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. 2023 Dec;61(1):1222-1233.
doi: 10.1080/13880209.2023.2241521.

Integrating network pharmacology and experimental validation to decipher the mechanism of the Chinese herbal prescription modified Shen-Yan-Fang-Shuai formula in treating diabetic nephropathy

Borui Yu  1   2 , Mengqi Zhou  1   2 , Zhaocheng Dong  1   2 , Huijuan Zheng  1   2 , Yuxue Zhao  1   2   3 , Jingwei Zhou  1 , Chao Zhang  1   2 , Fudong Wei  1   2 , Guoyong Yu  1 , Wei Jing Liu  1   2 , Hongfang Liu  1 , Yaoxian Wang  4
Affiliations

Integrating network pharmacology and experimental validation to decipher the mechanism of the Chinese herbal prescription modified Shen-Yan-Fang-Shuai formula in treating diabetic nephropathy

Borui Yu et al. Pharm Biol. 2023 Dec.

Abstract

Context: Diabetic nephropathy (DN) is the main cause of end-stage renal disease. Modified Shen-Yan-Fang-Shuai formula (M-SYFSF) has excellent clinical efficacy in treating diabetic kidney disease. However, the potential mechanism of M-SYFSF remains unknown.

Objective: To investigate the mechanism of M-SYFSF against DN by network pharmacological analysis and biological experiments.

Materials and methods: Utilizing a web-based pharmacology database, the potential mechanisms of M-SYFSF against DN were identified. In vivo experiments, male SD rats were injected with streptozotocin (50 mg/kg) and got uninephrectomy to construct a model of DN. M-SYFSF (11.34 g/kg/d) was gavaged once per day for 12 weeks after model establishment. In vitro experiments, human proximal tubular cells (HK-2) were performed with advanced glycation end-products (AGEs) (100 μg/mL), then intervened with M-SYFSF freeze-dried powder. Pathological staining, WB, IHC, ELISA were conducted to explore the mechanism of M-SYFSF against DN.

Results: Network pharmacological analysis showed that MAPK pathway was the potential pathway. Results showed that compared with the Model group, M-SYFSF significantly reduced 24h urine albumin, UACR, and serum creatinine levels (54.90 ± 26.67 vs. 111.78 ± 4.28, 8.87 ± 1.69 vs. 53.94 ± 16.01, 11.56 ± 1.70 vs. 118.70 ± 49.57, respectively), and improved renal pathological changes. Furthermore, the intervention of M-SYFSF reduced the expression of pro-inflammatory cytokines and inhibited the activation of MAPK pathway in AGEs-treated HK-2 cells.

Discussion and conclusion: M-SYFSF is likely to reduce inflammation in DN by inhibiting the MAPK pathway. It provides a theoretical basis for the clinical application of M-SYFSF in the treatment of DN.

Keywords: MAPK signaling pathway; Traditional Chinese medicine formula; inflammation; protein-protein interaction network.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Compounds found in M-SYFSF and their corresponding targets. (A) Compound-target network of M-SYFSF. (B) Veen diagram of compound targets of M-SYFSF and DN-related targets. (C) Compound-target network of M-SYFSF treating DN.
Figure 2.
Figure 2.
Overlapped gene-based enrichment analysis: (A-C) gene ontology (GO) enrichment analysis of overlapped genes: (A) cellular compound (CC); (B) biological process (BP) and (C) molecular function (MF); (D) KEGG enrichment analysis of overlapped genes; (E)The MAPK signaling pathway.
Figure 3.
Figure 3.
Screening of bioactive compounds in M-SYFSF by UPLC-ESI-MS/MS analysis.
Figure 4.
Figure 4.
Molecular docking of major compounds to core targets.
Figure 5.
Figure 5.
Effect of M-SYFSF on renal function and renal injury. (A) Blood glucose (mmol/L); (B) Serum creatinine (μmol/L); (C) 24h urinary albumin(mg); (D) Urine albumin-creatinine ratio (UACR) (ug/umol); *p < 0.05, **p < 0.01 compared to the so group; #p < 0.05 compared to the model group; (E) Hematoxylin and eosin (HE) staining; (F) Periodic acid-Schiff (PAS) staining; Magnification: 400×.
Figure 6.
Figure 6.
The effect of M-SYFSF on tubulointerstitial fibrosis. (A) Masson staining; (B) Col-I expression as determined by immunohistochemistry staining. Original magnification: 400×. (C) Western blot analysis of α-SMA expression (n = 3). *p < 0.05 compared to the so group; ##p < 0.01 compared to the model group.
Figure 7.
Figure 7.
The effect of M-SYFSF on cell viability in AGEs-treated HK 2 cells. (A) Screening for optimal concentration and duration of action of M-SYFSF. (B) Optimal therapeutic concentration of M-SYFSF on HK-2 cells treated with AGEs. **p < 0.01 compared to the control group; ##p < 0.01 compared to the model group.
Figure 8.
Figure 8.
The effect of M-SYFSF on the MAPK signaling pathway in HK-2 cells. (A) Western blot analysis of JNK and p-JNK expression (n = 3); (B) Western blot analysis of P38MAPK and p-P38MAPK expression (n = 3); (C) Western blot analysis of ERK and p-ERK expression (n = 3). *p < 0.05, **p < 0.01 compared to the control group; #p < 0.05, ##p < 0.01 compared to the model group.
Figure 9.
Figure 9.
The effect of M-SYFSF on inflammation in HK-2 cells. Western blot analysis of IL1β, IL 6, and TNF-α, n = 3. *p < 0.05, **p < 0.01 compared to the control group; #p < 0.05, ##p < 0.01 compared to the model group.
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Grants and funding

This study was supported by grants from the National Natural Science Foundation of China (82174342; 82004272; 81874443), Chinese Medicine Inheritance and Innovation Talent Project-Leading Talent Support Program of National Traditional Chinese Medicine (Grant No. 2018, No. 12) .