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.

Identification of lncRNA-miRNA-mRNA Networks in the Lenticular Nucleus Region of the Brain Contributes to Hepatolenticular Degeneration Pathogenesis and Therapy

“…Then, we performed gene expression profiling of Gan-Dou-Fu-Mu decoction (GDFMD)-treated TX-j 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 [ 53 ]. Moreover, we identified the circRNA/miRNA/mRNA network involved in kidney failure in HLD, which may serve as a potential biomarker for the pathogenesis of HLD [ 54 ].…”

Discussion on the Mechanism of Gandoufumu Decoction Attenuates Liver Damage of Wilson’s Disease by Inhibiting Autophagy through the PI3K/Akt/mTOR Pathway Based on Network Pharmacology and Experimental Verification

“…All herbs were supplied by the pharmacy of the First Hospital Affiliated to Anhui University of Chinese Medicine (Hefei, China). The medicinal herb mixture was immersed in water (10 times the weight of medicine) for 1 h, boiled twice for 1 h, and then concentrated to 0.2/mL to obtain GDFMD suspension [ 25 ].…”

Construction of a Novel circRNA/miRNA/mRNA Regulatory Network to Explore the Potential Pathogenesis of Wilson’s Disease

“…Male Toxic Milk (TX-J) mice were obtained from the Jackson Laboratory (United States). The genotype was confirmed via Sanger sequencing ( Wei et al, 2021 ). The TX-J model was prepared as described previously ( Wei et al, 2021 ) and the procedure conformed with the National Institutes of Health Animal Care and Welfare Guidelines.…”

“…The genotype was confirmed via Sanger sequencing ( Wei et al, 2021 ). The TX-J model was prepared as described previously ( Wei et al, 2021 ) and the procedure conformed with the National Institutes of Health Animal Care and Welfare Guidelines. All mice were sacrificed via intraperitoneal injection of sodium pentobarbital.…”