乌司他丁通过上调Cx43表达减轻缺氧/复氧诱导的心肌细胞铁死亡的作用机制

Ulinastatin attenuates hypoxia/reoxygenation-induced cardiomyocyte ferroptosis by up-regulating Cx43 expression

目的探讨乌司他丁(UTI)对缺氧/复氧(H/R)诱导的心肌细胞铁死亡的影响,并分析其保护机制。方法体外培养大鼠H9c2心肌细胞,分为对照组、H/R组、铁死亡诱导剂(Erastin)组、UTI组、UTI+Erastin组、UTI+小干扰RNA(siRNA)阴性对照(si-NC)组、UTI+连接蛋白43(Cx43)siRNA(si-Cx43)组,各组给予相应干预后,检测各组细胞活力、乳酸脱氢酶(LDH)活性、活性氧、Fe^(2+)、丙二醛、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性及Cx43、谷胱甘肽过氧化物酶4(GPX4)、铁蛋白重链1(FTH1)、酰基辅酶A合成酶长链家族成员4(ACSL4)mRNA和蛋白水平。结果与对照组比较,H/R组细胞活力、SOD、CAT活性和Cx43、GPX4、FTH1mRNA及蛋白水平显著降低,LDH活性、活性氧、丙二醛、Fe^(2+)和ACSL4mRNA及蛋白水平显著升高(P<0.01)。与H/R组比较,UTI组细胞活力[(71.40±8.05)%vs(42.63±6.71)%]、SOD、CAT活性和Cx43、GPX4、FTH1mRNA及蛋白水平显著升高,LDH活性、活性氧、丙二醛、Fe^(2+)和ACSL4mRNA及蛋白水平显著降低(P<0.05)。与UTI组比较,UTI+Erastin组和UTI+si-Cx43组细胞活力、SOD、CAT活性和Cx43、GPX4、FTH1mRNA及蛋白水平显著降低,LDH活性、活性氧、丙二醛、Fe^(2+)和ACSL4mRNA及蛋白水平显著升高(P<0.05)。结论UTI可能通过上调Cx43抑制H/R诱导的心肌细胞铁死亡。

Objective To investigate the effect of ulinastatin(UTI)on hypoxia/reoxygenation(H/R)induced iron death of cardiomyocytes and analyze its protective mechanism.Methods Rat cardiomyocytes H9c2were grouped into control group,H/R group,iron death inducer(Erastin)group,UTI group,UTI+Erastin group,UTI+siRNA negative control(si-NC)group,UTI+connexin 43(Cx43)small interfering RNA(si-Cx43)group.After corresponding interventions,cell viability,contents of reactive oxygen species(ROS),Fe^(2+)and malondialdehyde(MDA),activities of lactate dehydrogenase(LDH),superoxide dismutase(SOD)and catalase(CAT),and mRNA and protein levels of Cx43,glutathione peroxidase 4(GPX4),ferritin heavy chain 1(FTH1)and acyl coenzyme A synthetase long chain family member 4(ACSL4)were detected in each group.Results Cell viability,activities of SOD and CAT,and mRNA and protein levels of Cx43,GPX4 and FTH1were significantly decreased,while LDH activity,contents of ROS,MDA and Fe^(2+),and ACSL4mRNA and protein levels were obviously increased in the H/R group than the control group(P<0.01).UTI treatment resulted in notably enhanced cell viability[(71.40±8.05)%vs(42.63±6.71)%,P<0.05],stronger activities of SOD and CAT,up-regulated mRNA and protein levels of Cx43,GPX4and FTH1,reduced LDH activity,and declined contents of ROS,MDA and Fe^(2+),and reduced ACSL4mRNA and protein levels(P<0.05)when compared with the levels in the H/R group.Compared with the UTI group,cell vitality,SOD and CAT activities,and Cx43,GPX4,FTH1mRNA and protein levels were significantly decreased,while LDH activity,contents of ROS,MDA and Fe^(2+),and ACSL4mRNA and protein levels were significantly increased in the UTI+Erastin group and the UTI+si-Cx43group(P<0.05).Conclusion UTI may inhibit H/R induced ferroptosis in cardiomyocytes by up-regulating Cx43.