND AND AIMS: Chronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas. METHODS: Whole bone marrow was harvested from male β-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA. Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation. RESULTS: GFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3. CONCLUSIONS: This study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that

  10. An interesting case of Lucio phenomenon triggered by activation of hepatitis C infection

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    Jacob Mareen

    2016-01-01

    Full Text Available Lucio phenomenon (LP or erythema necroticans is a rare type of reaction pattern found in untreated patients with diffuse non-nodular leprosy. It is important to distinguish this from vasculonecrotic erythema nodosum because thalidomide with high-dose steroids is the mainstay of treatment for the latter, whereas LP shows no response to thalidomide. We report a case of a 60-year-old man who presented with purpuric patches, hemorrhagic blisters, and ulcers over extremities of 15 days duration. On cutaneous examination, there were multiple stellate purpuric patches, hemorrhagic bullae, and deep necrotic ulcers, mainly over extremities. Slit-skin smear examination from six sites revealed bacteriological index 6+ with globi, and morphological index 5%. Histopathology revealed diffuse infiltration of bacilli in epidermis, dermis, and endothelial cells along with neutrophilic and lymphocytic infiltrate. Fibrinoid necrosis and thrombosis of blood vessels was also noted. The above clinicohistopathological features helped in making the diagnosis of LP. Concomitantly he was found to be infected with hepatitis C virus. Many triggering factors have been described in literature; however, activation of hepatitis C as a trigger for Lucio phenomenon has not been reported. In addition, IgM and IgG anticardiolipin antibodies were found to be positive. The patient was started on high-dose steroids along with multibacillary antileprosy therapy and improved within 2 weeks.

  11. Arming drug carriers to disable the Hepatic Stellate Cell : the targeted delivery of apoptosis-inducing drugs to the fibrotic liver

    NARCIS (Netherlands)

    Hagens, Werner Ivo

    2006-01-01

    Chronic liver damage of various origins (e.g. viral hepatitis; chronic intoxication by alcohol, chemicals or drugs; Wilson’s disease) can eventually lead to liver cirrhosis, the end stage of liver fibrosis. This process is characterized by the accumulation of excessive amounts of scar tissue within

  12. The relationship between the morphous changes of hepatic stellate cells and liver microcirculatory disturbance in patients with chronic hepatitis B%慢性乙型肝炎肝星状细胞形态改变与肝脏微循环障碍的关系

    Institute of Scientific and Technical Information of China (English)

    汪念; 丁体龙; 马勇; 沈烈; 张文学; 于莉

    2012-01-01

    Objective To study the relationship between the morphous changes of hepatic stellate cells (HSCs) and liver microcirculatory disturbance in patients with chronic hepatitis B. Methods The number of lipid droplets and the changes of bulk density in hepatic stellate cells were observed by light microscope. Ultrastructure of HSCs and the changes of microcir-culation of sinus hepaticus were observed by transmission electron microscope (TEM). Results In patients with chronic hepatitis fl, the number of lipid droplets in hepatic stellate cells and typical HSCs reduced, while transitional HSCs increased. The surface of nuclear envelop showed anomalism; Rough endoplasmic reticulum intracytoplasm increased obviously, most of which expanded, and middling electron density floccule could be observed. Golgi complex became prosperous. Collagenous fibril a-round the HSCs turned more notably. Decreased sizes and reduced numbers of sinusoidal endothelial cells'(SECs) penestrate and collagen deposited in Disse space. Basal lamina could be found on SECs and WP ( Weibel-Palade) bodies were found in SECs. Conclusion The morphous changes of HSCs after being stimulated is an important promoting agent to liver microcircu-lation disturbance in patients with chronic hepatitis B.%目的 研究慢性乙型肝炎肝星状细胞形态改变与肝脏微循环障碍的关系.方法 采用光镜观察肝星状细胞内脂滴数和体密度的变化,同时采用透射电镜观察肝星状细胞超微结构的变化和肝窦微循环结构的改变.结果 慢性乙型肝炎肝星状内脂滴数减少,典型肝星状细胞数量减少,过渡型肝星状细胞数量增多,超微结构显示核被膜表面不规则,胞质内粗面内质网明显增多,多扩张,内有中等电子密度的絮状物质,高尔基复合体发达,细胞周围胶原原纤维量明显增多.肝窦内皮细胞窗孔减少变小,有的肝窦内皮细胞内出现WP( Weibel-Palade body)小体,狄氏腔中胶原纤维沉积增多,

  13. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of); Lim, Chaeseung [Department of Laboratory Medicine, Korea University Guro Hospital, Seoul 152-703 (Korea, Republic of); Kim, Jungho [Department of Life Science, Sogang University, Seoul 121-742 (Korea, Republic of); Cha, Dae Ryong [Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Gyeonggi do 425-020 (Korea, Republic of); Oh, Junseo, E-mail: ohjs@korea.ac.kr [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  14. Mechanisms of action of acetaldehyde in the up-regulation of the human α2(I) collagen gene in hepatic stellate cells: key roles of Ski, SMAD3, SMAD4, and SMAD7.

    Science.gov (United States)

    Reyes-Gordillo, Karina; Shah, Ruchi; Arellanes-Robledo, Jaime; Hernández-Nazara, Zamira; Rincón-Sánchez, Ana Rosa; Inagaki, Yutaka; Rojkind, Marcos; Lakshman, M Raj

    2014-05-01

    Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4-containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties.

  15. Effects of Ca2+/calmodulin-dependent protein kinase Ⅱ on PDGF-induced collagen α1(Ⅰ) production in human hepatic stellate cells%Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ在PDGF诱导肝星状细胞胶原合成中的作用

    Institute of Scientific and Technical Information of China (English)

    安萍; 刘亚玲; 全晓静; 刘璐; 罗和生

    2013-01-01

    目的 观察Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)对PDGF诱导下人肝星状细胞(hepatic stellate cell,HSC) collagen α1(Ⅰ)合成的影响.方法 CaMKⅡα siRNA转染对HSC内CaMKⅡα进行干扰,real-time PCR法检测HSC collagen α1(Ⅰ)及TIMP-1 mRNA的表达,Western blot法检测collagen α1(Ⅰ)及MMP-2、TIMP-1表达的变化,ELISA法检测HSC collagen α1(Ⅰ)分泌的变化.结果 CaMKⅡα siRNA可显著抑制PDGF诱导下HSC collagen α1(Ⅰ) 及MMP-2、TIMP-1的转录、蛋白表达以及collagen α1(Ⅰ)的分泌,差异均有统计学意义(P<0.05).结论 CaMKⅡα信号参与了PDGF诱导下HSC collagen α1(Ⅰ)的产生与分泌,同时通过抑制MMP-2、促进TIMP-1的表达而阻止胶原的降解,是肝纤维化发展过程中PDGF信号的重要调控分子和肝纤维化的潜在治疗靶点.%Objective To observe the effects of Ca2+/calmodulin-dependent protein kinase Ⅱ ( CaMK Ⅱ ) on PDGF-induced collagen α1 (Ⅰ) production in human hepatic stellate cells. Methods The knockdown of CaMK Ⅱ a was performed by CaMK Ⅱ α siRNA transient transfection. The mRNA and protein expression of collagen α1 (Ⅰ) , MMP-2 and TIMP-1 were determined by real-time PCR and Western blot, respectively. The secretion of collagen α1 (Ⅰ) in culture media was tested by ELISA. Results CaMK Ⅱ knockdown by CaMK Ⅱ a siRNA significantly inhibited collagen α1 (Ⅰ) expression and secretion in PDGF-induced HSCs. CaMK Ⅱ inhibition resulted in up-regulation of MMP-2 and down-regulation of TIMP-1. Conclusion CaMK Ⅱ α regulate PDGF-induced collagen α1 (Ⅰ) production and secretion, increase TIMP-1 expression and decrease MMP-2 expression. Our study shed light on CaMK Ⅱ as a crucial signal in PDGF-activated HSCs and a potential therapeutic target in hepatic fibrosis.

  16. Purinergic receptor X7 mediates leptin induced GLUT4 function in stellate cells in nonalcoholic steatohepatitis

    Science.gov (United States)

    Chandrashekaran, Varun; Das, Suvarthi; Seth, Ratanesh Kumar; Dattaroy, Diptadip; Alhasson, Firas; Michelotti, Gregory; Nagarkatti, Mitzi; Nagarkatti, Prakash; Diehl, Anna Mae; Chatterjee, Saurabh

    2015-01-01

    Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO mice showed marked decrease in Glut4 levels primarily in stellate cells. Mechanistically stellate cells showed increase in phosphorylated AKT, Glut4 protein and localization in the membrane following administration of P2X7r agonist or leptin+P2X7r agonist, while use of P2X7r antagonist or AKT inhibitor attenuated the response suggesting that leptin-P2X7r axis in concert but not leptin alone is responsible for the Glut4 induction and translocation. Finally P2X7r-agonist and leptin caused increase in intracellular glucose and consumption by increasing the activity of hexokinase. In conclusion, the study shows a novel role of leptin-induced P2X7r in modulating Glut4 induction and translocation in hepatic stellate cells, that are key to NASH progression. PMID:26474534

  17. Purinergic receptor X7 mediates leptin induced GLUT4 function in stellate cells in nonalcoholic steatohepatitis.

    Science.gov (United States)

    Chandrashekaran, Varun; Das, Suvarthi; Seth, Ratanesh Kumar; Dattaroy, Diptadip; Alhasson, Firas; Michelotti, Gregory; Nagarkatti, Mitzi; Nagarkatti, Prakash; Diehl, Anna Mae; Chatterjee, Saurabh

    2016-01-01

    Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO mice showed marked decrease in Glut4 levels primarily in stellate cells. Mechanistically stellate cells showed increase in phosphorylated AKT, Glut4 protein and localization in the membrane following administration of P2X7r agonist or leptin+P2X7r agonist, while use of P2X7r antagonist or AKT inhibitor attenuated the response suggesting that leptin-P2X7r axis in concert but not leptin alone is responsible for the Glut4 induction and translocation. Finally P2X7r-agonist and leptin caused an increase in intracellular glucose and consumption by increasing the activity of hexokinase. In conclusion, the study shows a novel role of leptin-induced P2X7r in modulating Glut4 induction and translocation in hepatic stellate cells, that are key to NASH progression.

  18. Influence of Ruangan Huajian granule on transforming signaling in hepatic stellate cells%软肝化坚颗粒药物血清对肝星状细胞信号传导通路的调节作用

    Institute of Scientific and Technical Information of China (English)

    杨莉; 刘莲; 赵连英; 叶立红; 侯军良; 戴二黑

    2013-01-01

    Objective:To study the influence of Ruangan Huajian granule on transforming signaling in the activation of hepatic stellate cells.Methods:Mouse medicated serum was prepared with Ruangan Huajian granule by gastrogavage.HSC-6 were divided into five groups:control group incubated with normal mouse serum (10%) ; Ruangan Huajian granule group incubated with Ruangan Huajian granule mouse medicated serum (5%,10%,20%) ; colchicine group incubated with colchicine mouse medicated serum (10%).Cells of each group were cultured for 24 hours,and cells were collected.The mRNA levels of CB1,FAK and ERK were determined by quantitative RT-PCR.Results:The levels of CB1 mRNA in colchicine group and Ruangan Huajian granule group (5%,10%,20%) were notably lower than those in control group,P value was 0.000,0.000,0.000 and 0.0000,respectively.The levels of CB1 mRNA in Ruangan Huajian granule group (10%,20%) were notably lower than those in colchicine group,P value was 0.000 and 0.000,respectively.The levels of CB1 mRNA in Ruangan Huajian granule group (10%,20%) were notably lower than those in Ruangan Huajian granule group (5%),P value was 0.000 and 0.000,respectively.The levels of CB1 mRNA in Ruangan Huajian granule group (20%) were notably lower than those in Ruangan Huajian granule group (10%),P value was 0.000.The levels of ERK mRNA in colchicine group and Ruangan Huajian granule group (5%,10%,20%) were notably lower than those in control group,P value was 0.000,0.002,0.000 and 0.000,respectively.The levels of ERK mRNA in Ruangan Huajian granule group (20%) were notably lower than those in Ruangan Huajian Particle group (5%),P value was 0.014.The levels of FAK mRNA in colchicine group and Ruangan Huajian granule group (5%,10%,20%) were notably lower than those in control group,P value was 0.001,0.000,0.000 and 0.000,respectively.Conclusion:Ruangan Huajian granule can inhibit the activation of HSC-T6 by ERK signal transduction and P13K

  19. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    2008312 Impact of hepatitis B virus infection on the activity of hematopoietic stem cell.SHI Yanmei(石雁梅),et al.Dept Infect Dis,1st Clin Coll,Harbin Med Univ,Harbin 150001.Chin J Infect Dis 2008;26(4):197-201.Objective To study the impact of hepatitis B virus (HBV)infection on the activity of cord hematopoieticstem cells.Methods CD34+cells were isolated from healthy human cord blood by mini MACS.Cells were

  20. Successful treatment of activated occult hepatitis B in a non-responder chronic hepatitis C patient

    OpenAIRE

    Emara Mohamed H; Radwan Mohamed I

    2011-01-01

    Abstract We reported a 23 years old male with chronic hepatitis C virus infection, discontinued from pegylated interferon/ribavirin combination therapy due to a lack of early virological response. He has developed activation of occult hepatitis B virus that was successfully treated by a one year of lamivudine therapy.

  1. Alterations of mast cells and TGF-β1 on the silymarin treatment for CCl4-induced hepatic fibrosis

    Institute of Scientific and Technical Information of China (English)

    Da-Hee Jeong; Gi-Ppeum Lee; Won-Il Jeong; Sun-Hee Do; Hai-Jie Yang; Dong-Wei Yuan; Ho-Yong Park; Kyu-Jong Kim; Kyu-Shik Jeong

    2005-01-01

    AIM: Silymarin is a potent antioxidant, antiinflammatory and anti-fibrogenic agent in the liver, which is mediated by alteration of hepatic Kupffer cell function, lipid peroxidation, and collagen production. Especially, in hepatic fibrogenesis, mast cells are expressed in chronic inflammatory conditions, and promote fibroblast growth and stimulate production of the extracellular matrix by hepatic stellate cells.METHODS: We examined the inhibitory mechanism of silymarin on CCl4-induced hepatic cirrhosis in rats. At 4, 8,and 12 wk, liver tissues were examined histopathologically for fibrotic changes produced by silymarin treatment.RESULTS: In the silymarin with CCl4-treated group,increase of hepatic stellate cells and TGF-β1 production were lower than in the CCl4-treated group at early stages.Additionally, at the late fibrogenic stage, expressions of TGF-β1 were weaker and especially not expressed in hepatocytes located in peripheral areas. Moreover, the number of mast cell in portal areas gradually increased and was dependent on the fibrogenic stage, but those of CCl4+silymarin-treated group decreased significantly.CONCLUSION: Anti-fibrotic and antiinflammatory effects of silymarin were associated with activation of hepatic stellate cells through the expression of TGF-β1 and stabilization of mast cells. These results suggest that silymarin prevent hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis.

  2. Functional and structural specific roles of activity-driven BDNF within circuits formed by single spiny stellate neurons of the barrel cortex

    Directory of Open Access Journals (Sweden)

    Qian-Quan eSun

    2014-11-01

    Full Text Available Brain derived neurotrophic factor (BDNF plays key roles in several neurodevelopmental disorders and actions of pharmacological treatments. However it is uncealr how specific BDNF’s effects are on diffeerent circuit components. Current studies have largely focused on the role of BDNF in modification of synaptic development. The precise roles of BDNF in the refinement of a functional circuit in vivo remain unclear. Val66Met polymorphism of BDNF may be associated with increased risk for cognitive impairments and is mediated at least in part by activity-dependent trafficking and/or secretion of BDNF. Using mutant mice that lacked activity-driven BDNF expression (bdnf-KIV, we previously reported that experience regulation of the cortical GABAergic network is mediated by activity-driven BDNF expression. Here, we demonstrate that activity-driven BDNF’s effects on circuits formed by the layer IV spiny stellate cells are highly specific. Structurally, dendritic but not axonal morphology was altered in the mutant. Physiologically, GABAergic but not glutamatergic synapses were severely affected. The effects on GABA transmission occurs via presynaptic alteration of calcium-dependent release probability. These results suggest that neuronal activity through activity-driven BDNF expression, can selectively regulate specific features of layer IV circuits in vivo. We postulate that the role of activity-dependent BDNF is to modulate the computational ability of circuits that relate to the gain control (i.e. feed-forward inhibition; whereas the basic wiring of circuits relevant to the sensory pathway is spared. Gain control modulation within cortical circuits has broad impact on cognitive processing and brain state-transitions. Cognitive behavior and mode is determined by brain states, thus the studying of circuit alteration by endogenous BDNF provides insights into the cellular and molecular mechanisms of diseases mediated by BDNF.

  3. MiR-21 simultaneously regulates ERK1 signaling in HSC activation and hepatocyte EMT in hepatic fibrosis.

    Directory of Open Access Journals (Sweden)

    Juan Zhao

    Full Text Available BACKGROUND: MicroRNA-21 (miR-21 plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on SPRY2 and HNF4α in modulating ERK1 signaling in hepatic stellate cells (HSCs and epithelial-mesenchymal transition (EMT of hepatocytes. METHODS: Quantitative RT-PCR was used to determine miR-21 and the expression of SPRY2, HNF4α and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes SPRY2 and HNF4α. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and SPRY2 or HNF4α. RESULTS: The serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. SPRY2 and HNF4α mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed SPRY2 and HNF4α expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting SPRY2 or HNF4α. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFβ1-treated hepatocytes. CONCLUSIONS: MiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating SPRY2 and HNF4α expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.

  4. Phenotypic changes in mouse pancreatic stellate cell Ca2+ signaling events following activation in culture and in a disease model of pancreatitis.

    Science.gov (United States)

    Won, Jong Hak; Zhang, Yu; Ji, Baoan; Logsdon, Craig D; Yule, David I

    2011-02-01

    The specific characteristics of intracellular Ca 2+ signaling and the downstream consequences of these events were investigated in mouse pancreatic stellate cells (PSC) in culture and in situ using multiphoton microscopy in pancreatic lobules. PSC undergo a phenotypic transformation from a quiescent state to a myofibroblast-like phenotype in culture. This is believed to parallel the induction of an activated state observed in pancreatic disease such as chronic pancreatitis and pancreatic cancer. By day 7 in culture, the complement of cell surface receptors coupled to intracellular Ca 2+ signaling was shown to be markedly altered. Specifically, protease-activated receptors (PAR) 1 and 2, responsive to thrombin and trypsin, respectively, and platelet-derived growth factor (PDGF) receptors were expressed only in activated PSC (aPSC). PAR-1, ATP, and PDGF receptor activation resulted in prominent nuclear Ca 2+ signals. Nuclear Ca 2+ signals and aPSC proliferation were abolished by expression of parvalbumin targeted to the nucleus. In pancreatic lobules, PSC responded to agonists consistent with the presence of only quiescent PSC. aPSC were observed following induction of experimental pancreatitis. In contrast, in a mouse model of pancreatic disease harboring elevated K-Ras activity in acinar cells, aPSC were present under control conditions and their number greatly increased following induction of pancreatitis. These data are consistent with nuclear Ca 2+ signaling generated by agents such as trypsin and thrombin, likely present in the pancreas in disease states, resulting in proliferation of "primed" aPSC to contribute to the severity of pancreatic disease.

  5. Mathematical modelling of interferon-gamma signalling in pancreatic stellate cells reflects and predicts the dynamics of STAT1 pathway activity.

    Science.gov (United States)

    Rateitschak, Katja; Karger, Anna; Fitzner, Brit; Lange, Falko; Wolkenhauer, Olaf; Jaster, Robert

    2010-01-01

    Signal transducer and activator of transcription (STAT) 1 is essentially involved in the mediation of antifibrotic interferon-gamma (IFN gamma) effects in pancreatic stellate cells (PSC). Here, we have further analysed the activation of the STAT1 pathway in a PSC line by combining quantitative data generation with mathematical modelling. At saturating concentrations of IFN gamma, a triphasic pattern of STAT1 activation was observed. An initial, rapid induction of phospho-STAT1 was followed by a plateau phase and another, long-lasting phase of further increase. The late increase occurred despite enhanced expression of the feedback inhibitor (SOCS1), and corresponded to increased levels of total STAT1 protein. If IFN gamma was applied at non-saturating concentrations, phospho-STAT1 and SOCS1 levels peaked and declined again over a 12 hour period, while STAT1 protein levels remained high. The mathematical model, based on a system of ordinary differential equations, describes temporal changes of the network components as a function of interactions and transport processes. The model reproduced activation profiles of all components of the STAT1 pathway that were experimentally analysed. Furthermore, it successfully predicted the dynamics of network components in additional experimental studies. Based on experimental findings and the results obtained from modelling, we suggest exhaustion of applied IFN gamma and STAT1 dephosphorylation by tyrosine phosphatases as limiting factors of STAT1 activation in PSC. In contrast, we did not obtain compelling evidence that SOCS1 acts as an efficient feedback inhibitor in our experimental system. We believe that further investigations into mathematical modelling of the STAT1 pathway will improve the understanding of the antifibrotic interferon action.

  6. Effect of caffeine on signaling transduction of TGF-β1 and CTGF in hepatic stellate cell-T6 stimulated by acetaldehyde%咖啡因对乙醛诱导的HSC-T6中TGF-β1,CTGF信号转导通路的影响

    Institute of Scientific and Technical Information of China (English)

    管文婕; 吕雄文; 杨万枝; 李俊

    2012-01-01

    Objective To explore the effect of caffeine on proliferation in hepatic stellate cell stimulated by acetaldehyde and its signaling pathway of TGF-β and CTGF. Methods Trials are divided into normal group( regular training ), model group, adenosine receptor group,which were given caffeine( 4 mmol · L-1 ),adenosine A2A receptor antagonist ZM241385( 1 μmol · L-1 ),adenosine A2A receptor agonists CGS21680( 1 μmol · L-1 ),caffeine + CGS21680,ZM241385 + CGS21680 and HSC-T6 respectively,stimulated by acetaldehyde after 1 h,before continueing to cultivate 48 h. The protein expression of a-SMA was analyzed by immunocytochemistry methods. The mR-NA expressions of TGF-β1 and CTGF were measured by RT-PCR. The protein expression of CTGF was analyzed by western blot methods. Results Caffeine or ZM241385 treatment inhibited the increase expressions of TGF-β1, CTGF, α-SMA in the HSC-T6,likewise,as with CGS21680 plus caffeine or ZM241385 groups, caffeine or ZM241385 prevented the adenosine A2A receptor agonist from stimulating an increase in hepatic stellate cell. Conclusion Caffeine can suppress the activation of α-SMA in HSC-T6 stimulated by acetaldehyde. Caffeine treatment inhibited the increase expressions of TGF-βl and CTGF in the HSC-T6,the mechanisms of which may be related to the expression of adenosine A2A receptor.%目的 探讨咖啡因(caffeine)对乙醛诱导的大鼠肝星状细胞系(Hepatic Stellate Cell-T6,HSC-T6)中转化生长因子-β1(Transforming Growth Factor-β1,TGF-β1),结缔组织生长因子(Connective Tissue Growth Factor,CTGF)信号转导通路的影响.方法 实验设正常组(常规培养),模型组及腺苷受体(Adenosine Receptor,AR)调节剂干预组.分别给予caffeine(4 mmol·L-1)[1-2],腺苷A2A受体拮抗剂ZM241385(1 μmol·L-1)[3],腺苷A2A受体激动剂CGS21680(1 μmol·L-1)[3],caffeine+CGS21680,ZM241385+CGS21680 与HSC-T6共同培养,1 h后加入终浓度200 μmol·L-1的乙醛刺激(每12 h补充1次),继续培养48 h.采

  7. The mechanism of abrupt transition between theta and hyper-excitable spiking activity in medial entorhinal cortex layer II stellate cells.

