| Objectives Explore the effect and elucidate the possible molecular mechanism of Rhein lysinate(RHL) against bile duct ligation(BDL) induced hepatic fibrosis in rats; and explore the possible mechanisms of RHL inhibiting human hepatic stellate cells(HSC)proliferation in vitro; to provide reference for RHL in prevention and treatment of cholestatic liver fibrosis.Methods 1 In vivo:(1) 42 rats were randomly divided into sham group, model group,35 mg·kg-1, 70 mg kg-1RHL treatment group, rhein group and lysine group, 7 rats in each group, the model of Cholestatic hepatic fibrosis rats were established by BDL.(2) The level of aspartate aminotransferase(AST), alanine aminotransferase(ALT), total bile acid(TBA) and total bilirubin(TBIL) in serum were detected by the automatic biochemical analyzer.(3) Pathological examination of liver tissue by HE staining; the level of fiber in liver tissue was observed by Masson staining; the level of hydroxyproline(Hyp) in liver tissue was measured by using microplate reader and Hyp kit; the expression and localization of α-smooth muscleactin(α-SMA) in liver tissue was observed by immunohistochemical staining(IHC).(4) The levels of m RNA and the expression of transforming growth factor-β1(TGF-β1) and α-SMA were detected by RT-PCR and Western blotting. 2 In vitro:(1) HSC-LX2 cells were cultured in vitro and the cells were divided into control group, 50 μmol·l-1, 100 μmol·l-1and150 μmol·l-1 RHL group.(2) The proliferation of LX-2 cells was detected by CCK-8 after treatment with different concentration of RHL.(3) The expression levels of α-SMA and TGF-β1 in LX-2 cells were detected with Western blotting after treatment with different concentration of RHL for 48 h.Results 1 In vivo:(1) Liver weight to body weight ratio and the serum levels of AST,ALT, TBA and TBIL were increased significantly of model group rats in comparison to the values of sham-operated rats(P<0.05); after HE staining, the histological structure of the liver was observed to be destroyed, bile duct proliferation and fibroplasia were frequently observed in the model group rats, the level of Hyp was increased significantly of model group rats in comparison to the values of sham-operated rats(P<0.05); IHC showed that α-SMA expressed on the cell membrane and the cytoplasm, RT-PCR and Western blotting showed that the levels of m RNA and the expression of TGF-β1 and α-SMA were increased significantly of model group rats in comparison to the values of sham-operated rats(P<0.05).(2) Liver weight to body weight ratio and the serum levels of AST, ALT, TBA and TBIL were decreased significantly of 35, 70 mg·kg-1RHL group and rhein group rats in comparison to the values of model group rats(P<0.05); after HE staining, the histological structure of the liver was observed to be partly destroyed, bile duct proliferation and fibroplasia were observed in rats of 35, 70 mg·kg-1RHL group and rhein group, the level of Hyp was decreased significantly of 35, 70 mg·kg-1RHL group and rhein group rats in comparison to the values of model group rats(P<0.05); RT-PCR and Western blotting showed that the levels of m RNA and the expression of TGF-β1 andα-SMA were decreased significantly of 35, 70 mg·kg-1RHL group and rhein group rats in comparison to the values of model group rats(P<0.05).(3) Liver weight to body weight ratio and the serum levels of AST, ALT, TBA and TBIL were decreased significantly of35, 70 mg·kg-1RHL group rats in comparison to the values of rhein group rats(P<0.05);after HE staining, bile duct proliferation and fibroplasia were decreased in rats of 35, 70mg·kg-1RHL group, the level of Hyp was decreased significantly of 35, 70 mg·kg-1RHL group rats in comparison to the values of rhein group rats(P<0.05); RT-PCR and Western blotting showed that the levels of m RNA and the expression of TGF-β1 and α-SMA were decreased significantly of 35, 70 mg·kg-1RHL group rats in comparison to the values of rhein group rats(P<0.05). 2 In vitro:(1) CCK-8 showed that compared with the control group, the proliferation of HSC-LX2 cells in 50 μmol·l-1, 100 μmol·l-1 and 150μmol·l-1RHL group were suppressed significantly(P<0.05).(2) Western blotting showed that compared with the cells of control group, the expressions of TGF-β1 and α-SMA of HSC-LX2 cells in 50 μmol·l-1, 100 μmol·l-1 and 150 μmol·l-1 RHL group were decreased,the differences were statistically significant(P<0.05).Conclusions 1 RHL can protect the liver and against the progression of hepatic fibrosis of BDL rats. 2 RHL can down-regulate the expression of TGF-β1 and α-SMA in rats, the effect of TGF-β1 inhibiting the proliferation of HSC may be the mechanism of RHL against hepatic fibrosis. 3 RHL can inhibit the activation and proliferation of LX-2 cells,the effect may through down-regulate the expression of TGF-β1. |
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