Elucidation of the potential mechanisms of di-2-ethylhexyl phthalate (DEHP)-associated osteoporosis by integrating network toxicology and in vitro validation.

Di-2-ethylhexyl phthalate (DEHP) is a plasticizer widely used in daily life and is known for its toxicity after exposure. However, its toxic effects and mechanisms in Osteoporosis (OP) have not been fully explored. This work explored the potential mechanisms of DEHP-associated OP by integrating network toxicology, molecular docking, and cell experiments. 32 potential targets were identified through multiple databases. Enrichment analysis suggested that DEHP might exert its effects primarily through estrogen and MAPK/PI3K-Akt signaling pathways, and cellular senescence. TNF, AKT1, MAPK3, and CYP17A1 were prioritized as hub targets through integrated computational screening and molecular docking, and qRT-PCR experiments showed that DEHP upregulated TNF while downregulating AKT1, MAPK3, and CYP17A1. In addition, DEHP exposure was associated with impaired osteogenic differentiation of MC3T3-E1 cells, as manifested by decreased ALP activity and mineralization capacity, and downregulation of key osteogenic markers, including Runx2, ALP, OPN, and OCN. Together, these findings suggest that DEHP exposure is associated with altered expression of these targets and impairs osteogenic differentiation, which may contribute to OP pathogenesis. This study offers a novel perspective on the potential impact of DEHP on OP, providing a theoretical and experimental basis for environmental health risk assessment.