Hederagenin protects against myocardial ischemia-reperfusion injury via attenuating ALOX5-mediated ferroptosis.

Hederagenin (HDG), a medical herb, is known for its beneficial activities against diverse diseases. The cardioprotective effect of HDG has been preliminarily disclosed, but the efficacy and underlying mechanism by which HDG protects against myocardial ischemia-reperfusion (MI/R) injury have not been elucidated yet. To simulate MI/R injury, the left anterior descending artery was occluded for 30 min and then reperfusion for 120 min in a rat model, and the cellular model of hypoxia-reoxygenation (H/R) injury was constructed in H9c2 cardiomyocytes. Hematoxylin-eosin, Prussian blue, and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining were conducted to assess the histological injury, iron deposition, and myocardial infarction. Myocardial enzymes and oxidative stress-related factors were detected using their commercial kits. Lipid peroxidation was measured using BODIPY581/591 probe, and iron content was detected. Cell counting kit (CCK)-8, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and flow cytometry assays were performed to assess cell viability and apoptosis. Protein levels were investigated by western blot. The interaction between HDG and 5-lipoxygenase (ALOX5) was verified using molecular docking. Our findings indicated that HDG significantly attenuated myocardial dysfunction by reducing infarction and myocardial injury. HDG significantly attenuated myocardial apoptosis in vitro and in vivo, as well as alleviating oxidative stress via reducing reactive oxygen species (ROS) and maintaining the balance between antioxidant and oxidant enzymes. Meanwhile, HDG inhibited I/R-induced ferroptosis in myocardium and cardiomyocytes, including reducing lipid peroxidation and iron level. Moreover, the binding relationship between HDG and ALOX5 was verified, and HDG could concentration dependently downregulate ALOX5. Furthermore, ALOX5 overexpression eliminated the inhibition of HDG on H/R-induced apoptosis, oxidative stress, and ferroptosis in H9c2 cardiomyocytes. HDG ameliorated myocardial dysfunction and cardiomyocyte injury by reducing apoptosis, oxidative stress, and ferroptosis through inhibiting ALOX5, providing a new perspective on the prevention and treatment of MI/R injury.