Multitarget Modulation of Ferroptosis by Tetramethylpyrazine in Myocardial Ischemia-Reperfusion Injury: An Integrative Network Pharmacology and Bioinformatics Approach

Abstract

Background and Objectives: This study employed an integrated approach combining network pharmacology and bioinformatics to systematically elucidate the multitarget mechanisms of tetramethylpyrazine (TMP) in ameliorating myocardial ischemia-reperfusion injury (MIRI), with a specific focus on its potential association with ferroptosis regulation. Methods: The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), PharmMapper, SwissTarget Prediction, and TargetNet databases were used to combine the data, which were subsequently used to identify TMP-related targets. Ferroptosis-associated targets were retrieved from the FerrDb database, whereas MIRI-related targets were screened via the GeneCards and DisGeNET databases. Venn diagram analysis was applied to identify overlapping targets among TMP, ferroptosis, and MIRI. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation pathway enrichment analyses were conducted via R software to elucidate the underlying mechanisms involved. The protein-protein interaction (PPI) network was constructed via the STRING database, and topological analysis was performed via Cytoscape software to identify key targets. AutoDock Vina and PyMol were used to validate molecular docking. For experimental validation, an in vitro MIRI model was established using H9C2 cardiomyocytes subjected to hypoxia/reoxygenation (4h/2h). The cells were treated with 20 μM TMP or the ferroptosis inhibitor ferrostatin-1 (Fer-1, 20 μM). Intracellular reactive oxygen species (ROS) levels were detected using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe and visualized by fluorescence microscopy, while the expression of the key ferroptosis-related protein GPX4 was assessed by Western blotting. Results: Twenty shared targets linked to ferroptosis were identified. Molecular docking validation revealed four important targets, namely, PPARG, MDM2, SIRT1, and GSK3B, which have favorable binding affinities for TMP (binding energy < -5.0 kcal/mol). The GO analysis indicated that TMP primarily regulates biological processes such as positive modulation of protein phosphorylation and activation of the MAPK cascade. The KEGG enrichment analysis highlighted key pathways, including proteoglycan (PG) signaling in cancer, lipid metabolism dysregulation, and efferocytosis. Consistently, in vitro experiments demonstrated that TMP treatment significantly attenuated the ischemia-reperfusion (I/R)-induced overproduction of ROS and counteracted the downregulation of GPX4 protein expression, exhibiting a protective effect comparable to Fer-1. Conclusions: These results suggest that TMP may reduce MIRI via synergistic and multipathway mechanisms, which may be related to the regulation of ferroptosis. Furthermore, this study provides new theoretical knowledge about the cardioprotective properties of TMP by validating its anti-inflammatory efficacy.