丹参通络解毒汤调控Nrf2-GPX4介导的铁死亡途径对心肌缺血再灌注损伤大鼠的保护作用研究

Study on the protective effect of DanShenTongLuo-detoxification decoction modulating Nrf2-GPX4-mediated ferroptosis pathway on myocardial ischaemia/reperfusion injury 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丹参通络解毒汤对大鼠心肌缺血/再灌注(I/R)损伤的保护作用。方法将SD大鼠随机分为空白组、假手术组、模型组(I/R)、中药组(I/R+丹参通络解毒汤)、Nrf2抑制剂+中药组(I/R+ML385+丹参通络解毒汤),每组8只。采用透射电镜观察心肌超微结构;酶联免疫吸附试验(ELISA)检测心肌组织的肌钙蛋白I(cTnI)、肌钙蛋白T(cTnT)和活性氧(ROS)水平;生化法检测心肌组织的超氧化物歧化酶(SOD)活性、丙二醛(MDA)、谷胱甘肽(GSH)和Fe^(2+)含量;Western blot检测心肌组织的GPX4、Nrf2的蛋白水平。结果与空白组或假手术组相比,模型组大鼠心肌细胞线粒体嵴断裂、减少甚至消失,心肌组织cTnI和cTnT水平、ROS、MDA、Fe^(2+)含量升高(P<0.05),SOD活性、GSH含量以及Nrf2和GPX4蛋白表达降低(P<0.05);与模型组相比,中药组、Nrf2抑制剂+中药组大鼠心肌细胞线粒体损伤改善显著,心肌组织cTnI、cTnT水平、ROS、MDA、Fe^(2+)含量降低(P<0.05),SOD活性、GSH含量以及GPX4和Nrf2蛋白表达升高(P<0.05);与中药组相比,Nrf2抑制剂+中药组大鼠心肌细胞线粒体损伤加重,心肌组织cTnI和cTnT水平、ROS、MDA、Fe^(2+)含量升高,SOD活性、GSH含量以及GPX4和Nrf2蛋白表达下降(P<0.05)。结论丹参通络解毒汤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 To explore the protective effect of DanShenTongLuo-detoxification decoction on myocardial ischaemia/reperfusion(I/R)injury in rats based on the nuclear factor E2-related factor 2(Nrf2)-glutathione peroxidase 4(GPX4)-mediated ferroptosis pathway.Methods SD rats were randomly divided into control group,sham-operated group,model group.Methods(I/R),traditional Chinese medicine group(I/R+DanShenTongLuo-detoxification decoction),and Nrf2 inhibitor+traditional Chinese medicine group(I/R+ML385+DanShenTongLuo-detoxification decoction),with 8 rats in each group.Transmission electron microscopy was used to observe the ultrastructure of myocardium;Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of troponin I(cTnI),troponin T(cTnT),and reactive oxygen species(ROS)in myocardial tissues;Biochemical methods were used to detect the activities of superoxide dismutase(SOD),malondialdehyde(MDA),glutathione(GSH),and Fe^(2+)content in myocardial tissues;Western blot detected the protein levels of GPX4 and Nrf2 in myocardial tissues.Results Compared with the control group or the sham-operated group,the mitochondrial cristae of rat cardiomyocytes in the model group were broken,reduced or even disappeared,and the myocardial tissue levels of cTnI and cTnT,ROS,MDA,and Fe^(2+)were elevated(P<0.05),and the activities of SOD,GSH content,and the expression of Nrf2 and GPX4 proteins were reduced(P<0.05);Compared with the model group,mitochondrial damage in rat cardiomyocytes was significantly improved in the traditional Chinese medicine group and the Nrf2 inhibitor+traditional Chinese medicine group,and the myocardial tissue cTnI and cTnT levels,ROS,MDA,and Fe^(2+)contents were reduced(P<0.05),and SOD activity,GSH content,as well as the protein expression of GPX4 and Nrf2 were elevated(P<0.05);Compared with the traditional Chinese medicine group In the Nrf2 inhibitor+Chinese medicine group,mitochondrial damage in rat cardiomyocytes was aggravated,and myocardial tissue cTnI and cTnT levels,ROS,MDA,and Fe^(2+)contents were elevated,and SOD activity,GSH content,as well as GPX4 and Nrf2 protein expression were decreased(P<0.05).Conclusion DanShenTongLuo-detoxification decoction may attenuate myocardial I/R injury by inhibiting ferroptosis,and its mechanism of action may be related to the activation of the Nrf2-GPX4 pathway.