Apoptosis Induced By Diallyl Disulfide In Rat Hepatic Stellate Cells And Its Mechanism

Objectives To observe the apoptosis induced by the diallyl disulfide in rat hepatic stellate cells, and to explore whether it exerts antifibrotic effects through inducing HSC-T6 apoptosis by downregulating bcl-2 and upregulating bax.Methods 1.CCK-8 method was used to observe the effects of DADS on cells proliferation. Cells were treated with DADS at different concentrations(0, 50, 100, 200, 400, 800μmol/L) for different time(24, 48, 72h) respectively, then the optimum concentration range and best treatment time were drew by the CCK-8 results. Lastly, the DADS concentrations in follow-up experiments were setted by the optimum concentration range and best time. 2.The cell morphology was observed by the optical inverted microscope, then the apoptosis morphological of cell nucleus stained by Hoechst33342 was observed by the fluorescence microscope. 3.Cells stained by PI were used to detecte the cell cycle and ones stained by Annexin-FITC/PI were used to detecte the cell apoptosis rate through the flow cytometry. 4.Both of immunohistochemistry and western blot were used to detecte the effects of DADS on the expression of Bcl-2 and Bax.Results 1.DADS inhibited the proliferation of HSC-T6 cells significantly in vitro in a dose-dependent manner. The best inhibition effects of DADS were observed from cells treated for 48 hours and its IC50 was 292μmol/L, according to which the DADS concentrations in subsequent experiments were setted at 0, 150, 300 and 600μmol/L. 2.DADS changed significantly the cell morphology. Compared with the control group, cells of drug groups were smaller and with karyopyknosis, sticked a wall unevenly, and even part of them decomposed into debrises. Those changes became more obvious as concentration increasing. 3.DADS blocked the cell cycle in S phase. With the increase of DADS concentration, the proportion of cells in S phase increased significantly and decreased in G0/G1 phase and G2/M phase, which suggested the cell cycle arrested in S phase. 4.DADS promoted cells apoptosis. Firstly, cell nucleus of drug groups stained by Hoechst33342 had apoptosis morphology changes, such as karyopyknosis like dense clumps with strong blue fluorescence, nuclear fragmentation, apoptotic body formation and so on. Secondly, the apoptosis rate of drug groups increasd as DADS concentration increasing. 5.Compared with the control group, both of immunohistochemistry and Western Blot confirmed the protein expression of Bcl-2 was decreased, while Bax increased in the drug groups.Conclusion 1.DADS significantly inhibits HSC-T6 proliferation in vitro. 2.DADS induces HSC-T6 cells apoptosis obviously, which may be related to the downregulation of bcl-2 and upregulation of bax. 3.DADS may play a role in anti-fibrosis by inhibiting HSC-T6 proliferation and inducing it apoptosis.