DINCH Exposure Contributes to Osteoarthritis Pathogenesis via TSPO-Mediated Chondrotoxicity: Evidence from In Silico Prediction to Experimental Validation.

Diisononyl cyclohexane-1,2-dicarboxylate (DINCH), a widely used plasticizer substitute, has been widely detected in human populations and has emerged as a new environmental pollutant of growing concern. Despite the established association between exposure to traditional plasticizer and an increased risk of osteoarthritis (OA), it remains unclear whether DINCH, as a major substitute, contributes to OA pathogenesis through direct chondrotoxicity. This study integrated network toxicology, single-cell transcriptomics, machine learning, and experimental validation to systematically investigate the role of DINCH exposure in the onset and progression of OA and its potential molecule mechanisms. Through network toxicology analysis, we identified 14 common targets shared by DINCH and OA, which were significantly enriched in pathways related to cellular senescence, mitochondrial, and stress responses. In vitro experiments confirmed that DINCH inhibited chondrocyte viability in a concentration- and time-dependent manner and mediated mitochondrial stress and chondrocyte senescence. Random Forest algorithm, a nomogram model based on four high-confidence predictive biomarker-TSPO, RAF1, STAT6, and CAPN2-was constructed to predict the progression of DINCH-exposed related OA. Molecular docking and molecular dynamics simulations suggested that DINCH may stably interact with the aforementioned targets in silico, with TSPO emerging as the candidate core target through multi-algorithm integration. Single-cell transcriptomic analysis revealed that TSPO is highly expressed in OA chondrocytes and specifically enriched in the hypertrophic chondrocyte subpopulation. Virtual gene knockout suggested that TSPO functional disruption was associated with extracellular matrix(ECM) homeostasis remodeling. Moreover, TRO-40303, as a TSPO-specific inhibitor, can effectively reverse TSPO upregulation-induced mitochondrial stress and chondrocyte senescence, thereby restoring the ECM metabolic balance in cartilage. Overall, this study elucidated the molecular mechanism by which DINCH exposure induced chondrotoxicity through TSPO targeting to drive OA development, providing new targets and theoretical foundations for the early diagnosis and intervention of environment-related OA.