Integrating Network Pharmacology and Experimental Validation to Elucidate the Mechanisms of Tang Shen Ping Decoction in Diabetic Kidney Disease - PubMed (original) (raw)
Integrating Network Pharmacology and Experimental Validation to Elucidate the Mechanisms of Tang Shen Ping Decoction in Diabetic Kidney Disease
Yi Zhou et al. ACS Omega. 2025.
Abstract
Diabetic kidney disease (DKD) is a severe complication of diabetes, characterized by chronic inflammation and fibrosis. Tang Shen Ping Decoction (TSPD), a traditional Chinese medicine formulation, has shown therapeutic efficacy in DKD, yet its molecular mechanisms remain to be fully elucidated. To explore the multitarget mechanisms of TSPD, this study integrated network pharmacology, transcriptomic analysis, molecular docking, and molecular dynamics simulations, followed by in vivo and in vitro validation. A total of 248 active compounds and 649 potential targets of TSPD were identified, among which network pharmacology and transcriptomic integration highlighted 21 key genes involved in DKD pathogenesis. Protein-protein interaction network analysis further identified ALB, CCL2, EGF, FN1, and PTGS2 as central targets. Molecular docking confirmed strong binding affinities between core TSPD compounds, including quercetin and kaempferol, and these targets, particularly CCL2. Molecular dynamics simulations validated the stability of these interactions, identifying CCL2 as a crucial therapeutic target. In vivo experiments demonstrated that TSPD significantly improved renal function, attenuated fibrosis, and down-regulated CCL2, NF-κB, and TGF-β1 expression in DKD rats. In vitro, TSPD effectively suppressed CCL2/NF-κB activation and reduced the secretion of inflammatory cytokines (TNF-α, IL-6, and IL-1β) in high-glucose-treated HK-2 cells. Functional analysis confirmed that CCL2 overexpression exacerbated inflammation, while its silencing enhanced the anti-inflammatory effects of TSPD. These findings reveal that TSPD exerts renoprotective effects by targeting the CCL2/NF-κB axis, offering mechanistic insights into its anti-inflammatory and antifibrotic actions and providing a theoretical foundation for its clinical application in DKD treatment.
© 2025 The Authors. Published by American Chemical Society.
Figures
1
TCMs, ingredients, and targets of TSPD. (A) Sankey diagram illustrating the relationship between the herbs and compounds of TSPD. (B) Network pharmacological analysis depicting the herb-compound-target network. The yellow nodes represent the 14 TCMs in TSPD, the orange nodes indicate the core compounds associated with TSPD, and the green nodes indicate the targets linked to TSPD.
2
Differential gene expression, hub gene screening, and PPI analysis. (A,B) Box plots of gene expression normalization in control and DKD groups. (C) Volcano plot of DEGs (|log2FC| > 1, p < 0.05). (D) Heatmap of the top 40 DEGs, with colors indicating expression levels. (E) Venn diagram of TSPD targets (orange), DKD_GeneCards DEGs (green), and DKD DEGs (blue). (F) PPI network of 21 hub genes, with nodes representing genes and edges indicating interactions.
3
GO and KEGG analyses of hub genes. (A,B) Chord diagrams of GO (A) and KEGG (B) analyses, showing gene counts, enrichment, and pathway categories. (C,E) Bubble plots of GO analysis for the top 15 downregulated (C) and upregulated (E) hub genes. (D,F) Sankey diagrams of KEGG pathways for the top 15 downregulated (D) and upregulated (F) hub genes.
4
GSEA and GSVA analyses of all genes. (A–F) GSEA of REACTOME (A), KEGG (B), GOBP (C), GOCC (D), GOMF (E), and Hallmark (F) data sets, showing the top 10 enriched pathways (p < 0.05). (G) Heatmap of GSVA results for significantly enriched genes (p < 0.05).
5
Molecular docking of core compounds and targets. (A) Heatmap of docking results for five core compounds and five core targets, represented by the AutoDock Vina scores. (B) Molecular docking visualization of ALB with tanshinone IIA. (C) Visualization of the docking of CCL2 with quercetin. (D) Docking visualization of EGF with luteolin. (E) Docking visualization of FN1 with baicalin. (F) Docking visualization of PTGS2 with kaempferol.
