Evaluation of the Mechanism of Jiedu Huazhuo Quyu Formula in Treating Wilson's Disease-Associated Liver Fibrosis by Network Pharmacology Analysis and Molecular Dynamics Simulation

Abstract

The Jiedu Huazhuo Quyu formula (JHQ) shows significant beneficial effects against liver fibrosis caused by Wilson's disease (WD). Hence, this study aimed to clarify the mechanisms of the JHQ treatment in WD-associated liver fibrosis. First, we collected 103 active compounds and 527 related targets of JHQ and 1187 targets related to WD-associated liver fibrosis from multiple databases. Next, 113 overlapping genes (OGEs) were obtained. Then, we built a protein-protein interaction (PPI) network with Cytoscape 3.7.2 software and performed the Gene Ontology (GO) term and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses with GENE DENOVO online sites. Furthermore, module analysis was performed, and the core target genes in the JHQ treatment of WD-associated liver fibrosis were obtained. Pathway and functional enrichment analyses, molecular docking studies, molecular dynamic (MD) simulation, and Western blot (WB) were then performed. The results indicated that 8 key active compounds including quercetin, luteolin, and obacunone in JHQ might affect the 6 core proteins including CXCL8, MAPK1, and AKT1 and 107 related signaling pathways including EGFR tyrosine kinase inhibitor resistance, Kaposi sarcoma-associated herpesvirus infection, and human cytomegalovirus infection signaling pathways to exhibit curative effects on WD-associated liver fibrosis. Mechanistically, JHQ might inhibit liver inflammatory processes and vascular hyperplasia, regulate the cell cycle, and suppress both the activation and proliferation of hepatic stellate cells (HSCs). This study provides novel insights for researchers to systematically explore the mechanism of JHQ in treating WD-associated liver fibrosis.

Figure 1

Schematic diagram of the network pharmacology approach to explore the mechanisms of JHQ in WD-associated liver fibrosis.

Figure 2

The number of overlapping ingredients and the herb-ingredient-target network relationships. (a) Venn diagram showing the number of overlapping ingredients among the six herbs. (b) Herb-ingredient-target network. Ellipses represent herbs in JHQ. Octagons represent active compounds in JHQ. Diamonds represent targets of active compounds. Edges represent interactions between compounds and targets vs. common targets of multiple herbs.

Figure 3

PPIs of 113 OGEs. (a) Venn diagram showing 113 common targets of JHQ and WD-associated liver fibrosis. (b) The PPI network: the larger the degree value is, the larger the node and the brighter the color. The larger the combined score is, the wider and brighter the line color is.

Figure 4

Enrichment analysis of 113 OGEs. (a) Top 20 enriched GO-CC terms. (b) Top 20 enriched GO-MF terms. (c) Top 20 enriched GO-BP terms. (d) Top 20 enriched KEGG pathways.

Figure 5

Identification of core target genes through module analysis adopted for gene set enrichment analysis of the 6 core target genes. (a) Module 2 consisted of 30 nodes and 378 edges as identified by the MCODE plugin in Cytoscape 3.7.2. (b) The top 6 core target genes identified by the CytoNCA plugin. (c) Top 20 enriched GO-CC terms. (d) Top 20 enriched GO-MF terms. (e) Top 20 enriched GO-BP terms. (f) Top 20 enriched KEGG pathways.

Figure 6

The herb-ingredient-target-pathway network relationship and the results of docking analysis and MD. (a) The herb-ingredient-target-pathway network. Octagons represent active compounds. Ellipses represent herbs. Rectangles represent pathways. Chevrons represent targets. (b) The molecular docking of CXCL8 to quercetin. (c) The molecular docking of MAPK1 to palmatine. (d) The molecular docking of AKT1 to bisdemethoxycurcumin. (e) The molecular docking of SRC to NSC122421. (f) The molecular docking of VEGFA to quercetin. (g) The molecular docking of IL-6 to quercetin. (h) RMSD plots for 3 protein-ligand complexes.

Figure 7

Effect of JHQ on core target proteins abundant in LX-2 cells after CuSO₄ treatment. ^∗∗∗P < 0.001 and ^∗∗P < 0.01 compared with the model group.