Synergistic Effect Of Nrf2 Pathway Inhibitor ML385 In Chemotherapy Of Hepatoma Cells In Vitro

Objective: To study the Nrf2 pathway specific inhibitor ML385 combined with the chemotherapeutic drug epirubicin to selectively enhance the killing effect of liver cancer cells,and to provide theoretical and experimental basis for further selective improvement of cancer chemotherapy effect and reduction of chemotherapy adverse reactions in clinical treatment.Methods: In this study,the method of in vitro cell experiment was used.The human normal liver cell line L-02 and the liver cancer cell line Hep G2 were selected,and then passaged and cultured them respectively.Poisoning according to the blank group,ML385 group,epirubicin group and combination group(ML385-Epirubicin)group.The drug treatment time of each group was 24 hours.The cell survival rate of each group was measured by SRB method,and the best combined dosing regimen was screen.DCFH-DA fluorescence staining method was used to detect intracellular ROS level,WST-8 method to detect intracellular total SOD content,TBA microplate method to detect MDA level,enzyme cycle method to detect intracellular GSH level and r GSH / GSSG ratio,and western blot detection the levels of Nrf2 protein,NQO1 protein,and PARP1 protein expression,to explore the selective synergistic effect and mechanism of ML385 in hepatocellular carcinoma chemotherapy system.Results: The combined dosing regimen is ML385: 5umol /L and epirubicin: 0.2umol/L.In this ratio,the killing ability of Hep G2 cells is higher than that of L-02 cells(p<0.05).The cell survival rate of the combined group,the blank and the single-acting group is statistically significant(p<0.01);Both L-02 and Hep G2 lines showed that the levels of ROS and MDA in the combined group were higher than those in the blank group and epirubicin group alone(p<0.05),and in Hep G2 cells,the ML385 combined epirubicintreated group were much higher than those in L-02 cells(p<0.05).The total SOD level of the two-cell combination group was higher than that of the single-administration group and the blank group,but the intracellular GSH / GSSG ratio was lower than that of the blank group,and the intracellular GSH / GSSG ratio of Hep G2 cells was significantly lower than L-02 Cells(p<0.05).In addition,in these cell lines,the expression levels of total Nrf2 protein and NQO1 protein in the combination group were lower than those in the blank group and the chemotherapy drug group alone(p<0.05),and Hep G2 cells were lower than L-02 cells(p<0.05).In the two cell lines,there was no statistically significant difference in the expression level of PARP1 protein between the combined drug administration group and the epirubicin group,but it increased compared with the blank group and the ML385 group(p<0.05),and Hep G2 cells were higher than L-02 cells(p<0.05).Conclusion: On the one hand,combined administration of ML385-epirubicin can selectively induce more ROS and MDA in hepatoma cells,on the other hand,can reduce glutathione reductase enzyme activity by inhibiting the Nrf2 signaling pathway,and as a result,the ability to scavenge ROS is reduced,thereby reducing the survival rate of liver cancer cells and improving the selective synergistic effect of chemotherapy.