Network pharmacology-based analysis in determining the mechanisms of Huoxin pill in protecting against myocardial infarction

Figures

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  Figure 1.. Integrated workflow for elucidating the mechanisms of HXP in the treatment of MI.

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  Figure 2.. (A) HPLC profiles corresponding to stellasterol (orange), kaempferol (gray), quercetin (yellow), and deoxycholic acid/taurine conjugate (blue), shown according to their intensity peaks at different retention times. (B) Construction of the HXP compound-putative target network. The compound-putative target network was constructed by linking candidate compounds from the ten herbs, which are constituents of HXP, to their putative targets. The nodes representing candidate compounds are shown as polychromatic triangles, and the targets are indicated by blue squares.

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  Figure 3.. (A) Overlapping genes among 342 compounds-related genes (I), 281 MI-related genes from DisGeNet database (II) and 4643 MI-related genes from Genecards database (III). (B) A PPI network of candidate HXP targets for MI treatment extracted from the interactive PPI network of HXP putative targets and known MI-related targets.

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  Figure 4.. Molecular docking heat map. Molecular docking heat map indicating the scores of binding affinities that were highly binding (red) or lowly binding (white) following bioactive compounds and protein targets.

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  Figure 5.. Molecular models of bioactive compounds that bind to their predicted protein targets. Green lines represent residues in the respective binding sites. Yellow dashed lines represent hydrogen bonds. Distance of interaction is indicated adjacent to the site of bonding. (A) 3D interaction diagrams of kaempferol in the active site of TNF (PDB ID 1A8M). (B) 3D interaction diagrams of stellasterol in the active site of RELA (PDB ID 1NFI). (C) 3D interaction diagrams of deoxycholic acid in the active site of AKT1 (PDB ID 3MV5). (D) 3D interaction diagrams of stellasterol in the active site of VEGFA (PDB ID 3BDY).

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  Figure 6.. Gene ontology analysis of therapeutic target genes for HXP on MI.

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  Figure 7.. Pathway enrichment analysis of candidate targets for HXP in MI.

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  Figure 8.. HXP prevents excessive inflammation after myocardial infarction. (A) Echocardiography parameters: ejection fraction (EF%), and fractional shortening (FS%), measured at 1 week post-MI in PBS-treated and HXP-treated (3 and 9 mg/mL) mice. *p < 0.05. **p < 0.01. n = 10 for each. (B) Representative M-mode echocardiography at 1 week post-MI showing LV end-systolic and end-diastolic dimensions in PBS-treated and HXP-treated (3 and 9 mg/mL) mice. (C) Real-time PCR results showing the relative expressions of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the infarct regions of PBS-treated and HXP-treated (3 and 9 mg/mL) mice at 1 week following MI. Error bars represent SEM of the fold changes in upper ΔCT values after normalisation with GAPDH. *p < 0.05. **p < 0.01. (D) Western blots showing the nuclear expression of NF-κB following TNF-α treatment at varying times in adult H9c2 cardiomyocytes. TBP, loading control. (E) Western blots showing the expressions of Iκb-α, p-Iκb-α and nuclear NF-κB following pre-treatment with HXP for 12 h and subsequent TNF-α treatment for 4 h in adult H9c2 cardiomyocytes. GAPDH: total loading control; TBP: nuclear loading control.