Integrating network toxicology, molecular docking and experimental validation reveals potential mechanisms of DEHP in osteoarthritis.

Di(2-ethylhexyl) phthalate (DEHP), a ubiquitous environmental endocrine disruptor, has been implicated in osteoarthritis (OA) pathogenesis, although the underlying molecular mechanisms remain poorly understood. This study integrates network toxicology, molecular docking, Mendelian randomization (MR), and experimental validation to elucidate how DEHP exposure contributes to cartilage degradation. By analyzing OA-related transcriptomic datasets (GSE55457, GSE169077, GSE113825) and DEHP targets from multiple databases (CTD, GeneCards, ChEMBL, STITCH, SwissTargetPrediction, and SuperPred), we identified 215 overlapping targets. Protein-protein interaction network analysis and topological screening revealed five hub genes (INS, IGF1, MMP2, MMP9, and EGFR) central to DEHP-induced OA. Functional enrichment highlighted oxidative stress, extracellular matrix degradation, and dysregulated AMPK and HIF-1 signaling. Machine learning-based diagnostic modeling demonstrated robust predictive efficacy for IGF1, MMP2, and MMP9 (AUC > 0.7). Two-sample MR analysis using eQTLGen cis-eQTL and UK Biobank OA GWAS data provided genetic evidence that higher MMP9 expression is causally associated with increased OA risk, while colocalization analysis suggested trans-regulatory mechanisms. Molecular docking confirmed strong binding affinities between DEHP and hub proteins (binding energies ranging from -6.1 to -8.1 kcal/mol). In vitro, DEHP exposure dose- and time-dependently reduced chondrocyte viability, upregulated catabolic markers (MMP9, ADAMTS5, MMP13), and downregulated the anabolic marker COL2A1, phenocopying IL-1β-induced cartilage degradation. Immunohistochemistry further validated elevated MMP9 in damaged human OA cartilage. Collectively, these findings demonstrate that DEHP disrupts cartilage homeostasis through multi-target interactions, with MMP9 serving as a genetically supported causal mediator. This work proposes novel biomarkers and therapeutic targets, establishing a mechanistic bridge between environmental toxicology and musculoskeletal pathology.