富马酸二甲酯对过氧化氢诱导心脏微血管内皮细胞铁死亡的影响

Effects of dimethyl fumarate on ferroptosis of cardiac microvascular endothelial cells induced by hydrogen peroxide and its mechanism

目的探讨富马酸二甲酯(DMF)对过氧化氢(H_(2)O_(2))诱导人心脏微血管内皮细胞(HCMEC)铁死亡的影响及其机制。方法体外培养血管内皮细胞,使用H_(2)O_(2)处理内皮细胞,并用不同浓度DMF干预内皮细胞,采用CCK⁃8法测细胞增殖;ELISA法检测细胞炎症水平;Western blot检测铁死亡蛋白GPX4和ACSL4,以及Nrf2/HO⁃1和JAK2/STAT1通路蛋白的改变。结果CCK8实验结果表明,与对照组相比,H_(2)O_(2)可诱导HCMEC增殖能力下降,而DMF成浓度依赖性促进HCMEC增殖(P<0.05)。H_(2)O_(2)诱导的HCMEC细胞炎症因子TNF⁃α、IL⁃1β和IL⁃18水平增加,DMF处理能抑制上述炎症因子的水平(P<0.05)。Western blot结果显示,H_(2)O_(2)处理引起细胞内GPX4蛋白下降,而ACSL4蛋白升高;DMF能抑制H_(2)O_(2)诱导的HCMEC细胞铁死亡(P<0.05)。H_(2)O_(2)可诱导HCMEC中Nrf2和HO⁃1蛋白表达下降,而JAK2和STAT1的磷酸化水平的增加(P<0.05);同H_(2)O_(2)组比,DMF可增加Nrf2和HO⁃1蛋白,并下调JAK2和STAT1的磷酸化水平(P<0.05)。使用Nrf2抑制剂ML385干预细胞,发现其可明显减弱DMF对细胞铁死亡的抑制和HCMEC损伤的保护作用。结论DMF能抑制H_(2)O_(2)诱导的HCMEC炎症和铁死亡。Nrf2/HO⁃1和JAK2/STAT1通路可能在DMF介导的HCMEC保护作用中起到关键作用。

Objective To explore the effects of dimethyl fumarate(DMF)on hydrogen peroxide(H_(2)O_(2))⁃induced ferroptosis of human cardiac microvascular endothelial cells(HCMEC)and its potential mechanism.Methods HCMEC were cultured in vitro,incubated with H_(2)O_(2),and then treated with different concentrations of DMF.Cell proliferation was measured by CCK⁃8 method.The level of cell inflammation was detected by ELISA.Western blot was used to detect the changes of ferroptosis⁃related proteins GPX4 and ACSL4,as well as Nrf2/HO⁃1 and JAK2/STAT1 pathway proteins.Results The results of CCK8 assay showed that compared those in the control group,H_(2)O_(2) significantly inhibited the proliferation of HCMEC cells,whereas,DMF treatment promoted H_(2)O_(2)⁃induced cell proliferation in a concentration dependent manner(P<0.05 for all).H_(2)O_(2)⁃induced increased levels of inflammatory cytokines TNF⁃α,IL⁃1βand IL⁃18 in HCMEC cells were reversed by DMF treatment(P<0.05).Western blot revealed that H_(2)O_(2) treatment caused the decrease in GPX4 protein and increase in ACSL4 protein;whereas DMF inhibited ferroptosis of HCMEC cells induced by H_(2)O_(2)(P<0.05).In addition,H_(2)O_(2) induced a decrease in Nrf2 and HO⁃1 expression,while an increase in JAK2 and STAT1 phosphorylation levels in HCMEC(P<0.05).Compared with those in the H_(2)O_(2) group,DMF increased Nrf2 and HO⁃1 proteins and down⁃regulated the phosphorylation levels of JAK2 and STAT1(P<0.05).Finally,it was found that Nrf2 inhibitor ML385 signifi⁃cantly weakened the inhibition of ferroptosis and the protective effect of HCMEC damage.Conclusion DMF inhib⁃its H_(2)O_(2)⁃induced inflammation of HCMEC and ferroptosis.Nrf2/HO⁃1 and JAK2/STAT1 pathways may play a key role in DMF⁃mediated protection of HCMEC.