Total glycosides of Yangyin Tongnao formula alleviate myocardial ischemia/reperfusion injury through regulation of PI3K/Akt signaling and ferroptosis: network pharmacology and experimental validation

Background Myocardial ischemia/reperfusion injury (MI/RI) is a major pathological process that limits the benefits of timely reperfusion therapy. Yangyin Tongnao formula (YYTN) is a traditional Chinese medicine for cerebrovascular diseases. Based on the Chinese traditional concept of brain-heart simultaneous treatment, we hypothesized that YYTN may exert protective effects against MI/RI. This study aimed to investigate the potential mechanisms underlying the protective effects of YYTN against MI/RI and to evaluate the cardioprotective effects of its glycoside-enriched fraction, the total glycosides of YYTN (YYTG). Methods Network pharmacology was employed to identify the potential active components, targets, and signaling pathways underlying the protective effects of YYTN against MI/RI. YYTG was prepared as a glycoside-enriched fraction of YYTN. An oxygen-glucose deprivation/reperfusion (OGD/R) model was established in H9c2 cardiomyocytes to investigate the cardioprotective effects of YYTG. HUVECs subjected to OGD/R were subsequently used as a complementary model to evaluate the protective effects on vascular endothelial injury. Results Network pharmacology analysis identified 29 active compounds and 231 YYTN-related targets associated with MI/RI, with nine core targets identified by PPI analysis. The PI3K/Akt signaling pathway emerged as a potential key pathway. In OGD/R-challenged H9c2 cells, YYTG significantly improved cell viability and attenuated oxidative stress, iron accumulation, and lipid peroxidation, as evidenced by reduced ROS, MDA, and Fe 2+ levels. YYTG also increased GPX4 and FTH1 expression and decreased TfR1 expression. Mechanistically, YYTG enhanced PI3K and Akt phosphorylation, whereas the PI3K inhibitor LY294002 attenuated its protective effects, including the preservation of GPX4 and FTH1 expression and cell viability. In OGD/R-challenged HUVECs, YYTG partially restored GSH, GSH-Px, and SOD levels and reduced ROS and Fe 2+ accumulation, further supporting its protective effects against endothelial injury. Conclusion YYTG protects H9c2 cardiomyocytes against OGD/R-induced injury through activation of PI3K/Akt signaling and modulation of oxidative stress and ferroptosis-related processes. YYTG also exerts protective effects against OGD/R-induced endothelial injury, suggesting broader cytoprotective activity during ischemia/reperfusion. These findings provide experimental evidence supporting further investigation of YYTN as a potential therapeutic candidate for MI/RI.