Comprehensive RNA-Seq Analysis of Potential Therapeutic Targets of Gan-Dou-Fu-Mu Decoction for Treatment of Wilson Disease Using a Toxic Milk Mouse Model

Abstract

Background: Gan-Dou-Fu-Mu decoction (GDFMD) improves liver fibrosis in experimental and clinical studies including those on toxic mouse model of Wilson disease (Model). However, the mechanisms underlying the effect of GDFMD have not been characterized. Herein, we deciphered the potential therapeutic targets of GDFMD using transcriptome analysis. Methods: We constructed a tx-j Wilson disease (WD) mouse model, and assessed the effect of GDFMD on the liver of model mice by hematoxylin and eosin, Masson, and immunohistochemical staining. Subsequently, we identified differentially expressed genes (DEGs) that were upregulated in the Model (Model vs. control) and those that were downregulated upon GDFMD treatment (compared to the Model) using RNA-sequencing (RNA-Seq). Biological functions and signaling pathways in which the DEGs were involved were determined by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses. A protein-protein interaction (PPI) network was constructed using the STRING database, and the modules were identified using MCODE plugin with the Cytoscape software. Several genes identified in the RNA-Seq analysis were validated by real-time quantitative PCR. Results: Total of 2124 DEGs were screened through the Model vs. control and Model vs. GDFMD comparisons, and dozens of GO and KEGG pathway terms modulated by GDFMD were identified. Dozens of pathways involved in metabolism (including metabolic processes for organic acids, carboxylic acids, monocarboxylic acids, lipids, fatty acids, cellular lipids, steroids, alcohols, eicosanoids, long-chain fatty acids), immune and inflammatory response (such as complement and coagulation cascades, cytokine-cytokine receptor interaction, inflammatory mediator regulation of TRP channels, antigen processing and presentation, T-cell receptor signaling pathway), liver fibrosis (such as ECM-receptor interactions), and cell death (PI3K-Akt signaling pathway, apoptosis, TGF-beta signaling pathway, etc.) were identified as potential targets of GDFMD in the Model. Some hub genes and four modules were identified in the PPI network. The results of real-time quantitative PCR analysis were consistent with those of RNA-Seq analysis. Conclusions: We performed gene expression profiling of GDFMD-treated WD model mice using RNA-Seq analysis and found the genes, pathways, and processes effected by the treatment. Our study provides a theoretical basis to prevent liver fibrosis resulting from WD using GDFMD.

Keywords: Gan–Dou–Fu–Mu decoction (GDFMD); RNA-sequencing; Wilson disease (WD); toxic milk mice (TX mice); traditional Chinese medicine (TCM).

FIGURE 1

GDFMD reversed DEGs with Model (A) The Venn diagram of GDFMD-revised gene with WD. down-DEGs (Model vs. CN and GDFMD vs. Model, down- and up-regulated, respectively) and up-DEGs (Model vs. CN and GDFMD vs. Model, up- and down-regulated, respectively) (B) Heatmap of down-DEGs and up-DEGS.

FIGURE 2

Bubbleplots of the GO enrichment analysis results using ClusterProfiler for down-DEGs (A) and down-DEGs (B) resulting for GDFMD-revised with WD.

FIGURE 3

Bubbleplots of the KEGG pathway enrichment analysis results using ClusterProfiler for down-DEGs ((A) and down-DEGs (B) resulting for GDFMD-revised with WD.

FIGURE 4

PPI network of the DEGs resulting from GDFMD treatment of WD. Node sizes correlate with node degree; The higher expression genes in WD group that compared with CN and GDFMD group were performed pink nodes, The lower expression genes in WD group that compared with CN and GDFMD group were performed green nodes; PPI, protein-rotein interaction; DEGs, differentially expressed genes.

FIGURE 5

Four key modules network (A–D) of PPI network for DEGs reversed by GDFMD with WD. Pink nodes denote up-regulated genes; PPI, protein-protein interaction; DEGs, differentially expressed genes.

FIGURE 6

Verification of DEGs by qRT-PCR. Expression of six genes in liver tissues was detected by qRT-PCR, and shown by the expression fold changes for Model and GDFMD vs. CN. Actb was used as the internal control.

FIGURE 7

Significantly enriched KEGG pathways in PPAR signaling pathway. Up-DEGs are marked in red. The pictures were drawn by KEGG Mapper (www.kegg.jp/kegg/tool/map_pathway2.html).

FIGURE 8

Significantly enriched KEGG pathways in PPAR signaling pathway.

FIGURE 9

Significantly enriched KEGG pathways in complement and coagulation cascades.

FIGURE 10

Significantly enriched KEGG pathways in ECM-receptors interaction.