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    Tilman Kispersky

    Full Text Available Recent studies have shown that stellate cells (SCs of the medial entorhinal cortex become hyper-excitable in animal models of temporal lobe epilepsy. These studies have also demonstrated the existence of recurrent connections among SCs, reduced levels of recurrent inhibition in epileptic networks as compared to control ones, and comparable levels of recurrent excitation among SCs in both network types. In this work, we investigate the biophysical and dynamic mechanism of generation of the fast time scale corresponding to hyper-excitable firing and the transition between theta and fast firing frequency activity in SCs. We show that recurrently connected minimal networks of SCs exhibit abrupt, threshold-like transition between theta and hyper-excitable firing frequencies as the result of small changes in the maximal synaptic (AMPAergic conductance. The threshold required for this transition is modulated by synaptic inhibition. Similar abrupt transition between firing frequency regimes can be observed in single, self-coupled SCs, which represent a network of recurrently coupled neurons synchronized in phase, but not in synaptically isolated SCs as the result of changes in the levels of the tonic drive. Using dynamical systems tools (phase-space analysis, we explain the dynamic mechanism underlying the genesis of the fast time scale and the abrupt transition between firing frequency regimes, their dependence on the intrinsic SC's currents and synaptic excitation. This abrupt transition is mechanistically different from others observed in similar networks with different cell types. Most notably, there is no bistability involved. 'In vitro' experiments using single SCs self-coupled with dynamic clamp show the abrupt transition between firing frequency regimes, and demonstrate that our theoretical predictions are not an artifact of the model. In addition, these experiments show that high-frequency firing is burst-like with a duration modulated by an M-current.

  8. miR-181b promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients.

    Science.gov (United States)

    Wang, Baocan; Li, Wenxi; Guo, Kun; Xiao, Yongtao; Wang, Yuqin; Fan, Jiangao

    2012-04-27

    MicroRNAs, as a kind of negative gene regulators, were demonstrated to be involved in many types of diseases. In this study, we found that transforming growth factor-beta 1 could induce the expression of miR-181a and miR-181b, and miR-181b increased in the much higher folds than miR-181a. Because of the important role of transforming growth factor-beta 1 in HSC activation and liver cirrhosis, we investigate the effect of miR-181a and miR-181b on HSC proliferation. The results showed that miR-181b could promote HSC-T6 cell proliferation by regulating cell cycle. Further study showed p27, the cell cycle regulator, was the direct target of miR-181b in HSC-T6 cell. But miR-181a had no effects on HSC-T6 cell proliferation and cell cycle, and did not target p27. Interestingly, miR-181b is elevated significantly in serum of liver cirrhosis cases comparing to that of normal persons, whereas miR-181a expression was in the similar level with that of normal persons. These results suggested that miR-181b could be induced by TGF-β1 and promote the growth of HSCs by directly targeting p27. The elevation of miR-181b in serum suggested that it may be potential diagnostic biomarkers for cirrhosis. As for miR-181a, it may work in TGF-β1 pathway by a currently unknown mechanism.

  9. [Hepatitis non-A, non-B: epidemiological significance in acute viral hepatitis and chronic active hepatitis of hepatological consultation].

    Science.gov (United States)

    Jmelnitzky, A C; Basualdo, J A; Belloni, P O; Ponce de León, H H; García, C; Curciarello, J

    1987-01-01

    157 acute viral hepatitis and 60 chronic active ones have been analyzed focusing on NANB etiology. HAV was implicated in 36.3% of the hole acute viral hepatitis sample, HBV in 29.3%, and HNANBV was presumed as etiology in 31.2%, 5 patients (3.2%) had acute infection by HAV, on previous one by HBV, except for Epstein-Barr virus, no other test for viruses were determined (CMV, HSV, etc.). Male/female ratio was 1.4:1, 1.9:1, and 1.4:1 for HAV, HBV and HNANBV acute hepatitis respectively; HAV was the main etiology in the 0-9 age group (72.2%) although it only represents 11.5% of the sample; small occurrence of HAV hepatitis were found in patients over 40 (8.8%); HBV was clearly prevalent in patients over 50 (65.2%); the highest concentration of NANB etiology was found between 20-39 years old, but it was represented in all age-groups. Out of 49 NANB acute hepatitis, 12.2% had related transfusional antecedents, 12.2% belonged to health care worker group, and 4.1% had a close family NANB hepatitis contact; 71.5% had no reported antecedent. Viral source was presumably implicated in 75.0% of chronic active hepatitis, 25.0% attributable to HNANBV. Results seem not feasible to transfer to general population due to the facts that most patients were of specialized consult, and pediatric assistance is unusual to the authors practice.

  10. [Chronic active hepatitis: clinical, biochemical, and histopathologic correlation].

    Science.gov (United States)

    Subauste, M C

    1989-01-01

    A retrospective study over 26 female patients with chronic active hepatitis was made. The mean age was 39 years old, the mean length of illness of 8 months; 5 patients had positive markers for hepatitis B. Patients were selected with the grade of histological activity: 8 patients had a mild form from disease (2A) and 16 with a severe one (2B). The predominant group was 2B. Severe inflammatory infiltration was the hallmark and multiobulillar necrosis, bridging, eosinophils and hiperplasia of kuppfer cells were found only in this group. Clinical features range from hepatic manifestations to systemic ones. Chronic active hepatitis may present with cholestasis, but the latter is not always related with the grade of activity. Group 2B had elevated aminotransferases and a low concentration for protrobine.

  11. 咖啡因对肝星形细胞活性和分泌功能的影响%Effects of caffeine on viability and secretion of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    李勇剑; 陈云扬; 施敏敏; 李宏为; 严佶祺

    2016-01-01

    Objective To investigate the effects of caffeine on viability and secretion of hepatic stellate cells (HSC) and the mechanisms. Methods Human HSC cell line LX-2 was used in in vitro study. LX-2 cells were incubated with caffeine at various concentrations (0, 5, 10, 20, 30 and 40 mM) and the expression of α-smooth muscle actin (α-SMA) and cleaved-PARP in HSC was detected by Western blot. The effect of caffeine on viability and apoptosis of LX-2 cell was analyzed by CCK-8 assay and flow cytometry respectively. Results Compared to control group, caffeine decreased the expression ofα-SMA in LX-2 cell significantly indicating the secretion of HSC was inhibited. Caffeine was shown by CCK-8 assay to inhibit the viability of LX-2 cell in a dose-and time-dependent manner. It was indicated by flow cytometry that caffeine increased the number of apoptotic cells dose-dependently. Furthermore, it was also showed by Western blot the significant increase in the expression of cleaved-PARP of LX-2 cell compared with control group, suggesting caffeine could induce the apoptosis of LX-2 cell. Conclusions The results demonstrate that caffeine inhibits the viability of HSC via inducing apoptosis of HSC and reduces the expression of α-SMA in HSC, which inhibits the secretion of collagen fiber in HSC.%目的:研究咖啡因对肝星形细胞活化状态和分泌功能的影响及其潜在机制。方法:选取人肝星形细胞LX-2进行体外实验。经浓度为0、5、10、20、30、40 mM的咖啡因处理后,通过Western 印迹法分别检测不同浓度的咖啡因对肝星形细胞中α-平滑肌肌动蛋白和凋亡蛋白cleaved-PARP表达量的影响;分别用CCK-8实验和流式细胞仪检测咖啡因对肝星形细胞活性和凋亡的影响。结果:在咖啡因作用下,肝星形细胞表达α平滑肌肌动蛋白的量与对照组相比显著减少,表示其分泌功能受到明显抑制。 CCK-8实验表明咖啡因可抑制肝星形细胞的活性,

  12. Colchicine Inhibited the Expression of Tissue Inhibitor of Metalloprotenase-1 and Interleukin-6 in Cultured Activated Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    LI Zesong; CAI Shaoxi; JIANG Yuan; GUO RuiJun; ZHANG Wen

    2006-01-01

    Cultured HSCs were treated colchicine with different concentrations for 12 h, respectively. The effects of colchicine on HSCs growth were measured by MTT assay. Effects of colchicine on gene expression of HSCs were analysed by using a self-made oligonucleotide microarray. Colchicine inhibited HSCs growth in a dose-dependent manner. After 12 h of treatment with 6.25 mg/L of colchicine, the expression of tissue inhibitor of metalloprotenase1 (TIMP-1) and the expression of interleukin-6 (IL-6) in HSCs were downregulated by 2.3 folds and 2.1 folds, respectively. These results suggest that colchicine's beneficial effects may, at least in part, owe to the inhibitory to the proliferation of HSCs and down-regulation of the expression of both TIMP1 and IL-6 in HSCs.

  13. Role of stellate cells in alcoholic liver fibrosis

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    Krzysztof Plewka

    2009-07-01

    Full Text Available Many different diseases and toxins can cause liver damage, which is diffi cult to treat and often leads to the development of liver fi brosis or even cirrhosis. The key event in this process is the activation of hepatic stellate cells (HSCs. During such activation, HSCs undergo a dramatic transformation in morphology and behavior, changing from a neuronal-like to a fi broblast-like morphology. After activation, HSCs increase their proliferation rate and extracellular matrix (ECM production. Overproduction of ECM, which contains mainly collagen type I, is a direct cause of liver disruption. HSCs also produce substances which inhibit protease activities, such as TIMPs, which enhance ECM deposition in the liver. On the molecular level, HSCs are activated by cytokines, growth factors, and oxidative stress, which are abundant in affl icted liver. These factors induce intracellular signals transmitted by many kinases, the most important of which are JNK, ERK1/2, p38, TAK-1, PKC, FAK, and P3IK. Signals transmitted via these pathways change the activities of transcription factors such as Smad, AP-1, and NF-κβ. This in turn causes changes In gene transcription and ultimately alters the whole cell’s behavior and morphology. The cell begins the production collagen type I, TIMP-1, and aSMA. Activated HSCs can sustain their own activation by producing growth factors such as PDGF and TGF-β. Despite the vast knowledge about the mechanisms causing liver fi brosis and cirrhosis, there is still no effective cure. Further studies are therefore needed to solve this problem.

  14. The effects of Angiotensin1-7 on Rho-Rock pathway of rat hepatic stellate cell%血管紧张素1-7对大鼠肝星状细胞Rho-Rock信号转导通路的影响

    Institute of Scientific and Technical Information of China (English)

    英嵩崧; 李旭; 张振书

    2013-01-01

    目的探讨血管紧张素Ⅱ(AngⅡ)和血管紧张素1-7(Ang1-7)对大鼠肝星状细胞Rho-Rock信号转导通路的影响。方法采用HSC-T6细胞株,分别给予AngⅡ、Ang1-7、AngⅡ+Ang1-7、Ang1-7+A77910μmol/L处理,逆转录聚合酶链反应(RT-PCR)检测Rho-Rock通路中Rock2(Rho kinase2)、RhoAGTP、RhoGEF (Rho Guanine Nucleotide Exchange Factors, RhoGEF)的表达。结果 AngⅡ处理组Rock2、RhoAGTP、RhoGEF mRNA的表达显著增强,Ang1-7组Rock2、RhoAGTP、RhoGEF mRNA的表达显著低于阴性对照组。结论 Ang1-7可抑制AngⅡ诱导的Rho-Rock信号通路的表达。%Objective To investigate the effects of Angiotensin1-7 on Rho-Rock pathway of rat hepatic stellate cell. Methods HSC-T6 cells were treated with 10μmol/L of AngII, Ang1-7,AngⅡ+Ang1-7, and Ang1-7+A779, respectively. RT-PCR was used to detect the expression of Rock2,RhoAGTP and RhoGEF in Ca2+-independent pathways mediated by Rho-kinase. Results The mRNA expression of Rock2, RhoAGTP, and RhoGEF was significantly increased after AngII treatment (P<0.01), but decreased following subsequent Ang1-7 treatment. Conclusion Ang1-7 could inhibit AngⅡ-induced activation of the Rho-Rock pathway.

  15. 肝细胞生长因子在肿瘤坏死因子相关凋亡诱导配体作用下对原代肝星状细胞增殖及凋亡的影响%Effect of hepatocyte growth factor on the proliferation and apoptosis of primary hepatic stellate cells under tumor necrosis factor-related apoptosis-inducing ligand

    Institute of Scientific and Technical Information of China (English)

    张君红; 姜海行; 覃山羽; 陆正峰; 孟云超; 宁琳; 杨文

    2012-01-01

    背景:活化的肝星状细胞是肝纤维化的关键因素,研究表明肝细胞生长因子能促进星状细胞凋亡,其具体机制可能与增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导星状细胞凋亡有关.目的:观察肿瘤坏死因子相关凋亡诱导配体作用下,肝细胞生长因子对原代肝星状细胞增殖、凋亡的影响并初步探讨其可能机制.方法:将SD 大鼠原代肝星状细胞复苏、传代,细胞增殖明显时用于实验.实验分为4 组:空白对照组为单纯肝星状细胞培养;肝细胞生长因子组:将100 μg/L 肝细胞生长因子作用于肝星状细胞;TRAIL 组:将2 mg/L 的TRAIL 作用于肝星状细胞;肝细胞生长因子+TRAIL 组:将肝细胞生长因子预先刺激肝星状细胞24 h,再加入2 mg/L TRAIL.结果与结论:MTT 检测显示肝细胞生长因子及TRAIL 分别在50~200 μg/L、0.5~1.5 mg/L 各浓度下对肝星状细胞增殖抑制率无影响,TRAIL 在2 mg/L 作用下对肝星状细胞有抑制作用.流式细胞仪检测肝细胞生长因子+TRAIL 组的中晚期凋亡率明显高于空白对照组及肝细胞生长因子组(P < 0.05);肝细胞生长因子+TRAIL组DR5荧光强度明显高于其他3组(P < 0.01).提示在TRAIL 作用下,肝细胞生长因子能促进肝星状细胞的凋亡、抑制其增殖.可能与肝细胞生长因子上调活化肝星状细胞表面DR5 表达有关.%BACKGROUND: Activated hepatic stellate cells play a key role in liver fibrosis. Research shows that hepatocyte growth factor (HGF) can promote the apoptosis of activated hepatic stellate cells and the specific mechanisms may have relationship with the apoptosis of enhanced-related apoptosis-inducing ligand (TRAIL)-induced stellate cells. OBJECTIVE: To investigate the role of HGF under the action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in primary hepatic stellate cells (HSCs) proliferation, apoptosis and to explore the possible mechanisms involved. METHODS

  16. Analysis of the effects of xanthohumol on hepatic homeostasis, inflammation, fibrosis and cancerogenesis

    OpenAIRE

    Dorn, Christoph Michael

    2009-01-01

    Xanthohumol is the major prenylated chalcone found in hops, and it has been shown to exhibit various biological effects. However, xanthohumol effects on liver cells or in liver diseases, respectively, are widely unknown. In the present work, first the effects of xanthohumol on hepatic stellate cells (HSC), the central mediators of liver fibrogenesis, were analyzed. Xanthohumol inhibited the activation of primary human HSC and induces apoptosis in activated HSC in vitro in a dose dependent ...

  17. Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation

    Directory of Open Access Journals (Sweden)

    Hsu Chen-Ming

    2009-08-01

    Full Text Available Abstract Background The pathogenesis of inflammation and fibrosis in chronic hepatitis C virus (HCV infection remains unclear. Transgenic mice with constitutive HCV core over-expression display steatosis only. While the reasons for this are unclear, it may be important that core protein production in these models begins during gestation, in contrast to human hepatitis C virus infection, which occurs post-natally and typically in adults. AIMS: To more realistically model the effect of core protein production in the adult liver, we developed a mouse with conditional expression of HCV core and examined the effect of core protein production in the adult liver. Methods Liver biopsy samples from transgenic mice with tetracycline(tet-regulated conditional core protein expression were evaluated immunohistologically. Microarray analysis of HCV core transgenic mice with steatohepatitis pointed to a role of the complement pathway. This was further explored by blocking complement activation by in vivo administration of CD55 (decay accelerating factor for complement, which inhibits activation of C3. Results Transgenic mice exhibited low, intermediate, or high HCV core protein expression when fed a permissive diet of standard chow. Aside from hepatic steatosis, hepatic inflammation and fibrosis were seen in mice with intermediate levels of core protein. Microarray analyses of inflamed liver demonstrated activation of both the complement (C3 up-regulation and coagulation pathways (fibrinogen B up-regulation. Administration of CD55 reduced hepatic inflammation. Conclusion Transgenic mice that conditionally express intermediate HCV core protein develop inflammation, steatosis, and fibrosis. These effects mediated by HCV core are reduced by administration of CD55, a regulator of the complement pathway. The model may be valuable in investigating the pathogenesis of liver inflammation in chronic hepatitis C.

  18. [Gallbladder motor activity in patients with virus hepatitis B].

    Science.gov (United States)

    Mamos, Arkadiusz; Wichan, Paweł; Chojnacki, Jan; Grzegorczyk, Krzysztof

    2003-12-01

    In acute stage of virus hepatitis B patients often complain of dyspeptic discomfort. They may be a consequence of alimentary tract motor activity disorders including these of gallbladder. Routine ultrasonography in an early phase of virus hepatitis often reveals gallbladder wall thickening what may confirm the above thesis. Thus, a group of 15 patients in an acute phase of virus hepatitis B was subjected to examinations. Gallbladder motor activity was assessed by ultrasonographic method determining its total volume and ejection fraction and volume after test meal stimulus. First examination was performed in the first week since the appearance of yellowing of the walls, successive in 4 and 8 week of the disease. Obtained results were compared to the values obtained in the group of 25 healthy volunteers. It was found out that gallbladder volume was significantly decreased and ejection fraction increased in the acute phase of virus hepatitis B than in the controls. This may speak for gallbladder hyperreactivity in patients in the course of virus hepatitis B. These disorders decreased during two-month observation but even in the 8 week the investigated parameters differed from those found in the control group.

  19. Novel perspectives on the origins of the hepatic myofibroblasts

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    Xu J

    2015-03-01

    Full Text Available Jun Xu,1,2 Xiao Liu,1,2 David A Brenner,1 Tatiana Kisseleva2 1Department of Medicine, 2Department of Surgery, University of California, San Diego, CA, USA Abstract: Liver fibrosis results from chronic liver injury that causes hepatocellular damage. Damaged hepatocytes apoptose, and release factors that facilitate recruitment of leukocytes to the site of injury, which in turn mediate recruitment and activation of liver- resident (Kupffer cells and bone marrow (BM-derived macrophages. Activated macrophages secrete TGF-ß1, the major profibrogenic cytokine, which activates hepatic myofibroblasts, which are not present in the liver under physiological conditions. Several sources of myofibroblasts have been identified, but it is believed that liver-resident hepatic stellate cells (HSCs and portal fibroblasts (PFs are the major source of hepatic myofibroblasts in fibrotic liver. Fibrocytes, designated as BM-derived collagen Type I producing cells, were also implicated in liver fibrosis; hence, their contribution to liver fibrosis remains controversial. Upon removal of the etiological agent, myofibroblasts either undergo apoptosis or inactivate into a quiescent-like state, followed by resorbtion of the fibrous scar. However, prolonged/repeated liver injury triggers irreversible cross-linking of collagen fibers that prevents fibrous scar from collagenase-mediated degradation. This review will discuss several types of fibrogenic cells contributing to the myofibroblast population, and the signaling pathways regulating their activation and collagen deposition. Keywords: Liver fibrosis, TGF-ß1 signaling, hepatic stellate cells, portal fibroblasts, fibrocytes, collagen Type I deposition

  20. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    1993-01-01

    930140 Hepatocyte stimulator peptide and itsclinical significance in viral hepatitis.ZHOUWeiping(周卫平),et al.Instit Viral Hepatitis,Chongqing Med Univ,630010.Chin J InternMed 1992;31(10):626-628.Hepatocyte stimulator peptide(HSP)is anewly developed hepatic stimulator substance.Its monoclonal antibodies have been obtained inour laboratory.In this study,HSP was deter-mined in the sera of 315 subjects including pa-

  1. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    2010349 Relationships between serum hepatitis B virus load in mothers,free maternal DNA in peripheral blood of newborns and hepatitis B virus infection of newborns. WEI Junni(魏俊妮),et al. Dept Epidemiol,Shanxi Med Univ,Taiyuan 030001. Chin J Infect Dis 2010;28(5):297-300. Objective To study the relationships between serum hepatitis B virus (HBV) DNA level

  2. Role of LncRNA-activated by transforming growth factor beta in the progression of hepatitis C virus-related liver fibrosis.

    Science.gov (United States)

    Fu, Na; Niu, Xuemin; Wang, Yang; Du, Huijuan; Wang, Baoyu; Du, Jinghua; Li, Ya; Wang, Rongqi; Zhang, Yuguo; Zhao, Suxian; Sun, Dianxing; Qiao, Liang; Nan, Yuemin

    2016-08-01

    Long non-coding RNA (LncRNA)-activated by transforming growth factor-beta (LncRNA-ATB) is a key regulator of transforming growth factor-beta (TGF-β) signaling pathway, and is positively correlated with the development of liver cirrhosis and vascular invasion of hepatocellular carcinoma (HCC). However, the role of LncRNA-ATB in hepatitis C virus (HCV)-related liver fibrosis remains largely unknown. In the present study, we confirmed a high expression level of LncRNA-ATB in the liver tissues and plasma samples of patients with HCV-related hepatic fibrosis, and the plasma level of LncRNA-ATB was significantly correlated with liver fibrosis stages. Furthermore, increased expression level of LncRNA-ATB was also present in activated hepatic stellate cells (HSCs), and knockdown of LncRNA-ATB inhibited the expression of alpha-smooth muscle actin (α-SMA) and alpha-1 type I collagen (Col1A1). LncRNA-ATB was found to share the common miRNA responsive element of miR-425-5p with TGF-β type II receptor (TGF-βRII) and SMAD2. Ectopic expression of LncRNA-ATB in HSCs could upregulate the protein expression of TGF-βRII and SMAD2 by inhibiting the endogenous miR-425-5p. Moreover, overexpression of miR-425-5p could partly abrogate the expression of TGF-βRII and SMAD2 induced by LncRNA-ATB. Hence, we conclude that LncRNA-ATB promotes HCV-induced liver fibrogenesis by activating HSCs and increasing collagen I production through competitively binding to miR-425-5p. LncRNA-ATB may be a novel diagnostic biomarker and a potential therapeutic target for HCV-related hepatic fibrosis.

