| Objective:The aim of this study was to investigate the dynamic changes of TGF-β1/Smad pathway in chronic liver diseases and the anti-fibrotic mechanism of cordyceps mycelia extracts (CME).Methods:1.168male SD rats were randomLy divided into4groups:control group, model group, CME (80mg/kg) treatment group and solvent group. Rats in control group were given0.9%normal saline orally by lOmL/kg five times a week till the14th weeks; rats in model group were given0.2%DEN (0.975g/mL) solution orally by10mg/kg according to body mass five times a week till the14th weeks; CME treatment group received the same disposal as the model group and then were given80mg/kg CME from the fifth week to the sixteenth week; solvent group received the same disposal as the model group and then were given the same volume of solvent as the CME treatment group.2. At the stage of hepatitis, liver fibrosis, liver cirrhosis and hepatoc-ellular carcinoma,12rats each group were sacrificed and whole blood was collected from the orbit to determine the liver function index ALT and AST. Liver samples were prepared for Hematoxylin Eosin staining and Masson staining to identify the liver damage degree.3. Primary HSC were isolated by the improved Nycodenz method; RT-PCR, QRT-PCR and Western blotting were also used to determine the expression of Smad3, Smad7.Results:1. The Scheuer formLa for HE and Masson staining score showed that at wk4,8,12,16, the rats were respectively at the stage of hepatitis, liver fibrosis, liver cirrhosis and early hepatocellular carcinoma; immuno-cytochemistry test showed that the cytoplasm of HSC can be stained to brown, the HSC can emit blue-green fluorescence light under ultraviolet excitation. 2. The expression of α-SMA, TGF-β1, Smad3, Smad7in model group showed dynamic changes.(1) The expression of α-SMA of model group in liver tissue and HSC at wk4,8,12,16were significantly higher than that of control group, and it peaked at wk12,16.(2) The expression of TGF-β1in liver tissue and HSC were inconsistent. The expression of TGF-(31of model group in liver tissue significantly increased at wk4,8,12compared to control group, while there were no significant difference between them at wk16; The expression of TGF-β1of model group in HSC significantly increased compared to control group at wk4,8,12,16, and peaked at wk8.(3)The expression of Smad3in liver tissue and HSC were consistent. The expression of Smad3mRNA and protein of model group in liver tissue at wk4,8,12were up-regulated compared to control group; Smad3mRNA expression of model group in liver tissue at wk16were up-regulated compared to control group, while there werer no significant difference of Smad3protein expression between the two group. Smad3mRNA expression of model group in HSC were up-regulated compared to control group at wk4,8,12,16, and peaked at wk8.(4) Smad7mRNA and protein expression of model group in liver tissue and HSC were consistent at wk4,8,12, it had no significant difference compared to control group. The expression of Smad7mRNA and protein in liver tissue significantly increased compared to control group at wk16, while the expression of Smad7mRNA and protein in HSC decreased compared to control group.3. CME could alleviate rats chronic liver disease progess obviously.(1) All indicators in solvent group had no significant difference with model group.(2) ALT and AST in CME treatment group were decreased compared to model group, HE and Masson staining score showed that CME could alleviate the liver inflammation activity, collagen deposition and liver cell damage degree at wk8,12,16.(3) Compared to model group, CME could significantly decrease the expression of α-SMA, TGF-β1, Smad3in liver tissue and a-SMA, Smad3in HSC and increase Smad7expression in liver tissue and HSC at wk8. CME could decrease α-SMA, TGF-β1, Smad3expression in liver tissue and a-SMA expression in HSC at wk12. CME could down-regulated a-SMA expression in liver tissue and up-regulated a-SMA expression in HSC at wk16.Conclusions:1. The high expression of TGF-β1, Smad3and low level of Smad7may be essential for the HSC activation; the high expression of TGF-β1, Smad3and low level of Smad7in liver tissue and HSC activation may be related to the progress of liver fibrosis, liver cirrhosis; the low expression of TGF-β1and high expression of Smad7may be associated with early hepatocellular carcinoma.2. CME can alleviate the DEN induced chronic liver injury possibly through suppressing the TGF-β1/Smad pathway, HSC activation and up-regulating the Smad7expression. |
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