Network toxicology based research into the effect and mechanism of plasticizers on infertility.

Introduction: To investigate the potential impact of plasticizers on infertility and elucidate the underlying mechanisms, we employed an integrated approach combining network toxicology, molecular docking, and in vitro experimental validation. Methods and results: Initially, we identified potential targets by intersecting plasticizer-related targets with infertility-associated targets, yielding 134 candidate targets. These targets were subsequently analyzed using the STRING database and Cytoscape software to construct protein-protein interaction (PPI) networks, from which 20 hub genes were identified. Functional enrichment analysis conducted using the R software revealed that these targets participate in multiple biological pathways. Molecular docking simulations performed with AutoDock Vina and visualized using PyMol demonstrated strong binding affinities between key hub targets and three representative plasticizers: diethyl phthalate (DEP), dimethyl phthalate (DMP), and dioctyl phthalate (DOP). For experimental validation, we examined the effects of plasticizers on the human ovarian granulosa cell lines. CCK-8 assays revealed significant inhibition of cell proliferation following exposure to plasticizer. Flow cytometry and western blot analyses further showed that plasticizer treatment upregulated the anti-apoptotic protein BCL-2 while downregulating pro-apoptotic BAX, as well as key reproductive markers, including CYP19A1, ER-α, and FSHR. Discussion: Collectively, these findings demonstrate that plasticizers may contribute to infertility through multiple molecular pathways, as predicted by network toxicology and confirmed by in vitro experiments. This integrated approach provides compelling evidence for the reproductive toxicity of plasticizers and offers insights into their potential mechanisms of action.