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

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.