Role Of Wnt/bata-catenin Singnaling Pathways In Rat Hepatic Stellate Cell Apoptosis Induced By Salinomycin

Objectives To explore whether salinomycin exerts antifibrotic effects in rat hepatic stellate cells.Methods(1) Activated HSC-T6 were treated with different concentritions of salinomycin(5?mol/l, 10?mol/l, 20?mol/l, 40?mol/l and 80?mol/l) for different time(12 h, 24 h and 48h). CCK-8 was used to determine cell proliferation. Through above the best concentration and best action about salinomycin was chosen for the follow-up experiments.(2) The cell morphology was observed by optical inverted fluorescence microscope, which was stained by AO/EB bifluorescence.(3) Cells stained by Annexin-FITC/PI were used to detect the cell apoptosis rate through flow cytometry.(4) The ?-catenin protein expression and location within the cell were used immunocytochemistry method to detect different concentrations under the action of salinomycin.(5) The expressions of ?-catenin and Survivin were measured by Western Blot and RT-PCR which were incubated with different concentrations of salinomycin.Results(1) Salinomycin inhibited the proliferation of HSC-T6 cells significantly in vitro, within a certain range in concentration and time dependence(P<0.05). At low concentration of salinomycin, within 24 hours of inhibition was no significant difference, when a large concentration of salinomycin, the inhibition rate of more than 24 hours and does not further increase with time. The best concentration of salinomycin was observed from 24 hours' IC50(27.87?mol/l), and according to which the salinomycin concentrations were setted at 15?mol/l, 30?mol/l and 60?mol/l.(2) Salinomycin promoted cells apoptosis:(1) Cell nucleus were observed by optical inverted fluorescence microscope, drug groups stained by AO/EB bifluorescence had apoptosis morphology changes such as the nucleus pycnosis, rupture and so on,and apoptosis cells were stained brightly and uniform. In the field of vision we can saw early apoptotic cells the nuclear chromatin were stained by green fluorescence and late apoptotic cells the nuclear chromatin were stained by orange fluorescence.Those changes became more obvious as concentration increasing.(2) The apoptosis rate of drug groups increasd as salinomycin concentration increasing, which were detected by flow cytometry, followed by Annexin-FITC/PI stained. Salinomycin main cause of early apoptotsis, late apoptotic effect was not obvious(P<0.05).(3) The expression of ?-catenin was found both inside and outside the nucleus which were used by immunocytochemistry, and the treated groups were lower than normal control(P<0.05).(4) Results from RT-PCR and Western Blot showed than the expression of ?-catenin and Survivin were lower in salinomycin treated group compared with normal control(P<0.05).Conclusion(1) Salinomycin suppresses the proliferation of HSC-T6 cells significantly in vitro in a dose- and time-dependent manner.(2) Salinomycin induces HSC-T6 apoptosis in a obvious dose-dependent manner, which may be related to the downregulation of Wnt/?-catenin signaling pathway.