Fig 1.
Workflow of this study.
Fig 2.
Identification and interaction of potential targets.
(A) Venn diagram showing the 96 overlapping targets between melatonin and cholestatic liver disease. (B) Protein-protein interaction (PPI) network of the overlapping targets, with nodes representing proteins and edges representing interactions.
Fig 3.
Topological analysis of targets for melatonin in treating cholestatic liver disease.
In the network, nodes represent potential targets of melatonin for treating cholestatic liver disease. A higher degree value is indicated by a darker node color (red), denoting greater importance of the target in the therapeutic mechanism.
Fig 4.
GO and KEGG enrichment analysis of 96 potential targets of melatonin against cholestatic liver disease.
(A) Top 10 significantly enriched terms in Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) categories of Gene Ontology (GO). (B) Bubble plot of the top 10 enriched KEGG pathways. The bubble size represents the number of target genes, and the color indicates the significance level (-log₁₀(P-value)).
Fig 5.
Target-pathway network of melatonin for treating cholestatic liver disease.
Green circles denote the key targets of melatonin for treating cholestatic liver disease, blue circles represent the key signaling pathways involved, and each edge indicates a target-pathway association.
Table 1.
Molecular docking results of melatonin with core targets.
Fig 6.
Molecular docking of melatonin with the top 10 core targets.
Binding modes of melatonin to the top 10 core targets (A-J: MMP9, ALB, PTGS2, SRC, ESR1, EGFR, AKT1, PPARG, IGF1, and CASP3). The left panel shows the overall view, with the protein structure colored in blue. The middle panel provides a detailed close-up view, where melatonin is shown in yellow, the interacting protein amino acid residues are colored in cyan, and yellow dashed lines indicate hydrogen bonds. The right panel displays the 2D ligand-interaction diagram, with pink circles denoting hydrophobic interactions.
Fig 7.
Liver morphology, body weight, and liver index across experimental groups.
(A) Gross appearance of liver tissue from mice in each group following intervention with different concentrations of melatonin. (B) Changes in body weight of mice in each group after intervention with different concentrations of melatonin. (C) Changes in liver index (liver weight/body weight × 100%) of mice in each group after intervention with different concentrations of melatonin.Data are presented as mean ± SD (n = 6). Significant differences compared to the DDC group are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Fig 8.
Melatonin ameliorates liver injury, fibrosis, and serum biomarkers in cholestatic mice.
(A) Representative hematoxylin and eosin (H&E)-stained liver sections from mice in each group at 100 × magnification. (B) Representative H&E-stained liver sections at 200 × magnification; red arrows indicate bile porphyrin deposits. (C) Representative Masson-stained liver sections from each group (×100), where blue color indicates collagen fibers. (D) Quantitative analysis of the collagen fiber area percentage in liver tissues. (E–I) Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA), and total bilirubin (TBIL) in each group. Data are presented as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001.
Fig 9.
Distribution of Targets in the Estrogen Signaling Pathway.
Fig 10.
Western blot analysis of key protein expression in liver tissues.
(A) Representative Western blot images of MMP9, EGFR, AKT, and p-AKT. (B–E) Quantitative analysis of the relative protein expression levels of MMP9, EGFR, AKT, and p-AKT, normalized to GAPDH (n = 3).Data are presented as mean ± SD. Significant differences compared to the DDC group are indicated (*P < 0.05, **P < 0.01, ***P < 0.001).