阿托伐他汀诱导的MIN6细胞铁死亡及相关机制研究

Study of atorvastatin induced ferroptosis in MIN6 cells and its related mechanisms

目的:探讨阿托伐他汀(atorvastatin,Ator)是否可诱导小鼠胰岛β细胞株MIN6细胞发生铁死亡,并探讨其可能的作用机制。方法:将MIN6细胞分为对照组、Ator组、Ator+凋亡抑制剂(Z-VAD-FMK)组、Ator+坏死抑制剂(necrostatin-1,Nec-1)组和Ator+铁死亡抑制剂(ferrostatin-1,Fer-1)组。采用CCK-8法检测细胞活力;透射电镜观察细胞超微结构;荧光显微镜观察活性氧(reactive oxygen species,ROS)和Fe^(2+)水平;酶联免疫吸附试验(enzyme-linked immuno sorbent assay,ELISA)检测丙二醛(malondialdehyde,MDA)和还原型谷胱甘肽(glutathione,GSH)含量;实时荧光定量PCR法(quantitative real-time PCR,RT-qPCR)检测凋亡基因半胱氨酸蛋白酶3(caspase-3)、坏死基因受体结合丝氨酸苏氨酸激酶3(receptor-interacting serine threonine kinase 3,Ripk3)、铁死亡相关基因长链酯酰辅酶A合成酶4(acyl-coA synthetase long-chain family member 4,Acsl4)、前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,Ptgs2)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,Gpx4)的mRNA表达水平;Western blot检测4-羟基壬烯醛(4-hydroxynonenal,4-HNE)和GPX4的蛋白表达水平。结果:与Ator组相比,Ator+Z-VAD-FMK组和Ator+Fer-1组细胞存活率更高(P均<0.01)。透射电镜下Ator组细胞可见凋亡、铁死亡和自噬相关的形态学特征。与对照组相比,Ator组细胞Fe^(2+)相对荧光强度、MDA水平和ROS相对水平均升高,GSH含量下降;caspase-3、Acsl4、Ptgs2的mRNA及4-HNE的蛋白表达增加(P均<0.05),GPX4的mRNA和蛋白表达减少(P<0.05)。与Ator组相比,Ator+Fer-1组Fe^(2+)相对荧光强度、MDA水平和ROS相对水平均下降,GSH含量上升;Acsl4的mRNA表达减少,Gpx4的mRNA表达增加(P均<0.05);4-HNE的蛋白表达减少而GPX4的蛋白表达增加,但差异无统计学意义。结论:Ator可能通过抑制甲羟戊酸途径下调GPX4表达,诱导MIN6细胞发生铁死亡。

Objective:To explore whether atorvastatin(Ator)can induce ferroptosis in pancreaticβ-cell line MIN6 cells and its possible mechanism.Methods:MIN6 cells were divided into control group,Ator group,Ator+apoptosis inhibito(r Z-VAD-FMK)group,Ator+necrostatin-1(Nec-1)group and Ator+ferrostatin-1(Fer-1)group.Cell viability was detected by cell counting kit-8(CCK-8)method.The ultrastructure of cells was observed by transmission electron microscopy.The levels of reactive oxygen species(ROS)and Fe^(2+)were observed by fluorescence microscopy.The contents of malondialdehyde(MDA)and glutathione(GSH)were detected by enzyme-linked immunosorbent assay(ELISA)method.Real-time quantitative PCR was used to detect the mRNA levels of caspase-3,receptor-interacting protein kinase 3(Ripk3),acyl-CoA synthetase long-chain family member 4(Acsl4),prostaglandin endoperoxidase synthase 2(Ptgs2)and glutathione peroxidase 4(Gpx4).Western blot was used to detect the proteins levels of 4-hydroxynonenal(4-HNE)and GPX4.Results:Compared with the Ator group,cell viability of MIN6 was higher in Ator+Z-VAD-FMK group and Ator+Fer-1 group(P<0.01).MIN6 cells,which were treated with Ator,exhibited the characteristic morphologic features associated with apoptosis,ferroptosis and autophagy under transmission electron microscopy.Compared with the control group,the levels of the intracellular Fe^(2+),MDA and ROS in the Ator group were increased and GSH was decreased.The mRNA relative expression levels of caspase-3,Acsl4 and Ptgs2 were increased,as well as the protein relative expression level of 4-HNE(all P<0.05).The mRNA and protein relative expression levels of GPX4 were decreased(P<0.05).Compared with the Ator group,the levels of the intracellular Fe^(2+),MDA and ROS in the Ator+Fer-1 group were decreased and GSH was increased.The mRNA relative expression level of Acsl4 was decreased and Gpx4 was increased(all P<0.05).The protein relative expression levels of 4-HNE was decreased and GPX4 was increased,though the changes were not statistically significant.Conclusion:Atorvastatin may induce ferroptosis in MIN6 cells by down-regulating GPX4 expression through inhibiting mevalonate pathway.