Synergistic Effect Of Deoxyschizandrin And Gemcitabine On The Proliferation Of HepG2 Human Hepatocellular Carcinoma Cell

Objective:To investigate the synergistic effect of deoxyschizandrin and GEM on the proliferation of human hepatocellular carcinoma cell and further explore its molecular mechanism.Methods:1.Effects of deoxyschizandrin,GEM and combination treatment on the proliferation and apoptosis of HepG2 cells:HepG2 cells were treated with different concentrations of deoxyschizandrin,GEM,and their combination for 48 h,respectively.The CCK-8 method and colony formation assay were used to detect the proliferation of HepG2 cells.The flow cytometry was used to evaluate the apoptosis of HepG2 cells in each group.2.Effects of deoxyschizandrin,GEM and their combination on regulating the expression of proteins which related with proliferation and apoptosis in HepG2 cells:HepG2 were treated with deoxyschizandrin,GEM,and their combination for 48 h,respectively.Western blot was performed to detect the expression of BCL2,BAX,pro-Caspase3,cleaved-Caspase3,pro-Caspase9and cleaved-Caspase9 in HepG2.3.Effects of deoxyschizandrin,GEM and their combination on the?-catenin/TCF-4 pathways in human HepG2 cells:The active site of?-catenin was identified by computer.Subsequently,molecular docking was performed to identify the binding site between deoxyschizandrin and the active pocket of?-catenin.HepG2 were treated with deoxyschizandrin,GEM,and their combination for 48 h,respectively.Western blot was used to detect the expression level of the proteins involved in?-catenin/TCF-4 pathway.Results:1.The results of CCK-8showed that deoxyschizandrin,GEM and their combination inhibited the proliferation of HepG2 cells?p<0.05?.The growth rates of deoxyschisandrin(15 mol·L^-1),GEM(5mol·L^-1)and their combination were?80.7±3.6?%,?75.7±3.6?%and?35.0±1.4?%,respectively.The results of plate cloning suggested that deoxyschizandrin,GEM and their combination inhibited the cell cloning of HepG2 cells?p<0.05?,and the number of cell clones of deoxyschisandrin,GEM,combination of them and the control group were?324±14?,?309±10?,?156±18?and?455±8?.The results of flow cytometry indicated that deoxyschisandrin monotherapy,GEM monotherapy and the combination therapy promoted the apoptosis of HepG2 cells?p<0.05?,while the apoptosis rates of the control group,deoxyschisandrin group,GEM group and the combination group were?2.04±0.19?%,?5.44±0.33?%,?6.17±0.20?%,and?20.68±0.39?%.2.After treatment with deoxyschisandrin,GEM and their combination,compared with the control group and single drug group,pro-Caspase3 and pro-Caspase9 had no significant effect,and the expression of BCL2 was significantly reduced?p<0.05?.The expression of apoptotic protein BAX,cleaved-Caspase3 and cleaved-Caspase9 was significantly increased?p<0.05?.3.The computer test results showed that deoxydschisandrin could be combined with?-catenin active site ser473;Western blot results showed that deoxyzchisandrin,GEM,and combined treatment of cells down-regulated the protein expression of?-catenin and TCF-4?p<0.05?.Conclusion:1.The combination of deoxyschizandrin and GEM significantly inhibited the proliferation of HepG2 cells,promoted their apoptosis.2.The combined application of deoxyschizandrin abd GEM regulated the expression of BCL2,BAX,Caspase3 and Caspase9 proteins,which affects the apoptosis of HepG2 cells.3.Deoxyschizandrin and GEM inhibited the?-catenin/TCF-4 pathway in HepG2 cells,thereby reducing cell proliferation and inducing apoptosis.