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[Effect of ursolic acid on proliferation and apoptosis of hepatic stellate cells in vitro].

OBJECTIVE: To investigate the effect of ursolic acid on proliferation and apoptosis of hepatic stellate cells (HSC) in vitro and explore the mechanisms of apoptosis of HSC induced by ursolic acid by studying the expressions of apoptosis-regulating proteins Bcl-2, Bax and Caspase 3 in HSC.

METHODS: Hepatic stellate cells HSC-T6 and hepatocytes L02 were incubated with different concentrations of ursolic acid (25, 50, 75, 100, 125 and 150 micromol/L) for 24 h, 48 h and 72 h. The effect of ursolic acid on the cell proliferation was studied by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The rate of HSC-T6 apoptosis was identified by flow cytometry (FCM) and the morphological change of apoptosis was observed with light microscopy. The expressions of apoptosis-regulating protein Bcl-2, Bax and Caspase 3 in HSC-T6 after apoptosis induced by ursolic acid were examined by immunocytochemical staining assay.

RESULTS: MTT analysis indicated administration of 25-150 micromol/L ursolic acid incubated with HSC-T6 for 24 h, 48 h and 72 h significantly inhibited HSC-T6 proliferation in a dose-dependent and time-dependent manner compared with the control group. Promotive effect of ursolic acid on proliferation of hepatocyte L02 was observed in the 25, 50, 75 micromol/L concentration groups. Ursolic acid inhibited L02 proliferation when its concentration was higher than 100 micromol/L and for 72 hours or longer. HE stained histological slides demonstrated morphologic changes of HSC-T6, including karyorrhexis and cytoplasm vacuolization, when they were treated with ursolic acid at 75 micromol/L concentrations for 48 h. FCM showed the apoptosis ratios of HSC-T6 were 10.30%+/-3.85%, 21.87%+/-4.46% and 31.33%+/-6.18% after treating HSC-T6 with ursolic acid at concentrations of 25, 50 and 75 micromol/L for 48 h. They were significantly higher than that of the control group 2.93%+/-1.60%. Immunocytochemistry also indicated the expressions of Bax and caspase 3 protein in HSC-T6 cells were up-regulated in a dose-dependent manner, but expressions of Bcl-2 protein were not significantly different from that of the blank control group (P more than 0.05).

CONCLUSIONS: Ursolic acid could significantly inhibit HSC proliferation and induce apoptosis in a dose-dependent and time-dependent manner. Ursolic acid in low concentration promotes proliferation of L02 cells, but in high concentrations (more than 100 micromol/L) it inhibits the growth of hepatocytes. Expressions of Bax and Caspase 3 in apoptotic HSC were increased; expressions of Bcl-2 protein were not significantly different from that of the control group, while Bcl-2/Bax ratio was reduced. Our results suggest that HSC-T6 cell apoptosis induced by ursolic acid occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.

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