DEHP exposure and cardiac fibrosis risk: An integrated network toxicology and molecular experimental investigation.

The environmental endocrine-disrupting chemical di-(2-ethylhexyl) phthalate (DEHP) has been epidemiologically linked to cardiac fibrosis, yet the underlying molecular mechanism remains poorly defined. To dissect the mechanism, we employed an integrative strategy combining network toxicology, molecular-dynamics (MD) simulations and in vitro experimental validation. Putative DEHP targets (n = 6439) were mined from the Comparative Toxicogenomics Database (CTD) and SwissTargetPrediction, while cardiac fibrosis-related genes (n = 2288) were curated from GeneCards and OMIM. Overlap analysis identified 1054 shared candidates. These were imported into STRING to construct a high-confidence protein-protein interaction (PPI) network, which was subsequently analyzed in Cytoscape, revealing a 12-hub core enriched in SRC, STAT3, EGFR and other key regulators. Functional enrichment underscored a prominent footprint in inflammatory responses and in the PI3K-Akt and MAPK signaling cascades. Molecular-docking analyses revealed high binding affinities between DEHP and the identified core targets, while MD simulations verified the structural stability of DEHP complexes with SRC, STAT3 and EGFR. In vitro, cardiomyocyte viability declined progressively with increasing DEHP concentrations. Exposure to 50 μg/mL DEHP elevated the BAX/BCL2 ratio, promoted cytochrome c release, and raised the number of TUNEL-positive cells, confirming that DEHP induces cardiomyocyte apoptosis. In cardiac fibroblasts, DEHP up-regulated transcription of Tgf-β1, Col1a1, and Acta2, driving their differentiation toward a myofibroblast phenotype. Western blot analyses revealed that DEHP activated SRC and STAT3 phosphorylation; pharmacologic inhibition of SRC or STAT3 phosphorylation attenuated DEHP-induced phenotypic conversion of cardiac fibroblasts. Collectively, DEHP exacerbates cardiac fibrosis by triggering cardiomyocyte apoptosis and by activating the SRC-STAT3 axis to promote the conversion of cardiac fibroblasts into myofibroblasts, providing clear evidence of DEHP-elicited cardiotoxicity and a theoretical basis for targeted intervention.