  3. Metformin Inhibits Glutaminase Activity and Protects against Hepatic Encephalopathy

    Science.gov (United States)

    Ampuero, Javier; Ranchal, Isidora; Nuñez, David; Díaz-Herrero, María del Mar; Maraver, Marta; del Campo, José Antonio; Rojas, Ángela; Camacho, Inés; Figueruela, Blanca; Bautista, Juan D.; Romero-Gómez, Manuel

    2012-01-01

    Aim To investigate the influence of metformin use on liver dysfunction and hepatic encephalopathy in a retrospective cohort of diabetic cirrhotic patients. To analyze the impact of metformin on glutaminase activity and ammonia production in vitro. Methods Eighty-two cirrhotic patients with type 2 diabetes were included. Forty-one patients were classified as insulin sensitizers experienced (metformin) and 41 as controls (cirrhotic patients with type 2 diabetes mellitus without metformin treatment). Baseline analysis included: insulin, glucose, glucagon, leptin, adiponectin, TNFr2, AST, ALT. HOMA-IR was calculated. Baseline HE risk was calculated according to minimal hepatic encephalopathy, oral glutamine challenge and mutations in glutaminase gene. We performed an experimental study in vitro including an enzymatic activity assay where glutaminase inhibition was measured according to different metformin concentrations. In Caco2 cells, glutaminase activity inhibition was evaluated by ammonia production at 24, 48 and 72 hours after metformina treatment. Results Hepatic encephalopathy was diagnosed during follow-up in 23.2% (19/82): 4.9% (2/41) in patients receiving metformin and 41.5% (17/41) in patients without metformin treatment (logRank 9.81; p = 0.002). In multivariate analysis, metformin use [H.R.11.4 (95% CI: 1.2–108.8); p = 0.034], age at diagnosis [H.R.1.12 (95% CI: 1.04–1.2); p = 0.002], female sex [H.R.10.4 (95% CI: 1.5–71.6); p = 0.017] and HE risk [H.R.21.3 (95% CI: 2.8–163.4); p = 0.003] were found independently associated with hepatic encephalopathy. In the enzymatic assay, glutaminase activity inhibition reached 68% with metformin 100 mM. In Caco2 cells, metformin (20 mM) decreased glutaminase activity up to 24% at 72 hours post-treatment (p<0.05). Conclusions Metformin was found independently related to overt hepatic encephalopathy in patients with type 2 diabetes mellitus and high risk of hepatic encephalopathy. Metformin

  4. Metformin inhibits glutaminase activity and protects against hepatic encephalopathy.

    Directory of Open Access Journals (Sweden)

    Javier Ampuero

    Full Text Available AIM: To investigate the influence of metformin use on liver dysfunction and hepatic encephalopathy in a retrospective cohort of diabetic cirrhotic patients. To analyze the impact of metformin on glutaminase activity and ammonia production in vitro. METHODS: Eighty-two cirrhotic patients with type 2 diabetes were included. Forty-one patients were classified as insulin sensitizers experienced (metformin and 41 as controls (cirrhotic patients with type 2 diabetes mellitus without metformin treatment. Baseline analysis included: insulin, glucose, glucagon, leptin, adiponectin, TNFr2, AST, ALT. HOMA-IR was calculated. Baseline HE risk was calculated according to minimal hepatic encephalopathy, oral glutamine challenge and mutations in glutaminase gene. We performed an experimental study in vitro including an enzymatic activity assay where glutaminase inhibition was measured according to different metformin concentrations. In Caco2 cells, glutaminase activity inhibition was evaluated by ammonia production at 24, 48 and 72 hours after metformina treatment. RESULTS: Hepatic encephalopathy was diagnosed during follow-up in 23.2% (19/82: 4.9% (2/41 in patients receiving metformin and 41.5% (17/41 in patients without metformin treatment (logRank 9.81; p=0.002. In multivariate analysis, metformin use [H.R.11.4 (95% CI: 1.2-108.8; p=0.034], age at diagnosis [H.R.1.12 (95% CI: 1.04-1.2; p=0.002], female sex [H.R.10.4 (95% CI: 1.5-71.6; p=0.017] and HE risk [H.R.21.3 (95% CI: 2.8-163.4; p=0.003] were found independently associated with hepatic encephalopathy. In the enzymatic assay, glutaminase activity inhibition reached 68% with metformin 100 mM. In Caco2 cells, metformin (20 mM decreased glutaminase activity up to 24% at 72 hours post-treatment (p<0.05. CONCLUSIONS: Metformin was found independently related to overt hepatic encephalopathy in patients with type 2 diabetes mellitus and high risk of hepatic encephalopathy. Metformin inhibits glutaminase

  5. Is autoimmune chronic active hepatitis a HCV-related disease?

    Science.gov (United States)

    Magrin, S; Craxì, A; Fiorentino, G; Fabiano, C; Provenzano, G; Pinzello, G B; Palazzo, U; Almasio, P; Pagliaro, L

    1991-07-01

    We evaluated the specificity and clinical relevance of anti-hepatitis C virus antibody positivity in 22 HBsAg-negative patients with autoimmune (anti-nuclear, anti-actin or anti-liver-kidney microsomal antibody positive) chronic active hepatitis. An ELISA anti-HCV test and a recombinant immunoblot assay (RIBA-HCV) were used. Thirteen patients (59%) were anti-HCV positive and five (23%) anti-HCV negative by both ELISA and RIBA-HCV tests. Four patients (18%) were borderline positive by ELISA (OD less than 1.0), and three of them (all with severe disease) were negative by RIBA. Histologic necroinflammation, AST/ALT and gamma-globulins levels were higher and response to prednisolone treatment was better in RIBA anti-HCV-negative than in anti-HCV-positive cases. We confirmed with both RIBA and ELISA tests the high prevalence of anti-HCV already reported by ELISA in anti-nuclear and anti-liver-kidney microsomal antibody positive chronic active hepatitis. False positive for anti-HCV (i.e., a positive ELISA test not confirmed by RIBA) occurred only among patients with severe disease. Since RIBA-negative subjects showed the best response to corticosteroid, they might represent the only subset of cases of 'true' autoimmune chronic active hepatitis.

  6. Successful Interferon Therapy Reverses Enhanced Hepatic Progenitor Cell Activation in Patients with Chronic Hepatitis C.

    Science.gov (United States)

    Noritake, Hidenao; Kobayashi, Yoshimasa; Ooba, Yukimasa; Matsunaga, Erika; Ohta, Kazuyoshi; Shimoyama, Shin; Yamazaki, Satoru; Chida, Takeshi; Kawata, Kazuhito; Sakaguchi, Takanori; Suda, Takafumi

    2015-12-01

    The enhanced accumulation of hepatic progenitor cells (HPCs) is related to the risk of progression to hepatocellular carcinoma (HCC). Interferon (IFN) treatment reduces HCC risk in patients with chronic hepatitis C virus (HCV) infection. However, the underlying mechanisms remain unclear. The aim of this study was to examine the effects of IFN treatment on HPC activation in HCV patients. Immunohistochemical detection and computer-assisted quantitative image analyses of cytokeratin 7 (CK7) were performed to evaluate HPC activation in paired pre- and post-treatment liver biopsies from 18 HCV patients with sustained virological response (SVR) to IFN-based therapy and from 23 patients without SVR, as well as normal liver tissues obtained from surgical resection specimens of 10 patients. Pretreatment HCV livers showed increased CK7 immunoreactivity, compared with normal livers (HCV: median, 1.38%; normal: median, 0.69%, P=0.006). IFN treatment reduced hepatic CK7 immunoreactivity (median, 1.57% pre-IFN vs. 0.69% post-IFN, P=0.006) in SVR patients, but not in non-SVR patients. The development of HCC following IFN treatment was encountered in 3 non-SVR patients who showed high post-IFN treatment CK7 immunoreactivity (>4%). Successful IFN therapy can reverse enhanced HPC activation in HCV patients, which may contribute to the reduced risk of HCC development in these patients.

  7. Relationship between differential hepatic microRNA expression and decreased hepatic cytochrome P450 3A activity in cirrhosis.

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    Raj Vuppalanchi

    Full Text Available BACKGROUND AND AIM: Liver cirrhosis is associated with decreased hepatic cytochrome P4503A (CYP3A activity but the pathogenesis of this phenomenon is not well elucidated. In this study, we examined if certain microRNAs (miRNA are associated with decreased hepatic CYP3A activity in cirrhosis. METHODS: Hepatic CYP3A activity and miRNA microarray expression profiles were measured in cirrhotic (n=28 and normal (n=12 liver tissue. Hepatic CYP3A activity was measured via midazolam hydroxylation in human liver microsomes. Additionally, hepatic CYP3A4 protein concentration and the expression of CYP3A4 mRNA were measured. Analyses were conducted to identify miRNAs which were differentially expressed between two groups but also were significantly associated with lower hepatic CYP3A activity. RESULTS: Hepatic CYP3A activity in cirrhotic livers was 1.7-fold lower than in the normal livers (0.28 ± 0.06 vs. 0.47 ± 0.07mL* min(-1*mg protein(-1 (mean ± SEM, P=0.02. Six microRNAs (miR-155, miR-454, miR-582-5p, let-7f-1*, miR-181d, and miR-500 had >1.2-fold increase in cirrhotic livers and also had significant negative correlation with hepatic CYP3A activity (range of r = -0.44 to -0.52, P <0.05. Notably, miR-155, a known regulator of liver inflammation, had the highest fold increase in cirrhotic livers (2.2-fold, P=4.16E-08 and significantly correlated with hepatic CYP3A activity (r=-0.50, P=0.017. The relative expression (2(-ΔΔCt mean ± SEM of hepatic CYP3A4 mRNA was significantly higher in cirrhotic livers (21.76 ± 2.65 vs. 5.91 ± 1.29, P=2.04E-07 but their levels did not significantly correlate with hepatic CYP3A activity (r=-0.43, P=0.08. CONCLUSION: The strong association between certain miRNAs, notably miR-155, and lower hepatic CYP3A activity suggest that altered miRNA expression may regulate hepatic CYP3A activity.

  8. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    1997-01-01

    970349 Primary structure and variability of partialsequences in nonstructural gene 5 region of hepatitis Gvirus, CHANG Jinhong(常锦红), et al. Hepatol Instis,People’s Hosp, Beijing Med Univ, Beijing, 100044. NatlMed J China 1997; 77(3): 178-182. Objective: To sequence partial genome of hepatitis G

  9. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    2009209 Effects of chronic hepatitis B virus infection on human hepatic cytochrome P450 2C9.ZHO Fuping(周福平),et al.Dept Infect Dis,Shanghai Changzheng Hosp,Shanghai 200003.Chin J Infect Dis,2009;27(2):94-98.

  10. Hepatitis

    Science.gov (United States)

    ... inflammation of the liver.” This inflammation can be caused by a wide variety of toxins, drugs, and metabolic diseases, as well as infection. There are at least 5 hepatitis viruses. Hepatitis A is contracted when a child eats food or drinks water that is contaminated with the virus or has ...

  11. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    1992-01-01

    920691 The determination of serum hepa-titis B virus DNA by polymerase chain rea-ction in hepatitis B patients treated withalpha-interferon. XU. Jianye(徐建业), et al.Centr Lab, Chongqing Cancer Instit, 630030.Chin J Intern Med, 1992; 31(5): 278-280. To clarify the status of HBV in serum of

  12. Didymin Alleviates Hepatic Fibrosis Through Inhibiting ERK and PI3K/Akt Pathways via Regulation of Raf Kinase Inhibitor Protein

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    Xing Lin

    2016-12-01

    Full Text Available Background: Didymin has been reported to have anti-cancer potential. However, the effect of didymin on liver fibrosis remains illdefined. Methods: Hepatic fibrosis was induced by CCl4 in rats. The effects of didymin on liver pathology and collagen accumulation were observed by hematoxylin-eosin and Masson's trichrome staining, respectively. Serum transaminases activities and collagen-related indicators levels were determined by commercially available kits. Moreover, the effects of didymin on hepatic stellate cell apoptosis and cell cycle were analyzed by flow cytometry. Mitochondrial membrane potential was detected by using rhodamine-123 dye. The expression of Raf kinase inhibitor protein (RKIP and the phosphorylation of the ERK/MAPK and PI3K/Akt pathways were assessed by Western blot. Results: Didymin significantly ameliorated chronic liver injury and collagen deposition. It strongly inhibited hepatic stellate cells proliferation, induced apoptosis and caused cell cycle arrest in G2/M phase. Moreover, didymin notably attenuated mitochondrial membrane potential, accompanied by release of cytochrome C. Didymin significantly inhibited the ERK/MAPK and PI3K/Akt pathways. The effects of didymin on the collagen accumulation in rats and on the biological behaviors of hepatic stellate cells were largely abolished by the specific RKIP inhibitor locostatin. Conclusion: Didymin alleviates hepatic fibrosis by inhibiting ERK/MAPK and PI3K/Akt pathways via regulation of RKIP expression.

  13. Curcumin attenuates diet-induced hepatic steatosis by activating AMP-activated protein kinase.

    Science.gov (United States)

    Um, Min Young; Hwang, Kwang Hyun; Ahn, Jiyun; Ha, Tae Youl

    2013-09-01

    Curcumin is a well-known component of traditional turmeric (Curcuma longa), which has been reported to prevent obesity and diabetes. However, the effect of curcumin on hepatic lipid metabolism remains unclear. The aim of this study was to examine the effects of curcumin on hepatic steatosis in high-fat/cholesterol diet (HFD)-induced obese mice. Male C57BL/6J mice were fed a normal diet (ND), HFD or HFD with 0.15% curcumin (HFD+C) for 11 weeks. We found that curcumin significantly lowered the body-weight and adipose tissue weight of mice in the HFD+C group compared with the findings for the HFD group (p cholesterol, fasting glucose and insulin in serum were decreased, and HFD-induced impairment of insulin sensitivity was improved by curcumin supplementation (p Curcumin protected against the development of hepatic steatosis by reducing hepatic fat accumulation. Moreover, curcumin activated AMP-activated protein kinase (AMPK) and elevated the gene expression of peroxisome proliferator-activated receptor alpha. By contrast, curcumin suppressed the HFD-mediated increases in sterol regulatory element-binding protein-1, acetyl-CoA carboxylase 1, fatty acid synthase and cluster of differentiation 36 expression. Taken together, these findings indicate that curcumin attenuates HFD-induced hepatic steatosis by regulating hepatic lipid metabolism via AMPK activation, suggesting its use as a therapeutic for hepatic steatosis.

  14. Role of Rho-Rock pathways induced by angiotensin Ⅱ in hepatic stellate cell contraction%Rho-Rock通路在血管紧张素Ⅱ诱导肝星状细胞收缩中的作用

    Institute of Scientific and Technical Information of China (English)

    张小兰; 肖冰; 李旭; 黄茂梁; 孟莹; 李鹰飞; 王媛媛; 宋卫兵

    2008-01-01

    Objective To investigate the mechanism of Ca2+-independent pathways mediated by Rho-kinase in contraction of hepatic steLlate cells (HSCs) induced by angiotonin Ⅱ (Ang )Ⅱ. Methods Human HSCs of the line HSC-T6 were cultured and randomly divided into 6 groups: negative control group, AngⅡ group treated by Ang Ⅱ 10 μmol/L for 15 min, Ang Ⅱ + irbesantan (Ang Ⅱ receptor inhibitor) group, exposed to irbesantan for 60 rain prior to Ang Ⅱ treatment, Ang Ⅱ + Y27632 (Rho kinase specific inhibitor) exposed to Y27632 for 60 min prior to Ang Ⅱ treatment, Ang Ⅱ + ML-7 (myosin fight chain kinase specific inhibitor) + saturo (protein kinase C specific inhibitor) group exposed to stauro for 60 min prior to Ang Ⅱ treatment, and Ang Ⅱ + Y27632 + ML-7 + stauro group, exposed to Y27632 and stauro for 60 min prior to Ang Ⅱ treatment. The cell contraction was detected by sillcone-tubber-membrane cultivation directly. The protein levels of MLC and phosphorylated MLC were detected by Western blotting 5, 15, 30, 60, and 120 min after Ang Ⅱ treatment. RT-PCR was used to detect the expression of Rock?., RhoAGTP, and RhoGEF in Ca2+- independent pathways mediated by Rho-kinage. Results The silicone-rubber- membrane covered by Ang Ⅱ treated HSCs showed obvious wrinkles indicating the contraction of HSCs. The ratios of phosphorylated MLC protein at the time pints 5, 15, 30, 60, and120 min of the Ang Ⅱ group to the control group (0 min)were 11.7±0. 1, 26.9±0.1, 11.2 ±0.1, 4.1 ±0. 1, and 1.0±0.1, showing that Ang Ⅱ increased the phosphorylated MLC protein level time-dependently with the peak level at the time point of 15 minutes. The levels of phosphorylated MLC protein of the Ang Ⅱ + irbesartan and Ang Ⅱ + Y27632 groups were (1.12±0.09)and (1.22±0.10) respectively, both significantly lower than that of the Ang Ⅱ group (1.33±0.06, both P<0.01). The level of phosphorylated MLC protein of the Ang Ⅱ + ML-7 + stauro group was (1.43 ± 0

  15. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    2005226 Characteristics of peripheral blood T lymphocyte subsets in hepatitis B patients. FAN Zhen-ping(范振平),et al. Center Bio Ther, Instit Infect Dis, 302 Hosp Chin PLA, Beijing 100039. World Chin J Digestol, 2005;13(2): 194-197. Objective: To characterize the T-lymphocyte subsets in peripheral blood of patients with acute and chronic hepatitis B, and to explore their relations with the disease state. Methods: Peripheral blood

  16. Serum arylesterase and paraoxonase activity in patients with chronic hepatitis

    Institute of Scientific and Technical Information of China (English)

    Suleyman Sirri Kilic; Suleyman Aydin; Nermin Kilic; Fazilet Erman; Suna Aydin; (I)lhami Celik

    2005-01-01

    AIM: To investigate the relationship between serum paraoxonase (PON1), AST, ALT, GGT, and arylesterase (AE) activity alterations and the degree of liver damage in patients with chronic hepatitis.METHODS: We studied 34 chronic hepatitis patients and 32 control subjects, aged between 35 and 65 years,in the Department of Infection and Clinical Microbiology at the Firat University School of Medicine. Blood samples were collected from subjects between 8:00 and 10:00 a.m. following a 12-h fast. Baseline and salt-stimulated PON1 activities were measured by the hydrolysis of paraoxon. Phenyl acetate was used as the substrate and formed phenol was measured spectrophotometrically at 270 nm after the addition of a 10-fold diluted serum sample in AE activity measurements.RESULTS: The results of this investigation revealed that the levels of AE activity decreased from 132±52 to 94±36 (29%), baseline PON1 activity from 452±112 to 164±67 (64%), salt-stimulated PON1 activity from 746±394 to 294±220 (61%), HDL from 58.4±5.1 to 47.2±5.6(20%), triglyceride from 133±51.2 to 86±34.0 (35%),while a slight increase in the level of LDL (from 163±54.1 to 177.3±56.0; 9%) and significant increases in the levels of AST (from 29±9.3 to 98±44), ALP (from 57.2±13.1 to 91±38.1), ALT (from 27.9±3.32 to 89±19.1), GGT (from 24.3±2.10 to 94±48.2), total bilirubin (from 0.74±0.02 to 1.36±0.06; 84%) and direct bilirubin (from 0.18±0.01 to 0.42±0.04; 133%) were detected.However, the levels of albumin, total protein, cholesterol,and uric acid were almost the same in chronic hepatitis and the control subjects.CONCLUSION: Low PON1 and AE activity may contribute to the increased liver dysfunction in chronic hepatitis patients by reducing the ability of HDL to retard LDL oxidation and might be clinically useful for monitoring the disease of chronic hepatitis.

  17. Effect of antihypertensive agents on stellate cells during liver regeneration in rats Efeito de agentes anti-hipertensivos sobre as células estreladas durante a regeneração hepática em ratos

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    Leandra N. Z. Ramalho

    2003-03-01

    Full Text Available BACKGROUND: Although most studies have focused on the hepatocytes, all the hepatic cells participate in the regenerative process, among them the stellate cells. The stellate cells are mesenchymal cells involved in local neurotransmission and paracrine regulation of several liver functions. Acute hepatic tissue loss promotes the proliferation and activation of stellate cells from a quiescent state to myofibroblast-like cells. AIM: Investigate the effects of antihypertensive agents on the stellate cell population during the liver regenerative phenomenon in rats. METHODS: Adult male Wistar rats received lisinopril, losartan, bradykinin, or saline solution in a proportional volume, intraperitoneally, before and after 70% partial hepatectomy. Animals from the experimental and saline groups were sacrificed at 36 hours after partial hepatectomy. The alpha-smooth muscle actin labelled stellate cells population was counted in the periportal and pericentral zones of the liver specimen. RESULTS: The labelled stellate cells were more numerous in the control group both in the periportal and pericentral zones at 36 hours after partial hepatectomy than at the other times. The population of stellate cells was significantly lower in the losartan group and higher in the bradykinin and lisinopril groups than in the control group. CONCLUSIONS: These results suggest that losartan can inhibit and bradykinin and lisinopril can stimulate the stellate cell population during liver regeneration in rats. These cells synthesize several substances to stimulate liver regeneration.RACIONAL: Embora a maioria dos estudos focalize os hepatócitos, todas as células hepáticas participam do processo regenerativo, entre elas as células estreladas, que são células mesenquimais envolvidas na regulação de uma série de funções hepáticas. A perda aguda de parênquima hepático induz proliferação e ativação destas células, a partir de estado de quiescência para fen

  18. [Hepatic manifestation of a macrophage activation syndrome (MAS)].

    Science.gov (United States)

    Nagel, Michael; Schwarting, Andreas; Straub, Beate K; Galle, Peter R; Zimmermann, Tim

    2017-04-04

    Background Elevated liver values are the most common pathological laboratory result in Germany. Frequent findings, especially in younger patients, are nutritive- or medicamentous- toxic reasons, viral or autoimmune hepatitis. A macrophage activation syndrome (MAS) may manifest like a viral infectious disease with fever, hepatosplenomegaly and pancytopenia and is associated with a high mortality. It is based on an enhanced activation of macrophages with increased cytokine release, leading to organ damage and multi-organ failure. In addition to genetic causes, MAS is commonly associated with infections and rheumatic diseases. We report the case of a 26-year-old female patient suffering from MAS as a rare cause of elevated liver enzymes. Methods Patient characteristics, laboratory values, liver histology, bone marrow and radiological imaging were documented and analyzed. Case Report After an ordinary upper airway infection with bronchitis, a rheumatic arthritis appeared and was treated with leflunomide und methotrexate. In the further course of the disease, the patient developed an acute hepatitis with fever, pancytopenia and massive hyperferritinemia. Immunohistochemistry of the liver biopsy revealed hemophagocytosis and activation of CD68-positive macrophages. In the radiological and histological diagnostics of the liver and bone marrow, an MAS was diagnosed as underlying disease of the acute hepatitis. Under therapy with prednisolone, the fever disappeared and transaminases and ferritin rapidly normalized. Conclusion Aside from the frequent causes of elevated liver values in younger patients, such as nutritive toxic, drug induced liver injury, viral or autoimmune hepatitis, especially in case of massive hyperferritinemia, a MAS should be considered as a rare cause of acute liver disease.

  19. Hepatitis B antigen in hepatocytes of chronic active liver disease.

    Science.gov (United States)

    Kawanishi, H

    1979-04-01

    To study the morphologic interrelation of hepatocytes with the replication of hepatitis B vius (HBV) and immunocompetent cells in chronic active liver disease(CALD), organ cultures were prepared from liver biopsy specimens. Replication of hepatitis B core antigen (HBcAg) appears to occur in the nucleus of the hepatocyte in close association with intranuclear electron-dense strands and sometimes intranucleolar matrixes (likely HBcAg genomes), and cytoplasmic maturation of the HBcAg takes place in the preautolytic condition of host hepatocytes. Immunocompetent cells became progressively autolyzed in the early period of cultures. No difference in progression of hepatocyte injury in tissues from normal subjects and from hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative patients with CALD may suggest that intracellular synthesis of HBV alone is not cytopathic to host hepatocytes. This model is promising for the study of HBV replication and development, and also for testing the efficacy of new antiviral agents against the virus.

  20. Inhibition of SIRT2 suppresses hepatic fibrosis.

    Science.gov (United States)

    Arteaga, Maribel; Shang, Na; Ding, Xianzhong; Yong, Sherri; Cotler, Scott J; Denning, Mitchell F; Shimamura, Takashi; Breslin, Peter; Lüscher, Bernhard; Qiu, Wei

    2016-06-01

    Liver fibrosis can progress to cirrhosis and result in serious complications of liver disease. The pathogenesis of liver fibrosis involves the activation of hepatic stellate cells (HSCs), the underlying mechanisms of which are not fully known. Emerging evidence suggests that the classic histone deacetylases play a role in liver fibrosis, but the role of another subfamily of histone deacetylases, the sirtuins, in the development of hepatic fibrosis remains unknown. In this study, we found that blocking the activity of sirtuin 2 (SIRT2) by using inhibitors or shRNAs significantly suppressed fibrogenic gene expression in HSCs. We further demonstrated that inhibition of SIRT2 results in the degradation of c-MYC, which is important for HSC activation. In addition, we discovered that inhibition of SIRT2 suppresses the phosphorylation of ERK, which is critical for the stabilization of c-MYC. Moreover, we found that Sirt2 deficiency attenuates the hepatic fibrosis induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). Furthermore, we showed that SIRT2, p-ERK, and c-MYC proteins are all overexpressed in human hepatic fibrotic tissues. These data suggest a critical role for the SIRT2/ERK/c-MYC axis in promoting hepatic fibrogenesis. Inhibition of the SIRT2/ERK/c-MYC axis represents a novel strategy to prevent and to potentially treat liver fibrosis and cirrhosis.

