The Inhibition Mechanism Of RHL On Liver Fibrosis In Rats And Proliferation Of LX-2 Cells

Objectives To explore the molecular mechanism of Rhein lysinate(RHL) on hepatic fibrosis induced by bile duct ligation(BDL) in vivo rats experiment. On this basis, to explore the molecular mechanism of RHL inhibit activation and proliferation of human hepatic stellate cells(HSC) LX-2 in vitro. To provide reference for RHL in prevention and treatment of cholestatic liver fibrosis.Methods 1 In vivo(1) 30 SD rats were randomly divded into four groups, the sham operation, the model group, the lysine group, 35 mg·kg-1 and 70 mg·kg-1 RHL group, 6 rats in each group, the model of Cholestatic hepatic fibrosis rats was established by bile duct ligation(BDL).(2) Pathological changes of livers in each group was assessed by Hematoxylin and eosin(HE) staining, the content of fiber in each group was observed by Masson staining.(3) The levels of m RNA of Smad2 and Smad3 in each liver tissues group were detected by q RT-PCR.(4) The expression of Smad2 and Smad3 in each liver tissues group were measured by Western blotting. 2 In vitro(1) Culturing the HSC LX-2 cells, the cells were divided into the control group, three groups with RHL(50, 100 and 150 μmol·l-1).(2) Methods the cell cycle and cell apoptosis were analysed by flow cytometry in each LX-2 cells group.(3) The expression and localization of Smad2 and Smad3 were measured by immunohistochemical analysis.(4) The levels of m RNA of Smad2 and Smad3 in each LX-2 cells group were detected by q RT-PCR.(5) The expression of Smad2 and Smad3 in each LX-2 cells group were measured by Western blotting.Results 1 In vivo(1) Compared to the sham operation group, the structure of the hepatic lobule in the model group was almostly damaged, a lot of collagen fibers located in portal area. Compared to the model group, the injury of the hepatic lobule and the fibroplasias in the RHL groups were decreased, the animal model was successfully built.(2) Compared to the sham operation group, the levels of m RNA and the expression of Smad2 and Smad3 were observably increased in the model group, the difference was statistically significant(P<0.05). Compared to the model group, the levels of m RNA and the expression of Smad2 and Smad3 were observably decreased in the RHL groups, the difference was statistically significant(P<0.05). 2 In vitro(1)The result of flow cytometry showed, compared to the control group, the percentage of LX-2 cells at G0/G1 phase apparent increased, while the percentage of LX-2 cells at S phases was apparent decreased in the RHL groups, the difference was statistically significant(P<0.05). Compared to the control group, the percentages of early and total apoptosis significantly increased in the RHL groups, the difference was statistically significant(P<0.05).(2) The immunohidtochemical analysis showed that the Smad2 and Smad3 were expressed in LX-2 cytoplasm, compared to the control group, the RHL groups markedly decreased the Smad2 and Smad3 expression, the difference was statistically significant(P<0.05).(3) The difference was statistically significant(P<0.05), compared to the control group, the levels of m RNA and the expression of Smad2 and Smad3 were observably decreased in the RHL groups,Conclusions 1 The suppression of TGF-β1/Smads inhibiting the proliferation of HSC may be the mechanism of RHL against hepatic fibrosis. 2 RHL may promote cell apoptosis to inhibit the proliferation of LX-2 cells.3 RHL may inhibit the TGF-β1/Smads signal transduction, to inhibit the excitation and proliferation of LX-2 cells.