[Electroacupuncture improves ischemic myocardial injury by activating Nrf2/HO-1 signaling pathway to inhibit ferroptosis in rats].

To explore the role of nuclear factor E2 related factor 2 (Nrf2) / heme oxygenase (HO-1) signal pathway in electroacupuncture (EA) induced improvement of acute myocardial ischemia (AMI) and its relationship with ferroptosis in rats.Male SD rats were randomly and equally divided into sham operation, model, EA and EA+ML385 (inhibitor of Nrf2) groups (n=8). The rat model of AMI was established by ligating the descending anterior branch of the left coronary artery. EA (2 Hz/100 Hz) was applied to bilateral "Shenmen"(HT7) and "Tongli"(HT5) for 20 min, once daily for 7 days. The electrocardiogram (ECG) of standard Ⅱ (ECG ST) lead and heart rate (HR) in each group was recorded and analyzed before and after modeling and after treatment by using PowerLab physiological recorder system. Histopathological changes of myocardial tissue were observed by H.E. staining, and the ultrastructure of myocardiocytes of cardiac apical tissue was observed under transmission electron microscope. The contents of Fe2+ and glutathione (GSH) in the myocardial tissue were measured by chromato-metry. The protein expression levels of Nrf2, HO-1, glutathione peroxidase 4 (GPX4), ferritin heavy chain polypeptide 1 (FTH1) and long chain acyl CoA synthase 4 (ACSL4) in the myocardial tissue were detected by Western blot.Compared with the sham operation group, the HR, ECG ST, Fe2+ content, expression levels of Nrf2, HO-1, FTH1 and ACSL4 proteins in myocardial tissues were significantly increased (P<0.01), while GSH content and GPX4 protein expression considerably decreased (P<0.01) in the model group. Compared with the model group, both EA and EA+ML385 groups had an obvious decrease in HR, Fe2+ content, and ACSL4 levels (P<0.01), and an increase in the expression levels of GPX4 and FTH1 proteins (P<0.01), EA (rather than EA+ML385) effectively down-regulated ECG ST, and up-regulated GSH, Nrf2 and HO-1 (P<0.01), whereas EA+ML385 apparently down-regulated expression levels of Nrf2 and HO-1 (P<0.01). It shows that ML385 pronouncedly weaken the effects of EA in slowing down ECG ST and HR, down-regulating Fe2+ content and ACSL4 expression (P<0.01), up-regulating GSH content, Nrf2, HO-1, GPX4 and FTH1 expressions (P<0.01). H.E. staining showed disordered arrangement and hyperplasia of myocardiocytes, enlarged myocardial fiber gap, agglomerated and deeply stained myoplasma, and some broken myocardial fibers with irregular mass and local tissue fibrosis in the model group, which was relatively milder in both EA and EA+ML385 groups. Compared with the sham operation group, the model group showed decreased mitochondrial atrophy, increased membrane density, and disappearance or reduction of cristae in myocardial cells，which was improved in the EA group.EA of HT7 and HT5 has a protective effect on ischemic myocardium in rats, which may be related to its effects in reducing oxidative stress by regulating Nrf2/HO-1 signaling pathway, and inhibiting "iron death" of myocardial cells.目的：探讨核因子E2相关因子2(Nrf2)/血红素氧合酶(HO-1)信号通路在电针减轻急性心肌缺血(AMI)中的作用及其与铁死亡的关系。方法：SD大鼠随机分为假手术组、模型组、电针组、电针+抑制剂组，每组8只。采用结扎冠状动脉左前降支法制备AMI大鼠模型。电针组电针“神门”“通里”，每次20 min，电针+抑制剂组大鼠每日电针前30 min予以Nrf2抑制剂ML385(3 mg/100 g)腹腔注射，均连续治疗7 d。运用PowerLab多导生理记录仪记录各组大鼠造模前后及治疗后心率和ST段电压，HE染色法观察大鼠心肌细胞形态变化，透射电镜观察心肌组织超微结构，比色法测定大鼠心肌组织Fe2+、谷胱甘肽(GSH)含量，Western blot法检测大鼠心肌组织Nrf2、HO-1、谷胱甘肽过氧化物酶(GPX)4、铁蛋白重链(FTH)1、酰基辅酶A合成酶长链家族成员(ACSL)4蛋白表达。结果：造模后模型组心率、ST段电位均较假手术组显著升高(P<0.01)。治疗后，电针组较模型组、电针+抑制剂组大鼠心率、ST段电位显著降低(P<0.01)。与假手术组比较，模型组大鼠心肌细胞排列紊乱，心肌纤维大片断裂、间隙扩大，心肌细胞增生，肌浆凝聚深染，局部心肌组织纤维化；与模型组、电针+抑制剂组比较，电针组心肌细胞排列、心肌纤维断裂情况有所改善，胞质深染程度减轻。透射电镜下观察，与假手术组比较，模型组大鼠心肌细胞线粒体萎缩变小、膜密度增高、嵴消失或减少；与模型组比较，电针组心肌线粒体萎缩变小、膜密度增高、嵴消失或减少有所改善。与假手术组比较，模型组心肌组织Fe2+含量升高、GSH含量降低(P<0.01)。与模型组比较，电针组心肌组织Fe2+含量降低、GSH含量升高(P<0.01)；电针+抑制剂组心肌组织Fe2+含量降低(P<0.01)。与假手术组比较，模型组心肌组织Nrf2、HO-1、ACSL4、FTH1蛋白表达升高(P<0.01)，GPX4蛋白表达降低(P<0.01)。与模型组比较，电针组Nrf2、HO-1、GPX4、FTH1蛋白表达升高(P<0.01), ACSL4蛋白表达降低(P<0.01)；电针+抑制剂组Nrf2、HO-1、ACSL4蛋白表达降低(P<0.01)，FTH1、GPX4蛋白表达升高(P<0.01)。与电针+抑制剂组比较，电针组心肌组织Fe2+含量降低(P<0.01)，GSH含量升高(P<0.01)，Nrf2、HO-1、GPX4、FTH1蛋白表达升高(P<0.01), ACSL4蛋白表达降低(P<0.01)。结论：电针心经“神门”“通里”对AMI大鼠心肌保护作用可能与激活Nrf2/HO-1信号通路调节氧化应激和相关蛋白抑制心肌细胞“铁死亡”相关。.