  1. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    2008449 A cross-sectional survey of occult hepatitis B virus infection in HIV-infected patients. MA Jianxin(马建新), et al.Dept Infect Dis, Shanghai Public Health Clin Center, Shanghai 201508. Chin J Intern Med 2008;47(7):574-577. Objective To assess the prevalence of occult HBV infection in HIV-infected patients.

  2. HCV virological response during treatment of chronic hepatitis C is associated with liver histological improvement in patients with HCV/HIV co-infection.

    Science.gov (United States)

    Castro, Gleusa; Ramalho, Leandra Naira Zambelli; Zucoloto, Sérgio; Martinelli, Ana de Lourdes Candolo; Figueiredo, José Fernando de Castro

    2008-06-01

    Liver histological improvement after treatment for chronic hepatitis C in patients co-infected with human immunodeficiency virus-1 (HIV-1) has been described. Paired liver biopsies in twenty six HCV/HIV co-infected patients were compared to determine factors possibly associated with histological improvement. The patients were submitted to a liver biopsy before treatment for hepatitis C and 25 months after the end of treatment. Fragments of the liver biopsy obtained before and after treatment were compared regarding the following parameters: histological activity index (HAI) and degree of fibrosis (Knodell); intensity of collagen deposits (Sirius Red staining) and degree of stellate cell activation (alpha-smooth muscle actin labeling). The ratios of the post and pre-treatment variables were related through logistic regression to body mass index (BMI), alcohol ingestion, HCV genotype, HCV viremia, presence of hepatic iron and pre-treatment hepatic steatosis. A negative RNA test in the 24th week of treatment was associated with improvement in fibrosis, collagen deposits and stellate cell numbers. The other variables analyzed did not correlate to an improvement in hepatic histology after hepatitis C treatment. Reduction in HCV viremia during treatment may result in reduced hepatic fibrosis even in patients without a sustained virological response.

  3. HCV virological response during treatment of chronic hepatitis C is associated with liver histological Improvement in patients with HCV/HIV co-infection

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    Gleusa Castro

    2008-06-01

    Full Text Available Liver histological improvement after treatment for chronic hepatitis C in patients co-infected with human immunodeficiency virus-1 (HIV-1 has been described. Paired liver biopsies in twenty six HCV/HIV co-infected patients were compared to determine factors possibly associated with histological improvement. The patients were submitted to a liver biopsy before treatment for hepatitis C and 25 months after the end of treatment. Fragments of the liver biopsy obtained before and after treatment were compared regarding the following parameters: histological activity index (HAI and degree of fibrosis (Knodell; intensity of collagen deposits (Sirius Red staining and degree of stellate cell activation (alpha-smooth muscle actin labeling. The ratios of the post and pre-treatment variables were related through logistic regression to body mass index (BMI, alcohol ingestion, HCV genotype, HCV viremia, presence of hepatic iron and pre-treatment hepatic steatosis. A negative RNA test in the 24th week of treatment was associated with improvement in fibrosis, collagen deposits and stellate cell numbers. The other variables analyzed did not correlate to an improvement in hepatic histology after hepatitis C treatment. Reduction in HCV viremia during treatment may result in reduced hepatic fibrosis even in patients without a sustained virological response.

  4. Anti-hepatitis B virus active constituents from Swertia chirayita.

    Science.gov (United States)

    Zhou, Ning-Jia; Geng, Chang-An; Huang, Xiao-Yan; Ma, Yun-Bao; Zhang, Xue-Mei; Wang, Ju-Le; Chen, Ji-Jun

    2015-01-01

    Four new compounds swertiachiralatone A (1), swertiachoside A (2), swertiachirdiol A (3) and swertiachoside B (4), together with twenty-six known ones were isolated from the ethanol extract of Swertia chirayita. Their structures were elucidated by extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, UV, IR and [α]D). All compounds were evaluated for anti-hepatitis B virus (anti-HBV) activities on HepG 2.2.15 cells line in vitro, of which compounds 14 and 19 showed inhibitory activity on hepatitis B surface antigen (HBsAg) secretion with IC50 values of 0.31 ± 0.045 and 1.49 ± 0.033 mM; compounds 14 and 28 exhibited activity against hepatitis B e antigen (HBeAg) secretion with IC50 values of 0.77 ± 0.076 and 5.92 ± 1.02 mM; and eight compounds (8,9,13,14,24-26,29) possessed activity against HBV DNA replication with IC50 values of 0.07-0.33 mM. In particular (+)-cycloolivil-4'-O-β-d-glucopyranoside (14) exhibited inhibition not only on the secretions of HBsAg and HBeAg with IC50 values of 0.31 ± 0.045 mM (SI=4.29) and 0.77 ± 0.076 mM (SI=1.75), respectively, but also on HBV DNA replication with an IC50 value of 0.29 ± 0.034 mM (SI=4.66).

  5. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    2008079 Relationship of HBV genotype and bcp and pc mutations with HBV DNA rebound after lamivudine therapy. SU Minghua(苏明华), et al. Dept Infect Dis Clin Hosp, Guangxi Med Univ, Nanning 530027. World Chin J Digestol 2007;15(33):3507-3513. Objective To investigate the relationship of HBV gene mutations with HBV DNA rebound after lamivudine therapy. Methods Twenty-seven hepatitis B patients with HBV DNA rebound after

  6. IL28B polymorphism correlates with active hepatitis in patients with HBeAg-negative chronic hepatitis B.

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    I-Cheng Lee

    Full Text Available BACKGROUND AIMS: The clinical relevance of single nucleotide polymorphisms (SNPs near the IL28B gene is controversial in patients with hepatitis B virus (HBV infection. This study aimed to investigate the role of viral and host factors, including IL28B genotypes, in the natural course of chronic hepatitis B (CHB. METHODS: The study enrolled consecutive 115 treatment-naive CHB patients. HBV viral loads, genotypes, precore and basal core promotor mutations, serum hepatitis B surface antigen (HBsAg and interferon-gamma inducible protein 10 (IP-10 levels as well as four SNPs of IL28B were determined. Serial alanine transaminase (ALT levels in the previous one year before enrollment at an interval of three months were recorded. Factors associated with active hepatitis, defined as persistent ALT >2× upper limit of normal (ULN or a peak ALT level >5× ULN, were evaluated. RESULTS: The prevalence of rs8105790 TT, rs12979860 CC, rs8099917 TT, and rs10853728 CC genotypes were 88.3%, 87.4%, 88.4% and 70.9%, respectively. In HBeAg-positive patients (n = 48, HBV viral load correlated with active hepatitis, while in HBeAg-negative patients (n = 67, rs10853728 CC genotype (p = 0.032 and a trend of higher IP-10 levels (p = 0.092 were associated with active hepatitis. In multivariate analysis, high viral load (HBV DNA >10(8 IU/mL, p = 0.042, odds ratio = 3.946 was significantly associated with HBeAg-positive hepatitis, whereas rs10853728 CC genotype (p = 0.019, odds ratio = 3.927 was the only independent factor associated with active hepatitis in HBeAg-negative population. CONCLUSIONS: HBV viral load and IL28B rs10853728 CC genotype correlated with hepatitis activity in HBeAg-positive and HBeAg-negative CHB, respectively. Both viral and host factors play roles in disease activity during different phases of CHB.

  7. Effect of small interfering RNA targeting transforming growth factor β receptor Ⅰ gene on the collagen synthesis of hepatic stellate cells in vitro%转化生长因子βⅠ型受体基因的靶向小干扰RNA抑制肝星状细胞合成胶原的体外研究

    Institute of Scientific and Technical Information of China (English)

    俞富军; 楼迪栋; 林镯; 董培红; 陈永平

    2010-01-01

    目的 观察大鼠转化生长因子βⅠ型受体(TβR Ⅰ)基因的靶向小干扰RNA(siRNA)表达质粒对肝星状细胞(HSC)合成胶原的影响.方法 根据大鼠Tβ R Ⅰ的基因序列,设计并构建3个大鼠TβR Ⅰ基因的靶向siRNA表达质粒,以脂质体作转染试剂,将siRNA表达质粒分别转染至HSC-T6细胞中,RT-PCR和Western印迹技术分析TβRⅠ mRNA及蛋白表达水平,噻唑蓝(MTT)法检测细胞增殖,放射免疫法测HA、PCⅢ含量.采用LSD法进行统计学处理.结果 酶切证实siRNA的目的 基因片段已成功克隆入载体中.与空白对照组比较,转染siRNA表达质粒后,3组HSC-T6细胞TβR Ⅰ mRNA表达水平均抑制,其中以psiRNA2组抑制作用最强(psiRNA1组:t=7.354,P<0.01;psiRNA2组:t=9.214,P<0.01;psiRNA3组:t=5.967,P<0.01).3组TβRⅠ蛋白表达水平均降低,以psiRNA2组降低最明显(psiRNA1组:t=6.324,P<0.01;psiRNA2组:t=8.741,P<0.01;psiRNA3组:t=4.128,P<0.01).3组HSC-T6细胞增殖活性均下降,合成胶原均减少,以psiRNA2组最明显;而转染无关siRNA组无明显变化.结论 构建的TβR Ⅰ siRNA表达质粒可抑制HSC-T6细胞合成胶原,为肝纤维化基因治疗提供新的靶点.%Objective To observe the effect of small interfering RNA(siRNA)expression plasmids targeting transforming growth factor p receptor(TαR)Ⅰ gene on the collagen synthesis of hepatic stellate cells(HSCs).Methods Three siRNA expression plasmids were designed and constructed according to TBR Ⅰ sequence.Then the plasmids were transfected into HSC-T6 using 1ipofectamine2000 reagent. The mRNA and protein expressions of TβR Ⅰ were analyzed by reverse transcription polymerase chain reaction(RT-PCR)and Western blot technique, respectively. The cell proliferation was detected using methylthiazo-lyldiphenyl-tetrazolium bromide(MTT)methods. Concentrations of haluronic acid and type Ⅲ pro-collagen in the supernatants were determined by radioimmunoassay. The data were analyzed using

  8. Steatosis recovery after treatment with a balanced sunflower or olive oil-based diet: Involvement of perisinusoidal stellate cells

    Institute of Scientific and Technical Information of China (English)

    Raquel Hernández; Esther Martínez-Lara; Ana Ca(n)uelo; Ma Luisa del Moral; Santos Blanco; Eva Siles; Ana Jiménez; Juan (A)ngel Pedrosa; Ma (A)ngeles Peinado

    2005-01-01

    AIM: To analyze the relationship between perisinusoidal stellate cell (PSC) activation and the dietary fat quantity and composition in the treatment of hepatic steatosis.METHODS: Using an experimental rat model of steatosis based on the intake of a hyperlipidic diet (14% fat as olive oil or sunflower oil, HL-O and HL-S, respectively), we analyzed the liver's capability of recovery after the treatment with a normal-lipidic diet (5% fat as olive oil or sunflower oil, NL-O and NL-S, respectively) by immunocytochemical and Western blot analysis of glial fibrillary acidic protein (GFAP) expression in PSCs, collagen quantification and serum aminotransferase determination.RESULTS: The fatty infiltration in the steatotic livers decreased after the treatment with both NL diets, indicating liver recovery. This decrease was accompanied with a lower collagen deposition and aminotransferase level as well as changes in the PSC population that increased the GFAP expression. The above-mentioned effects were more pronounced in animals fed on NL-O based diet. CONCLUSION: Treatment with a balanced dietenriched in olive oil contributes to the liver recovery from a stea totic process. The PSC phenotype is a marker of this hepatic-recovery model.

  9. Effects of phenoxyherbicides and glyphosate on the hepatic and intestinal biotransformation activities in the rat.

    Science.gov (United States)

    Hietanen, E; Linnainmaa, K; Vainio, H

    1983-08-01

    The effects of phenoxyacid herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and MCPA (4-chloro-2-methylphenoxyacetic acid), clofibrate, and glyphosate on hepatic and intestinal drug metabolizing enzyme activities were studied in rats intragastrically exposed for 2 weeks. The hepatic ethoxycoumarin O-deethylase activity increased about 2-fold with MCPA. Both 2,4-D and MCPA increased the hepatic epoxide hydrolase activity and decreased the hepatic glutathione S-transferase activity. MCPA also increased the intestinal activities of ethoxycoumarin O-deethylase and epoxide hydrolase. Glyphosate decreased the hepatic level of cytochrome P-450 and monooxygenase activities and the intestinal activity of aryl hydrocarbon hydroxylase. Clofibrate decreased the hepatic activities of UDPglucuronosyltransferase with p-nitrophenol or methylumbelliferone as the substrate. Also 2,4-D decreased the hepatic activity of UDPglucuronosyltransferase with p-nitrophenol as the substrate. MCPA decreased the intestinal activities of UDPglucuronosyltransferase with either p-nitrophenol or methylumbelliferone as the substrate. The results indicate that phenoxyacetic acids, especially MCPA, may have potent effects on the metabolism of xenobiotics. Glyphosate, not chemically related to phenoxyacids, seems to inhibit monooxygenases. Whether these changes are related to the toxicity of these xenobiotics remains to be clarified in further experiments.

  10. Cannabinoids reduce markers of inflammation and fibrosis in pancreatic stellate cells.

    Directory of Open Access Journals (Sweden)

    Christoph W Michalski

    Full Text Available BACKGROUND: While cannabinoids have been shown to ameliorate liver fibrosis, their effects in chronic pancreatitis and on pancreatic stellate cells (PSC are unknown. METHODOLOGY/PRINCIPAL FINDINGS: The activity of the endocannabinoid system was evaluated in human chronic pancreatitis (CP tissues. In vitro, effects of blockade and activation of cannabinoid receptors on pancreatic stellate cells were characterized. In CP, cannabinoid receptors were detected predominantly in areas with inflammatory changes, stellate cells and nerves. Levels of endocannabinoids were decreased compared with normal pancreas. Cannabinoid-receptor-1 antagonism effectuated a small PSC phenotype and a trend toward increased invasiveness. Activation of cannabinoid receptors, however, induced de-activation of PSC and dose-dependently inhibited growth and decreased IL-6 and MCP-1 secretion as well as fibronectin, collagen1 and alphaSMA levels. De-activation of PSC was partially reversible using a combination of cannabinoid-receptor-1 and -2 antagonists. Concomitantly, cannabinoid receptor activation specifically decreased invasiveness of PSC, MMP-2 secretion and led to changes in PSC phenotype accompanied by a reduction of intracellular stress fibres. CONCLUSIONS/SIGNIFICANCE: Augmentation of the endocannabinoid system via exogenously administered cannabinoid receptor agonists specifically induces a functionally and metabolically quiescent pancreatic stellate cell phenotype and may thus constitute an option to treat inflammation and fibrosis in chronic pancreatitis.

  11. Potential of Cannabidiol for the Treatment of Viral Hepatitis

    Science.gov (United States)

    Lowe, Henry I. C.; Toyang, Ngeh J.; McLaughlin, Wayne

    2017-01-01

    Viral hepatitis B (HBV) and hepatitis C (HCV) pose a major health problem globally and if untreated, both viruses lead to severe liver damage resulting in liver cirrhosis and cancer. While HBV has a vaccine, HCV has none at the moment. The risk of drug resistance, combined with the high cost of current therapies, makes it a necessity for cost-effective therapeutics to be discovered and developed. The recent surge in interest in Medical Cannabis has led to interest in evaluating and validating the therapeutic potentials of Cannabis and its metabolites against various diseases including viruses. Preliminary screening of cannabidiol (CBD) revealed that CBD is active against HCV but not against HBV in vitro. CBD inhibited HCV replication by 86.4% at a single concentration of 10 μM with EC50 of 3.163 μM in a dose-response assay. These findings suggest that CBD could be further developed and used therapeutically against HCV. SUMMARY Cannabidiol exhibited in vitro activity against viral hepatitis C. Abbreviations Used: CB2: Cannabis receptor 2, CBD: Cannabidiol, DNA: Deoxyribonucleic acid, HBV: Hepatitis B virus, HCV: Hepatitis C virus, HIV/AIDS: Human immunodeficiency virus/acquired immune deficiency syndrome, HSC: Hepatic stellate cells, MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, PCR: Polymerase chain reaction PMID:28250664

  12. The slowed brain: cortical oscillatory activity in hepatic encephalopathy.

    Science.gov (United States)

    Butz, Markus; May, Elisabeth S; Häussinger, Dieter; Schnitzler, Alfons

    2013-08-15

    Oscillatory activity of the human brain has received growing interest as a key mechanism of large-scale integration across different brain regions. Besides a crucial role of oscillatory activity in the emergence of other neurological and psychiatric diseases, recent evidence indicates a key role in the pathophysiology of hepatic encephalopathy (HE). This review summarizes the current knowledge on pathological alterations of oscillatory brain activity in association with liver dysfunction and HE in the context of spontaneous brain activity, motor symptoms, sensory processing, and attention. The existing literature demonstrates a prominent slowing of the frequency of oscillatory activity as shown for spontaneous brain activity at rest, with respect to deficits of motor behavior and motor symptoms, and in the context of visual attention processes. The observed slowing extends across different subsystems of the brain and has been confirmed across different frequency bands, providing evidence for ubiquitous changes of oscillatory activity in HE. For example, the frequency of cortico-muscular coherence in HE patients appears at the frequency of the mini-asterixis (⩽12Hz), while cirrhotics without overt signs of HE show coherence similar to healthy subjects, i.e. at 13-30Hz. Interestingly, the so-called critical flicker frequency (CFF) as a measure of the processing of an oscillating visual stimulus has emerged as a useful tool to quantify HE disease severity, correlating with behavioral and neurophysiological alterations. Moreover, the CFF reliably distinguishes patients with manifest HE from cirrhotics without any signs of HE and healthy controls using a cut-off frequency of 39Hz. In conclusion, oscillatory activity is globally slowed in HE in close association with HE symptoms and disease severity. Although the underlying causal mechanisms are not yet understood, these results indicate that pathological changes of oscillatory activity play an important role in the

  13. The multiple functions of T stellate/multipolar/chopper cells in the ventral cochlear nucleus.

    Science.gov (United States)

    Oertel, Donata; Wright, Samantha; Cao, Xiao-Jie; Ferragamo, Michael; Bal, Ramazan

    2011-06-01

    Acoustic information is brought to the brain by auditory nerve fibers, all of which terminate in the cochlear nuclei, and is passed up the auditory pathway through the principal cells of the cochlear nuclei. A population of neurons variously known as T stellate, type I multipolar, planar multipolar, or chopper cells forms one of the major ascending auditory pathways through the brainstem. T Stellate cells are sharply tuned; as a population they encode the spectrum of sounds. In these neurons, phasic excitation from the auditory nerve is made more tonic by feedforward excitation, coactivation of inhibitory with excitatory inputs, relatively large excitatory currents through NMDA receptors, and relatively little synaptic depression. The mechanisms that make firing tonic also obscure the fine structure of sounds that is represented in the excitatory inputs from the auditory nerve and account for the characteristic chopping response patterns with which T stellate cells respond to tones. In contrast with other principal cells of the ventral cochlear nucleus (VCN), T stellate cells lack a low-voltage-activated potassium conductance and are therefore sensitive to small, steady, neuromodulating currents. The presence of cholinergic, serotonergic and noradrenergic receptors allows the excitability of these cells to be modulated by medial olivocochlear efferent neurons and by neuronal circuits associated with arousal. T Stellate cells deliver acoustic information to the ipsilateral dorsal cochlear nucleus (DCN), ventral nucleus of the trapezoid body (VNTB), periolivary regions around the lateral superior olivary nucleus (LSO), and to the contralateral ventral lemniscal nuclei (VNLL) and inferior colliculus (IC). It is likely that T stellate cells participate in feedback loops through both medial and lateral olivocochlear efferent neurons and they may be a source of ipsilateral excitation of the LSO.

  14. Repressor and activator protein accelerates hepatic ischemia reperfusion injury by promoting neutrophil inflammatory response.

    Science.gov (United States)

    Li, Chang Xian; Lo, Chung Mau; Lian, Qizhou; Ng, Kevin Tak-Pan; Liu, Xiao Bing; Ma, Yuen Yuen; Qi, Xiang; Yeung, Oscar Wai Ho; Tergaonkar, Vinay; Yang, Xin Xiang; Liu, Hui; Liu, Jiang; Shao, Yan; Man, Kwan

    2016-05-10

    Repressor and activator protein (Rap1) directly regulates nuclear factor-κB (NF-κB) dependent signaling, which contributes to hepatic IRI. We here intended to investigate the effect of Rap1 in hepatic ischemia reperfusion injury (IRI) and to explore the underlying mechanisms. The association of Rap1 expression with hepatic inflammatory response were investigated in both human and rat liver transplantation. The effect of Rap1 in hepatic IRI was studied in Rap1 knockout mice IRI model in vivo and primary cells in vitro. Our results showed that over expression of Rap1 was associated with severe liver graft inflammatory response, especially in living donor liver transplantation. The results were also validated in rat liver transplantation model. In mice hepatic IRI model, the knockout of Rap1 reduced hepatic damage and hepatic inflammatory response. In primary cells, the knockout of Rap1 suppressed neutrophils migration activity and adhesion in response to liver sinusoidal endothelial cells through down-regulating neutrophils F-Actin expression and CXCL2/CXCR2 pathway. In addition, the knockout of Rap1 also decreased production of pro-inflammatory cytokines/chemokines in primary neutrophils and neutrophils-induced hepatocyte damage. In conclusion, Rap1 may induce hepatic IRI through promoting neutrophils inflammatory response. Rap1 may be the potential therapeutic target of attenuating hepatic IRI.

  15. Saturated fatty acids activate ERK signaling to downregulate hepatic sortilin 1 in obese and diabetic mice.

    Science.gov (United States)

    Bi, Lipeng; Chiang, John Y L; Ding, Wen-Xing; Dunn, Winston; Roberts, Benjamin; Li, Tiangang

    2013-10-01

    Hepatic VLDL overproduction is a characteristic feature of diabetes and an important contributor to diabetic dyslipidemia. Hepatic sortilin 1 (Sort1), a cellular trafficking receptor, is a novel regulator of plasma lipid metabolism and reduces plasma cholesterol and triglycerides by inhibiting hepatic apolipoprotein B production. Elevated circulating free fatty acids play key roles in hepatic VLDL overproduction and the development of dyslipidemia. This study investigated the regulation of hepatic Sort1 in obesity and diabetes and the potential implications in diabetic dyslipidemia. Results showed that hepatic Sort1 protein was markedly decreased in mouse models of type I and type II diabetes and in human individuals with obesity and liver steatosis, whereas increasing hepatic Sort1 expression reduced plasma cholesterol and triglycerides in mice. Mechanistic studies showed that the saturated fatty acid palmitate activated extracellular signal-regulated kinase (ERK) and inhibited Sort1 protein by mechanisms involving Sort1 protein ubiquitination and degradation. Consistently, hepatic ERK signaling was activated in diabetic mice, whereas blocking ERK signaling by an ERK inhibitor increased hepatic Sort1 protein in mice. These results suggest that increased saturated fatty acids downregulate liver Sort1 protein, which may contribute to the development of dyslipidemia in obesity and diabetes.

  16. Antiviral activity of glycyrrhizin against hepatitis C virus in vitro.

    Directory of Open Access Journals (Sweden)

    Yoshihiro Matsumoto

    Full Text Available Glycyrrhizin (GL has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc. To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp, replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD, respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2. We found that group 1B PLA2 (PLA2G1B inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release.

