Identification of key targets and mechanisms of isoorientin in osteoporosis treatment through integrated network pharmacology and experimental validation.

Osteoporosis is a prevalent metabolic bone disorder characterized by diminished bone mineral density and elevated fracture susceptibility. Although isoorientin (ISO) has emerged as a promising candidate for osteoporosis treatment, its molecular mechanisms remain unclear. In this study, a comprehensive network pharmacology approach was employed to identify potential therapeutic targets by systematically mining the GeneCards and DisGeNET databases. ISO-target interactions were predicted through an integrated analysis of multiple chemoinformatic platforms, including Super-Pred, SwissTargetPrediction, PharmMapper, and ChemMapper. Hub targets were identified via protein-protein interaction (PPI) network analysis, complemented by functional enrichment assessments and molecular docking simulations. The computational findings were experimentally validated using an ovariectomy (OVX)-induced osteoporotic murine model. Network pharmacological analysis revealed 332 putative ISO targets, 45 of which significantly overlapped with 610 osteoporosis-associated targets. Functional enrichment analysis highlighted the critical involvement of these genes in hormone-mediated signaling pathways and cellular responses to nutrient levels. KEGG pathway analysis further implicated these targets in key regulatory cascades, including the MAPK, relaxin signaling, and lipid metabolism-associated atherosclerosis pathways. Molecular docking simulations demonstrated strong binding affinities between ISO and pivotal targets, including MAPK14, TLR4, and ESR1. In vivo validation using OVX mice confirmed ISO's capacity to attenuate bone loss by suppressing osteoclast activation, as evidenced by micro-CT analysis and histomorphometric quantification. Further in vitro studies demonstrated that ISO inhibits RANKL-induced osteoclastogenesis via suppression of the MAPK pathway. This study elucidates the key targets and pathways through which ISO exerts anti-osteoporotic effects, highlighting its therapeutic potential in osteoporosis management.