Deciphering the Potential Pharmaceutical Mechanism of GUI-ZHI-FU-LING-WAN on Systemic Sclerosis based on Systems Biology Approaches

Abstract

Systemic sclerosis (SSc; scleroderma) is a complicated idiopathic connective tissue disease with seldom effective treatment. GUI-ZHI-FU-LING-WAN (GFW) is a classic Traditional Chinese Medicine (TCM) formula widely used for the treatment of SSc. However, the mechanism of how the GFW affects SSc remains unclear. In this study, the system biology approach was utilized to analyze herb compounds and related targets to get the general information of GFW. The KEGG enrichment analysis of 1645 related targets suggested that the formula is involved in the VEGF signaling pathway, the Toll-like receptor signaling pathway, etc. Quantitative and qualitative analysis of the relationship among the 3 subsets (formula targets, drug targets and disease genes) showed that the formula targets overlapped with 38.0% drug targets and 26.0% proteins encoded by disease genes. Through the analysis of SSc related microarray statistics from the GEO database, we also validated the consistent expression behavior among the 3 subsets before and after treatment. To further reveal the mechanism of prescription, we constructed a network among 3 subsets and decomposed it into 24 modules to decipher how GFW interfere in the progress of SSc. The modules indicated that the intervention may come into effect through following pathogenic processes: vasculopathy, immune dysregulation and tissue fibrosis. Vitro experiments confirmed that GFW could suppress the proliferation of fibroblasts and decrease the Th1 cytokine (TNF-α, MIP-2 and IL-6) expression for lipopolysaccharide (LPS) and bleomycin (BLM) stimulation in macrophages, which is consistent with previous conclusion that GFW is able to relieve SSc. The systems biology approach provides a new insight for deepening understanding about TCM.

Figure 1

The descriptions of herbs, compounds and targets. (a) The detailed number of compounds in each herbs of GFW formula. (b) The 4 herb association pairs shared with the same compounds, the width of the edge represents the number of the common compounds. (c) The network is constructed with compounds sharing the same targets between compounds. The size of the node represents the number of the shared common targets. (d) Distribution of targets in GFW formula, drug targets and disease genes.

Figure 2

Relationship between formula targets and disease genes. The ratio of formula targets and disease genes in biological processes. The network was generated with ClueGO in Cytoscape.

Figure 3

Relationship between formula targets and drug targets. The ratio of formula targets and drug targets in biological processes. The network was generated with ClueGO in Cytoscape.

Figure 4

The PPI network with GFW formula targets, disease genes, drug targets and other targets. The network was constructed by ClueGO in Cytoscape. (red: Formula targets; yellow: Disease genes; green: drug targets; orange: Formula targets and disease genes; purple: Formula targets and drug targets; gray: other genes).

Figure 5

Quantitative comparison of GFW targets, drug targets and SSc disease genes. (a) The numbers of DEGs before and after treatment. (b) The distribution of GFW targets, drug targets and SSc genes before treatment. (c) The distribution of GFW targets, drug targets and SSc genes after treatment.

Figure 6

Effect of GFW on the viability and cytotoxicity of MRC-5 cells. MRC-5 cells incubated with different concentrations (0, 50, 250, 625, and 1250 μg/ml) of GFW freeze-dried powder for 48 h. (a) The cell inhibition rate of MRC-5 cells was detected using CCK8 assay. (b) The cell viability of MRC-5 cells was measured by LDH release assays. ****P < 0.0001.

Figure 7

The KEGG enrichment analysis of PPIs networks downloaded from online website KEGG (https://www.kegg.jp/kegg/).

Figure 8

The effect of GFW serum on the mRNA expression levels in Raw264.7 cells. Raw264.7 were pretreated with 10% GFW serum for 24 h.Then the cells were incubated with low concentration (50 ng/ml) or high concentration (100 ng/ml) of bleomycin (BLM) in the presence of lipopolysaccharide (LPS, 100 ng/ml) for 12 h.The expression levels of TNF-α, MIP-2 and IL-6 mRNAs were measured using RT-qPCR. (a–c) The effects of 10% GFW serum on the expression levels of TNF-α, MIP-2 and IL-6 mRNAs at low concentration of BLM in presence of LPS. (d–f) The effects of 10% GFW serum on the expression levels of TNF-α, MIP-2 and IL-6 mRNAs at high concentration of BLM in presence of LPS. *P < 0.05, **P < 0.01, ***P < 0.001.