Yishen Qingli Heluo Granule in the Treatment of Chronic Kidney Disease: Network Pharmacology Analysis and Experimental Validation

Abstract

Background: Chronic kidney disease (CKD) is considered a global public health problem with high morbidity and mortality. Yishen Qingli Heluo granule (YQHG) is representative traditional Chinese medicine (TCM) remedy for clinical treatment of CKD. This study aims to explore the mechanism of YQHG on CKD through network pharmacology and experimental validation.

Methods: Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and wide-scale literature mining were applied to screen active compounds of YQHG. Multiple bioinformatic tools and online databases were applied by us to obtain relevant targets of YQHG and CKD. The intersection targets between YQHG and CKD were considered as candidate targets. The compound-target, herb-candidate target and protein-protein interaction networks were constructed and visualized for topological analyses. GO and KEGG enrichment analyses were conducted to determine the biological processes and signaling pathways. Molecular docking was used to verify the reliability of network pharmacology. Finally, pharmacological evaluation was performed to explore the mechanism of YQHG against CKD on a 5/6 nephrectomy model.

Results: Seventy-nine candidate targets, ten core biological processes and one key signaling pathway (p53) were screened. PTGS2 was identified as a key target based on H-CT network. The molecular docking showed that Quercetin, Kaempferol, Luteolin were three key compounds with the best binding activity. In addition, IL6 and Quercetin could form a stable complex with high binding affinity (-7.29 kcal/mol). In vivo experiment revealed that YQHG improved kidney function and fibrosis in 5/6 nephrectomized rats. Moreover, the decreased expression of PTGS2, IL6, and the increased expression of p53 were observed in kidney tissue. Notably, the gut microbiota of rats treated with YQHG was reshaped, which was characterized by a reduced ratio of Firmicutes/Bacteroidota.

Conclusion: Our results predicted and verified the potential targets of YQHG on CKD from a holistic perspective, and provided valuable direction for the further research of YQHG.

Keywords: 5/6 nephrectomy; Yishen Qingli Heluo granule; chronic kidney disease; gut microbiota; network pharmacology.

Figure 1

The work flow of the study. (A) Compounds screening and targets fishing. (B) Multi-level data analysis. (C) Experimental validation.

Figure 2

Venn diagrams. (A) The targets of CKD. CKD-related targets obtained from five databases (OMIM, DisGeNET, GeneCards, MalaCards, TTD). (B) Candidate targets of YQHG in the treatment of CKD.

Figure 3

Network visualization. (A) The 10 herbs of YQHG. (B) C-T network of YQHG. The C-T network is constructed by the active compounds (circle) and their corresponding potential targets (rhombus). (C) H-CT network of YQHG. The 79 candidate targets (circles) of YQHG against CKD are connected with their corresponding herbs (octagons). The color of the target represents their corresponding herbs. (D) PPI network of candidate targets.

Figure 4

GO and KEGG enrichment analysis for candidate targets of YQHG against CKD. (A) GO enrichment analysis. GO terms are classified according to biological process (BP), molecular function (MF) and cellular component (CC). The top 10 terms of each one are presented. (B) KEGG enrichment analysis. The top 40 KEGG signaling pathways are presented. The X-axis represents the rich factor, bubble size represents the count of targets enriched in terms and the color represents the p value.

Figure 5

The binding mode of protein with compound. (A) The binding mode of ESR1 protein with Luteolin. (B) The binding mode of MAPK1 protein with Quercetin. (C) The binding mode of TP53 protein with Luteolin. (D) The binding mode of AKT1 protein with Kaempferol. (E) The binding mode of RELA protein with Quercetin. (F) The binding mode of IL6 protein with Quercetin. Each picture shows three areas: (I) The 3D structure of complex. (II) The surface of active site. (III) The detail binding mode of complex. The backbone of protein is rendered in tube and colored in green. Compound is rendering by yellow. Yellow dash represents hydrogen bond distance or π-stacking.

Figure 6

YQHG improves kidney function and fibrosis in 5/6 nephrectomized rats. (A) Construction of 5/6 nephrectomy model and YQHG dosing schedule. (B) The kidney of the rats was photographed (n=6). (C) The body weight of the rats was measured (n=6). (D–F) Effects of YQHG treatment on levels of Scr, BUN, and urinary protein in 5/6 nephrectomized rats (n=6). (G) Representative images of H&E (40×Magnification, Scale bar 20μm) staining and Masson (40×Magnification, scale bar 20μm) staining of kidney tissues (n=6). (H–I) Quantitative analysis of glomerular fibrosis area and tubulointerstital fibrosis area based on Masson staining (n=6). All data are expressed as mean±SEM. For normally distributed data (body weight, Scr, urinary protein, glomerular fibrosis area, tubulointerstital fibrosis area), one-way ANOVA followed by Tukey’s test was used. For non-normally distributed data (BUN), Kruskal–Wallis test followed by non-parametric Wilcoxon rank-sum test was used. **P < 0.01 vs the sham group; ^#P < 0.05, ^##P < 0.01 vs the model group.

Figure 7

YQHG reshaped gut microbiota by reducing Firmicutes/Bacteroidota ratio in 5/6 nephrectomized rats. (A) PCoA on phylum level (n=6). (B) Relative abundance of gut microbiota on phylum level (n=6). (C and D) Relative abundance of Firmicutes and Bacteroidota on phylum level (n=6). (E) The ratio of Firmicutes/Bacteroidota on phylum level (n=6). (F) PCoA on genus level (n=6). (G) Relative abundance of gut microbiota on genus level (n=6). (H) Circos diagram of species-sample relationship on genus level (n=6). The small semicircle (left half circle) indicates the composition of the gut microbiota in each group, the color of the outer ribbon represents which group it comes from, the color of the inner ribbon represents the gut microbiota, and the length represents the relative abundance of gut microbiota in the corresponding group; the large semicircle (right half circle) indicates the distribution ratio of gut microbiota in different groups on genus level. The outer color band represents the gut microbiota, the inner color band represents different groups, and the length represents the distribution ratio of the group in a certain gut microbiota. All data are expressed as mean±SEM. For normally distributed data (Firmicutes, Bacteroidota), one-way ANOVA followed by Tukey’s test was used. For non-normally distributed data (Firmicutes/Bacteroidota), Kruskal–Wallis test followed by non-parametric Wilcoxon rank-sum test was used. *P < 0.05, **P < 0.01 vs the sham group; ^#P < 0.05, ^##P < 0.01 vs the model group.

Figure 8

YQHG regulates the expression of PTGS2, p53 and IL-6 in 5/6 nephrectomized rats. (A) Representative Western blots for PTGS2, p53 and GAPDH protein expression in kidney tissue (n=6). (B) Relative expression of PTGS2 (n=6). (C) Relative expression of p53 (n=6). (D) Expression of IL6 in kidney tissue was determined by ELISA. All data are expressed as mean±SEM. For non-normally distributed data (PTGS2, p53, IL6), Kruskal–Wallis test followed by non-parametric Wilcoxon rank-sum test was used. **P < 0.01 vs the sham group; ^#P < 0.05, ^##P < 0.01 vs the model group.