Integrating proteomics and network pharmacology to explore the relevant mechanism of Huangkui capsule in the treatment of chronic glomerulonephritis

Abstract

Introduction: Chronic glomerulonephritis (CGN) is a common glomerular disease with multifactorial pathogenesis. Huangkui capsule (HKC), a traditional Chinese herbal formulation, demonstrates therapeutic potential in CGN; however, its molecular mechanisms remain insufficiently characterized. This study aimed to clarify the therapeutic mechanisms of HKC in CGN by integrating proteomic analysis with network pharmacology.

Methods: We employed liquid chromatography-mass spectrometry (LC-MS) to identify the active components of HKC. A CGN rat model was established and treated with HKC. Renal function parameters and serum inflammatory cytokines were assessed. Histopathological alterations and IgG deposition in kidney tissues were examined using hematoxylin-eosin (HE) staining and immunofluorescence, respectively. Proteomic profiling of renal tissue was conducted, and network pharmacology analysis was applied to identify potential therapeutic targets of HKC.

Results: A total of 39 active compounds were identified in HKC. HKC administration significantly improved renal function and mitigated glomerular injury in CGN rats. Proteomic analysis revealed 2,079 differentially expressed proteins, predominantly associated with oxidoreductase activity. Network pharmacology identified 462 targets related to HKC and 1,835 targets associated with CGN, with 13 overlapping targets, including STAT3, PIK3R1, AKT1, HIF-1α, and VEGF, which were downregulated following HKC treatment.

Conclusion: HKC exerts renoprotective effects in CGN by regulating multiple signaling pathways, notably HIF-1, VEGF, PI3K-Akt, MAPK, and PPAR. Through attenuation of inflammatory and oxidative responses, HKC alleviates renal pathological damage and supports kidney function, offering mechanistic insight into its multi-target therapeutic potential.

Keywords: Huangkui capsule; chronic glomerulonephritis; mechanism; network pharmacology; proteomics.

FIGURE 1

Determination of 39 compounds in Huangkui capsule by LC-MS/MS.18. Rutin; 20. Hyperoside; 21. Isoquercetin.; 25. Hibifolin; 33. Myricetin; 36. Quercetin. (A) Structural formulae of Rutin, Hyperoside, Isoquercetin, Hibifolin, Myricetin and Quercetin (B).

FIGURE 2

Schematic diagram of CGN rat model establishment (A). 24hUP, PCR, BUN, and Scr levels in each group of rats (n = 8). Compared with the NOR group, the 24-h urine protein content (B), PCR (C), BUN (D) and Scr (E) values in the model group were significantly higher (P < 0.05); compared with the MOD group, the 24-h urine protein content, PCR, Scr and BUN values in the LST, HK-M and HK-H groups were significantly lower (P < 0.05). ^#p<0.05 vs NOR group; *p<0.05 vs MOD group.

FIGURE 3

Statistical results of relative intensity of HE staining, immunofluorescence staining and IgG immunofluorescence (n = 8). (A) HE staining showed that the glomeruli of rats in the NOR group were structurally intact and the size of the capsule was normal, while the glomeruli of rats in the MOD group were enlarged in size and the basement membrane was thickened to different degrees. Compared with the MOD group, the increase in glomerular volume and the thickening of basement membrane of rats in the NOR group were reduced after treatment with Huangkui capsule and chlorosartan potassium. (B, C) At the same time, immunofluorescence results showed that there was almost no IgG deposition in the renal tissues of rats in the NOR group, while in the MOD group, IgG was obviously seen to be diffusely deposited in the glomerular mesangium or basement membrane, and the phenomenon of IgG deposition in the glomeruli of the rats in all groups was significantly reduced after treatment with Huangkui Capsule and Losartan Potassium. ^#p < 0.05 vs. NOR group; *p < 0.05 vs. MOD group.

FIGURE 4

Results of SOD\MDA\IL-6 and TNF-α content in rat serum (n = 8). Compared with the NOR group, the serum TNF-α (D), IL-6 (C) and MDA (B) levels in the MOD group were significantly higher (P < 0.05), and the SOD (A) activity was significantly lower (P < 0.05); and compared with the MOD group, the serum TNF-α (D), IL-6 (C) and MDA (B) levels in the LST, HK-M and HK-H groups were significantly lower (P < 0.05), and the SOD (C) activity was significantly higher (P < 0.05). levels were significantly decreased (P < 0.05) and SOD (C) activity was significantly increased (P < 0.05). ^#p < 0.05 vs. NOR group; *p < 0.05 vs. MOD group.

FIGURE 5

Network pharmacology target analysis. (A) Chronic glomerulonephritis target prediction and drug-disease target collection. (B) PPI network diagram of the overlapping targets.

FIGURE 6

Network pharmacology target analysis. PPI network diagram of the drug composition-target (A). PPI network diagram of disease-target (B). Venn of the CGN and HKC (C). PPI network diagram of the overlapping targets (D).

FIGURE 7

Statistical analysis of differential proteins in NOR, MOD and HK groups. (A) Differential protein analysis Veen plots of NOR vs. MOD vs. HK intersecting targets. (B) Statistics of differential proteins in the kidney tissues of each group of rats. Volcano plots of NOR vs. MOD (C, D), NOR vs. HK (E, F) and MOD vs. HK (G, H) with heat maps of differential protein hierarchical clustering analysis.

FIGURE 8

Screening of differential proteins and GO and KEGG enrichment analysis. GO functional enrichment analysis of differential proteins among NOR, MOD and HK groups (A), and the top 20 differential proteins were selected for KEGG enrichment analysis (B) Differential protein PPI network construction and core target screening (C).

FIGURE 9

Analysis of the targets and related signaling pathways of CGN treatment by Huangkui capsule (n = 8). (A) Veen diagram of core targets of Huangkui capsule based on proteomics and network pharmacology. (B, C) Comparison of AKT1, STAT3 and PIK3R1 protein expression levels in the kidney tissues of rats in each group (immunohistochemistry × 400). (D, E) Comparison of HIF-1α and VEGF protein expression levels in rat kidney tissues of each group (immunohistochemistry × 400). ^#p < 0.05 vs. NOR group; *p < 0.05 vs. MOD group.

FIGURE 10

The main possible targets of Huangkui capsule.