Complete list of enriched KEGG pathways.

The aim of this study was to investigate the mechanism underlying the action of melatonin (MT) in treating cholestatic liver disease. Melatonin and therapeutic targets for cholestatic liver disease were screened. A protein–protein interaction network was constructed using intersecting targets. Core targets were subjected to GO and KEGG enrichment analyses. We evaluated core target affinity through molecular docking. Biochemical indicators were measured in a mouse model of cholestasis to determine the pathological changes in liver tissue. The expression of core targets (MMP9, EGFR, and AKT) was detected through western blotting. The core targets for treating cholestatic liver disease included ALB, AKT1, ESR1, CASP3, PPARG, MMP9, PTGS2, SRC, EGFR, and IGF1. The biological processes included lipopolysaccharide stress response, bacterial molecular response, nutrient level response, and regulation of inflammatory response. Additionally, the estrogen, tumor necrosis factor-alpha, and VEGF signaling pathways were enriched in cholestatic liver disease. Molecular docking showed that MT had a strong binding affinity for MMP9, EGFR, and AKT1. Animal experiments demonstrated that melatonin alleviated inflammation and fibrosis in cholestatic liver disease, downregulated MMP9 expression, and upregulated the expression of EGFR, AKT, and phosphorylated AKT. Network pharmacology predictions suggested that these targets are closely associated with the estrogen signaling pathway. In conclusion, the protective effect of melatonin against cholestatic liver injury is likely mediated through the downregulation of MMP9 and upregulation of EGFR/AKT.