白藜芦醇调控Nrf2-GPX4通路对H_(2)O_(2)诱导肺泡上皮细胞铁死亡的影响

Influence of resveratrol on H_(2)O_(2)-induced ferroptosis in alveolar epithelial cells by regulating the Nrf2-GPX4 pathway

目的探索白藜芦醇(RES)调控核因子E2相关因子2(Nrf2)-谷胱甘肽过氧化物酶4(GPX4)通路对H_(2)O_(2)诱导A549细胞铁死亡的影响。方法将人肺泡上皮A549细胞随机分为对照组、H_(2)O_(2)组、RES低浓度(RES-L,50μmol/L)组、RES中浓度(RES-M,100μmol/L)组、RES高浓度(RES-H,150μmol/L)组、ML385(Nrf2抑制剂,2μmol/L ML385)组、RES-H+ML385(150μmol/L RES+2μmol/L ML385)组。除对照组外,其余各组均经H_(2)O_(2)处理细胞后,再使用相应剂量的药物或抑制剂进行干预。干预结束后,MTT法测定各组细胞活力,试剂盒检测细胞内铁离子含量,酶联免疫吸附试验(ELISA)检测丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)水平;透射电子显微镜观察线粒体结构;Western blot法检测Nrf2、GPX4、血红素加氧酶1(HO-1)蛋白表达水平。结果与对照组相比,H_(2)O_(2)组线粒体形态较受损严重,细胞存活率、SOD、GSH含量以及Nrf2、GPX4、HO-1蛋白表达均降低,铁离子、MDA含量增加(P<0.05);与H_(2)O_(2)组相比,随着RES浓度的增加,A549细胞线粒体形态损伤得到改善,细胞存活率、SOD、GSH含量以及Nrf2、GPX4、HO-1蛋白表达逐渐增加,铁离子、MDA含量逐渐下降(P<0.05),但ML385组细胞线粒体损伤加重,细胞存活率、SOD、GSH含量以及Nrf2、GPX4、HO-1蛋白表达均降低,铁离子、MDA含量增加(P<0.05),且ML385处理可逆转RES对H_(2)O_(2)诱导A549细胞的保护作用。结论RES可能通过激活Nrf2-GPX4信号通路降低H_(2)O_(2)诱导的A549细胞内氧化应激水平,抑制铁死亡。

Objective To explore the influence of resveratrol(RES)on H_(2)O_(2)-induced ferroptosis in A549 cells by regulating nuclear factor E2-related factor 2(Nrf2)-glutathione peroxidase 4(GPX4)pathway.Methods Human alveolar epithelial cells A549 were randomly grouped into the control group,the H_(2)O_(2)group,the RES low(RES-L,50μmol/L),the RES medium(RES-M,100μmol/L)and the RES high(RES-H,150μmol/L)concentration groups,the ML385(Nrf2 inhibitor,2μmol/L ML385)group and the RES-H+ML385(150μmol/L RES+2μmol/L ML385)group.Except for the control group,the other groups were treated with H_(2)O_(2),and then intervened with corresponding doses of drugs and inhibitors.After the intervention,MTT method was performed to determine the viability of A549 cells in each group.The kit was applied to measure intracellular iron content.ELISA method was performed to measure levels of oxidative stress in A549 cells,including malondialdehyde(MDA),superoxide dismutase(SOD)and reduced glutathione(GSH).Transmission electron microscopy was applied to observe mitochondrial structure.Western blot assay was performed to measure the protein expression levels of Nrf2,GPX4 and heme oxygenase 1(HO-1)in A549 cells.Results Compared with the control group,mitochondrial morphology was seriously damaged in the H_(2)O_(2)group,and cell viability,SOD and GSH contents,and the Nrf2,GPX4 and HO-1 protein expression were significantly decreased,and the iron ion and MDA content were significantly increased(P<0.05).Compared with the H_(2)O_(2)group,with the increase of the RES concentration,the mitochondrial morphological damage of A549 cells was improved,the cell viability,SOD and GSH contents,and the Nrf2,GPX4 and HO-1 protein expression gradually increased,while the iron ion and MDA content gradually decreased(P<0.05).However,the mitochondrial damage of A549 cells was aggravated in the ML385 group,and cell viability,SOD and GSH contents,and the Nrf2,GPX4 and HO-1 protein expression were significantly decreased,and iron ion and MDA content were significantly increased(P<0.05).ML385 was able to reverse the protective effect of RES on H_(2)O_(2)-induced A549 cells.Conclusion RES can reduce the level of oxidative stress in A549 cells induced by H_(2)O_(2)and inhibit ferroptosis,which may be related to the activation of Nrf2-GPX4 signaling pathway.