黄芩苷诱导肝癌细胞发生铁死亡的作用机制研究

Study on the mechanism of baicalin inducing ferroptosis in hepatocellular carcinoma cells

目的探讨黄芩苷诱导肝癌HepG-2细胞发生铁死亡的可能机制。方法该研究采用不同浓度(10、20、30、40、50、60、70、80、90,100μmol/L)的黄芩苷处理肝癌HepG-2细胞24 h,采用四甲基偶氮唑蓝比色法检测细胞活力并计算半数抑制浓度(IC_(50))。根据IC_(50)将细胞分为对照组(黄芩苷浓度=0μmol/L)、25μmol/L黄芩苷处理组和50μmol/L黄芩苷处理组,使用谷胱甘肽(GSH)试剂盒测定分组后细胞内GSH水平;采用Western blot试验检测分组后细胞内铁死亡相关蛋白胱氨酸-谷氨酸的反向转运体亚基(xCT)、谷胱甘肽过氧化物酶4(GPX4)、二价金属转运蛋白1(DMT1)表达水平;采用DCFH-DA探针检测细胞内活性氧(ROS)水平。随后本研究测定了同时使用铁死亡抑制剂Ferrostatin-1和黄芩苷处理后的细胞活力、GSH水平变化。采用Pearson相关对肝癌HepG-2细胞内GSH水平与黄芩苷浓度的相关性进行分析。结果肝癌HepG-2细胞活力随着黄芩苷作用浓度增加而明显降低。黄芩苷能减少肝癌HepG-2细胞内GSH水平,下调细胞中xCT和GPX4蛋白表达水平,上调DMT1表达水平和升高ROS水平。Ferrostatin-1可有效抑制黄芩苷诱导的细胞铁死亡,主要表现为细胞活力和GSH水平恢复。Pearson相关分析结果显示,肝癌HepG-2细胞内GSH水平与黄芩苷浓度呈负相关(P<0.05)。结论黄芩苷可通过下调GPX4和xCT蛋白表达水平,上调DMT1表达水平,诱导ROS大量生成,并导致肝癌HepG-2细胞铁死亡。

Objective To investigate the possible mechanism of ferroptosis induced by baicalin in hepatocellular carcinoma HepG-2 cells.Methods This research adopted the different concentrations(10,20,30,40,50,60,70,80,90,100 mol/L)of baicalin HepG-2 of liver cancer cells within 24 h,determined by tetramethyl azazole blue colorimtric method was used to detect the cell vitality and calculate the half inhibitory concentration(IC_(50)).According to IC_(50),the cells were divided into control group(baicalin concentration=0μmol/L),25μmol/L baicalin treatment group and 50μmol/L baicalin treatment group.Glutathione(GSH)level was measured by GSH kit.Western blot test was used to detect the expression levels of ferroptosis-related proteins cystine-glutamate antiporter subunit(xCT),glutathione peroxidase 4(GPX4)and divalent metal transporter 1(DMT1)in after each group.The intracellular reactive oxygen species(ROS)level was detected by DCFH-DA probe.The changes of cell viability and GSH level after co-treatment with baicalin and ferroptosis inhibitor Ferrostatin-1 were determined.Pearson correlation was used to analyze the correlation between the intracellular GSH level and baicalin concentration in HepG-2 cells.Results The viability of HepG-2 cells was significantly decreased with the increase of baicalin concentration.Baicalin could reduce the GSH level in HepG-2 cells,down-regulate the expression levels of xCT and GPX4 proteins,up-regulate the expression level of DMT1 and increase the level of ROS.Ferrostatin-1 could effectively inhibit the ferroptosis induced by baicalin,which was mainly reflected in the recovery of cell viability and GSH level.Pearson correlation analysis showed that intracellular GSH level was negatively correlated with baicalin concentration in HepG-2 cells(P<0.05).Conclusion Baicalin can down-regulate the protein expression levels of GPX4 and xCT and up-regulate the expression level of DMT1,induce a large amount of ROS and lead to ferroptosis in hepatocellular carcinoma HepG-2 cells.