ANPEP as a potential mediator linking aspartame exposure to MAFLD: insights from network toxicology and multi-omics analyses

Background: Aspartame (APM) is a widely consumed artificial sweetener that has recently been implicated in the pathogenesis of metabolic disorders. However, the molecular mechanisms linking APM to metabolic-associated fatty liver disease (MAFLD) remain poorly understood. Materials and Methods: This study employed a comprehensive analytical strategy, integrating network toxicology, molecular docking, molecular dynamics simulations, machine learning, bulk RNA sequencing and single-cell transcriptomics, Mendelian randomization, and histological validation, to systematically investigate APM-associated molecular targets and their potential role in MAFLD. Results: The ADMETlab3.0 prediction indicated that APM exhibits strong hepatotoxic potential. Network toxicology analysis identified 80 overlapping genes, among which ANPEP demonstrated the lowest binding energy to APM (−8.2 kcal/mol). Molecular dynamics simulations further confirmed the stability of this interaction. Bulk RNA-seq data revealed that ANPEP was significantly upregulated in MAFLD liver tissues (P < 0.001) and achieved robust diagnostic performance (AUC = 0.84, 95% CI: 0.69–1.00, P < 0.01). In mouse models, Anpep expression was significantly elevated (P = 0.005). Mendelian randomization analysis indicated that elevated ANPEP levels were significantly associated with a higher risk of MAFLD (OR = 1.069, 95% CI: 1.017–1.124, P = 0.008). Single-cell sequencing and immunofluorescence analyses consistently confirmed that ANPEP was predominantly localized in CD68⁺ myeloid cells and was markedly upregulated as early as the fibrotic initiation stage of MAFLD (P < 0.01). Conclusion: ANPEP was identified as a potential target that links aspartame exposure to MAFLD, and was further associated with inflammation, lipid dysregulation, and fibrosis. These findings suggest that aspartame may not represent a safe strategy for weight management and could increase the risk of liver injury and MAFLD progression.