Aflatoxin B1 Induces Anxiety-related Neurotoxicity Through Brain-region-Specific EGFR/AKT Signaling: Evidence from Integrative Network Toxicology and Mendelian Randomization.

Aflatoxin B1 (AFB1) is a widespread foodborne mycotoxin with well-established hepatotoxic and carcinogenic effects; however, its potential role in neuropsychiatric disorders remains unclear. Emerging evidence suggests that AFB1 can cross the blood-brain barrier, inducing oxidative stress and neuronal dysfunction. Whether these alterations contribute to anxiety-related molecular changes in the human brain remains poorly understood. An integrative approach combining network toxicology, brain-region-specific two-sample Mendelian randomization (MR), molecular docking, and experimental validation was employed. AFB1-associated targets were identified via STITCH, ChEMBL, and SwissTargetPrediction, and intersected with anxiety-related genes from GeneCards and GSEA. Protein-protein interaction (PPI) analysis, topological screening, and functional enrichment were used to identify hub genes and key pathways. MR analysis utilized cortex and cerebellum expression quantitative trait loci (eQTL) data from GTEx v8. Molecular docking evaluated binding interactions between AFB1 and key proteins. SH-SY5Y cells were used to validate alterations in oxidative stress and signaling. A total of 196 shared targets were identified, and 24 hub genes were screened based on the intersection of the top 40 genes ranked by degree, betweenness, and closeness centrality analyses. Enrichment analysis highlighted oxidative stress, apoptosis, MAPK, PI3K-Akt signaling, and monoaminergic synaptic pathways. MR analysis revealed region-specific associations: cortical EGFR expression positively correlated with anxiety risk, while cerebellar PPARG expression exhibited a protective effect. Additional genes (AKT1, KRAS, ABL1, PRKACB) showed positive cerebellar associations. Docking analysis indicated stable binding between AFB1 and key proteins. Experimental validation confirmed increased EGFR and AKT1 expression, reduced PPARG expression, and elevated ROS levels in SH-SY5Y cells. AFB1 promotes anxiety-related neurotoxicity in a brain-region-specific manner, supported by Mendelian randomization evidence, by inducing oxidative stress, disrupting EGFR/AKT signaling, and suppressing PPARG-mediated neuroprotection. These findings elucidate a mechanistic link between AFB1 exposure and neuropsychiatric risk, and highlight the need for stricter food safety regulation and incorporation of mycotoxin exposure into anxiety risk assessment and prevention strategies.