Role of Nrf2-Gpx4 signaling pathway in Shenmai injection-induced reduction of myocardial ischemia-reperfusion injury: relationship with ferroptosis in rats

Objective To evaluate the role of nuclear factor erythroid 2-related factor 2 (Nrf2)-glutathione peroxidase 4 (GPX4) signaling pathway in Shenmai injection-induced reduction of myocardial ischemia-reperfusion (I/R) injury and the relationship with ferroptosis in rats. Methods Forty-eight SPF healthy adult male Sprague-Dawley rats, weighing 220-250 g, were divided into 4 groups (n=12 each) by a random number table method: sham operation group (S group), myocardial I/R group (IR group), Shenmai injection group (SM group), and Shenmai injection plus Nrf2 inhibitor group (SMM group). Acute myocardial I/R injury was induced by ligating the anterior descending brach of the left coronary artery for 30 min followed by 120-min reperfusion in anesthetized rats.In SM group, Shenmai injection 9 ml/kg was intravenously injected immediately before reperfusion.In group SMM, Nrf2 inhibitor ML385 30 mg/kg was intraperitoneally injected at 30 min before ischemia, and Shenmai injection 9 ml/kg was intravenously injected immediately before reperfusion.Six rats in each group were selected at 120 min of reperfusion, blood samples were collected from the left ventricle, the rats were then sacrificed, specimens were obtained from cardiac apex for examination of the ultrastructure and for determination of serum cardiac tropomin I( cTnI) level (by enzyme-linked immunosorbent assay), contents of Fe2+ and malondialdehyde (MDA) and superoxide dismutase (SOD) activity (by colometry), and expression of Nrf2, GPX4 and AcylCoA synthetase longchain family member 4 (ACSL4) (by Western blot). Another 6 rats in each group were selected to measure the myocardial infarct size. Results Compared with group S, the myocardial infarct size, serum cTnI concentrations, and myocardial Fe2+ and MDA contents were significantly increased, the activity of SOD was decreased, the expression of Nrf2 and GPX4 was down-regulated, and the expression of ACSL4 was up-regulated in IR, SM and SMM groups (P 0.05). Compared with group SM, the myocardial infarct size, serum cTnI concentrations, and myocardial Fe2+ contents were significantly increased, the activity of SOD was decreased, the expression of Nrf2 and GPX4 was down-regulated, and the expression of ACSL4 was up-regulated in group SMM (P<0.05). Conclusion Activation of Nrf2-Gpx4 signaling pathway is involved in Shenmai injection-induced reduction of myocardial I/R injury and is related to inhibiting ferroptosis in rats. Key words: NF-E2-related factor 2; Glutathione peroxidase; SAPONINS; Myocardial reperfusion injury; Cell death