  17. Protective effects of L-carnosine on CCl4 -induced hepatic injury in rats.

    Science.gov (United States)

    Alsheblak, Mehyar Mohammad; Elsherbiny, Nehal M; El-Karef, Amro; El-Shishtawy, Mamdouh M

    2016-03-01

    The present study was undertaken to investigate the possible protective effect of L-carnosine (CAR), an endogenous dipeptide of alanine and histidine, on carbon tetrachloride (CCl4)-induced hepatic injury. Liver injury was induced in male Sprague-Dawley rats by intraperitoneal (i.p.) injections of CCl4, twice weekly for six weeks. CAR was administered to rats daily, at dose of 250 mg/kg, i.p. At the end of six weeks, blood and liver tissue specimens were collected. Results show that CAR treatment attenuated the hepatic morphological changes, necroinflammation and fibrosis induced by CCl4, as indicated by hepatic histopathology scoring. In addition, CAR treatment significantly reduced the CCl4-induced elevation of liver-injury parameters in serum. CAR treatment also combatted oxidative stress; possibly by restoring hepatic nuclear factor erythroid 2-related factor 2 (Nrf-2) levels. Moreover, CAR treatment prevented the activation of hepatic stellate cells (HSCs), as indicated by reduced α-smooth muscle actin (α-SMA) expression in the liver, and decreased hepatic inflammation as demonstrated by a reduction in hepatic tumor necrosis factor-α (TNF-α) and restoration of interleukin-10 (IL-10) levels. In conclusion, CCl4-induced hepatic injury was alleviated by CAR treatment. The results suggest that these beneficial, protective effects are due, at least in part, to its anti-oxidant, anti-inflammatory and anti-fibrotic activities.

  18. 大鼠结缔组织生长因子基因miRNA表达质粒的构建及其稳定转染大鼠肝星状细胞系的建立%Construction of miRNA expression vector for rat connective tissue growth factor and establishment of stably transfected rat hepatic stellate cell line

    Institute of Scientific and Technical Information of China (English)

    阳宏; 李孝生; 向颖; 邢旎旎; 沈艳

    2013-01-01

    目的 构建大鼠结缔组织生长因子(Connective tissue growth factor,CTGF)基因miRNA表达质粒,并建立稳定转染大鼠肝星状细胞(Hepatic stellate cell,HSC)系.方法 根据大鼠CTGF基因mRNA序列,设计并合成3对寡聚单链DNA X191-1、X191-2和X191-3及1对阴性对照序列DNA X191-4,将4对寡聚单链DNA退火成双链后,分别与载体pcDNA6.2-GW/EmGFP-miR连接,构建CTGF基因miRNA重组表达质粒,分别转染HSC-T6细胞,荧光显微镜观察细胞的转染效率,RT-PCR检测转染细胞中CTGF基因mRNA的转录水平;取干扰效率最高的重组质粒及阴性对照质粒,分别转染HSC-T6细胞,经杀稻瘟菌素持续加压筛选.结果 经测序鉴定,重组表达质粒构建正确,插入片段的碱基序列与设计相符;细胞的瞬时转染效率约为50%;3组干扰质粒转染的HSC-T6细胞中,CTGF基因mRNA的转录水平明显低于空白对照组(P<0.01),其中X191-2质粒对CTGF基因转录的干扰效率最高;获得了稳定转染的HSC-T6细胞.结论 成功构建了CTGF基因miRNA表达质粒,并获得了稳定转染的肝星状细胞系,为进一步研究肝纤维化的形成机制及其治疗奠定了基础.

  19. Effect of caffeine on proliferation,apoptosis in hepatic stellate cell-T6 stimulated by acetaldehyde and its partial mechanisms%咖啡因对乙醛刺激的大鼠肝星状细胞HSC-T6增殖、凋亡的影响及部分作用机制代

    Institute of Scientific and Technical Information of China (English)

    代雪飞; 吕雄文; 管文婕; 杨万枝; 李俊

    2011-01-01

    目的 探讨咖啡因(CAF)对乙醛刺激的大鼠肝星状细胞(HSC)-T6增殖、凋亡的影响及部分作用机制.方法用不同浓度的CAF(0.5、1、2、4、8 mmol/L)对乙醛刺激的HSC-T6进行处理,四甲基偶氮唑蓝(MTT)法检测细胞增殖;流式细胞仪检测细胞凋亡及细胞周期分布;RT-PCR法检测HSC-T6中平滑肌肌动蛋白(α-SMA)、肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体DR4、DR5的mRNA表达.结果 CAF对乙醛刺激的大鼠HSC-T6增殖具有抑制作用,阻滞细胞于G0/G1期;能够明显下调HSC-T6中α-SMA的mRNA表达,上调DR4、DR5的mRNA表达.结论 CAF对乙醛刺激的HSC-T6增殖具有抑制作用,并能够促进其凋亡,其机制可能与TRAIL受体DR4、DR5的表达有关.%Objective To explore the effect of caffeine on proliferation, apoptosis in hepatic stellate cell-T6( HSCT6 ) stimulated by acetaldehyde and its partial mechanisms. Methods HSC-T6 stimulated by acetaldehyde was incubated with different doses of caffeine( 0. 5 ,1 .2 ,4 ,8 mmol/L ) , cell proliferation was analyzed by MTT colorimetric assay, apoptosis rate and the cell cycle distribution were analyzed by flow cytometry( FCM ). The mRNA expressions of a-SMA.TRAIL receptor DR4 and DR5 were measured by RT-PCR. Results Caffeine could inhibit HSCT6 stimulated by acetaldehyde,the cells were accumulated in the G0/G1 phase. The mRNA expression levels of αSMA decreased,but DR4 and DR5 increased. Conclusion Caffeine can inhibit the growth of HSC-T6 stimulated by acetaldehyde and promote the apoptosis,the mechanisms may be related to the expression of TRAIL receptor DR4 and DR5.

  20. Overexpression of hepatic plasminogen activator inhibitor type 1 mRNA in rabbits with fatty liver

    Institute of Scientific and Technical Information of China (English)

    Jian-Gao Fan; Liang-Hua Chen; Zheng-Jie Xu; Min-De Zeng

    2001-01-01

    @@ INTRODUCTION Plasminogen activator inhibitor type 1 ( PAI-I ), an approximately Mr 50000 glycoprotein, is the major physiological inhibitor of plasminogen activators. It is not only the priming factor for atherosclerosis and coronary thrombosis[1-3] , but also participates in the genesis of chronic hepatitis and liver fibrosis[4-11] . However, there has been no available report yet about the research of hepatic PAl-1 gene expression in hyperlipidemia and fatty liver. The present study aimed to explore the change of hepatic PAl-1 mRNA and its plasma activity by means of animal model.

  1. Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor

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    Kumi Kimura

    2016-03-01

    Full Text Available Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity.

  2. Chronic viral hepatitis C in pediatric age group; assessment of viral activity and hepatic fibrosis by 1H magnetic resonance spectroscopy and diffusion weighted imaging in asymptomatic

    Directory of Open Access Journals (Sweden)

    Shereen Mansour Galal

    2016-09-01

    Conclusion: Early diagnosis of asymptomatic chronic hepatitis C is essential to prevent or delay end stage chronic parenchymal liver disease. 1H MRS may be a potential noninvasive helpful diagnostic tool in the assessment of staging and fibrosis of asymptomatic chronic hepatitis C. The increase in metabolites were correlated with histopathological changes. DW-MRI can be considered as an effective predictor in the assessment of activity in chronic hepatitis C.

  3. Role of YAP and TAZ in pancreatic ductal adenocarcinoma and in stellate cells associated with cancer and chronic pancreatitis.

    Science.gov (United States)

    Morvaridi, Susan; Dhall, Deepti; Greene, Mark I; Pandol, Stephen J; Wang, Qiang

    2015-11-16

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic and inflammatory microenvironment that is formed primarily by activated, myofibroblast-like, stellate cells. Although the stellate cells are thought to contribute to tumorigenesis, metastasis and drug resistance of PDAC, the signaling events involved in activation of the stellate cells are not well defined. Functioning as transcription co-factors, Yes-associated protein (YAP) and its homolog transcriptional co-activator with PDZ-binding motif (TAZ) modulate the expression of genes involved in various aspects of cellular functions, such as proliferation and mobility. Using human tissues we show that YAP and TAZ expression is restricted to the centroacinar and ductal cells of normal pancreas, but is elevated in cancer cells. In particular, YAP and TAZ are expressed at high levels in the activated stellate cells of both chronic pancreatitis and PDAC patients as well as in the islets of Langerhans in chronic pancreatitis tissues. Of note, YAP is up regulated in both acinar and ductal cells following induction of acute and chronic pancreatitis in mice. These findings indicate that YAP and TAZ may play a critical role in modulating pancreatic tissue regeneration, neoplastic transformation, and stellate cell functions in both PDAC and pancreatitis.

  4. 2-octynoic acid inhibits hepatitis C virus infection through activation of AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Darong Yang

    Full Text Available Many chronic hepatitis C virus (HCV-infected patients with current therapy do not clear the virus. It is necessary to find novel treatments. The effect of 2-octynoic acid (2-OA on HCV infection in human hepatocytes was examined. The mechanism of 2-OA antiviral activity was explored. Our data showed that 2-OA abrogated lipid accumulation in HCV replicon cells and virus-infected hepatocytes. It suppressed HCV RNA replication and infectious virus production with no cytotoxicity to the host cells. 2-OA did not affect hepatitis B virus replication in HepG2.2.15 cells derived from HepG2 cells transfected with full genome of HBV. Further study demonstrated that 2-OA activated AMP-activated protein kinase (AMPK and inhibited acetyl-CoA carboxylase in viral-infected cells. Compound C, a specific inhibitor of AMPK, inhibited AMPK activity and reversed the reduction of intracellular lipid accumulation and the antiviral effect of 2-OA. Knockdown of AMPK expression by RNA interference abolished the activation of AMPK by 2-OA and blocked 2-OA antiviral activity. Interestingly, 2-OA induced interferon-stimulated genes (ISGs and inhibited microRNA-122 (miR-122 expression in virus-infected hepatocytes. MiR-122 overexpression reversed the antiviral effect of 2-OA. Furthermore, knockdown of AMPK expression reversed both the induction of ISGs and suppression of miR-122 by 2-OA, implying that activated AMPK induces the intracellular innate response through the induction of ISGs and inhibiting miR-122 expression. 2-OA inhibits HCV infection through regulation of innate immune response by activated AMPK. These findings reveal a novel mechanism by which active AMPK inhibits HCV infection. 2-OA and its derivatives hold promise for novel drug development for chronic hepatitis C.

  5. Female hepatology: Favorable role of estrogen in chronic liver disease with hepatitis B virus infection

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Chronic hepatitis B virus (HBV) infection is the most common cause of hepatic fibrosis and hepatocellular carcinoma (HCC), mainly as a result of chronic necroinflammatory liver disease. A characteristic feature of chronic hepatitis B infection, alcoholic liver disease and nonalcoholic fatty liver disease (NAFLD) is hepatic steatosis. Hepatic steatosis leads to an increase in lipid peroxidation in hepatocytes, which, in turn, activates hepatic stellate cells (HSCs). HSCs are the primary target cells for inflammatory and oxidative stimuli, and these cells produce extracellular matrix components.Chronic hepatitis B appears to progress more rapidly in males than in females, and NAFLD, cirrhosis and HCC are predominately diseases that tend to occur in men and postmenopausal women. Premenopausal women have lower hepatic iron stores and a decreased production of proinflammatory cytokines. Hepatic steatosis has been observed in aromatase-deficient mice, and has been shown to decrease in animals after estradiol treatment. Estradiol is a potent endogenous antioxidant which suppresses hepatic fibrosis in animal models, and attenuates induction of redox sensitive transcription factors, hepatocyte apoptosis and HSC activation by inhibiting a generation of reactive oxygen species in primary cultures. Variant estrogen receptors are expressed to a greater extent in male patients with chronic liver disease than in females. These lines of evidence suggest that the greater progression of hepatic fibrosis and HCC in men and postmenopausal women may be due, at least in part, to lower production of estradiol and a reduced response to the action of estradiol. A better understanding of the basic mechanisms underlying the sex-associated differences in hepatic fibrogenesis and carciogenesis may open up new avenues for the prevention and treatment of chronic liver disease.

  6. Genetic abolishment of hepatocyte proliferation activates hepatic stem cells.

    Directory of Open Access Journals (Sweden)

    Yoko Endo

    Full Text Available Quiescent hepatic stem cells (HSCs can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1, an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

  7. Effects of PI3K/Akt signaling pathway on acetaldehyde-stimulated proliferation of hepatic stellate cells HSC-T6%PI3K/Akt 信号传导通路在乙醛刺激下大鼠肝星状细胞株 HSC-T6增殖中的作用

    Institute of Scientific and Technical Information of China (English)

    张迪; 程敏; 康馨丹; 陈卫刚; 郑勇

    2016-01-01

    目的:探讨磷脂酰肌醇3激酶/蛋白激酶 B(PI3K/Akt)信号传导通路在乙醛刺激的大鼠肝星状细胞株HSC-T6增殖中的作用。方法将大鼠 HSC-T6随机分为6组:空白对照组(A 组):采用10%胎牛血清的 DMEM培养液培养;乙醛组(B 组):在 A 组基础上加乙醛,使终浓度为200μmol/L;实验组在 B 组基础上加入不同浓度(25、50、75、100μmol/L)的 PI3K/Akt 通路抑制剂 LY294002,分别称为 C、D、E、F 组。用 CCK-8法检测大鼠 HSC-T6增殖活力(用 A 值表示)。再随机将 HSC-T6分为3组:空白对照组(10%胎牛血清的 DMEM培养液培养)、乙醛组(在空白对照组基础上加乙醛,使终浓度为200μmol/L)、乙醛+LY294002组(在乙醛组的基础上加入50μmol/L LY294002),用 Western blotting 法检测大鼠 HSC-T6中 PI3K、p-Akt 蛋白的表达情况。结果 A、B、C、D、E、F 组 A 值分别为0.5450±0.0244、0.9523±0.0455、0.9273±0.0419、0.8658±0.0289、0.8143±0.0247、0.7593±0.2200。与 A 组比较,其他各组 A 值均升高;与 B、C 组比较,D、E、F 组降低(P 均<0.01)。与空白对照组比较,乙醛组、乙醛+LY294002组 PI3K、p-AKT 蛋白相对表达量高(P 均<0.01),与乙醛组比较,乙醛+LY294002组降低(P <0.05)。结论 PI3K/Akt 信号传导通路部分参与调控乙醛刺激的大鼠 HSC-T6增殖。%Objective To investigate the effects of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway spe-cific inhibitor LY294002 on the proliferation of rat hepatic stellate cells (HSC-T6)stimulated by acetaldehyde.Methods The rat HSC-T6 cells were randomly divided into six groups:the blank control group (group A):we used DMEMculture solution with 10% fetal bovine serum (FBS)to culture,the acetaldehyde group (group B):we added acetaldehyde on the basis of the blank control group and made the final

  8. Ultrastructural changes in hepatic sinusoidal endothelial cells acutely exposed to colloidal iron.

    Science.gov (United States)

    Bassett, Mark L; Dahlstrom, Jane E; Taylor, Matthew C; Koina, Mark E; Maxwell, Lesley; Francis, Douglas; Jain, Sanjiv; McLean, Allan J

    2003-07-01

    Hepatic sinusoidal endothelial cells form an important interface between the vascular system, represented by the sinusoids, and the space of Disse that surrounds the hepatocyte microvilli. This study aimed to assess the light microscopic and ultrastructural effects of acute exposure of hepatic sinusoidal endothelial cells to colloidal iron by injection of rats with iron polymaltose. Eight minutes after a single intravenous injection of iron polymaltose sinusoidal endothelial cells showed defenestration, and thickening and layering as assessed by transmission electron microscopy. Kupffer cells and stellate cells appeared activated. These changes were not observed in control animals, experiments using equivalent doses of maltose, or experiments using colloidal carbon except for Kupffer cell activation due to colloidal carbon. No significant light microscopic changes were seen in study or control animals. The findings indicate that acute exposure to colloidal iron causes changes in hepatic sinusoidal endothelial cells, stellate cells and Kupffer cells. This may be the result of a direct toxic effect of iron or increased production of reactive oxygen species. These observations suggest a possible mechanism for defenestration of sinusoidal endothelial cells in ageing and in disease states.

  9. Chronic active hepatitis in transgenic mice expressing interferon-gamma in the liver.

    OpenAIRE

    1994-01-01

    Interferon-gamma may play an important role in the immune response and in inflammatory diseases, including chronic active hepatitis. To understand the role of interferon-gamma in the regulation of inflammation and to establish a mouse model of chronic active hepatitis, we produced transgenic mice in which the mouse interferon-gamma gene was regulated by a liver-specific promoter, the serum amyloid P component gene promoter. Four transgenic mouse lines were generated, and two of these lines ex...

  10. The hepatoselective glucokinase activator PF-04991532 ameliorates hyperglycemia without causing hepatic steatosis in diabetic rats.

    Directory of Open Access Journals (Sweden)

    Derek M Erion

    Full Text Available Hyperglycemia resulting from type 2 diabetes mellitus (T2DM is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that

  11. The hepatoselective glucokinase activator PF-04991532 ameliorates hyperglycemia without causing hepatic steatosis in diabetic rats.

    Science.gov (United States)

    Erion, Derek M; Lapworth, Amanda; Amor, Paul A; Bai, Guoyun; Vera, Nicholas B; Clark, Ronald W; Yan, Qingyun; Zhu, Yimin; Ross, Trenton T; Purkal, Julie; Gorgoglione, Matthew; Zhang, Guodong; Bonato, Vinicius; Baker, Levenia; Barucci, Nicole; D'Aquila, Theresa; Robertson, Alan; Aiello, Robert J; Yan, Jiangli; Trimmer, Jeff; Rolph, Timothy P; Pfefferkorn, Jeffrey A

    2014-01-01

    Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase

  12. Thalamic pain alleviated by stellate ganglion block

    Science.gov (United States)

    Liao, Chenlong; Yang, Min; Liu, Pengfei; Zhong, Wenxiang; Zhang, Wenchuan

    2017-01-01

    Abstract Rationale: Thalamic pain is a distressing and treatment-resistant type of central post-stroke pain. Although stellate ganglion block is an established intervention used in pain management, its use in the treatment of thalamic pain has never been reported. Patient concerns: A 66-year-old woman presented with a 3-year history of severe intermittent lancinating pain on the right side of the face and the right hand. The pain started from the ulnar side of the right forearm after a mild ischemic stroke in bilateral basal ganglia and left thalamus. Weeks later, the pain extended to the dorsum of the finger tips and the whole palmar surface, becoming more severe. Meanwhile, there was also pain with similar characteristics emerging on her right face, resembling atypical trigeminal neuralgia. Diagnoses: Thalamic pain was diagnosed. Interventions: After refusing the further invasive treatment, she was suggested to try stellate ganglion block. Outcomes: After a 3-day period of pain free (numerical rating scale: 0) postoperatively, she reported moderate to good pain relief with a numerical rating scale of about 3 to 4 lasting 1 month after the first injection. Pain as well as the quality of life was markedly improved with less dose of analgesic agents. Lessons: Stellate ganglion block may be an optional treatment for thalamic pain. PMID:28151918

  13. Ultrastructure of oval cells in children with chronic hepatitis B, with special emphasis on the stage of liver fibrosis: The first pediatric study

    Institute of Scientific and Technical Information of China (English)

    Maria Elzbieta Sobaniec-Lotowska; Joanna Maria Lotowska; Dariusz Marek Lebensztejn

    2007-01-01

    AIM: To investigate the ultrastructure of oval ceils in children with chronic hepatitis B, with special emphasis on their location in areas of collagen fibroplasia.METHODS: Morphological investigations were conducted on biopsy material obtained from 40 children,aged 3-16 years with chronic hepatitis B. The stage of fibrosis was assessed histologically using the arbitrary semiquantitative numerical scoring system proposed by Ishak et al. The material for ultrastructural investigation was fixed in glutaraldehyde and paraformaldehyde and processed for transmission-electron microscopic analysis.RESULTS: Ultrastructural examination of biopsy specimens obtained from children with chronic hepatitis B showed the presence of two types of oval cells, the hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis (at least stage 2 according to Ishak et al) and in the vicinity of the limiting plate of the lobule. The activated nonparenchymal hepatic cells, i.e. transformed hepatic stellate cells and Kupffer cells were seen in close proximity to the intermediate hepatic-like cells.CONCLUSION: We found a distinct relationship between the prevalence of oval cells (hepatic progenitor cells and intermediate hepatocyte-like cells) and fibrosis stage in pediatric patients with chronic hepatitis B.

  14. Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2*

    Science.gov (United States)

    Hogan, Meghan F.; Ravnskjaer, Kim; Matsumura, Shigenobu; Huising, Mark O.; Hull, Rebecca L.; Kahn, Steven E.; Montminy, Marc

    2015-01-01

    Under fasting conditions, increases in circulating concentrations of glucagon maintain glucose homeostasis via the induction of hepatic gluconeogenesis. Triggering of the cAMP pathway in hepatocytes stimulates the gluconeogenic program via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where it contributes to the attendant hyperglycemia. Whether selective activation of the hepatic CREB/CRTC pathway is sufficient to trigger metabolic changes in other tissues is unclear, however. Modest hepatic expression of a phosphorylation-defective and therefore constitutively active CRTC2S171,275A protein increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A-expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice, demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis. PMID:26342077

  15. Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2.

    Science.gov (United States)

    Hogan, Meghan F; Ravnskjaer, Kim; Matsumura, Shigenobu; Huising, Mark O; Hull, Rebecca L; Kahn, Steven E; Montminy, Marc

    2015-10-23

    Under fasting conditions, increases in circulating concentrations of glucagon maintain glucose homeostasis via the induction of hepatic gluconeogenesis. Triggering of the cAMP pathway in hepatocytes stimulates the gluconeogenic program via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where it contributes to the attendant hyperglycemia. Whether selective activation of the hepatic CREB/CRTC pathway is sufficient to trigger metabolic changes in other tissues is unclear, however. Modest hepatic expression of a phosphorylation-defective and therefore constitutively active CRTC2S171,275A protein increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A-expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice, demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis.

  16. Modulation of catecholamine-synthesizing enzymes in adrenal medulla and stellate ganglia by treadmill exercise of stressed rats.

    Science.gov (United States)

    Gavrilovic, Ljubica; Spasojevic, Natasa; Dronjak, Sladjana

    2012-03-01

    The sympatho-adrenal system represents one of the main systems involved in the response to stressful events because its stress-induced activation results in an increased release of catecholamines. Exercise training acts as an important modulator of sympatho-adrenal system, adrenal medulla and stellate ganglia being two components of this system. This study aimed at investigating physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase in the adrenal medulla and stellate ganglia of chronically psychosocially stressed adult rats exposed daily to 20-min treadmill exercise for 12 weeks, using TaqMan RT-PCR assay. Chronic psychosocial stress decreased gene expression of the examined enzymes in the adrenal medulla and treadmill exercise did not lead to further modulation of the corresponding gene expression. On the other hand, chronic psychosocial stress produced a significant increase of TH (about 51%) and DBH (about 103%) gene expression in stellate ganglia, while treadmill exercise decreased gene expression of these enzymes to control levels in psychosocially stressed rats. Our data indicate that treadmill exercise leads to a decreased gene transcription of catecholamine biosynthetic enzymes in stellate ganglia and attenuation of cardiac noradrenaline production in stressful situations. Reduction of catecholamine synthesis in stellate ganglia may be linked to the beneficial effects of treadmill exercise on cardiovascular system in stressed animals.

  17. Identification of 6-octadecynoic acid from a methanol extract of Marrubium vulgare L. as a peroxisome proliferator-activated receptor γ agonist.

    Science.gov (United States)

    Ohtera, Anna; Miyamae, Yusaku; Nakai, Naomi; Kawachi, Atsushi; Kawada, Kiyokazu; Han, Junkyu; Isoda, Hiroko; Neffati, Mohamed; Akita, Toru; Maejima, Kazuhiro; Masuda, Seiji; Kambe, Taiho; Mori, Naoki; Irie, Kazuhiro; Nagao, Masaya

    2013-10-18

    6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Fibrogenesis caused by hepatic stellate cells is inhibited by PPARγ whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPARγ agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPARγ agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPARγ in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPARγ-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPARγ agonists.

