| Liver cirrhosis and portal hypertension have a high incidence around the world especially in china, and a high mortality rate when complicating esophageal and gastric variceal bleeding.The main causes of liver fibrosis in industrialized countries include chronic HBV/HCV infection, alcohol abuse, and nonalcoholic steatohepatitis (NASH). Considering hepatic fibrosis is reversible while liver cirrhosis is still irreversible, thus it is very important and emergent to treat hepatic fibrosis to prevent it from developing to liver cirrhosis. It is widely considered that activated HSCs play an essential role in the development of hepatic fibrogenesis, however, there are many debates recently that hepatocytes may also contribute greatly to hepatic fibrogenesis.A direct contribution of hepatocytes to liver fibrosis is considered to be relatively minor, even though hepatocytes perform the majority of liver associated functions and constitute more than 80% of the liver cellular mass .More and more research demonstrate that in the fibrotic liver, fibroblasts not only derive from activated hepatic stellate cells but also from hepatocytes which undergo a epithelial-mesenchymal transition in the development of hepatic fibrogenesis.Toll-like receptors (TLRs),which play an important role in the natual immune response as an essential kind of pattern recognition receptors, also contribute greatly to the development and progress of many hepatic diseases such as alcoholic liver disease, viral hepatitis, hepatic fibrosis and so on.In this study, we focused on the role of TLRs played in epithelial-mesenchymal transition of rat hepatocytes induced by TGF-β. These results indicated unrevealed roles of TLRs in regulating epithelial-mesenchymal transition of rat hepatocytes, providing a novel therapeutic strategy during progression of hepatic fibrosis.Methods一,Isolation, identification and culture of HepatocytesHepatocytes were purified from normal male Sprague-Dawley rats (150 g-250 g) by sequential digestion of the liver with collagenaseⅣ, followed by brachytely centrifugation .Isolated hepatocytes were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), standard antibiotics, and 2 mM L-glutamine in a 95% air, 5% CO2-humidified atmosphere at 37°C. Growth medium was exchanged every other day. The survival rate of HSC was evaluated by Trypan blue staining.二,EMT of rat hepatocytes was induced by treatment with TGF-βWhen the hepatocytes were adherent,plate and formed small clusters of parenchymal cells in the dishes and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and TGF-β(5 ng/mL) 48h. Hepatocytes were assessed and shot by inverted phase contrast microscope. Control group was the hepatocytes without TGF-β. RNAs were obtained from hepatocytes with and without TGF-β. E-cad and vimentin mRNA expressions were examined by RT-PCR.三,The expression of TLR2 in EMT of rat hepatocytes Firstly,the mRNA expression of vimentin and E-cadherin were examined and then the mRNA expression of TLR2 were detected by RT-PCR.四,Statistical analysis All data were expressed as the means±S.E of at least three independent experiments. The changes of two groups was evaluated by student's-t and the correlations were evaluated by line regression analysis. The significance of changes was evaluated by One-Way ANOVA. When the variance is equal, LSD will be used. Or not, Dunnett's will be used. A P value of≤0.05 was considered statistically significant . Results一,Identification of Hepatocytes More than about 95% fresh HSC survived evaluated by trypan blue staining.二,EMT of rat hepatocytes was induced by treatment with TGF-β(一) Effects of TGF-βon the morphology of hepatocytesCell detached and changed from plate to spindle when hepatocytes were treatment with TGF-βafter 48h.(二) Effects of TGF-βon EMT of hepatocytes TGF-β-stimulated hepatocytes increased expression of vimentin mRNA(P<0.05). and decreased expression of E-cadherin mRNA(P<0.05).三,mRNA expression of TLR2 in EMT of hepatocytesTLR2 mRNA expression was increased (P<0.05). TLR2/β-actin mRNA ratio was positively correlated to vimentin/β-actin mRNA and negatively correlated to E-cadherin/β-actin mRNA.ConclusionTGF-βcan induce EMT of hepatocytes and TLR2 may play an important role in EMT of hepatocytes.
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