Mechanism of Chaihuang-Yishen formula to attenuate renal fibrosis in the treatment of chronic kidney disease: Insights from network pharmacology and experimental validation

Abstract

Renal fibrosis represents a pivotal characteristic of chronic kidney disease (CKD), for which effective interventions are currently lacking. The Src kinase activates the phosphatidylinositol-3 kinases (PI3K)/Akt1 pathway to promote renal fibrosis, casting a promising target for anti-fibrosis treatment. Chaihuang-Yishen formula (CHYS), a traditional Chinese medicinal prescription, has a validated efficacy in the treatment of CKD, however, with the underlying mechanism unresolved. This study aimed to uncover the pharmacological mechanisms mediating the effect of CHYS in treating renal fibrosis using network pharmacology followed by experimental validation. The chemical compounds of CHYS were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database or published literature, followed by the prediction of their targets using SwissTargetPrediction software. Disease (CKD/renal fibrosis)-related targets were retrieved from the Genecards database. Protein-protein interaction (PPI) network was generated using the drug-disease common targets and visualized in Cytoscape software. The drug-disease targets were further subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses by Metascape software. Additionally, the compound-target-pathway network was established in Cytosape to identify key compounds, targets, and pathways. Network pharmacology analysis screened out 96 active compounds and 837 potential targets within the 7 herbal/animal medicines of CHYS, among which 237 drug-disease common targets were identified. GO and KEGG analysis revealed the enrichment of fibrosis-related biological processes and pathways among the 237 common targets. Compound-target-pathway network analysis highlighted protein kinases Src and Akt1 as the top two targets associated with the anti-renal fibrosis effects of CHYS. In UUO mice, treatment with CHYS attenuates renal fibrosis, accompanied by suppressed expression and phosphorylation activation of Src. Unlike Src, CHYS reduced Akt1 phosphorylation without affecting its expression. In summary, network pharmacology and in vivo evidence suggest that CHYS exerts its anti-renal fibrosis effects, at least in part, by inhibiting the Src/Akt1 signaling axis.

Keywords: AKT1; Chaihuang-Yishen formula; Chronic kidney disease; Network pharmacology; Renal fibrosis; SRC.

Fig. 1

Study design and CHYS-compound-target network analysis. (A) The flowchart illustrates the comprehensive methodology encompassing network pharmacology and animal experiment undertaken in this study. (B) The CHYS-compound-target network. Active compounds are depicted as green rectangles, wherein the intensity of color corresponds to the number of gene targets linked to each active compound. The associated herbal/animal medicines are positioned centrally within the compounds. Gene targets are represented by red rectangles. Notably, duplicate and triplicate compounds among HQ, CH, DG, and SW are delineated separately (orange rectangles) to ensure clarity and avoid redundancy within the network visualization.

Fig. 2

PPI network analysis with drug-disease targets. (A) Venn diagram to show the intersection of targets among CHYS, CKD, and renal fibrosis. (B) PPI network comprising 237 drug-disease targets. Targets are represented by rectangles, with darker colors indicating higher degrees of centrality within the network. (C) Second round of PPI network analysis using the top 20-ranked targets identified in panel B.

Fig. 3

Enrichment analysis and compound-target-pathway network. (A) Top 20 enriched biological processes (BP) identified through GO analysis using the 237 drug-disease targets. (B) Top 20 enriched pathways identified at the summary level in KEGG analysis with the 237 drug-disease targets. (C) Expansion of 20 enriched pathways derived from five higher-level renal fibrosis-related KEGG pathways. (D) Compound-target-pathway network. Circles stand for compounds, arrows for pathways, and rectangles for targets. The intensity of red color corresponds to the degree of centrality of the respective target.

Fig. 4

CHYS treatment attenuates renal fibrosis in UUO mice. (A) Representative images of HE, PAS, and Sirius red staining of kidney sections, respectively. (B) Quantitation of tubular injury score based on PAS staining. (C) Quantitation of positive area of Sirius red staining. n = 6 animals for each group in B and C. ***p < 0.001 versus Sham. ^###p < 0.001 versus UUO. IRB denotes irbesartan.

Fig. 5

CHYS treatment suppresses the expression of fibrotic marker genes in UUO mice. (A–C) RT-PCR to quantitate the mRNA levels of fibronectin, α-SMA, and Col1a1. n = 6 animals for each group. (D) Western blot to analyze the protein expression of fibronectin, α-SMA, and collagen I. E to G are quantitation of corresponding protein bands in D. n = 4 animals for each group. (H) Representative images of IHC staining of fibronectin, α-SMA, and collagen I in kidney sections. I to K are quantitation of positive staining area for each fibrosis marker proteins in H, respectively. n = 6 animals for each group. ***p < 0.001 versus Sham. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus UUO. IRB stands for irbesartan.

Fig. 6

CHYS treatment suppresses Src and Akt1 in UUO kidneys. (A and B) RT-PCR to detect the mRNA expression of Src and Akt1 in kidney tissues. n = 6 animals in each group. (C) Western blot to detect the protein expression of p-Src, Src, p-Akt1, and Akt1 in kidney tissues. (D–G) Quantitation of corresponding proteins in C. n = 6 animals in each group. (H) Representative images of immunohistochemistry of Src, p-Src, Akt, and p-Akt in kidney tissues. (I–L) Quantitation of positive staining area in H. n = 6 animals in each group. *p < 0.05 and ***p < 0.001 versus Sham. ^#p < 0.05 and ^###p < 0.001 versus UUO.