Pubescenoside D Ameliorates Myocardial Ischemia–Reperfusion Injury via Preventing the Dissociation of HK2 and Promoting Mitophagy by Targeting GSK‐3β

ABSTRACT Myocardial ischemia–reperfusion injury (MI/RI) is a critical challenge for acute myocardial infarction therapy, as there is currently no ideal drug available. Glycogen synthase kinase 3 beta (GSK‐3β) serves as an promising therapeutic target for treating MI/RI. Our previous studies have demonstrated that Ilex pubescens ameliorates MI/RI. The purpose of this study is to evaluate the therapeutic efficacy and potential mechanism of the screened GSK‐3β inhibitor from Ilex pubescens against MI/RI. Three‐dimensional‐quantitative structure–activity relationship (3D‐QSAR) modeling, molecular docking, the oxygen and glucose deprivation/reperfusion (OGD/R) and left anterior descending (LAD) artery ligation‐induced MI/RI mice model, and western blotting analysis were used to screen and investigate the myocardial protective efficacy and mechanism. Here, we screened Pubescenoside D (PBD) as a GSK‐3β inhibitor with an IC50 value of 0.3769 μM from Ilex pubescens, using 3D‐QSAR modeling, molecular docking, and kinase assay verification. Ile217, Leu88, Phe93, and Phe67 are the key binding sites for PBD and GSK‐3β. PBD protects cardiomyocytes against MI/RI in vitro and in vivo. Further mechanism studies show that PBD inhibits mitochondrial permeability transition pore (mPTP) opening by preventing GSK‐3β‐mediated the dissociation of hexokinase2 (HK2) from the outer membrane of the mitochondria and enhances mitophagy by suppressing GSK‐3β activity, subsequently reducing cardiomyocyte apoptosis. Our findings shed light on the efficacy of PBD as a promising therapeutic agent in the treatment of MI/RI targeting GSK‐3β.