阿托伐他汀诱导的MIN6细胞铁死亡及相关机制研究
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南京医科大学第一附属医院内分泌科,江苏 南京 210029

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国家自然科学基金(81974103,82370828)


Study of atorvastatin induced ferroptosis in MIN6 cells and its related mechanisms
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Department of Endocrinology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029 ,China

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    摘要:

    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Fe2+ 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 Fe2+ 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Fe2+ 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。

    Abstract:

    Objective:To explore whether atorvastatin(Ator)can induce ferroptosis in pancreatic β- cell line MIN6 cells and its possible mechanism. Methods:MIN6 cells were divided into control group,Ator group,Ator+apoptosis inhibitor(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 Fe2+ 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 Fe2+ ,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 Fe2+ ,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.

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魏倩影,陈欣,秦瑶,李雨潇,秦璐,张梅.阿托伐他汀诱导的MIN6细胞铁死亡及相关机制研究[J].南京医科大学学报(自然科学版),2024,(8):1044-1050

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  • 收稿日期:2024-03-04
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  • 在线发布日期: 2024-08-09
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