  18. Variability of human hepatic UDP-glucuronosyltransferase activity

    NARCIS (Netherlands)

    Little, JM; Lester, R; Kuipers, F; Vonk, R; Mackenzie, PI; Drake, RR; Frame, L; Radominska-Pandya, A

    1999-01-01

    The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases usi

  19. Inhibitory effect of liposomal quercetin on acute hepatitis and hepatic fibrosis induced by concanavalin A

    Directory of Open Access Journals (Sweden)

    Y. Wan

    2014-08-01

    Full Text Available Immune response plays an important role in the development of hepatic fibrosis. In the present study, we investigated the effects of quercetin on hepatitis and hepatic fibrosis induced by immunological mechanism. In the acute hepatitis model, quercetin (2.5 mg/kg was injected iv into mice 30 min after concanavalin A (Con A challenge. Mice were sacrificed 4 or 24 h after Con A injection, and aminotransferase tests and histopathological sections were performed. Treatment with quercetin significantly decreased the levels of alanine aminotransferase (ALT and aspartate aminotransferase (AST. Consistent with this observation, treatment with quercetin markedly attenuated the pathologic changes in the liver. A hepatic fibrosis model was also generated in mice by Con A challenge once a week for 6 consecutive weeks. Mice in the experimental group were treated with daily iv injections of quercetin (0.5 mg/kg. Histopathological analyses revealed that treatment with quercetin markedly decreased collagen deposition, pseudolobuli development, and hepatic stellate cells activation. We also examined the effects of quercetin on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB and transforming growth factor beta (TGF-β pathways by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR. NF-κB and TGF-β production was decreased after treatment with quercetin, indicating that the antifibrotic effect of quercetin is associated with its ability to modulate NF-κB and TGF-β production. These results suggest that quercetin may be an effective therapeutic strategy in the treatment of patients with liver damage and fibrosis.

  20. 肝星状细胞条件培养基对肝癌细胞PLC/PRF/5耐药性的影响及其机制%Effect of hepatic stellate cell condition medium on chemo-resistance of hepatocellular carcinoma PLC/PRF/5 cells and its mechanism

    Institute of Scientific and Technical Information of China (English)

    喻国锋; 井莹莹; 寇兴瑞; 李蓉; 吴孟超; 卫立辛

    2013-01-01

    目的:探讨肝星状细胞条件培养基(hepatic stellate cell conditioned medium,HSC-CM)对人肝癌PLC/PRF/5细胞耐药性的影响及其可能的机制.方法:用无血清RPMI 1640培养肝星状细胞LX-2,使其在缺营养的环境下活化,收集其条件培养上清即为HSC-CM.PLC/PRF/5细胞在HSC-CM条件下培养24 h后,顺铂处理12 h或24 h,采用流式细胞术检测PLC/PRF/5细胞的凋亡情况,MTT法检测PLC/PRF/5细胞的增殖,real-time PCR检测PLC/PRF/5细胞上皮间质转化(epithelial mesenchymal transition,EMT)相关基因的表达水平.结果:顺铂组12和24h两个时间点PLC/PRF/5细胞的凋亡率为(22.34±1.12)%和(26.78±1.56)%;HSC-CM+顺铂组细胞的凋亡率为(17.22±1.42)%和(21.52±1.76)%,顺铂组细胞凋亡率显著高于HSC-CM+顺铂组(P<0.05).同在这两个时间点,顺铂组和HSC-CM+顺铂组PLC/PRF/5细胞的增殖活性分别为(68.65±2.56)%和(79.47 ±1.43)%,(46.54 ±3.65)%和(62.77±2.89)%,HSC-CM+顺铂组细胞增殖活性均高于顺铂组(P<0.05).Real-time PCR结果显示,与顺铂组相比较,HSC-CM+顺铂组PLC/PRF/5细胞中上皮标记物钙黏蛋白(E-cadherin)的表达下降(P<0.05),而间质细胞标记物神经黏附素(N-cadherin)、波形蛋白(vimentin)以及EMT相关转录因子Snail和ZEB1的表达显著上调(P<0.01).结论:HSC-CM可能通过诱导PLC/PRF/5细胞发生EMT,从而增强PLC/PRF/5细胞对顺铂的抵抗作用.

  1. Angiotensin (1-7) inhibits angiotensin Ⅱ-stimulated expression of connective tissue growth factor mRNA in hepatic stellate cells%血管紧张素(1-7)对血管紧张素Ⅱ诱导肝星状细胞表达结缔组织生长因子的抑制作用

    Institute of Scientific and Technical Information of China (English)

    李旭; 黄茂梁; 黄珊; 张文雍; 宁佐伟; 孟莹

    2012-01-01

    结合后对AngⅡ激活的CTGF mRNA的表达具有抑制作用.%Objective To explore the angiotensin peptide [Ang (1-7)]-mediated inhibition of Ang Ⅱ in human hepatic stellate cells (HSCs) and determine the involvement of the ACE2-Ang (1-7)-Mas axis.Methods The human HSC line,LX2,was used in all experiments,and divided into control (unstimulated)and Ang Ⅱ-stimulated (10-6 mol/L) groups.The Ang Ⅱ-stimulated cells were further divided among several pretreatment (prior to Ang Ⅱ) groups:ROCK-inhibited (Y27632 blocking agent,10-6mol/L); irbesartan-inhibited (AT-1 receptor antagonist,10-6mol/L); and Mas receptor-inhibited (A779 Mas receptor antagonist,10-6mol/L).To explore the potential inhibitory effects of various Ang family members,the Ang Ⅱ-stimulated and pre-treated LX2 cells were exposed to Ang (1-7) (10-6 mol/L) for 24h.Western blot,reverse transcription-polymerase chain reaction (RT-PCR),and QuantiGene assay were used to assess changes in protein and mRNA expression levels of RhoA,ROCK,and connective tissue growth factor (CTGF).Results Compared with the control group,Ang Ⅱ-stimulated cells showed significantly increased levels of RhoA protein (0.337±0.074 vs.0.870±0.093),ROCK2 mRNA (0.747±0.061 vs.0.368±0.023),and CTGF mRNA (0.262±0.007 vs.0.578±0.028) (all,P<0.01).Pre-treatment with irbesartan 0r Y27632 eliminated these responses.Ang (1-7) inhibited the Ang Ⅱ-stimulated up-regulation of RhoA,ROCK,and CTGF.Conclusion Ang (1-7) can inhibit the Ang Ⅱ-stimulated up-regulation of RhoA,ROCK and CTGF in hepatic stellate cells,indicating that the ACE2-Ang (1-7)-Mas axis,an important branch of the renin-Ang-aldosterone system is involved in the occurrence and development of liver fibrosis.

  2. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    Energy Technology Data Exchange (ETDEWEB)

    Shlomai, Amir, E-mail: amirsh@tasmc.health.gov.il [Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel); Institute for Gastroenterology and Liver disease, Tel-Aviv Sourasky Medical Center, 6 Weizmann street, Tel-Aviv (Israel); Shaul, Yosef [Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel)

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  3. 单次大剂量照射对大鼠肝星形细胞、细胞TGF-β及PI3K/Akt信号通路分子p-Akt(S473)的影响%Effect of single high-dose irradiation on rat hepatic stellate cells,transforming growth factor β,and PI3K/Akt signaling pathway molecule p-Akt(S473)

    Institute of Scientific and Technical Information of China (English)

    肖蕾; 吴戈; 王云莲; 程纬; 艾尼瓦尔・艾木都拉; 毛睿; 张华; 包永星

    2016-01-01

    Objective To investigate the effect of single high-dose irradiation on rat hepatic stellate cells (HSCs),transforming growth factor β(TGF-β),and PI3K/Akt signaling pathway.Methods A total of 40 healthy male Sprague-Dawley rats were randomly divided into model group (30 rats) and control group (10 rats).The rats in the model group were given single 6 MV X-ray irradiation of the right liver at a dose of 30 Gy,and those in the control group were given sham irradiation with the same method.At 3,5,and 10 days after irradiation,10 rats in the model group and 3-4 rats in the control group were randomly selected to measure the changes in the serum levels of aspartate aminotransferase ( AST),alanine aminotransferase (ALP),alkaline phosphatase (ALP),total bilirubin (TB),and direct bilirubin ( DB).Hematoxylin-eosin (HE) staining and Masson staining were applied for liver tissues to observe the pathomorphological changes, and immunohistochemistry was used to measure the expression of transforming growth factor-beta 1(TGF-β1 ),alpha-smooth muscle actin (α-SMA),and p-Akt (S473) in liver tissues.Results At 3,5,and 10 days after irradiation,the model group showed increases in the serum levels of AST,ALT,ALP,TB,and DB, and had significantly higher serum levels of these indices than the control group.The HE staining and Masson staining showed that compared with the control group,the rats with acute liver injury induced by radiation in the model group had a significant formation of collagen fibers around the hepatic sinusoids. Immunohistochemistry showed that with the prolonged time after irradiation,the expression of TGF-β1 ,α-SMA,and p-Akt ( S473) was gradually enhanced after irradiation, and IPP analysis showed significant differences between the two groups ( P<0.000).Conclusions During the process of acute radiation-induced liver injury induced by single high-dose irradiation,with the activation of HSCs,the secretion of TGF-β1 is increased and the PI3K/Akt signaling

  4. Correlation of biological serum markers with the degree of hepatic fibrosis and necroinflammatory activity in hepatitis C and schistosomiasis patients

    Directory of Open Access Journals (Sweden)

    Clarice Neuenschwander Lins de Morais

    2010-07-01

    Full Text Available Liver biopsy is the gold-standard method to stage fibrosis; however, it is an invasive procedure and is potentially dangerous. The main objective of this study was to evaluate biological markers, such as cytokines IL-13, IFN-γ, TNF-α and TGF-β, platelets, bilirubins (Bil, alanine aminotransferase (ALT and aspartate aminotransferase (AST, total proteins, γ-glutamil transferase (γ-GT and alkaline phosphatase (AP, that could be used to predict the severity of hepatic fibrosis in schistosomiasis and hepatitis C (HC as isolated diseases or co-infections. The following patient groups were selected: HC (n = 39, HC/hepatosplenic schistosomiasis (HSS (n = 19, HSS (n = 22 and a control group (n = 13. ANOVA and ROC curves were used for statistical analysis. P < 0.05 was considered significant. With HC patients we showed that TNF-α (p = 0.020 and AP (p = 0.005 could differentiate mild and severe fibrosis. With regard to necroinflammatory activity, AST (p = 0.002, γ-GT (p = 0.034 and AP (p = 0.001 were the best markers to differentiate mild and severe activity. In HC + HSS patients, total Bil (p = 0.008 was capable of differentiating between mild and severe fibrosis. In conclusion, our study was able to suggest biological markers that are non-invasive candidates to evaluate fibrosis and necroinflammatory activity in HC and HC + HSS.

  5. P0525 : N-Acetylated alpha smooth muscle actin levels are increased in hepatic fibrosis but decreased in hepatocellular carcinoma

    DEFF Research Database (Denmark)

    Nielsen, M.J.; Nielsen, Signe Holm; Hansen, N.U.B.

    2015-01-01

    Alpha Smooth Muscle Actin (a-SMA) is upregulated together with extracellular matrix (ECM) during activation of Hepatic Stellate Cells (HSCs) in fibrosis. Histone deacetylase (HDAC) remove acetylations and regulate the expression of genes, which is associated with cancers. There is a close...... relationship between cirrhosis and hepatocellular carcinoma (HCC), and markers enabling identification of patients in risk of developing HCC with cirrhosis is a major unmet clinical need. We developed an ELISA for the assessment of acetylated a-SMA (Aca- SMA) in serum. The objective was to investigate...

  6. Activation of peroxisome proliferator-activated receptor gamma by rosiglitazone increases sirt6 expression and ameliorates hepatic steatosis in rats.

    Directory of Open Access Journals (Sweden)

    Soo Jin Yang

    Full Text Available BACKGROUND: Sirt6 has been implicated in the regulation of hepatic lipid metabolism and the development of hepatic steatosis. The aim of this study was to address the potential role of Sirt6 in the protective effects of rosiglitazone (RGZ on hepatic steatosis. METHODS: To investigate the effect of RGZ on hepatic steatosis, rats were treated with RGZ (4 mg·kg⁻¹·day⁻¹ by stomach gavage for 6 weeks. The involvement of Sirt6 in the RGZ's regulation was evaluated by Sirt6 knockdown in AML12 mouse hepatocytes. RESULTS: RGZ treatment ameliorated hepatic lipid accumulation and increased expression of Sirt6, peroxisome proliferator-activated receptor gamma coactivtor-1-α (Ppargc1a/PGC1-α and Forkhead box O1 (Foxo1 in rat livers. AMP-activated protein kinase (AMPK phosphorylation was also increased by RGZ, accompanied by alterations in phosphorylation of LKB1. Interestingly, in free fatty acid-treated cells, Sirt6 knockdown increased hepatocyte lipid accumulation measured as increased triglyceride contents (p = 0.035, suggesting that Sirt6 may be beneficial in reducing hepatic fat accumulation. In addition, Sirt6 knockdown abolished the effects of RGZ on hepatocyte fat accumulation, mRNA and protein expression of Ppargc1a/PGC1-α and Foxo1, and phosphorylation levels of LKB1 and AMPK, suggesting that Sirt6 is involved in RGZ-mediated metabolic effects. CONCLUSION: Our results demonstrate that RGZ significantly decreased hepatic lipid accumulation, and that this process appeared to be mediated by the activation of the Sirt6-AMPK pathway. We propose Sirt6 as a possible therapeutic target for hepatic steatosis.

  7. Decreased peripheral natural killer cells activity in the immune activated stage of chronic hepatitis B.

    Directory of Open Access Journals (Sweden)

    Yuan Li

    Full Text Available BACKGROUND & AIMS: The natural course of chronic hepatitis B virus (HBV infection is characterized by different immune responses, ranging from immune tolerant (IT to immune activated (IA stages. In our study, we investigated the natural killer (NK cells activity in patients at different immunological stages of chronic HBV infection. METHODS: Blood samples obtained from 57 HBeAg positive patients with chronic hepatitis B (CHB, including 15 patients in the immune tolerant (IT stage, 42 patients in the immune activated (IA stage, and 18 healthy individuals (HI. The analyses included flow cytometry to detect NK cells, the determination of cytokine levels as well as of surface receptor expression and cytotoxicity. RESULTS: NK cells in peripheral blood were significantly lower in patients in the IA stage of CHB compared to HI (p<0.05. Patients in the IA stage of CHB had lower levels of NK cells activating receptor NKp30 and NKG2D expression, cytokine interferon-γ (IFN-γ and tumor necrosis factor-α (TNF-α production, as compared to patients in the IT stage and HI, respectively (p<0.05. Cytotoxicity of NK cells was lower in patients in the IA stage of CHB compared to patients in the IT stage and HI, respectively (p<0.05. The level of IFN-γ but not level of TNF-α and cytotoxicity of NK cells was inversely correlated with serum HBV load in patients with CHB. Peripheral NK cells activity did not correlate with ALT level. CONCLUSION: NK cells activity was lower in CHB patients, especially in those in the IA stage.

  8. Adeno-associated virus mediated interferon-gamma inhibits the progression of hepatic fibrosis in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Miao Chen; Guang-Ji Wang; Yong Diao; Rui-An Xu; Hai-Tang Xie; Xin-Yan Li; Jian-Guo Sun

    2005-01-01

    AIM: To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosisin vitro and in vivo.METHODS: We constructed the recombinant AAV encoding human INF-γ (rAAV- INF-γ) and took the primary rat hepatic stellate cells and carbon tetrachloride induced rats as the experimental hepatic fibrosis model in vitro and in vivo. Immunocytochemistry analysis was used to reveal the expression of α-SMA, the marker protein expressed in hepatic stellate cells. The mRNA expression of TGF-β, TIMP-L, and MMP-13 were analyzed by RT-PCR method. In vivo study, the hydroxyproline content in liver and serum AST, ALT were also detected.RESULTS: In vitro study, AAV vector could mediated efficient expression of human INF-γ,, which inhibit the activation of hepatic stellate cells, decrease the expression of α-SMA and mRNA of TIMP-1, TGF-β, with the MMP-13unchanged. In vivo study, the histological examination revealed that rAAV- INF-γ could inhibit the progression of the hepatic fibrosis. In the rAAV-INF-γ induced group,the hydroxyproline content and serum AST, ALT level were decreased to 177±28 μg/g wet liver, 668.5±140.0,458.4±123.5 U/L, compare with the fibrosis control group 236±31 μg/g wet liver, 1 019.1±276.3, 770.5±154.3 U/L,respectively (P<0.01). mRNA expression of TIMP-1 in the rAAV-INF-γ induced rat liver was decreased while no significant change was observed in TGF-β and MMP-13.CONCLUSION: All these results indicated that rAAV-INF-γhas potential effects for gene therapy of hepatic fibrosis,which could inhibit the progression of hepatic fibrosis.

  9. Protective Immunity to Hepatitis B and Streptococcus Pneumoniae in Active Duty Women Versus Men: Prevalence and Responses to Preventive Immunization

    Science.gov (United States)

    1996-04-01

    Protective Immunity to Hepatitis B and Streptococcus Pneumoniae in Active Duty Women Versus Men: Prevalence and Responses to Preventive Immunization...April 1996 I Final (1 Dec 94 - 31 Dec 95) 4. TITLE AND SUBTITLE Prot~ecti•ve Inmnunity¥ to Hepat~it~is B and 6. FUNDING NUMBERS Streptococcus Pneumoniae in...pneumococcal vaccine is not included in the standard vaccinations for active duty military. The prevalence of immunity to pathogenic Streptococcus pneumoniae in

  10. Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2

    DEFF Research Database (Denmark)

    Hogan, Meghan F; Ravnskjaer, Kim; Matsumura, Shigenobu

    2015-01-01

    and dephosphorylation of the cAMP regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where...... accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis....... increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite...

  11. Metformin Reduces Desmoplasia in Pancreatic Cancer by Reprogramming Stellate Cells and Tumor-Associated Macrophages

    OpenAIRE

    Incio, Joao; Suboj, Priya; Chin, Shan M.; Vardam-Kaur, Trupti; Liu,Hao; Hato, Tai; Babykutty, Suboj; Chen, Ivy; Deshpande, Vikram; Jain, Rakesh K.; Fukumura, Dai

    2015-01-01

    Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with a dismal prognosis for most patients. Fibrosis and inflammation are hallmarks of tumor desmoplasia. We have previously demonstrated that preventing the activation of pancreatic stellate cells (PSCs) and alleviating desmoplasia are beneficial strategies in treating PDAC. Metformin is a widely used glucose-lowering drug. It is also frequently prescribed to diabetic pancreatic cancer patients and has been sho...

  12. 应用抑制性消减杂交技术筛选转化生长因子β1刺激LX02细胞的反式调节基因%Screening and cloning genes transactivated by TGF beta 1 in hepatic stellate cells using suppression subtractive hybridization technique

    Institute of Scientific and Technical Information of China (English)

    肖琳; 张跃新; 张建龙; 李燕; 成军; 郭江; 张黎颖; 洪源; 伦永志; 蓝贤勇; 武会娟; 张丽娟

    2008-01-01

    目的 构建转化生长因子(TGF)β1刺激大鼠肝星状细胞(LX02)反式调节基因的cDNA消减文库,筛选并克隆TGF β1反式调节相关基因,以阐明TGF β1介导肝纤维化的分子生物学机制.方法 以TGF β1刺激LX02细胞,同时以磷酸盐缓冲液刺激的LX02细胞作为对照.提取mRNA并逆转录为cDNA,经Rsa Ⅰ酶切后,将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性多聚酶链反应.将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增;随机挑选克隆经PCR扩增后进行测序及同源性分析. 结果成功构建了TGF β1刺激LX02细胞反式调节基因的cDNA消减文库.文库扩增后得到146个200~1000bp插入片段的阳性克隆;随机挑取其中35个克隆进行测序,30个列序成功,并通过生物信息学分析发现有28个与已知基因序列和2个与未知功能基因序列高度同源.结论 应用抑制性消减杂交技术成功构建了TGF β1刺激LX02细胞反式调节基因的cDNA消减文库,筛选到一些与细胞生长调节、蛋白质合成,信号传导、细胞外基质代谢、扰脂质过氧化等密切相关的蛋白质编码基因,为进一步阐明TGF β1介导肝纤维化的分子生物学机制提供了线索.%Objectives To construct a eDNA subtractive library of genes transactivated by TGF beta 1 in LX02 hepatic stellate cells (HSC); to screen and to clone the regulated genes transactivated by TGF beta 1; and to elucidate the molecular biological mechanism of hepatic fibrosis mediated by TGF beta 1. Methods mRNA was isolated from HSC treated with TGF beta 1 or with PBS (as controls). Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated

  13. Stellate ganglion blockade for analgesia following upper limb surgery.

    LENUS (Irish Health Repository)

    McDonnell, J G

    2012-01-31

    We report the successful use of a stellate ganglion block as part of a multi-modal postoperative analgesic regimen. Four patients scheduled for orthopaedic surgery following upper limb trauma underwent blockade of the stellate ganglion pre-operatively under ultrasound guidance. Patients reported excellent postoperative analgesia, with postoperative VAS pain scores between 0 and 2, and consumption of morphine in the first 24 h ranging from 0 to 14 mg. While these are preliminary findings, and must be confirmed in a clinical trial, they highlight the potential for stellate ganglion blockade to provide analgesia following major upper limb surgery.

  14. On $k$-stellated and $k$-stacked spheres

    OpenAIRE

    Bagchi, Bhaskar; Datta, Basudeb

    2012-01-01

    We introduce the class $\\Sigma_k(d)$ of $k$-stellated (combinatorial) spheres of dimension $d$ ($0 \\leq k \\leq d + 1$) and compare and contrast it with the class ${\\cal S}_k(d)$ ($0 \\leq k \\leq d$) of $k$-stacked homology $d$-spheres. We have $\\Sigma_1(d) = {\\cal S}_1(d)$, and $\\Sigma_k(d) \\subseteq {\\cal S}_k(d)$ for $d \\geq 2k - 1$. However, for each $k \\geq 2$ there are $k$-stacked spheres which are not $k$-stellated. The existence of $k$-stellated spheres which are not $k$-stacked remains...

  15. Vitamin A-induced cholestatic hepatitis: a case report

    DEFF Research Database (Denmark)

    Becker, P.; Maurer, B.; Schirrmacher, P.

    2007-01-01

    showed an acute toxic liver injury with focal parenchymal necrosis, sinusoidal lesions, inflammatory infiltrate (round cells, macrophages), and activation and proliferation of stellate cells. The hepatic vitamin A concentration was found to be significantly elevated. There were no signs of intrahepatic......We report a case of intrahepatic cholestasis due to chronic vitamin A supplementation. A 70-year-old woman was admitted to the hospital for jaundice and reduced nutritional and general status with a 2-month history of increasing cholestasis. Some years previously she had suffered from breast...... and ovarian cancer with subsequent surgery and chemotherapy. Chemotherapy was terminated one month before elevated serum transaminase activities and cholestatic serum markers were noted. Following the chemotherapy, supportive care included weekly vitamin A injections (100,000 IU per injection). Liver biopsy...

  16. Retinoic acid-induced pancreatic stellate cell quiescence reduces paracrine Wnt-beta-catenin signaling to slow tumor progression

    NARCIS (Netherlands)

    Froeling, F.E.; Feig, C.; Chelala, C.; Dobson, R.; Mein, C.E.; Tuveson, D.A.; Clevers, H.; Hart, I.R.; Kocher, H.M.

