Multi-omics integration reveals macrophage polarization and ferroptosis as key mechanisms underlying kaempferol's therapeutic efficacy in peripheral artery disease.

Peripheral artery disease (PAD) is a chronic ischemic condition characterized by vascular occlusion and metabolic-immune dysregulation. To elucidate the molecular mechanisms underlying PAD and the therapeutic effects of Kaempferol, we integrated bulk transcriptomics, single-cell RNA sequencing, network pharmacology, and experimental validation. Transcriptomic analysis identified 1,144 differentially expressed genes in PAD, highlighting extensive transcriptional remodeling. WGCNA revealed distinct gene modules significantly correlated with PAD, suggesting key networks associated with vascular inflammation and immune dysregulation. Network pharmacology identified kaempferol as the core active component of Ruan Jian Qing Mai formula that is clinically effective in treating PAD, targeting 87 PAD-related genes enriched in immune and inflammatory pathways, predominantly expressed in macrophages. Integration of kaempferol-associated targets from RNA-seq and multi-database prediction uncovered 70 overlapping targets implicated in macrophage activation, lipid metabolism, and atherosclerosis signaling. CIBERSORTx and single-cell analysis further pinpointed macrophages as major cellular mediators, with kaempferol-target genes (e.g., TNF, SYK, LCK, IL4I1) specifically enriched in macrophages. Macrophages were stratified into high-inflammatory and low-inflammatory subsets; PAD lesions exhibited prominent enrichment of high-inflammatory macrophages, which concurrently displayed elevated inflammatory signatures and enhanced ferroptosis transcriptional activity, and represented the primary cellular population responsive to kaempferol. In silico gene knockout analysis further verified that SYK and SNX5 serve as core immune signaling regulators in macrophages, while TNF and LCK lack macrophage-specific immune regulatory functions. Functional experiments confirmed that kaempferol significantly suppressed LPS-induced M1 macrophage polarization and inflammatory cytokine expression in RAW264.7 cells. Ferroptosis scoring revealed enhanced ferroptosis susceptibility in PAD macrophages, with kaempferol-target genes correlating positively with ferroptosis drivers. Molecular docking further validated strong binding affinities between kaempferol and key macrophage-related targets (IL4I1, LCK, TNF, SYK). Using a zebrafish vascular regeneration injury model, we further verified that high-dose kaempferol rescues impaired angiogenesis under vascular ischemic damage. Collectively, these multi-omics and experimental findings uncover macrophage polarization and ferroptosis modulation as central mechanisms mediating kaempferol's therapeutic efficacy in PAD, providing mechanistic insights and potential molecular targets for precision intervention.