6
Molecular dynamics simulation analysis of key compounds and targets. (A) RMSD plots of backbone atoms over simulation time for each key compound–target complex. (B) RMSF plots showing residue-level flexibility of the target proteins. (C) Radius of gyration (R g) plots depicting the overall compactness of the target–compound complexes along different axes. (D) Hydrogen-bond analysis displaying the number of hydrogen bonds formed between key compounds and targets during the simulation. The pink areas indicate the proportion of hydrogen bonds with a donor–acceptor distance ≤3.5 Å.
7
Urinary levels of Alb, Cr, and UACR in different groups of rats. (A) UAlb levels at week 4. (B) UAlb levels at week 12. (C) UCr levels at week 4. (D) UCr levels at week 12. (E) UACR values at week 4. (F) UACR values at week 12. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001 represent significant differences between the HFD/STZ and Normal groups. #p < 0.05, ##p < 0.01, and ###p < 0.001 indicate significant differences between all treatment groups and the HFD/STZ group. †p < 0.05 signifies significant differences between the medium-dose and high-dose groups compared to the low-dose group.
8
Renal hypertrophy indices, blood glucose changes, and blood biochemical indices in different groups of rats. (A) Histogram of the renal hypertrophy index among the different groups. (B) Histogram of UP among the different groups. (C) Line graph of blood glucose changes up to week 12 in different groups. (D) Histogram of insulin levels among the different groups. (E) Histogram of HbA1c among the groups. (F) Histogram of Scr among the different groups. (G) Histogram of BUN among the different groups. Statistical significance is indicated as follows: *p < 0.05 and **p < 0.01 represent significant differences between the HFD/STZ and Normal groups. #p < 0.05 and ##p < 0.01 indicate significant differences between all treatment groups and the HFD/STZ group. †p < 0.05 signifies significant differences between the medium-dose and high-dose groups compared to the low-dose group.
9
Histopathological staining, immunohistochemistry, and Western blot analysis in different rat groups. (A) HE and PAS staining of the different groups (400× magnification). (B) IHC of TGF-β1 and CCL2 in different groups (400× magnification). (C) Quantification of the glomerular area (HE). (D) Quantification of the PAS-positive area fraction (PAS). (E) Quantification of the TGF-β1-positive area fraction (IHC). (F) Quantification of the CCL2-positive area fraction (IHC). (G) Western blot analysis of NF-κB, TGF-β1, and CCL2. (H) ELISA for TNF-α in the rat serum. (I) ELISA for IL-6 in the rat serum. (J) ELISA for IL-1β in the rat serum. Statistical significance is indicated as follows: *p < 0.05 and **p < 0.01 represent significant differences between the HFD/STZ and Normal groups. #p < 0.05 and ##p < 0.01 indicate significant differences between all treatment groups and the HFD/STZ group. †p < 0.05 signifies significant differences between the medium-dose and high-dose groups compared to the low-dose group.
10
TSPD modulates the CCL2/NF-κB signaling pathway and reduces inflammatory cytokine expression in different treatment groups. (A) Relative expression of CCL2 mRNA in the control, high glucose (HG), HG + TSPD, HG + oe-CCL2, and HG + oe-CCL2 + TSPD groups, as determined by quantitative PCR. (B) Relative expression of CCL2 mRNA in the control, HG, HG + TSPD, HG + si-CCL2, and HG + si-CCL2 + TSPD groups, as assessed by qPCR. The data are presented as the means ± SDs, with *p < 0.05 indicating statistically significant differences between groups. (C) Western blot analysis of p-NF-κB p65, NF-κB p65, and CCL2 protein levels across different treatment groups, with GAPDH used as a loading control. The adjacent bar graph presents the quantitative analysis of protein expression. The data are expressed as the means ± SDs. (D–F) ELISA results showing the concentrations of TNF-α (D), IL-6 (E), and IL-1β (F) in the supernatants of different treatment groups. The data are expressed as the means ± SDs. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001 denote significant differences between the HG group and the control group. ^p < 0.05 and ^^p < 0.01 represent differences before and after TSPD treatment. #p < 0.05 and ##p < 0.01 indicate differences between HG + oe-CCL2 or HG + si-CCL2 groups and the HG group before and after treatment. †p < 0.05 denotes significant differences in the HG + oe-CCL2 and HG + si-CCL2 groups before versus after treatment.
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