基于Nrf2-GPX4铁死亡途径探讨芒果苷对大鼠心肌缺血再灌注损伤的作用机制

Effect of Mangiferin on Myocardial Ischemia-reperfusion Injury in Rats Based on Nrf2-GPX4 Ferroptosis Pathway

目的:探讨芒果苷对大鼠心肌缺血再灌注损伤(MIRI)的保护机制。方法:60只无特定病原体(SPF)级健康雄性SD大鼠随机分为假手术组、MIRI组、芒果苷低剂量组、芒果苷高剂量组、芒果苷+核因子E2相关因子2(Nrf2)抑制剂(ML385)组,每组12只。除假手术组外,其余组大鼠通过结扎左冠状动脉前降支建立MIRI模型。再灌注4 h后,采用酶联免疫吸附测定法(ELISA)测定血清肌钙蛋白I(cTnI)、乳酸脱氢酶(LDH)、肌酸激酶(CK)水平;苏木精-伊红(HE)染色观察心肌组织病理学变化;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)分析心肌细胞凋亡情况;二氢乙锭(DHE)荧光法检测心肌组织活性氧(ROS)水平;比色法检测心肌组织超氧化物歧化酶(SOD)活性、丙二醛(MDA)、谷胱甘肽(GSH)和亚铁离子(Fe^(2+))含量;实时荧光定量逆转录聚合酶链式反应(qRT-PCR)和蛋白免疫印迹法(Western Blot)检测心肌组织Nrf2、谷胱甘肽过氧化物酶4(GPX4)、铁蛋白重链1(FTH1)、长链脂酰辅酶A合成酶4(ACSL4)的基因和蛋白表达。结果:与假手术组比较,MIRI组大鼠血清cTnI、CK和LDH水平,心肌细胞凋亡率,ROS、MDA、Fe^(2+)含量,ACSL4 mRNA和蛋白水平升高;SOD活性和GSH含量,Nrf2、GPX4、FTH1 mRNA和蛋白水平降低(P<0.05);心肌细胞肿胀,心肌纤维排列紊乱,大量炎性细胞浸润。与MIRI组比较,芒果苷低剂量组、芒果苷高剂量组血清cTnI、CK和LDH水平,心肌细胞凋亡率,ROS、MDA和Fe^(2+)含量,ACSL4 mRNA和蛋白水平降低;SOD活性和GSH含量,Nrf2、GPX4、FTH1 mRNA和蛋白水平升高(P<0.05);心肌细胞水肿减轻,少量心肌纤维断裂和炎性细胞浸润。使用Nrf2抑制剂ML385抑制Nrf2的核易位可明显阻断芒果苷对心肌铁死亡的抑制作用(P<0.05)。结论:芒果苷可通过激活Nrf2-GPX4轴抑制铁死亡对MIRI发挥保护作用。

Objective:To investigate the protective effect of mangiferin on myocardial ischemia-reperfusion injury(MIRI)in rats.Methods:Sixty healthy male SD rats without specific pathogen(SPF)grade were randomly divided into sham operation group,MIRI group,low-dose mangiferin group,high-dose mangiferin group,and mangiferin+Nrf2 inhibitor(ML385)group,with 12 rats in each group.Except the sham group,the MIRI model was established by ligation of the anterior descending branch of the left coronary artery.After 4 h of reperfusion,serum troponin I(cTnI),lactate dehydrogenase(LDH)and creatine kinase(CK)levels were determined by enzyme-linked immunosorbent assay(ELISA).The cardiac histopathological changes were observed by hematoxylin-eosin(HE)staining.Myocardial cell apoptosis was analyzed by terminal deoxyribonucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)method.Dihydroethidium(DHE)fluorescence assay was used to detect the level of reactive oxygen species(ROS)in myocardial tissue.Superoxide dismutase(SOD)activity,malondialdehyde(MDA),glutathione(GSH),and ferrous ion(Fe^(2+))contents were detected by colorimetry.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western Blot were used to detect the gene and protein expression of nuclear factor E2-related factor 2(Nrf2),glutathione peroxidase 4(GPX4),ferritin heavy chain 1(FTH1),and long chain fatty acyl-CoA synthetase 4(ACSL4)in myocardial tissue.Results:Compared with sham group,serum levels of cTnI,CK and LDH,cardiomyocyte apoptosis rate,ROS,MDA,Fe^(2+)content,ACSL4 mRNA and protein levels of rats in MIRI group increased,SOD activity and GSH content,Nrf2,GPX4,FTH1 mRNA and protein levels decreased(P<0.05),cardiomyocytes were swollen,myocardial fibers were disordered,and a large number of inflammatory cells were infiltrated.Compared with MIRI group,serum cTnI,CK and LDH levels,cardiomyocyte apoptosis rate,ROS,MDA and Fe^(2+)contents,ACSL4 mRNA and protein levels in low-dose mangiferin group,high-dose mangiferin group decreased,SOD activity and GSH content,Nrf2,GPX4,FTH1 mRNA and protein levels increased(P<0.05),cardiomyocyte edema reduced,with a small amount of myocardial fiber rupture and inflammatory cell infiltration.Inhibition of nuclear translocation of Nrf2 by Nrf2 inhibitor ML385 could significantly block the inhibitory effect of mangiferin on cardiac ferroptosis(P<0.05).Conclusion:Mangiferin can protect MIRI by inhibiting ferroptosis through activation of Nrf2-GPX4 axis.