富马酸二甲酯调控Nrf2-GPX4介导的铁死亡途径对大鼠心肌缺血/再灌注损伤的保护作用研究

Study on the protective effect of dimethyl fumarate on myocardial ischemia/reperfusion injury by regulating the iron death pathway mediated by Nrf2-GPX4 in rats

目的基于核因子E2相关因子2(Nrf2)-谷胱甘肽过氧化物酶4(GPX4)介导的铁死亡途径探讨富马酸二甲酯(DMF)对心肌缺血/再灌注(I/R)损伤的保护机制。方法将72只SD大鼠随机分为假手术组(Sham组)、I/R组、Ferrostatin-1组(Fer-1组)、DMF低剂量组(DMF-L组,25 mg/kg)、DMF高剂量组(DMF-H组,50 mg/kg)、DMF+ML385组(50 mg/kg DMF+30 mg/kg ML385),每组12只,给予相应的药物干预,1次/d,连续7 d;同时结扎左冠状动脉前降支建立心肌I/R大鼠模型。再灌注12 h后,酶联免疫吸附试验(ELISA)检测血清乳酸脱氢酶(LDH)、肌钙蛋白I(c TnI)、肌酸激酶同工酶MB(CK-MB)水平;苏木精-伊红(HE)染色观察心肌组织病理学变化;TUNEL染色检测心肌细胞凋亡;DHE荧光染色法检测心肌组织活性氧(ROS)水平;生化法检测心肌组织匀浆中丙二醛(MDA)、谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)活性;比色法检测心肌组织Fe^(2+)含量;Western blot检测心肌组织Nrf2、GPX4、铁蛋白重链1(FTH1)、转铁蛋白受体1(TfR1)蛋白水平。结果与Sham组相比,I/R组大鼠血清CK-MB、c TnI、LDH水平,心肌细胞凋亡率,ROS、MDA、Fe^(2+)含量,TfR1蛋白水平显著升高(P<0.05),GSH含量,SOD活性以及Nrf2、GPX4、FTH1蛋白水平显著降低(P<0.05);与I/R组相比,Fer-1组、DMF-L组和DMF-H组大鼠血清CK-MB、cTnI、LDH水平,心肌细胞凋亡率,ROS、MDA、Fe^(2+)含量,TfR1蛋白水平显著降低(P<0.05),GSH含量,SOD活性以及Nrf2、GPX4、FTH1蛋白水平显著升高(P<0.05);与DMF-H组相比,DMF+ML385组大鼠血清CK-MB、c TnI、LDH水平,心肌细胞凋亡率,ROS、MDA、Fe^(2+)含量,TfR1蛋白水平显著升高(P<0.05),GSH含量,SOD活性以及Nrf2、GPX4、FTH1蛋白水平显著下降(P<0.05);Nrf2抑制剂ML385可明显减弱DMF对铁死亡的抑制和心肌I/R损伤的保护作用。结论DMF可能通过抑制铁死亡减轻心肌I/R损伤,且其作用机制可能与激活Nrf2-GPX4通路有关。

Objective reperfusion(I/R)injury based on the iron death pathway mediated by nuclear factor E2 related factor 2(Nrf2)-glutathione peroxidase 4(GPX4).Methods A total of 72 SD rats were randomly divided into the sham operation group,the I/R group,the Ferrostatin-1 group(2 mg/kg),the DMF low-dose group(25 mg/kg),the DMF high-dose group(50 mg/kg)and the DMF+ML385 group(50 mg/kg DMF+30 mg/kg ML385),with 12 rats in each group.Rats were given corresponding drug intervention once a day for 7 consecutive days.At the same time,the rat model of myocardial I/R was established by ligating the left anterior descending coronary artery.After 12 h of reperfusion,the serum levels of lactate dehydrogenase(LDH),troponin I(cTnI)and creatine kinase isoenzyme MB(CK-MB)were detected by ELISA.HE staining was used to observe the pathological changes of myocardium.Cardiomyocyte apoptosis was detected by TUNEL staining.The level of reactive oxygen species(ROS)in myocardial tissue was detected by DHE fluorescence staining.Malondialdehyde,glutathione content and superoxide dismutase activity in myocardial tissue homogenate were detected by biochemical method.Fe^(2+)content in myocardial tissue was detected by colorimetry.The protein levels of Nrf2,GPX4,ferritin heavy chain 1(FTH1)and transferrin receptor 1(TfR1)in myocardial tissue were detected by Western blot assay.Results Compared with the sham group,the serum levels of CK-MB,c TnI,LDH,cardiomyocyte apoptosis rate,ROS and MDA,Fe^(2+)content,TfR1 protein level were significantly increased in the I/R group(P<0.05).GSH content,SOD activity and Nrf2,GPX4,FTH1 protein level were significantly decreased(P<0.05).Compared with the I/R group,the serum levels of CK-MB,c TnI,LDH,myocardial cell apoptosis rate,ROS,MDA,Fe^(2+)content and TfR1 protein were significantly decreased in the Fer-1 group,the DMF-L group and the DMF-H group(P<0.05),while GSH and SOD activities,and Nrf2,GPX4,and FTH1 protein levels were significantly increased(P<0.05).Compared with the DMF-H group,the serum levels of CK-MB,cTnI,LDH,cardiomyocyte apoptosis rate,ROS and MDA,Fe^(2+)content and TfR1 protein level were significantly increased in the DMF+ML385 group(P<0.05).GSH and SOD and protein levels of Nrf2,GPX4 and FTH1 were significantly decreased(P<0.05).The Nrf2 inhibitor ML385 could significantly attenuate the inhibition of DMF on ferroptosis and the protective effect of myocardial I/R injury.Conclusion DMF may alleviate myocardial I/R injury by inhibiting iron death,and its mechanism may be related to the activation of Nrf2-GPX4 pathway.