Abstract
BACKGROUND: As public awareness of environmental issues grows and research on ecological pollutants advances, mounting evidence indicates that Methyl-4-hydroxybenzoate (MEP) is associated with the development of various diseases. This study aims to uncover the key genes and underlying molecular mechanisms by which MEP influences osteoporosis (OP). METHODS: This study integrates network toxicology, molecular docking, and molecular dynamics simulation approaches. Potential targets of the environmental contaminant were identified using the Comparative Toxicogenomics Database (CTD). In contrast, osteoporosis-related targets were retrieved from the Gene Expression Omnibus (GEO), GeneCards, and Online Mendelian Inheritance in Man (OMIM) databases. The intersection between MEP and OP targets was subsequently analyzed using PPI networks and functional enrichment analyses to identify core targets and pathways. For experimental validation, Sprague-Dawley rats were administered MEP via gavage for three months. Bone tissues were then collected for Micro-CT analysis and Hematoxylin and eosin (H&E) staining to evaluate the impact of MEP on bone mass. Further mechanistic investigations were conducted using immunofluorescence staining, western blotting (WB), and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Bioinformatics analysis identified 379 overlapping genes between MEP and OP, with 31 core genes (e.g., AKT1, HSP90AA1) implicated in cell cycle regulation, oxidative stress, and DNA damage. Molecular docking confirmed stable binding of MEP to AKT1 (- 4.39 kcal/mol), Beclin1 (- 18.77 kcal/mol), and LC3B (- 3.55 kcal/mol). Molecular dynamics simulations revealed that the MEP-Beclin1 complex stabilized after 85 ns (RMSD ≈ 2.2 Å), with persistent hydrogen bond interactions. In vivo experiments confirmed that MEP disrupts bone microstructure in a dose-dependent manner. Both micro-computed tomography (Micro-CT) and H&E staining revealed trabecular fractures and enlarged bone marrow cavities. Immunofluorescence analysis demonstrated significantly reduced LC3B puncta (P < 0.0001). Further validation by WB and RT-qPCR showed significantly upregulated expression of the autophagy-related protein AKT1 (P < 0.01), whereas downregulated expression of autophagy-related proteins (LC3B, Beclin1) and osteogenic markers (RUNX2, BMP2) (P < 0.01). CONCLUSIONS: Collectively, this study demonstrates that the environmental pollutant methylparaben (MEP) induces OP by suppressing AKT1-mediated autophagy signaling. Our findings provide novel mechanistic insights into how ecological contaminants may trigger OP pathogenesis, thereby establishing a theoretical foundation for the development of preventive and therapeutic strategies against pollutant-associated bone disorders.