    2011-01-01

    BACKGROUND & AIMS: Patients with pancreatic ductal adenocarcinoma are deficient in vitamin A, resulting in activation of pancreatic stellate cells (PSCs). We investigated whether restoration of retinol to PSCs restores their quiescence and affects adjacent cancer cells. METHODS: PSCs and cancer cell

  17. The vagal nerve stimulates activation of the hepatic progenitor cell compartment via muscarinic acetylcholine receptor type 3.

    Science.gov (United States)

    Cassiman, David; Libbrecht, Louis; Sinelli, Nicoletta; Desmet, Valeer; Denef, Carl; Roskams, Tania

    2002-08-01

    In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver.

  18. Endogenous n-3 polyunsaturated fatty acids attenuate T cell-mediated hepatitis via autophagy activation

    Directory of Open Access Journals (Sweden)

    Yanli Li

    2016-09-01

    Full Text Available Omega-3 polyunsaturated fatty acids (n-3 PUFAs exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A was administered intravenously to wild-type (WT and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase (ALT activity, and inhibited production of pro-inflammatory cytokines (e.g. TNF-α, IL-6, IL-17A and IFN-γ. In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism, and could be exploited as a new therapeutic approach for autoimmune hepatitis.

  19. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation

    Science.gov (United States)

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis. PMID:27679638

  20. Metformin reduces hepatic resistance and portal pressure in cirrhotic rats.

    Science.gov (United States)

    Tripathi, Dinesh M; Erice, Eva; Lafoz, Erica; García-Calderó, Héctor; Sarin, Shiv K; Bosch, Jaime; Gracia-Sancho, Jordi; García-Pagán, Juan Carlos

    2015-09-01

    Increased hepatic vascular resistance is the primary factor in the development of portal hypertension. Metformin ameliorates vascular cells function in several vascular beds. Our study was aimed at evaluating the effects, and the underlying mechanisms, of metformin on hepatic and systemic hemodynamics in cirrhotic rats and its possible interaction with the effects of propranolol (Prop), the current standard treatment for portal hypertension. CCl4-cirrhotic rats received by gavage metformin 300 mg/kg or its vehicle once a day for 1 wk, before mean arterial pressure (MAP), portal pressure (PP), portal blood flow (PBF), hepatic vascular resistance, and putative molecular/cellular mechanisms were measured. In a subgroup of cirrhotic rats, the hemodynamic response to acute Prop (5 mg/kg iv) was assessed. Effects of metformin ± Prop on PP and MAP were validated in common bile duct ligated-cirrhotic rats. Metformin-treated CCl4-cirrhotic rats had lower PP and hepatic vascular resistance than vehicle-treated rats, without significant changes in MAP or PBF. Metformin caused a significant reduction in liver fibrosis (Sirius red), hepatic stellate cell activation (α-smooth muscle actin, platelet-derived growth factor receptor β polypeptide, transforming growth factor-βR1, and Rho kinase), hepatic inflammation (CD68 and CD163), superoxide (dihydroethidium staining), and nitric oxide scavenging (protein nitrotyrosination). Prop, by decreasing PBF, further reduced PP. Similar findings were observed in common bile duct ligated-cirrhotic rats. Metformin administration reduces PP by decreasing the structural and functional components of the elevated hepatic resistance of cirrhosis. This effect is additive to that of Prop. The potential impact of this pharmacological combination, otherwise commonly used in patients with cirrhosis and diabetes, needs clinical evaluation.

  1. Comparative azo reductase activity of red azo dyes through caecal and hepatic microsomal fraction in rats.

    Science.gov (United States)

    Singh, S; Das, M; Khanna, S K

    1997-09-01

    In order to study the rate of formation of toxic aromatic amines, anaerobic reduction of four red azo dyes viz. amaranth, carmoisine, fast Red E and ponceau 4R was investigated by incubating caecal content and hepatic microsomal fraction of rats with 37.5 microM concentration of dyes in sodium phosphate buffer pH 7.4 using NADPH generating system, glucose oxidase system and nitrogen as the gaseous phase. Caecal suspension exhibited higher azo reductase activity than that of hepatic microsomal fraction using any of the 4 azo dyes. Caecal microbes showed maximal azo reductase activity when ponceau 4R was used as a substrate followed by fast Red E and carmoisine, while with amaranth the activity was minimum. Similarly ponceau 4 R exhibited maximum hepatic microsomal azo reductase activity followed by fast Red E and carmoisine whereas, amaranth had minimum activity. Caecal flora possessed almost 17 fold higher degradative capability of ponceau 4 R and fast Red E colourants than the hepatic microsomal fraction. The higher reductive ability through caecal flora for ponceau 4R and fast Red E signifies the formation of more aromatic amines which may be re-absorbed through the intestine to be either eliminated through urine as conjugates or retained in the target tissues to elicit toxic effects.

  2. Macromolecular (pro)drugs with concurrent direct activity against the hepatitis C virus and inflammation.

    Science.gov (United States)

    Wohl, Benjamin M; Smith, Anton A A; Jensen, Bettina E B; Zelikin, Alexander N

    2014-12-28

    Macromolecular prodrugs (MPs) are a powerful tool to alleviate side-effects and improve the efficacy of the broad-spectrum antiviral agent ribavirin. In this work, we sought an understanding of what makes an optimal formulation within the macromolecular parameter space--nature of the polymer carrier, average molar mass, drug loading, or a good combination thereof. A panel of MPs based on biocompatible synthetic vinylic and (meth)acrylic polymers was tested in an anti-inflammatory assay with relevance to alleviating inflammation in the liver during hepatitis C infection. Pristine polymer carriers proved to have a pronounced anti-inflammatory activity, a notion which may prove significant in developing MPs for antiviral and anticancer treatments. With conjugated ribavirin, MPs revealed enhanced activity but also higher toxicity. Therapeutic windows and therapeutic indices were determined and discussed to reveal the most potent formulation and those with optimized safety. Polymers were also tested as inhibitors of replication of the hepatitis C viral RNA using a subgenomic viral replicon system. For the first time, negatively charged polymers are revealed to have an intracellular activity against hepatitis C virus replication. Concerted activity of the polymer and ribavirin afforded MPs which significantly increased the therapeutic index of ribavirin-based treatment. Taken together, the systematic investigation of the macromolecular space identified lead candidates with high efficacy and concurrent direct activity against the hepatitis C virus and inflammation.

  3. Altered hyperlipidemia, hepatic steatosis, and hepatic peroxisome proliferator-activated receptors in rats with intake of tart cherry.

    Science.gov (United States)

    Seymour, E Mitchell; Singer, Andrew A M; Kirakosyan, Ara; Urcuyo-Llanes, Daniel E; Kaufman, Peter B; Bolling, Steven F

    2008-06-01

    Elevated plasma lipids, glucose, insulin, and fatty liver are among components of metabolic syndrome, a phenotypic pattern that typically precedes the development of Type 2 diabetes. Animal studies show that intake of anthocyanins reduces hyperlipidemia, obesity, and atherosclerosis and that anthocyanin-rich extracts may exert these effects in association with altered activity of tissue peroxisome proliferator-activated receptors (PPARs). However, studies are lacking to test this correlation using physiologically relevant, whole food sources of anthocyanins. Tart cherries are a rich source of anthocyanins, and whole cherry fruit intake may also affect hyperlipidemia and/or affect tissue PPARs. This hypothesis was tested in the Dahl Salt-Sensitive rat having insulin resistance and hyperlipidemia. For 90 days, Dahl rats were pair-fed AIN-76a-based diets supplemented with either 1% (wt:wt) freeze-dried whole tart cherry or with 0.85% additional carbohydrate to match macronutrient and calorie provision. After 90 days, the cherry-enriched diet was associated with reduced fasting blood glucose, hyperlipidemia, hyperinsulinemia, and reduced fatty liver. The cherry diet was also associated with significantly enhanced hepatic PPAR-alpha mRNA, enhanced hepatic PPAR-alpha target acyl-coenzyme A oxidase mRNA and activity, and increased plasma antioxidant capacity. In conclusion, physiologically relevant tart cherry consumption reduced several phenotypic risk factors that are associated with risk for metabolic syndrome and Type 2 diabetes. Tart cherries may represent a whole food research model of the health effects of anthocyanin-rich foods and may possess nutraceutical value against risk factors for metabolic syndrome and its clinical sequelae.

  4. Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis.

    Directory of Open Access Journals (Sweden)

    Jan eGörtzen

    2015-12-01

    Full Text Available Introduction:In liver fibrosis activation of hepatic stellate cells (HSC comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.Methods:Liver fibrosis was induced in rats using bile duct ligation (BDL, thioacetamide (TAA or carbon tetrachloride (CCl4 models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1 and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1kPa and 12kPa or on plastic to mimic non-fibrotic, fibrotic or cirrhotic environments, then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.Results:Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12kPa compared to 1kPa gels. LX2 cells cultured on 12kPa gels showed upregulation of RHOA, COL1A1 and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis

  5. Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

    NARCIS (Netherlands)

    A.J.M. Verhoeven (Adrie); B.P. Neve (Bernadette); H. Jansen (Hans)

    1999-01-01

    textabstractHuman hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylat

  6. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    Science.gov (United States)

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  7. An application of multilayer neural network on hepatitis disease diagnosis using approximations of sigmoid activation function

    Directory of Open Access Journals (Sweden)

    Onursal Çetin

    2015-06-01

    Full Text Available Objective: Implementation of multilayer neural network (MLNN with sigmoid activation function for the diagnosis of hepatitis disease. Methods: Artificial neural networks (ANNs are efficient tools currently in common use for medical diagnosis. In hardware based architectures activation functions play an important role in ANN behavior. Sigmoid function is the most frequently used activation function because of its smooth response. Thus, sigmoid function and its close approximations were implemented as activation function. The dataset is taken from the UCI machine learning database. Results: For the diagnosis of hepatitis disease, MLNN structure was implemented and Levenberg Morquardt (LM algorithm was used for learning. Our method of classifying hepatitis disease produced an accuracy of 91.9% to 93.8% via 10 fold cross validation. Conclusion: When compared to previous work that diagnosed hepatitis disease using artificial neural networks and the identical data set, our results are promising in order to reduce the size and cost of neural network based hardware. Thus, hardware based diagnosis systems can be developed effectively by using approximations of sigmoid function.

  8. Angiotensin receptor blockade recovers hepatic UCP2 expression and aconitase and SDH activities and ameliorates hepatic oxidative damage in insulin resistant rats.

    Science.gov (United States)

    Montez, Priscilla; Vázquez-Medina, José Pablo; Rodríguez, Rubén; Thorwald, Max A; Viscarra, José A; Lam, Lisa; Peti-Peterdi, Janos; Nakano, Daisuke; Nishiyama, Akira; Ortiz, Rudy M

    2012-12-01

    Metabolic syndrome (MetS) is commonly associated with elevated renin-angiotensin system, oxidative stress, and steatohepatitis with down-regulation of uncoupling proteins (UCPs). However, the mechanisms linking renin-angiotensin system, steatosis, and UCP2 to hepatic oxidative damage during insulin resistance are not described. To test the hypothesis that angiotensin receptor activation contributes to decreased hepatic UCP2 expression and aconitase activity and to increased oxidative damage after increased glucose intake in a model of MetS, lean and obese Long Evans rats (n = 10/group) were randomly assigned to the following groups: 1) untreated Long Evans Tokushima Otsuka (lean, strain control), 2) untreated Otsuka Long Evans Tokushima Fatty (OLETF) (MetS model), 3) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg·d × 6 wk), 4) OLETF + high glucose (HG) (5% in drinking water × 6 wk), and 5) OLETF + ARB + HG (ARB/HG × 6 wk). HG increased body mass (37%), plasma triglycerides (TGs) (35%), plasma glycerol (87%), plasma free fatty acids (28%), and hepatic nitrotyrosine (74%). ARB treatment in HG decreased body mass (12%), plasma TG (15%), plasma glycerol (23%), plasma free fatty acids (14%), and hepatic TG content (42%), suggesting that angiotensin receptor type 1 (AT1) activation and increased adiposity contribute to the development of obesity-related dyslipidemia. ARB in HG also decreased hepatic nitrotyrosine and increased hepatic UCP2 expression (59%) and aconitase activity (40%), as well as antioxidant enzyme activities (50-120%), suggesting that AT1 activation also contributes to protein oxidation, impaired lipid metabolism, and antioxidant metabolism in the liver. Thus, in addition to promoting obesity-related hypertension, AT1 activation may also impair lipid metabolism and antioxidant capacity, resulting in steatosis via decreased UCP2 and tricarboxylic acid cycle activity.

  9. Anti-hepatitis B activity of isoquinoline alkaloids of plant origin.

    Science.gov (United States)

    Aljofan, Mohamad; Netter, Hans J; Aljarbou, Ahmed N; Hadda, Taibi Ben; Orhan, Ilkay Erdogan; Sener, Bilge; Mungall, Bruce A

    2014-05-01

    Hepatitis B virus (HBV) is the causative agent of B-type hepatitis in humans, a vaccine-preventable disease. Despite the availability of effective vaccines, globally, 2 billion people show evidence of past or current HBV infection, of which 350 million people are persistently infected, with an estimated annual increase of 1 million. There is no cure for chronic HBV infections, which are associated with cirrhotic liver failure and with an increased risk of developing hepatocellular carcinoma. Hepatitis antiviral research has focused primarily on the development of inhibitors of viral polymerase through the use of nucleoside analogues. Therefore, there is an urgent need for the development of non-nucleoside compounds to be used as an alternative or to complement the current therapy. To address this need, 18 isoquinoline alkaloids were evaluated for their potential antiviral activity against HBV in vitro.

  10. Temporal expression of chemokines dictates the hepatic inflammatory infiltrate in a murine model of schistosomiasis.

    Directory of Open Access Journals (Sweden)

    Melissa L Burke

    Full Text Available Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of granulomas and fibrosis will be essential for understanding how schistosome antigens interact with the host environment. We report here the first whole genome microarray analysis of the murine liver during the progression of Schistosoma japonicum egg-induced granuloma formation and hepatic fibrosis. Our results reveal a distinct temporal relationship between the expression of chemokine subsets and the recruitment of cells to the infected liver. Genes up-regulated earlier in the response included T- and B-cell chemoattractants, reflecting the early recruitment of these cells illustrated by flow cytometry. The later phases of the response corresponded with peak recruitment of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs and the expression of chemokines with activity for these cells including CCL11 (eotaxin 1, members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12 and the Hepatic Stellate Cell/Fibrocyte chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14 and markers of alternatively activated macrophages (e.g. Retnla during this later phase provides further evidence of a role for these cells in schistosome-induced pathology. Additionally, we demonstrate that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggest the involvement of neutrophils in S. japonicum-induced hepatic fibrosis. These results further our understanding of the immunopathogenic and, especially, chemokine signalling pathways that regulate the development of S. japonicum-induced granulomas and fibrosis and may provide correlative insight into the pathogenesis of other

  11. FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis.

    Science.gov (United States)

    Kir, Serkan; Beddow, Sara A; Samuel, Varman T; Miller, Paul; Previs, Stephen F; Suino-Powell, Kelly; Xu, H Eric; Shulman, Gerald I; Kliewer, Steven A; Mangelsdorf, David J

    2011-03-25

    Fibroblast growth factor (FGF) 19 is an enterokine synthesized and released when bile acids are taken up into the ileum. We show that FGF19 stimulates hepatic protein and glycogen synthesis but does not induce lipogenesis. The effects of FGF19 are independent of the activity of either insulin or the protein kinase Akt and, instead, are mediated through a mitogen-activated protein kinase signaling pathway that activates components of the protein translation machinery and stimulates glycogen synthase activity. Mice lacking FGF15 (the mouse FGF19 ortholog) fail to properly maintain blood concentrations of glucose and normal postprandial amounts of liver glycogen. FGF19 treatment restored the loss of glycogen in diabetic animals lacking insulin. Thus, FGF19 activates a physiologically important, insulin-independent endocrine pathway that regulates hepatic protein and glycogen metabolism.

  12. IL28B Polymorphism Correlates with Active Hepatitis in Patients with HBeAg-Negative Chronic Hepatitis B

    OpenAIRE

    2013-01-01

    BACKGROUND AIMS: The clinical relevance of single nucleotide polymorphisms (SNPs) near the IL28B gene is controversial in patients with hepatitis B virus (HBV) infection. This study aimed to investigate the role of viral and host factors, including IL28B genotypes, in the natural course of chronic hepatitis B (CHB). METHODS: The study enrolled consecutive 115 treatment-naive CHB patients. HBV viral loads, genotypes, precore and basal core promotor mutations, serum hepatitis B surface antigen ...

  13. Non-linear Membrane Properties in Entorhinal Cortical Stellate Cells Reduce Modulation of Input-Output Responses by Voltage Fluctuations.

    Science.gov (United States)

    Fernandez, Fernando R; Malerba, Paola; White, John A

    2015-04-01

    The presence of voltage fluctuations arising from synaptic activity is a critical component in models of gain control, neuronal output gating, and spike rate coding. The degree to which individual neuronal input-output functions are modulated by voltage fluctuations, however, is not well established across different cortical areas. Additionally, the extent and mechanisms of input-output modulation through fluctuations have been explored largely in simplified models of spike generation, and with limited consideration for the role of non-linear and voltage-dependent membrane properties. To address these issues, we studied fluctuation-based modulation of input-output responses in medial entorhinal cortical (MEC) stellate cells of rats, which express strong sub-threshold non-linear membrane properties. Using in vitro recordings, dynamic clamp and modeling, we show that the modulation of input-output responses by random voltage fluctuations in stellate cells is significantly limited. In stellate cells, a voltage-dependent increase in membrane resistance at sub-threshold voltages mediated by Na+ conductance activation limits the ability of fluctuations to elicit spikes. Similarly, in exponential leaky integrate-and-fire models using a shallow voltage-dependence for the exponential term that matches stellate cell membrane properties, a low degree of fluctuation-based modulation of input-output responses can be attained. These results demonstrate that fluctuation-based modulation of input-output responses is not a universal feature of neurons and can be significantly limited by subthreshold voltage-gated conductances.

  14. Does limited virucidal activity of biocides include duck hepatitis B virucidal action?

    Directory of Open Access Journals (Sweden)

    Sauerbrei Andreas

    2012-10-01

    Full Text Available Abstract Background There is agreement that the infectivity assay with the duck hepatitis B virus (DHBV is a suitable surrogate test to validate disinfectants for hepatitis B virucidal activity. However, since this test is not widely used, information is necessary whether disinfectants with limited virucidal activity also inactivate DHBV. In general, disinfectants with limited virucidal activity are used for skin and sensitive surfaces while agents with full activity are more aggressive. The present study compares the activity of five different biocides against DHBV and the classical test virus for limited virucidal activity, the vaccinia virus strain Lister Elstree (VACV or the modified vaccinia Ankara strain (MVA. Methods Virucidal assay was performed as suspension test according to the German DVV/RKI guideline. Duck hepatitis B virus obtained from congenitally infected Peking ducks was propagated in primary duck embryonic hepatocytes and was detected by indirect immunofluorescent antigen staining. Results The DHBV was inactivated by the use of 40% ethanol within 1-min and 30% isopropanol within 2-min exposure. In comparison, 40% ethanol within 2-min and 40% isopropanol within 1-min exposure were effective against VACV/MVA. These alcohols only have limited virucidal activity, while the following agents have full activity. 0.01% peracetic acid inactivated DHBV within 2 min and a concentration of 0.005% had virucidal efficacy against VACV/MVA within 1 min. After 2-min exposure, 0.05% glutardialdehyde showed a comparable activity against DHBV and VACV/MVA. This is also the case for 0.7% formaldehyde after a contact time of 30 min. Conclusions Duck hepatitis B virus is at least as sensitive to limited virucidal activity as VACV/MVA. Peracetic acid is less effective against DHBV, while the alcohols are less effective against VACV/MVA. It can be expected that in absence of more direct tests the results may be extrapolated to HBV.

  15. Aldosterone stimulates hepatic stellate cells contraction via Ca2+-independent pathways%醛固酮对介导大鼠肝星状细胞收缩的非钙离子依赖信号通路的影响

    Institute of Scientific and Technical Information of China (English)

    张小兰; 肖冰; 孟莹; 李旭

    2011-01-01

    Objective To investigate the mechanisms of Aldosterone stimulating hepatic stellate cells(HSCs) contraction via Ca2+-independent pathways. Methods HSC-T6 cell line was pre-disposed with Aldo 10 μmol/L. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The concentration variation of intracellular free calcium in rat HSC was observed by laser confocal microscopy.Besides, HSC-T6 cell line was under pre-disposal treatment with the blocking agents of Aldo receptor -antisterone, protein kinase C (PKC) special blocking agent-Stauro, Rho kinase blocking agent-Y27632 and MLCK special blocking agent-ML-7 respectively prior to stimulation with aldosterone. RT-PCR was used to detect the expression of Rock2, RhoAGTP and RhoGEF in Ca2+- independent pathways mediated by Rho-kinase. Results Aldo could induce HSCs contraction. The concentration of intracellular free calcium in rat HSCs had no change after pre-disposal treatment with Aldo. The mRNA expressions of Rock2, RhoAGTP and RhoGEF increased significantly after treatment with Aldo (0.770 ± 0.049, 0.960 ± 0.096, 0.180 ±0.006, P < 0.01).When inhibited with antisterone, the mRNA expressions of the three elements were (0.440 ± 0.166, 0.370 ± 0.180 and 0.050 ± 0.001, P < 0.01), lower than that of Aldo group, but higher in ML-7+Stauro + Aldo groups (0.940 ± 0.066, 1.330 ±0.192 and 0.160 ± 0.007, P < 0.05) as compared to the control group (0.140 ± 0.023,0.540 ± 0.111 and 0.110 ± 0.012). In the Y27632 + ML-7 + Stauro+Aldo group, the mRNA expression of RhoGEF (0.290 ± 0.004, P < 0.01)was higher than that of the ML-7 + Stauro + Aldo group (0.160 ± 0.007). Conclusion Aldo could induce HSCs contraction via Ca2+-independent pathways and Rho-Rock pathway involved in the process.%目的 探讨醛固酮(Aldo)对介导大鼠肝星状细胞(HSC)收缩的非Ca2+依赖性信号传导通路的影响.方法 对HSC-T6细胞给予Aldo 10 μmol/L处理,用聚硅酮膜法检测HSC-T6细胞的收缩性;

  16. 抗纤复方I号对乙醛刺激的肝星状细胞的细胞外基质及细胞因子分泌的干预作用%KangXian FuFang I inhibits acetaldehyde-stimulated extracellular matrix synthesis and cytokine secretion in cultured hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    林红; 黄玉红; 李异玲; 傅宝玉; 王炳元

    2011-01-01

    AIM: To investigate the effect of KangXian Fu-Fang I (KXI) on acetaldehyde-stimulated extracellular matrix synthesis and cytokine secretion in cultured hepatic stellate cells (HSCs).METHODS: HSCs were isolated from rats and then primarily cultured and subcultured in vitro. Serum containing KXI was collected from normal rats intragastrically given the drug. HSCs were treated with acetaldehyde and the drug-containing serum. The contents of hyaluronic acid (HA) and transforming growth factor β1 (TGF-β1) in the media and expression of type I collagen al mRNA in stimulated HSCs were measured by radioimmunoassay, ELISA and reverse transcription polymerase chain reaction (RT-PCR), respectively.RESULTS: After HSCs were stimulated with acetaldehyde (100 μmol/L), the expression level of type I collagen al mRNA (1.193 ± 0.0344 vs 0.988 ± 0.0208, P < 0.05) in HSCs and secreted levels of HA (1243.22 ng/L ± 58.13 ng/L vs 602.33 ng/L ± 194.06 ng/L, P < 0.05) and TGF-β1 (2734.43 ng/L ± 787.12 ng/L vs 559.92 ng/L ± 97.63 ng/L, P < 0.01) in the media significantly increased. Drug-containing serum (10%) significantly inhibited acetaldehyde-stimulated expression of type I collagen al mRNA (0.973 ± 0.0605 vs 1.193 ± 0.0344, P < 0.05) and secretion of HA (1032.667 ng/L ± 77.65 ng/ L vs 1243.22 ng/L ± 58.13 ng/L, P < 0.05) and TGF-β1 (759.62 ng/L ± 205.34 ng/L vs 2734.43 ng/L ± 787.12 ng/L, P < 0.01).CONCLUSION: KXI could inhibit acetaldehyde-stimulated secretion of HA and TGF-β1 and expression of type I collagen al mRNA in cultured HSCs.%目的:研究乙醛对肝星状细胞(HSC) 细胞外基质和细胞因子分泌的影响及中药抗纤复方I号(KXI) 的干预作用.方法:采用大鼠肝脏原位灌流消化法获得并原代及传代培养HSC,大鼠灌以KXI 制备药物血清,以乙醛和药物血清作用于HSC,通过RTPCR 测定细胞内α1(I) 型胶原mRNA 的表达,以放射免疫法和ELISA 法分别测定培养上清中透明质酸(HA)

  17. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    Institute of Scientific and Technical Information of China (English)

    Yoshiyuki Takahara; Mitsuo Takahashi; Hiroki Wagatsuma; Fumihiko Yokoya; Qing-Wei Zhang; Mutsuyo Yamaguchi; Hiroyuki Aburatani; Norifumi Kawada

    2006-01-01

    AIM: To determine the gene expression profile data for the whole liver during development of dimethylnitrosamine (DMN)-induced hepatic fibrosis.METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells),and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells.RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSCspecific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis,suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocytespecific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis.CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis.

