重楼皂苷Ⅱ诱导肝癌HepG2细胞铁死亡作用机制

Mechanism of PolyphyllinⅡin Induction of Ferroptosis in Hepatocellular Carcinoma HepG2 Cells

目的:探讨重楼皂苷Ⅱ(PPⅡ)诱导肝癌HepG2细胞发生铁死亡作用及其机制。方法:采用噻唑蓝(MTT)比色法检测PPⅡ(0、1.5、3.0、4.5、6.0、9.0、18.0 mg·L^(-1))对HepG2细胞体外增殖能力的影响;平板克隆实验检测HepG2细胞克隆形成能力;划痕实验检测HepG2细胞迁移能力;利用试剂盒检测HepG2细胞中乳酸脱氢酶(LDH)的含量;借助荧光倒置显微镜观察HepG2细胞的活性氧(ROS)水平;利用试剂盒检测HepG2细胞中丙二醛(MDA)、谷胱甘肽(GSH)和游离Fe^(2+)的含量;利用透射电镜观察HepG2细胞的线粒体超微结构;采用蛋白免疫印迹法(Western blot)检测HepG2细胞中铁死亡相关蛋白p53、溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GPX4)、长链脂酰辅酶A合成酶4(ACSL4)及转铁蛋白受体1(TFR1)表达的情况。结果:与空白组比较,PPⅡ组HepG2细胞存活率显著下降,并呈浓度依赖性(P<0.01),细胞克隆数显著减少(P<0.01),划痕愈合距离明显缩短,迁移距离和药物浓度呈反比(P<0.01),细胞LDH的泄漏显著增多(P<0.01),细胞内ROS相对荧光强度显著增强,细胞内脂质过氧化物MDA积累量显著增多(P<0.01),细胞内GSH含量随着药物浓度的增大而显著减少(P<0.01),细胞FeRhoNox-1荧光强度显著增强(P<0.01),细胞出现空泡,线粒体明显皱缩,线粒体嵴减少甚至消失。与空白组比较,PPⅡ组细胞中p53、ACSL4、TFR1蛋白表达明显上调,SLC7A11、GPX4蛋白表达明显下调(P<0.05)。结论:综上,PPⅡ通过调控p53/SLC7A11/GPX4信号通路轴,促进ACSL4表达和细胞摄取Fe3+,使抗氧化系统失衡,诱导HepG-2细胞铁死亡。

Objective:To investigate the induction of ferroptosis by polyphyllinⅡ(PPⅡ)in hepatocellular carcinoma HepG2 cells and its underlying mechanism.Method:The effect of PPⅡ(0,1.5,3.0,4.5,6.0,9.0,18.0 mg·L^(-1))on the in vitro proliferation of HepG2 cells was assessed using the methyl thiazolyl tetrazolium(MTT)assay.Colony formation ability of HepG2 cells was evaluated through a colony formation assay.Cell migration ability was assessed via a scratch assay.Lactate dehydrogenase(LDH)content in HepG2 cells was measured using a kit.Reactive oxygen species(ROS)levels in HepG2 cells were observed using a fluorescence inverted microscope.Malondialdehyde(MDA),glutathione(GSH),and free Fe^(2+)content in HepG2 cells were detected using respective kits.The mitochondrial ultrastructure in HepG2 cells was observed by transmission electron microscopy.The expression of ferroptosis-related proteins p53,solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4),long-chain acyl-CoA synthetase 4(ACSL4),and transferrin receptor 1(TFR1)in HepG2 cells was detected using Western blot.Result:Compared with the control group,the PPⅡtreatment groups showed significantly decreased survival rate of HepG2 cells in a dosedependent manner(P<0.01),significantly reduced number of cell colonies(P<0.01),significantly shortened scratch healing distance,inverse correlation of the migration distance with drug concentration(P<0.01),significantly increased LDH leakage in cells(P<0.01),significantly enhanced relative fluorescence intensity of intracellular ROS,and significantly increased accumulation of lipid peroxide MDA(P<0.01),decreased intracellular GSH content with increasing drug concentration(P<0.01),and significantly enhanced fluorescence intensity of FeRhoNox-1 in cells(P<0.01).Moreover,cells exhibited vacuolation,and mitochondria showed significant shrinkage with reduced or even disappeared cristae.Compared with the results in the control group,the expression of p53,ACSL4,and TFR1 proteins significantly increased,while the expression of SLC7A11 and GPX4 proteins significantly decreased in the PPⅡtreatment groups(P<0.05).Conclusion:In summary,PPⅡinduces ferroptosis in HepG2 cells by regulating the p53/SLC7A11/GPX4 signaling axis,promoting ACSL4 expression and Fe3+uptake,leading to an imbalance in the antioxidant system.