Multi-target modulation of the homologous recombination pathway by resveratrol promotes DNA damage repair in POI: integrated network pharmacology, molecular dynamics simulation, and experimental validation.

Premature ovarian insufficiency (POI) is characterized by gonadotropin elevation, estrogen deficiency, and follicular loss. Resveratrol (RSV), a natural polyphenol with antioxidant and anti-aging properties, shows therapeutic promise for POI, but its molecular targets and mechanisms remain unclear. Network pharmacology analysis was used to identify overlapping targets of RSV and POI, followed by Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, protein-protein interaction (PPI) network construction, and hub gene screening. Molecular docking and dynamics simulations were performed to characterize the affinity and binding stability. In vivo, a cyclophosphamide-induced POI rat model was established to evaluate the protective effects of RSV on ovarian morphology and hormone levels. In vitro, a 4-hydroperoxycyclophosphamide-induced granulosa cell model was used to assess DNA damage and homologous recombination (HR) activity through TUNEL staining, Western blotting, and nuclear foci analysis, with RAD51 inhibition applied to verify pathway dependence. About 609 overlapping genes between RSV- and POI-related targets were identified. GO and KEGG enrichment analyses revealed significant involvement in reproductive system development, DNA repair complex, and cellular senescence. PPI and topological analysis identified three core genes - ATM, BRCA1, and RAD51 - significantly enriched in the HR pathway. Molecular docking and dynamic simulations indicate that RSV has a strong affinity and stable binding mode with these three targets. In vivo, RSV ameliorated cyclophosphamide-induced ovarian injury, increasing serum anti-Müllerian hormone levels and secondary follicle counts. Mechanistically, in the POI cell model, RSV upregulated RAD51 and downregulated γH2AX expression, thereby promoting HR pathway activation and DNA double-strand break repair. The protective effect of RSV was abolished by the RAD51 inhibitor RI-1. Immunofluorescence foci analysis further verified that RSV enhanced the recruitment of RAD51 to DNA damage sites and reduced nuclear γH2AX accumulation. This study provides structural and experimental evidence for the target selection, structural optimization, and molecular mechanism of RSV in the treatment of POI.