Identification and experimental validation of biomarkers associated with macrophages in myocardial ischemia-reperfusion injury

Myocardial ischemia-reperfusion injury (MIRI) is a complex process leading to substantial myocardial damage. Macrophages play a pivotal role in tissue development and homeostasis. This study aimed to identify macrophage-associated biomarkers in MIRI and explore the molecular mechanisms underlying their involvement. Macrophage-related genes (MRGs) were retrieved from public databases, and differentially expressed genes (DEGs) were identified using transcriptomic data. MRGs and DEGs were integrated to identify candidate genes. Biomarkers were identified through protein-protein interaction (PPI) analysis and gene expression validation. Enrichment analysis, immune infiltration analysis, construction of molecular regulatory networks, drug prediction, and molecular docking were conducted to investigate the functional mechanisms of the identified biomarkers. Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed. Finally, single-cell data were analyzed to assess the distribution of biomarkers across annotated cell types. Tlr7, Tlr1, Tlr6, and Il33 were identified as key biomarkers. Enrichment analysis revealed that the "Ribosome," "Oxidative phosphorylation," and "Spliceosome" pathways were significantly enriched in these biomarkers. Immune infiltration analysis demonstrated distinct differences in the presence of macrophages, M1 macrophages, and M2 macrophages between MIRI and control groups. Notably, Tlr7, Tlr1, and Tlr6 exhibited strong positive correlations with macrophages, M1, and M2 macrophages. Biomarkers were regulated by several transcription factors (TFs) (e.g., MYC, TBP) and microRNAs (miRNAs) (e.g., -mmu-miR-15a-5p). Drug prediction and molecular docking analyses indicated that IMIQUIMOD and Acetaminophen might serve as potential therapeutic targets for MIRI. RT-qPCR confirmed significant upregulation of Tlr7, Tlr1, and Tlr6 in the MIRI group, consistent with bioinformatics findings, whereas Il33 showed no significant difference between the two groups. Five major cell types were identified, including pericytes, M2 macrophages, and M1 macrophages. Notably, Tlr7 and Tlr1 were predominantly expressed in M2 macrophages. Tlr7, Tlr1, Tlr6, and Il33 were identified as macrophage-associated biomarkers in MIRI, providing valuable insights for MIRI diagnosis and potential therapeutic interventions.