Study On The Effect Of Sinomenine On Human Hepatic Stellate Cells In Vitro And Liver Fibrosis In Vivo

BackgroundLiver fibrosis is the necessary pathologic process of a variety of chronic liver disease progress to cirrhosis,which is due to the metabolic imbalance of liver ECM, increased generation and decreased degradation. Study showed that HSC is the main source of ECM, and TGF-β1 plays an important role in the promotion of HSC activation, proliferation and production of ECM process. It has been reported that high dose Sinomenine could decreased α-SMA and TGF-β1 expression in epithelial cells of renal tubular,inhibit epithelial-mesenchymal transitaion, alleviate the development of renal interstitial fibrosis. but the effects of sinomenine on liver fibrosis and HSC proliferation were rarely reported.Aims:This study aimed to investigate the effects of sinomenine on human hepatic stellate cells (LX-2) proliferation, apoptosis in vitro and liver fibrosis in mice induced by CCL4 in vivo.Methods(1) When the LX-2 cells were treated with different concentration sinomenine 12 and 24 hours, proliferation and apoptosis induction studies were conducted by MTS and flow cytometry analysis.Then the Bcl-2 and Bax protein expression in LX-2 were detected by western blot.(2) 40 male C57BL/6 mice were randomly divided into normal control group, liver fibrosis model group, sinomenine intervention group (low dose group and high dose group) 10 mice every group and normal control group given olive oil by intraperitoneal injection of 5 ml/kg, the other groups were given 10% of CCL4 olive oil mixture 5 ml/kg, twice a week, total of 6 weeks, After 4 weeks, the control group and model group was given saline lavages and the intervention group were given different doses of sinomenine by intragastric administration per day for two weeks and then were sacrificed.The Liver hydroxyproline was measured using a hydroxyproline detection kit according to the manufacturer’s instructions. The expression of COL1A1 and TGF-β1 were detected by western blot. pathologic changes were observed after H&E.and Masson staining.Results(1)Sinomenine reduced LX-2 proliferation by MTS assay and resulted higher levels apoptosis by FCM assay. Western blot results showed that sinomenine could increase the expression of Bax and reduce the ratio of Bcl-2 to Bax.(2) Compared to normal control group, the liver hydroxyproline and expression of TGF-β1and COL1A1 of liver fibrosis model group mice were increased (P< 0.05); liver tissue H&E staining showed severe cell necrosis, masson staining showed more collagen deposition.Compared to liver fibrosis model group, sinomenine intervention group mice showed that hydroxyproline and the expression of COL1Aland TGF-β1were reduced, especially in high dose group, and the cell necrosis and collagen deposition were alleviated.Conclusions(1) Sinomenine could inhibit the LX-2 proliferation and induce apoptosis in vitro.(2)Sinomenine could alleviate the mice liver fibrosis process in vivo, which may be related to sinomenine could lower the expression of TGF-β1, reduce the activation of hepatic stellate cells and induce apoptosis of hepatic stellate cells.