Effects Of Salinomycin On The Proliferation And Extracellular Matrix Secretion Of Rat Hepatic Stellate Cells

Objectives This study was designed to determine the effects of salinomycin on proliferation and extracellular matrix secretion of rat hepatic stellate cells in vitro, and to investigate whether it exerts effects through inhibition of Wnt/?-catenin signaling pathway.Methods HSC-T6, the research object, was treated with different concentrations of salinomycin and its solvent dimethyl sulfoxide(DMSO) for different period time. The CCK-8 method was used to test the effects of salinomycin on cells' proliferation, then the optimum concentration range and best treatment time of salinomycin can be drew. According to these, we setted the salinomycin concentrations in follow-up experiments. The cell morphology changes among normal group, solvent control group and salinomycin treatment groups were observed by optical inverted microscope. The levers of type ? procollagen(PC?) and hyaluronic acid(HA),which are leading marks of liver fibrosis,were assessed by chemiluminescence immunoassay. Flow cytometry(stained by PI) was used to detecte cell cycle. The m RNA and protein levels of ?-catenin, cyclin D1 and c-Myc, which are key molecules of Wnt/?-catenin signaling pathway, were measured by RT-PCR and Western Blot respectively.Results Salinomycin inhibited the proliferation of HSC-T6 cells in vitro in a dose- and time-dependent manner within limits, and its IC50 was 27.7?mol/L, according to which the salinomycin concentrations in subsequent experiments were setted at 15?mol/L, 30?mol/L and 60?mol/L. Salinomycin changed cell morphology. Compared with the control groups, cells of treatment groups were smaller and with karyopyknosis, and a part of them decomposed into debrises. Those changes became more obvious as the salinomycin concentration increased. Chemiluminescence immunoassay indicated that salinomycin can reduce the levels of PC?,HA in a dose-dependent manner. Salinomycin arrested HSC-T6 in G1 phase. With the increase of salinomycin concentration, the ratios of cells in G1 phase upregulated and the ratios of cells in S phase downregulated. RT-PCR results showed that the m RNA levels of ?-catenin, cyclin D1 and c-Myc were inhibited by salinomycin.Western Blot results indicated that salinomycin downregulated the protein levels of ?-catenin, cyclin D1 and c-Myc in a dose-dependent manner.Conclusion Salinomycin may inhibite the proliferation of HSC-T6 cells and reduce the levels of extracellular matrix by inhibiting the Wnt/?-catenin signaling pathway.