The Research Of LY2109761 Inhibits Proliferation And Activation Of HSC-T6 Cells Through TGF-β1/Smad Pathway

Objective:To explore the effect of LY2109761 on the proliferation and activation of rat hepatic stellate cells(HSC-T6),and to research the possible role of LY2109761 on the TGF-β1/Smad pathway.Methods:1.HSC-T6 cells were cultured in Duibecco’s Modified Eagie’s Medium(DMEM)containing 10% Fetal Bovine Serum(FBS),100 U/m L penicillin and 100 μg/m L streptomycin in an incubator at 37 °C and 5% CO2.Trypsin digestion was performed when the cell growth reached 70%-80%.The cells were divided into the control group,the LY2109761 group and the positive group.The control group was cultured in the complete medium all the time,while the LY2109761 group was treated with different concentrations of LY2109761 and the positive group was treated with colchicine for 12,24 and 36 h.The influence of LY2109761 on the proliferation of rat hepatic stellate cells(HSC-T6)were detected by Cell Counting KIT 8(CCK-8)method in which the drug concentration and time of subsequent experiments will be selected.Cells divided into the control group,the LY2109761 group and the positive group,24 h after processing,cell scratch method are used to observe cell migration ability,flow cytometry to detect cell apoptosis rate,western blot method LY2109761 of HSC-T6 cells activation key protein α-SMA and Collagen protein expression level,the influence of I observe LY2109761 affect HSC-T6 cells proliferation.2.In order to investigate the mechanism of LY2109761 inhibiting HSC-T6 cells proliferation and activation,the effects of LY2109761 on TGF-β1 protein expression level and Smad2 phosphorylation level in TGF-β1/Smad pathway of HSC-T6 cells were detected by Western blot.In order to further verify the mechanism of action,the effects of LY2109761 on the protein expression levels of α-SMA and Collagen I and the phosphorylation level of Samd2 in HSC-T6 cells were investigated after TGF-β1preparation in TGF-β1/Smad pathway was administered.Results:1.Compared with the control group,HSC-T6 cells were treated with LY2109761 and colchicine at different concentrations for 12 h,24 h and 36 h respectively.After12 h treatment,10 μmol/L and 20 μmol/L LY2109761 had no significant effect on the survival rate of HSC-T6 cells(P >0.05),and the survival rate of other groups was significantly decreased(P <0.05,P <0.01,P <0.01).The survival rate of HSC-T6 cells was not significantly affected by 10 μmol/L LY2109761(P >0.05),and the survival rate of cells in other groups was significantly decreased(P <0.05,P <0.01,P<0.01).After 36 h treatment,the survival rate of cells in the colchicine and LY2109761 groups was significantly decreased(P <0.05,P <0.01,P <0.01).The50% inhibitory concentration(IC50)of HSC-T6 cells treated with LY2109761 for 24 h was 53 μmol/L.LY2109761 with a concentration of 53 μmol/L was selected for 24 h for subsequent experiments.Compared with the control group,both the LY2109761 group and the positive group significantly decreased the cell migration ability of HSC-T6 cells(P <0.05,P <0.01),significantly increased the cell apoptosis rate of HSC-T6 cells(P <0.01),and significantly down-regulated the protein expression levels of α-SMA and Collagen I(P <0.05,P <0.01).Compared with the positive group,there were no significant differences in the migration ability,apoptosis rate,α-SMA and Collagen I protein expression levels of HSC-T6 cells in LY2109761(P>0.05).2.Compared with the control group,the expression levels of α-SMA and Collagen I and the phosphorylation level of Smad2 in the LY2109761 group were significantly increased(P <0.05,P <0.01).TGF-β1 significantly reversed the effect of LY2109761 on the expression levels of α-SMA and Collagen I and the phosphorylation level of Smad2.Conclusions:LY2109761 can effectively inhibit the proliferation,activation and migration of HSC-T6 cells,and effectively induce the apoptosis of HSC-T6 cells,and the mechanism is related to its inhibition of TGF-β1/Smad pathway.