Network Pharmacology and Experimental Verification Strategies to Illustrate the Mechanism of Jian-Pi-Yi-Shen Formula in Suppressing Epithelial-Mesenchymal Transition

Abstract

Jian-Pi-Yi-Shen formula (JPYSF), a traditional Chinese medicine, has been recommended to treat renal fibrosis for decades. Previous studies had shown that JPYSF could inhibit epithelial-mesenchymal transition (EMT), an important regulatory role in renal fibrosis. However, the mechanism of JPYSF action is largely unknown. In this study, network pharmacology and experimental verification were combined to elucidate and identify the potential mechanism of JPYSF against renal fibrosis by suppressing EMT at molecular and pathway levels. Network pharmacology was first performed to explore the mechanism of JPYSF against renal fibrosis targeting EMT, and then a 5/6 nephrectomy (5/6 Nx)-induced rat model of renal fibrosis was selected to verify the predictive results by Masson's trichrome stains and western blot analysis. Two hundred and thirty-two compounds in JPYSF were selected for the network approach analysis, which identified 137 candidate targets of JPYSF and 4,796 known therapeutic targets of EMT. The results of the Gene Ontology (GO) function enrichment analysis included 2098, 88, and 133 GO terms for biological processes (BPs), molecular functions (MFs), and cell component entries, respectively. The top 10 enrichment items of BP annotations included a response to a steroid hormone, a metal ion, oxygen levels, and so on. Cellular composition (CC) is mainly enriched in membrane raft, membrane microdomain, membrane region, etc. The MF of JPYSF analysis on EMT was predominately involved in proximal promoter sequence-specific DNA binding, protein heterodimerization activity, RNA polymerase II proximal promoter sequence-specific DNA binding, and so on. The involvement signaling pathway of JPYSF in the treatment of renal fibrosis targeting EMT was associated with anti-fibrosis, anti-inflammation, podocyte protection, and metabolism regulation. Furthermore, the in vivo experiments confirmed that JPYSF effectively ameliorated interstitial fibrosis and inhibited the overexpression of α-SMA, Wnt3a, and β-catenin, and increased the expression of E-cadherin by wnt3a/β-catenin signaling pathway in 5/6 Nx-induced renal fibrosis rats. Using an integrative network pharmacology-based approach and experimental verification, the study showed that JPYSF had therapeutic effects on EMT by regulating multi-pathway, among which one proven pathway was the Wnt3a/β-catenin signaling pathway. These findings provide insights into the renoprotective effects of JPYSF against EMT, which could suggest directions for further research of JPYSF in attenuating renal fibrosis by suppressing EMT.

Keywords: Jian-Pi-Yi-Shen formula; Wnt3a/β-catenin signaling pathway; epithelial–mesenchymal transition; network pharmacology; renal fibrosis.

FIGURE 1

The interaction of shared targets between Jian-Pi-Yi-Shen formula (JPYSF) active ingredients and epithelial–mesenchymal transition (EMT). (A). The Venn diagram; (B) 87 connected nodes in the network; and (C) The top 30 targets in action frequency.

FIGURE 2

The top 10 pathways for the GO enrichment analysis based on the common targets of the Jian-Pi-Yi-Shen formula (JPYSF) and epithelial–mesenchymal transition (EMT).

FIGURE 3

The top 10 pathways for the KEGG pathway are enriched from the common targets between JPYSF and epithelial–mesenchymal transition (EMT).

FIGURE 4

The “chemicals-shared target genes-signal pathway "(C-T-S) network. The blue fonts represent the components of JPYSF, the red fonts represent the targets of JPYSF components and epithelial–mesenchymal transition (EMT), and the rose fonts represent the signaling pathway.

FIGURE 5

The core targets and compounds in the “chemicals-shared target genes-signal pathway” (C-T-S) network. The nodes with a larger area have a larger degree value.

FIGURE 6

JPYSF administration significantly reduces renal fibrosis by alleviating EMT in 5/6 nephrectomy (Nx) rats. (A) Representative images of renal tissue are shown by Masson staining from the sham group, model group, the JPYSF 2.73 g/kg group, and the JPYSF 10.89 g/kg group (200×). (B) Quantitative analysis of the fibrotic area. Data were presented as the mean ± SEM, n = 10 rats per group (^###, p < 0.001, compared with the sham group; ***,p < 0.001, compared with the model group). (C) Relative protein expression of α-SMA, E-cadherin, Wnt3a, and β-catenin in the kidney cortex tissues of the sham, model, 2.73, and 10.89 g/kg Jian-Pi-Yi-Shen formula (JPYSF) groups by western blot analysis. Representative bands show the relative expression of α-SMA, E-cadherin, Wnt3a, and β-catenin. (D) Quantitative analysis of protein expressions of α-SMA, E-cadherin, Wnt3a, and β-catenin by densitometry based on western blot images. ^###, p < 0.001, compared with the Sham group; ***, p < 0.001, compared with the model group.

FIGURE 7

The workflow of the system’s pharmacology analysis and experimental verification of Jian-Pi-Yi-Shen formula (JPYSF) in suppressing epithelial–mesenchymal transition (EMT).