Effect Of Ursolic Acid On Proliferation And Apoptosis Of Hepatic Stellate Cells In Vitro

Background: Hepatic fibrosis occurs in the course of wound healing in response to chronic liver injury. This response represents the final common pathway for many chronic liver diseases. Nowdays,research about hepatic fibrosis has focused on the hepatic stellate cell (HSC), a kind of mesenchymal cell responsible for fibrosis. The perisinusoidal HSC is largely responsible for the increase in extracellular matrix(ECM) deposition. Prolonged liver injury results in hepatocyte damage and secretion of many fibrogenic cytokines such as TGF-beta1, angiotensin II, and leptin, which triggers activation of hepatic stellate cells by different intracellular signalling pathways. During the recovery of hepatic fibrosis, diminution of activated HSC is mainly associated with apoptosis but not its phenotype transforming from activated state to quiescent state. Consequently inhibiting the activation and proliferation of HSC and inducing apoptosis of activated HSC may provide a novel therapeutic approach to the treatment of advanced hepatic fibrosis. Recently, we selected several bioactive components that have been isolated from Chinese herbs as part of our search for anti-liver fibrosis materials. We found that Ursolic acid can inhibits hepatic fibrosis,The aim of our present research was to investigate effect of Ursolic acid on proliferation and apoptosis of hepatic stellate cells and mechanisms of its anti-liver fibrosis.Objective:To investigate the effect of Ursolic acid on proliferation and apoptosis of HSC in vitro and explore the mechanisms of Ursolic acid inducing apoptosis of HSC by studying the expressions of apoptosis-regulating proteins Bcl-2,Bax and Caspase3 in HSC.Methods: Hepatic stellate cells (HSC-T6) and hepatocytes (L02) was incubated with different concentrations of Ursolic acid(25μM,50μM,75μM,100μM,125μM and150μM) for different times(24h,48h,72h). The effect of Ursolic acid on cell proliferation was observed by MTT colormetric assay. The rate of HSC-T6 apoptosis was identified by flow cytometry (FCM) and the morphological change of apoptosis was observed by light microscopy. And then the expression of apoptosis-regulating protein Bcl-2,Bax and Caspase3 in HSC-T6 after apoptosis induced by Ursolic acid were examined by immunocytochemical staining assay . Result:①MTT analysis: Administration of 25-150μM Ursolic acid incubated with HSC-T6 for 24h, 48h and 72h could significantly inhibit HSC-T6 proliferation in dose-dependent and time-dependent manner compared with the control group(p<0.05); Promotive effect of Ursolic acid on proliferation of hepatocytes (L02) was observed in 25μM,50μM,75μM concentration groups. when its concentration is more than 100μM for 72 hours or longer, Ursolic acid could inhibit L02 proliferation.②HE staining: Treatment with 75μM concentrations of Ursolic acid for 48h resulted in morphologic changes of HSC-T6, including karyorrhexis and cytoplasm vacuolization.③Flow cytometry quantitation: After treating HSC-T6 with Ursolic acid at concentration of 25, 50, 75μM for 48h, the apoptosis ratio of HSC-T6 were (10.30±3.85)%, (21.87±4.46)%, (31.33±6.18)% respectively, which were significantly higher than that of control group(2.93±1.60)%(P<0.01).④Immunocytochemistry: the expressions of Bax and caspase3 protein in HSC-T6 cells were up-regulated(P<0.05) in dose-dependent manner,,but expressions of bcl-2 protein were not significantly different from that of blank control group (P﹥0.05).Conclusion:①Ursolic acid could significantly inhibit HSC proliferation and induce apoptosis in dose-dependent and time-dependent manner.②Promotive effect of Ursolic acid on proliferation of L02 cell was observed in low concentration groups, but it could inhibite hepatocytes growth in high concentration groups(≥100μM)③Expressions of Bax and Caspase3 in apoptotic HSC were increased , expressions of bcl-2 protein were not significantly different from that of control group,while Bcl2/Bax ratio was reduced.④Our results suggested that HSC-T6 cells apoptosis induced by Ursolic acid occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins. The study also indicated that Ursolic acid might be a potential Chinese medical component for inhibiting liver fibrosis.