Abstract

Background : Myocardial ischemia/reperfusion injury (MIRI) remains a significant challenge in the field of cardiovascular treatment. Ferroptosis is a key driver in the pathogenesis of MIRI. Shenhong Tongluo Formula (SHTLF), a Chinese formula, has been clinically proven effective in treating cardiovascular diseases such as angina pectoris and acute coronary syndrome. However, its potential mechanism concerning ferroptosis in MIRI remains unclear. Purpose : To investigate the inhibitory effect of SHTLF on ferroptosis in MIRI and its underlying molecular mechanism. Methods : First, the chemical constituents of SHTLF were characterized using UPLC–Q-Orbitrap–MS. SHTLF was then evaluated for its cardioprotective and anti-ferroptotic effects in a rat MIRI model and in vitro cardiomyocyte models, including oxygen–glucose deprivation/reoxygenation (OGD/R) and an erastin-induced ferroptosis model. RNA sequencing combined with bioinformatic analyses was performed to identify ferroptosis-related pathways and key genes regulated by SHTLF. Based on the absorbed constituents detected in vivo, reverse network pharmacology, molecular docking, and molecular dynamics simulations were used to explore compound–target interactions. Finally, functional manipulation of spermidine/spermine N¹-acetyltransferase 1 (SAT1), together with RT–qPCR, Western blotting, and immunofluorescence analyses, was conducted to validate the mechanistic role of SHTLF in regulating ferroptosis. Results : UPLC–Q-Orbitrap–MS analysis demonstrated good batch-to-batch consistency of SHTLF and identified multiple potentially bioactive constituents. In both in vivo and in vitro experiments, SHTLF pretreatment ameliorated ferroptosis-associated markers. To elucidate the underlying mechanism, RNA-seq combined with bioinformatic analyses suggested that SAT1 and prostaglandin-endoperoxide synthase 2 (PTGS2) are key nodes in SHTLF-mediated regulation of ferroptosis during MIRI. Reverse network pharmacology based on s