地黄苷A通过调节AKT/Nrf2/GPX4信号通路介导的铁死亡改善大鼠心肌缺血再灌注损伤

Rehmannioside A Improves Myocardial Ischemia-Reperfusion Injury in Rats by Regulating Ferroptosis Mediated by AKT/Nrf2/GPX4 Signaling Pathway

目的:探讨地黄苷A(ReA)通过调节蛋白激酶B(AKT)/核因子红细胞系2相关因子2(Nrf2)/谷胱甘肽过氧化酶4(GPX4)信号通路介导的铁死亡对大鼠心肌缺血再灌注损伤(MI/RI)的影响。方法:将90只大鼠随机分为假手术组(Sham组)、模型组(Model组)、ReA低剂量组(ReA-L组)、ReA高剂量组(ReA-H组)、铁死亡抑制剂ferrostatin-1(Fer-1)组(Fer-1组)及ReA高剂量+AKT抑制剂perifosine组(ReA-H+perifosine组),每组15只。ReA-L组、ReA-H组大鼠分别腹腔注射40、80 mg/kg ReA,Fer-1组大鼠腹腔注射2 mg/kg ferrostatin-1,ReA+perifosine组大鼠腹腔注射80 mg/kg ReA及20 mg/kg perifosine,Sham组和Model组大鼠腹腔注射等量生理盐水,每日1次,连续给药2周。除Sham组外,各组大鼠在末次给药1 h后采用左冠状动脉前降支结扎法建立MI/RI大鼠模型。试剂盒检测血清心肌酶水平;氯代三苯基四氮唑(TTC)染色测定心肌梗死面积;苏木素-伊红(HE)染色观察心肌组织病理形态学变化;原位末端标记测定法(TUNEL)染色检测心肌细胞凋亡率;试剂盒测定心肌组织抗氧化能力;Fe^(2+)含量检测试剂盒检测心肌组织Fe^(2+)含量;蛋白免疫印迹法(Western Blot)检测AKT/Nrf2/GPX4信号通路相关蛋白表达。结果:与Sham组比较,Model组大鼠血清乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(cTnI)水平及心肌组织中丙二醛(MDA)和Fe^(2+)含量上升,组织中谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性及p-AKT/AKT、Nrf2和GPX4蛋白水平下降,转铁蛋白受体1(TfR1)蛋白水平升高,心肌细胞凋亡率及心肌梗死面积增加,心肌组织损伤严重(P<0.05);与Model组相比,ReA-L组、ReA-H组、Fer-1组大鼠血清LDH、CK-MB和cTnI水平及心肌组织中MDA和Fe^(2+)含量下降,组织中GSH-Px、SOD活性及p-AKT/AKT、Nrf2和GPX4蛋白水平升高,TfR1蛋白水平降低,心肌细胞凋亡率及心肌梗死面积减少,心肌组织损伤减轻(P<0.05);perifosine减弱了高剂量ReA对MI/RI大鼠铁死亡的抑制作用。结论:ReA可能通过激活AKT/Nrf2/GPX4信号通路,抑制铁死亡,改善MI/RI大鼠心肌组织损伤。

Objective:To investigate the influence of rehmannioside A(ReA)on myocardial ischemia-reperfusion injury(MI/RI)in rats by regulating ferroptosis mediated by protein kinase B(AKT)/nuclear factor erythroid 2-related factor 2(Nrf2)/glutathione peroxidase 4(GPX4)signaling pathway.Methods:Ninety rats were randomly divided into sham operation group(sham group),model group,ReA low-dose group(ReA-L group),ReA high-dose group(ReA-H group),ferroptosis inhibition ferrostatin-1(Fer-1)group(Fer-1 group),and ReA high-dose+AKT inhibitor perifosine group(ReA-H+perifosine group),with 15 rats in each group.The rats in ReA-L group and ReA-H group were injected intraperitoneally with 40 mg/kg and 80 mg/kg ReA,respectively.The rats in Fer-1 group were intraperitoneally injected with 2 mg/kg ferrostatin-1.The rats in ReA+perifosine group were intraperitoneally injected with 80 mg/kg ferrostatin-1 and 20 mg/kg perifosine.The rats in sham group and model group were intraperitoneally injected with the same volume of normal saline,once a day,for 2 weeks.Except for the sham group,MI/RI rat models were established by ligation of the left anterior descending coronary artery 1 h after the last administration.The kit was applied to detect serum myocardial enzyme level.Triphenyltetrazolium chloride(TTC)staining was used to determine myocardial infarct size.Hematoxylin(HE)staining was applied to observe the pathological changes of myocardial tissue.Terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)staining was applied to detect the apoptosis rate of cardiomyocytes.The kit was used to measure the antioxidant capacity of myocardial tissue.Fe^(2+)content detection kit was applied to detect Fe^(2+)content in myocardial tissue.Western Blot was applied to detect the expression of AKT/Nrf2/GPX4 signaling pathway related protein.Results:Compared with sham group,the serum levels of lactate dehydrogenase(LDH),creatine kinase isoenzyme MB(CK-MB)and cardiac troponin I(cTnI),the contents of malondialdehyde(MDA)and Fe^(2+)in myocardial tissue were increased in model group,the activities of glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD),and the levels of p-AKT/AKT,Nrf2,and GPX4 proteins in the tissue decreased,the level of transferrin receptor 1(TfR1)protein increased,the apoptosis rate of myocardial cells and myocardial infarction area were increased,and myocardial tissue damage was serious(P<0.05).Compared with model group,the serum levels of LDH,CK-MB,and cTnI and the contents of MDA and Fe^(2+)in myocardial tissue decreased in ReA-L group,ReA-H group,and Fer-1 group,the activities of GSH-Px and SOD,and the levels of p-AKT/AKT,Nrf2,and GPX4 proteins in the tissue increased,the level of TfR1 protein decreased,the apoptosis rate of myocardial cells and myocardial infarction area were decreased,and myocardial tissue damage greatly alleviated(P<0.05).Perifosine attenuated the inhibitory effect of high-dose ReA on ferroptosis in MI/RI rats.Conclusion:ReA may inhibit ferroptosis and improve myocardial tissue damage in MI/RI rats by activating AKT/Nrf2/GPX4 signaling pathway.