  18. Monocytes infiltrate the pancreas via the MCP-1/CCR2 pathway and differentiate into stellate cells.

    Directory of Open Access Journals (Sweden)

    Kazuko Ino

    Full Text Available Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP(+CD45(- cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl4. Because the vast majority of EGFP(+CD45(- cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs. EGFP(+ PaSCs were also observed in CCl4-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1 and angiotensin II (Ang II increased in the pancreas of CCl4-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2 and Ang II type 1 receptor (AT1R, were expressed on Ly6C(high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP(+F4/80(+CCR2(+ monocytic cells and EGFP(+ PaSCs in the pancreas of CCl4-treated chimeric mice receiving EGFP(+ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP(+ PaSCs in injured mice. We propose that CCR2(+ monocytes migrate into the pancreas possibly via the

  19. 血吸虫病肝纤维化小鼠IL-22表达及肝星状细胞衰老的意义%Dynamic expressions of IL-22 and hepatic stellate cells senescence in mice in-fected with Schistosoma japonicum

    Institute of Scientific and Technical Information of China (English)

    李兰; 孙颖; 王洪武; 黄宇; 黄加权; 宁琴

    2014-01-01

    Objective To investigate the dynamic expressions of interleukin-22(IL-22),Interleukin-22 receptor 1(IL-22R1),and hepatic stellate cells(HSC)senescence in mice with Schistosoma japonicum infection. Methods A murine model of S. japonicum infection was established and the serum samples and liver tissues were collected 4,6,8,12 weeks post-infection. The serum samples were detected for the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST). The pathological changes and proliferation of hepatic collagen fibers in the liver tissue were observed after HE staining and Masson staining. The HSC senescence in fibrotic livers was determined by the detection of senescence-associatedβ-galactosidase(SA-β-Gal). Sandwich ELISA was used to measure the expressions of IL-22,and Real-time PCR was used to test the mRNA levels of IL-22 and IL-22R1. The control group without S. japonicum infection was set up. Results The serum levels of ALT and AST signifi-cantly increased 8 weeks and 12 weeks after the infection(vs. 0 week,all P<0.05). The level of IL-22 increased 4 weeks and 6 weeks after the infection(vs. 0 week,both P<0.05),but reduced 8 weeks post-infection,and was even lower 12 weeks post-in-fection(vs. 4 weeks and 6 weeks,both P<0.01). Being consistent with the dynamic expression of IL-22 protein,the mRNA ex-pression of IL-22 began to increase 4 weeks and reached the peak 6 weeks after the infection(vs. 0 week,both P<0.05),and continuously declined 8 weeks and 12 weeks post-infections(vs. 6 weeks,both P<0.05). The increase of the expression of IL-22R1 mRNA was correlated with the progression of fibrosis,and the peak was in 12 weeks post-infections(vs. 0 week and 6 weeks,both P<0.05). The number of senescence-associated beta-galactosidase-positive HSCs was reduced with the decreasing expression of IL-22 in the advanced liver fibrosis. Conclusion IL-22 and IL-22R1 are involved in the pathogenesis of schistoso-miasis liver fibrosis. As an inflammation factor

  20. Atorvastatin dose-dependently decreases hepatic lipase activity in type 2 diabetes - Effect of sex and the LIPC promoter variant

    NARCIS (Netherlands)

    Berk-Planken, IIL; Hoogerbrugge, N; Stolk, RP; Bootsma, AH; Jansen, H

    2003-01-01

    OBJECTIVE - Hepatic lipase (HL) is involved in the metabolism of several lipoproteins and may contribute to the atherogenic lipid profile in type 2 diabetes. Little is known about the effect of cholesterol synthesis inhibitors on HL activity in relation to sex and the hepatic lipase gene, the LIPC p

  1. Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy.

    Science.gov (United States)

    Sinha, Rohit Anthony; You, Seo-Hee; Zhou, Jin; Siddique, Mobin M; Bay, Boon-Huat; Zhu, Xuguang; Privalsky, Martin L; Cheng, Sheue-Yann; Stevens, Robert D; Summers, Scott A; Newgard, Christopher B; Lazar, Mitchell A; Yen, Paul M

    2012-07-01

    For more than a century, thyroid hormones (THs) have been known to exert powerful catabolic effects, leading to weight loss. Although much has been learned about the molecular mechanisms used by TH receptors (TRs) to regulate gene expression, little is known about the mechanisms by which THs increase oxidative metabolism. Here, we report that TH stimulation of fatty acid β-oxidation is coupled with induction of hepatic autophagy to deliver fatty acids to mitochondria in cell culture and in vivo. Furthermore, blockade of autophagy by autophagy-related 5 (ATG5) siRNA markedly decreased TH-mediated fatty acid β-oxidation in cell culture and in vivo. Consistent with this model, autophagy was altered in livers of mice expressing a mutant TR that causes resistance to the actions of TH as well as in mice with mutant nuclear receptor corepressor (NCoR). These results demonstrate that THs can regulate lipid homeostasis via autophagy and help to explain how THs increase oxidative metabolism.

  2. TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

    Science.gov (United States)

    Zambirinis, Constantinos P; Levie, Elliot; Nguy, Susanna; Avanzi, Antonina; Barilla, Rocky; Xu, Yijie; Seifert, Lena; Daley, Donnele; Greco, Stephanie H; Deutsch, Michael; Jonnadula, Saikiran; Torres-Hernandez, Alejandro; Tippens, Daniel; Pushalkar, Smruti; Eisenthal, Andrew; Saxena, Deepak; Ahn, Jiyoung; Hajdu, Cristina; Engle, Dannielle D; Tuveson, David; Miller, George

    2015-11-16

    Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis.

  3. Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake.

    Directory of Open Access Journals (Sweden)

    Marilyne Labrie

    Full Text Available Transgenic mice (Tg overexpressing human apolipoprotein D (H-apoD in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA, the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver.

  4. Eastern region represents a worrying cluster of active hepatitis C in Algeria in 2012.

    Science.gov (United States)

    Bensalem, Aïcha; Selmani, Karima; Hihi, Narjes; Bencherifa, Nesrine; Mostefaoui, Fatma; Kerioui, Cherif; Pineau, Pascal; Debzi, Nabil; Berkane, Saadi

    2016-08-01

    Algeria is the largest country of Africa, peopled with populations living a range of traditional/rural and modern/urban lifestyles. The variations of prevalence of chronic active hepatitis care poorly known on the Algerian territory. We conducted a retrospective survey on all patients (n = 998) referred to our institution in 2012 and confirmed by us for an active hepatitis C. Half of the hepatitis C virus (HCV) isolates were genotyped. Forty Algerian regions out of the 48 were represented in our study. Three geographical clusters (Aïn-Temouchent/SidiBelAbbes, Algiers, and a large Eastern region) with an excess of active hepatitis C were observed. Patients coming from the Eastern cluster (Batna, Khenchela, Oum el Bouaghi, and Tebessa) were strongly over-represented (49% of cases, OR = 14.5, P < 0.0001). The hallmarks of Eastern region were an excess of women (65% vs. 46% in the remaining population, P < 0.0001) and the almost exclusive presence of HCV genotype 1 (93% vs. 63%, P = 0.0001). The core of the epidemics was apparently located in Khenchela (odds ratio = 24.6, P < 0.0001). This situation is plausibly connected with nosocomial transmission or traditional practices as scarification (Hijama), piercing or tattooing, very lively in this region. Distinct hepatitis C epidemics are currently affecting Algerian population. The most worrying situation is observed in rural regions located east of Algeria. J. Med. Virol. 88:1394-1403, 2016. © 2016 Wiley Periodicals, Inc.

  5. Molecular therapy for hepatic injury and fibrosis: Where are we?

    Institute of Scientific and Technical Information of China (English)

    Colette C Prosser; Roy D Yen; Jian Wu

    2006-01-01

    Hepatic fibrosis is a wound healing response, involving pathways of inflammation and fibrogenesis. In response to various insults, such as alcohol, ischemia, viral agents,and medications or hepatotoxins, hepatocyte damage will cause the release of cytokines and other soluble factors by Kupffer cells and other cell types in the liver.These factors lead to activation of hepatic stellate cells,which synthesize large amounts of extracellular matrix components. With chronic injury and fibrosis, liver architecture and metabolism are disrupted, eventually manifesting as cirrhosis and its complications. In addition to eliminating etiology, such as antiviral therapy and pharmacological intervention, it is encouraging that novel strategies are being developed to directly address hepatic injury and fibrosis at the subcellular and molecular levels. With improvement in understanding these mechanisms and pathways, key steps in injury,signaling, activation, and gene expression are being targeted by molecular modalities and other molecular or gene therapy approaches. This article intends to provide an update in terms of the current status of molecular therapy for hepaticinjury and fibrosis and how far we are from clinical utilization of these new therapeutic modalities.

  6. Hepatitis D in Chronic Active Hepatitis B: Prevalence, Liver Enzyme Levels and Histopathology- an Epidemiological Study in Shiraz, Southern Iran, 2003-2004

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    Farnaz Khademolhosseini

    2008-12-01

    Full Text Available Background and Aims: At least 5% of hepatitis B carriers worldwide are infected with Hepatitis D virus (HDV. This study aims to determine the prevalence, transaminase levels and histopathological findings of HDV among patients with chronic active hepatitis B in southern Iran.Methods: During 2003-2004, 93 patients >15 years with chronic active hepatitis B were enrolled from referrals to Shiraz University of Medical Sciences in southern Iran.Results: Nine (9.7% patients were seropositive for the anti HDV antibody. 76.3% of patients were male and among the HDV positive group, all subjects were male too. A significantly higher AST and more advanced grade and stage of liver disease were observed in the HDV positive group. The most common mode of transmission in the positive group was intravenous drug use.Conclusions: The risk of liver disease progression in chronic hepatitis B appears to be higher in HDV infected patients. Intravenous drug abuse is an important risk factor for acquiring HDV infection. Checking anti-HDV is suggested in any patient with positive HBsAg, especially in males or those with history of intravenous drug abuse.

  7. Signal transduction pathways in liver and the influence of hepatitis C virus infection on their activities

    Institute of Scientific and Technical Information of China (English)

    Magdalena M Dabrowska; Anatol Panasiuk; Robert Flisiak

    2009-01-01

    In liver, the most intensively studied transmembrane and intracellular signal transduction pathways are the Janus kinase signal transduction pathway, the mitogen-activated protein kinases signal transduction pathway, the transforming growth factor b signal transduction pathway, the tumor necrosis factor a signal transduction pathway and the recently discovered sphingolipid signal transduction pathway. All of them are activated by many different cytokines and growth factors. They regulate specific cell mechanisms such as hepatocytes proliferation, growth, differentiation, adhesion, apoptosis, and synthesis and degradation of the extracellular matrix. The replication cycle of hepatitis C virus (HCV) is intracellular and requires signal transduction to the nucleus to regulate transcription of its genes. Moreover, HCV itself, by its structural and nonstructural proteins, could influence the activity of the second signal messengers. Thus, the inhibition of the transmembrane and intracellular signal transduction pathways could be a new therapeutic target in chronic hepatitis C treatment.

  8. Deficiency of DJ-1 Ameliorates Liver Fibrosis through Inhibition of Hepatic ROS Production and Inflammation

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    Yu, Yingxue; Sun, Xuehua; Gu, Jinyang; Yu, Chang; Wen, Yankai; Gao, Yueqiu; Xia, Qiang; Kong, Xiaoni

    2016-01-01

    Liver fibrosis is a global health problem and previous studies have demonstrated that reactive oxygen species (ROS) play important roles in fibrogenesis. Parkinson disease (autosomal recessive, early onset) 7 (Park7) also called DJ-1 has an essential role in modulating cellular ROS levels. DJ-1 therefore may play functions in liver fibrogenesis and modulation of DJ-1 may be a promising therapeutic approach. Here, wild-type (WT) and DJ-1 knockout (DJ-1 KO) mice were administrated with carbon tetrachloride (CCl4) to induce liver fibrosis or acute liver injury. Results showed that DJ-1 depletion significantly blunted liver fibrosis, accompanied by marked reductions in liver injury and ROS production. In the acute CCl4 model, deficiency of DJ-1 showed hepatic protective functions as evidenced by decreased hepatic damage, reduced ROS levels, diminished hepatic inflammation and hepatocyte proliferation compared to WT mice. In vitro hepatic stellate cells (HSCs) activation assays indicated that DJ-1 has no direct effect on the activation of HSCs in the context of with or without TGFβ treatment. Thus our present study demonstrates that in CCl4-induced liver fibrosis, DJ-1 deficiency attenuates mice fibrosis by inhibiting ROS production and liver injury, and further indirectly affecting the activation of HSCs. These results are in line with previous studies that ROS promote HSC activation and fibrosis development, and suggest the therapeutic value of DJ-1 in treatment of liver fibrosis.

  9. Role of the receptor for advanced glycation end products in hepatic fibrosis

    Institute of Scientific and Technical Information of China (English)

    Christina Lohwasser; Daniel Neureiter; Yury Popov; Michael Bauer; Detlef Schuppan

    2009-01-01

    AIM: To study the role of advanced glycation end products (AGE) and their specific receptor (RAGE) in the pathogenesis of liver fibrogenesis. METHODS: In vitro RAGE expression and extracellular matrix-related gene expression in both rat and human hepatic stellate cells (HSC) were measured after stimulation with the two RAGE ligands, advanced glycation end product-bovine serum albumin (AGEBSA) and Nε-(carboxymethyl) lysine (CML)-BSA, or with tumor necrosis factor-α (TNF-α). In vivo RAGE expression was examined in models of hepatic fibrosis induced by bile duct ligation or thioacetamide. The effects of AGE-BSA and CML-BSA on HSC proliferation, signal transduction and profibrogenic gene expression were studied in vitro. RESULTS: In hepatic fibrosis, RAGE expression was enhanced in activated HSC, and also in endothelial cells, inflammatory cells and activated bile duct epithelia. HSC expressed RAGE which was upregulated after stimulation with AGE-BSA, CML-BSA, and TNF-α. RAGE stimulation with AGE-BSA and CML-BSA did not alter HSC proliferation, apoptosis, fibrogenic signal transduction and fibrosis- or fibrolysis-related gene expression, except for marginal upregulation of procollagen α1(Ⅰ) mRNA by AGE-BSA. CONCLUSION: Despite upregulation of RAGE in activated HSC, RAGE stimulation by AGE does not alter their fibrogenic activation. Therefore, RAGE does not contribute directly to hepatic fibrogenesis.

  10. Treatment with 4-methylpyrazole modulated stellate cells and natural killer cells and ameliorated liver fibrosis in mice.

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    Hyon-Seung Yi

    Full Text Available Accumulating evidence suggests that retinol and its metabolites are closely associated with liver fibrogenesis. Recently, we demonstrated that genetic ablation of alcohol dehydrogenase 3 (ADH3, a retinol metabolizing gene that is expressed in hepatic stellate cells (HSCs and natural killer (NK cells, attenuated liver fibrosis in mice. In the current study, we investigated whether pharmacological ablation of ADH3 has therapeutic effects on experimentally induced liver fibrosis in mice.Liver fibrosis was induced by intraperitoneal injections of carbon tetrachloride (CCl4 or bile duct ligation (BDL for two weeks. To inhibit ADH3-mediated retinol metabolism, 10 μg 4-methylpyrazole (4-MP/g of body weight was administered to mice treated with CCl4 or subjected to BDL. The mice were sacrificed at week 2 to evaluate the regression of liver fibrosis. Liver sections were stained for collagen and α-smooth muscle actin (α-SMA. In addition, HSCs and NK cells were isolated from control and treated mice livers for molecular and immunological studies.Treatment with 4-MP attenuated CCl4- and BDL-induced liver fibrosis in mice, without any adverse effects. HSCs from 4-MP treated mice depicted decreased levels of retinoic acids and increased retinol content than HSCs from control mice. In addition, the expression of α-SMA, transforming growth factor-β1 (TGF-β1, and type I collagen α1 was significantly reduced in the HSCs of 4-MP treated mice compared to the HSCs from control mice. Furthermore, inhibition of retinol metabolism by 4-MP increased interferon-γ production in NK cells, resulting in increased apoptosis of activated HSCs.Based on our data, we conclude that inhibition of retinol metabolism by 4-MP ameliorates liver fibrosis in mice through activation of NK cells and suppression of HSCs. Therefore, retinol and its metabolizing enzyme, ADH3, might be potential targets for therapeutic intervention of liver fibrosis.

  11. Lymphocyte activation and hepatic cellular infiltration in immunocompetent mice infected by dengue virus.

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    Chen, Hsuen-Chin; Lai, Show-Yun; Sung, Jui-Min; Lee, Shu-Hwae; Lin, Yu-Chin; Wang, Wei-Kung; Chen, Yee-Chun; Kao, Chuan-Liang; King, Chwan-Chuen; Wu-Hsieh, Betty A

    2004-07-01

    Activation and expansion of dengue virus-specific T cells and abnormal liver functions in dengue patients have been documented. However, it remains to be determined whether T cells are involved in the pathogenic mechanism of dengue virus infection. In this study, immunocompetent C57BL/6 mice were employed to study dengue virus-induced T cell activation. Mice were inoculated with 10(8) PFU dengue virus serotype 2 strain 16681 by the intravenous route. Dengue viral core RNA was detected by RT-PCR in mouse serum, liver, spleen, and brain at different time points after infection. Splenic T cells were activated as evidenced by their expression of CD69 and O-glycosylated CD43 at as early as day 3 after infection. Splenic T cell expression of O-glycosylated CD43 and IFN-gamma production coordinately peaked at day 5. Coincided with the peak of splenic T cell activation was hepatic lymphocyte infiltration and elevation of liver enzymes. Flow cytometric analysis revealed the infiltrating CD8(+) T cell to CD4(+) T cell ratio was 5/3. After a second inoculation of dengue virus, hepatic T cell infiltration and liver enzyme levels increased sharply. The infiltrating hepatic CD8(+) T cell to CD4(+) T cell ratio increased to 5.8/1. A strong correlation was found between T cell activation and hepatic cellular infiltration in immunocompetent mice infected with dengue virus. The kinetics of liver enzyme elevation also correlated with that of T cell activation. These data suggest a relationship between T cell infiltration and elevation of liver enzymes.

  12. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

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    Qin Feng

    2013-01-01

    Full Text Available Qushi Huayu Decoction (QHD, a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK in vivo and in vitro. Nonalcoholic fatty liver (NAFL model was duplicated with high-fat diet in rats and with free fatty acid (FFA in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c, alanine aminotransferase (ALT, and aspartate aminotransferase (AST levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1 and carbohydrate-responsive element-binding protein (ChREBP in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro.

  13. Regulation of Hepatic Triacylglycerol Metabolism by CGI-58 Does Not Require ATGL Co-activation

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    Caleb C. Lord

    2016-07-01

    Full Text Available Adipose triglyceride lipase (ATGL and comparative gene identification 58 (CGI-58 are critical regulators of triacylglycerol (TAG turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes.

  14. Hepatitis A vaccine associated with autoimmune hepatitis

    Institute of Scientific and Technical Information of China (English)

    PA Berry; G Smith-Laing

    2007-01-01

    To describe a case of probable relapsing autoimmune hepatitis associated with vaccination against hepatitis A virus (HAV). A case report and review of literature were written concerning autoimmune hepatitis in association with hepatitis A and other hepatotropic viruses. Soon after the administration of formalin-inactivated hepatitis A vaccine, a man who had recently recovered from an uncharacterized but self-limiting hepatitic illness,experienced a severe deterioration (AST 1687 U/L, INR 1.4). Anti-nuclear antibodies were detectable, and liver biopsy was compatible with autoimmune hepatitis. The observation supports the role of HAV as a trigger of autoimmune hepatitis. Studies in helper T-cell activity and antibody expression against hepatic proteins in the context of hepatitis A infection are summarized, and the concept of molecular mimicry with regard to other forms of viral hepatitis and autoimmunity is briefly explored.

  15. 外源性转化生长因子β3对HSC-T6细胞内源性转化生长因子β3表达的影响%Effects of exogenous TGF-β3 on the expression of endogenous TGF-β3 in hepatic stellate cell-T6 (HSC-T6)

    Institute of Scientific and Technical Information of China (English)

    李莹; 邓亮; 钱伟; 周建宁; 徐可树

    2011-01-01

    Objective To investigate the effects of exogenous TGF-β3 on the expression of endogenous TGF-β3 in hepatic stellate cell (HSC).Methods HSCs were cultured and divided into two groups:TGF-β3group and blank control group,the cells of TGF-β3 group were exposed to TGF-β3 (10 ng/ml),whereas the blank control group was not treated.The cells were incubated in the presence of exogenous TGF-β3 and then (1)were harvested at 0h,lh,2h,4h,12h,24h,and real time PCR was performed to detect the mRNA expression of endogenous TGF-β3.(2) The cells were collected at 0h,lh,6h,12h,and western-blot was used to detect the protein synthesis of endogenous TGF-β3 in HSC; (3) The cell culture supematant was harvested at 0h,lh,2h,4h,8h,14h,24h,and ELISA was performed to measure the total protein of extracellular TGF-β3; HSCs were treated with TGF-β3 (10ng/ml) for 2h.The cells were then incubated in serum-free medium and the cell culture supernatant was harvested at 2.25h,2.5h,3h,4h,6h,10h and 14h.ELISA was used to detect the extracellular secret ion of endogenous TGF-β3 by HSCs.Results (1) Exogenous TGF-β3 treatment induced a marked increase in TGF-β3 mRNA expression.By 2h of exogenous TGF-β3 treatment,maximal TGF-β3 mRNA expression levels (2.796 ± 0.518) of 2.74 fold above control values (1.022 ± 0.038) was reached (P <0.05).Thereafter,TGF-β3 mRNA expression level declined,and the expression level was maintained at level of 1.45-fold for at least 10h and was 1.18-fold above control values by 24h TGF-β3 treatment (P < 0.05); (2)No significant difference about the intracellular protein expression level of endogenous TGF-β3 was found between two groups.(P > 0.05); (3) The total expression level of TGF-β3 reached a peak [(18.931 ± 2.904)ng/ml] at 4h after TGF-β3 treatment (1.89-fold higher than basic TGF-β3 (10ng/ml).After that,it slowly declined.The expression peak [(0.835 ± 0.027) ng/ml] induction of extracellular secreted TGF-β3 was at 3h (32.12-fold higher than

  16. Hepatoprotective activity of Tridax procumbens against d-galactosamine/lipopolysaccharide-induced hepatitis in rats.

    Science.gov (U