Network pharmacology and experimental validation to investigate the mechanism of action of Zhilong Huoxue Tongyu capsule in the prevention and treatment of diabetic cardiomyopathy

Figures

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  Fig 1. The active ingredients-targets diagram of ZL capsule The red circle represents the drug name, the magenta hexagon denotes the target of huangqi (HQ), the purple quadrilateral represents the target of guizhi (GZ), the green triangle denotes the target of dilong (DL), the orange hexagon indicates the target of shuizhi (SZ), the light green square represents the target of daxueteng (DXT), and the gray inverted triangle represents the common target of all drugs. The central blue rhombus represents the gene target.

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  Fig 2. Identification of the core targets for ZL against DCM. (A) Compilation (A) Compilation of DCM-related predicted targets. (B) Venn diagram depicting the intersection of targets associated with ZL and those related to DCM.

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  Fig 3. The drug-target network interactions for ZL against DCM. The The drug-target network comprises 166 nodes and 298 edges. The blue diamond-shaped nodes represent potential targets of DCM. Within the blue square nodes, HQ stands for huangqi, GZ for Guizhi, SZ for shuizhi, DL for dilong, DXT for daxueteng, and CF represents the common component shared by guizhi and daxueteng. The external magenta-colored nodes reflect the 45 bioactive substances of ZL. The larger the node size, the greater the degree of the bioactive compound within the network.

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  Fig 4. Hub gene target screening process. (A) (A) PPI network of predicted targets for the treatment of DCM using ZL. (B) Key proteins from the PPI network were extracted from (A). (C) Three key proteins for the treatment ofDCM were extracted from (B).

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  Fig 5. GO and KEGG pathway enrichment analyses. (A) (A) Top 10 results from GO enrichment analysis in Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). (B) Sankey diagram of KEGG pathways for analysis of ZL targets in DCM therapy. The left rectangle represents the target, the right rectangle represents the target, and the line represents the pathway in which the target acts. (C) The drug-target-pathway network. Orange circles represent the bioactive components of ZL, blue diamonds represent cross-targets, and green triangles represent the top 20 KEGG pathways..

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  Fig 6. Distribution of targets in the MAPK signaling pathway.

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  Fig 7. Molecular docking results for the main chemical components of ZL.

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  Fig 8. Effect of ZL on myocardial histopathologic changes in DCM mice. H H&E staining analysis and Masson staining analysis of the effect of ZL on myocardial tissue in DCM mice (X200).

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  Fig 9. ZL up-regulates the expression of p-P38MAPK, TNF- α, α-SMA, and Collagen-I in DCM cardiomyocytes. (A) The relative (A) The relative mRNA expression level of TNF-α (A1), α-SMA (A2), Collagen-I (A3). (B - C) Western blot analysis of p-P38MAPK (C1), TNF-α (C2). Data are presented as means±SD, from 3 independent experiments. (*P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the DCM group, n = 3).

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  Fig 10. The effect of ZL on myocardial tissue fibrosis in DCM mice. (A) Immunofluorescence analysis of p-P38MAPK expression in DCM mice (X400). (B) Immunofluorescence analysis of TNF-α expression in DCM mice (X400). (C) Immunofluorescence analysis α-SMA expression in DCM mice (X400). (D) Immunofluorescence analysis of Collagen-I expression in DCM mice (X400). (E-H) Quantitative analysis of immunofluorescence of p-P38MAPK, TNF-α, α-SMA, and Collagen-I expression in myocardial tissue. Data are presented as means±SD, from 3 independent experiments. (*P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the DCM group, n = 3).