Identification of GSK3β as a molecular target of Alternaria mycotoxin-induced reproductive toxicity using integrated network toxicology and surface plasmon resonance (SPR) validation

Alternaria mycotoxins are emerging foodborne contaminants associated with reproductive toxicity, yet the molecular mechanisms remain poorly understood. In this study, we integrated network toxicology, molecular docking, and surface plasmon resonance (SPR) to identify potential molecular targets of reproductive toxicity induced by two Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME). The CCK-8 assay revealed that both toxins induced time- and dose-dependent cytotoxicity in TM3 mouse Leydig cells and HTR-8/SVneo human trophoblast cells. Network toxicology identified 76 overlapping targets associated with mycotoxin-induced reproductive toxicity. Enrichment analysis revealed that these targets were significantly enriched in pathways related to cancer and viral infection. A protein-protein interaction network was constructed and analyzed in Cytoscape. Based on this network, the robust rank aggregation method was employed to prioritize ten core targets: CASP3, DNMT1, ABL1, ESR1, RELA, CDK2, GSK3β, CDK4, CHEK1, and SRC. Molecular docking showed favorable binding between AOH/AME and these targets, with GSK3β displaying the lowest binding energy scores for both toxins. Subsequently, SPR analysis confirmed direct molecular interactions between GSK3β and both AOH and AME, with dissociation constants in the micromolar range. This study provides experimental evidence of direct binding of AOH/AME to GSK3β, suggesting that GSK3β is a candidate molecular target mediating the reproductive toxicity induced by Alternaria mycotoxins and providing a mechanistic basis for future risk assessment.