Network toxicology and in vitro data suggest PPARA and NFE2L2 as potential key targets in TCDD-induced non-alcoholic fatty liver disease.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant linked to metabolic disorders, but its role in non-alcoholic fatty liver disease (NAFLD) remains unclear. This study integrated network toxicology, transcriptomics, and in vitro experiments to investigate the molecular mechanisms linking TCDD exposure to NAFLD. Using multi-source databases (CTD, PubChem, STITCH, SwissTargetPrediction) and two liver transcriptome datasets (GSE126848, GSE213621), we identified 176 common targets between TCDD and NAFLD, enriched in lipid metabolism, oxidative stress, inflammation, and PPAR/AhR pathways. Protein‑protein interaction network and cytoHubba algorithms prioritized ten hub genes, including PPARA, NFE2L2, IL6, and CYP1A1. Molecular docking predicted strong binding affinities of TCDD to PPARA (- 8.5 kcal/mol) and NFE2L2 (- 8.1 kcal/mol). In vitro experiments using HepG2 cells showed that TCDD dose‑dependently downregulated PPARA, NFE2L2, and their downstream targets CPT1A and NQO1, while upregulating IL6. TCDD also increased malondialdehyde (MDA) and triglyceride (TG) levels, indicating oxidative stress and lipid accumulation. These experimental results are consistent with the computational predictions. We propose a mechanistic framework in which TCDD may impair PPARA‑mediated fatty acid oxidation and NFE2L2‑mediated antioxidant defense, while promoting inflammation, thereby contributing to NAFLD. This study provides a systematic understanding of TCDD‑induced NAFLD and suggests PPARA and NFE2L2 as potential key targets for environmental risk assessment and therapeutic intervention.