Cardioprotective Effects of Calycosin-7-O-β-D-glucoside against Myocardial Ischemia-Reperfusion Injury by Orchestrating PIM1/CTSD-Mediated T cells Responses

• Calycosin-7-O-β-D-glucoside (C7G) showed significant protective effect on MIRI in rats. • Integrated bioinformatics and machine learning identified six high-confidence targets, with CTSD and PIM1 specifically enriched in cardiac T cells. • Single-cell RNA-seq revealed T cells constitute 43% of post-MIRI immune infiltrates, where CTSD and PIM1 expression correlated with Treg and CD8⁺ T-cell subsets, respectively. • Molecular docking and dynamics simulations confirmed high-affinity binding of C7G to both proteins, illustrating that C7G can bind to CTSD and PIM1. • C7G significantly reverses the expression changes of Ctsd and Pim1 caused by MIRI. Immune dysregulation plays a central role in myocardial ischemia–reperfusion injury (MIRI). This study investigates the protective mechanism of calycosin-7-O-β-D-glucoside (C7G), a major isoflavone from Astragalus membranaceus , in a rat MIRI model. All three doses of C7G (7.5, 15, 30 mg/kg) showed significant protective effects on MIRI in rats. Among them, medium-dose pretreatment achieved 100% seven-day survival, the largest reduction in infarct size, and the best preservation of myocardial architecture. Integrated bioinformatics and machine learning identified six high-confidence targets, with CTSD and PIM1 specifically enriched in cardiac T cells. Single-cell RNA-seq revealed T cells constitute 43% of post-MIRI immune infiltrates, where CTSD and PIM1 expression correlated with Treg and CD8⁺ T-cell subsets, respectively. Molecular docking and dynamics simulations confirmed high-affinity binding of C7G to both proteins, illustrating that C7G can bind to CTSD and PIM1. Importantly, C7G significantly reverses the expression changes of Ctsd and Pim1 caused by MIRI. Thus, C7G mitigates MIRI by directly modulating CTSD and PIM1 in T cells to reprogram immunity, supporting its clinical development.