Integrating Network Pharmacology and in vivo Validation to Explore the Mechanisms of Buyang Huanwu Decoction in Myocardial Ischemia-Reperfusion Injury

Figure 1. Flowchart of the integrated strategy for investigating BuYang HuanWu Decoction (BYHWD) against myocardial ischemia/reperfusion injury (MI/RI). (A) Venn diagram for BYHWD-related targets and MI/RI-related targets; (B) Protein-protein interaction (PPI) network and top 10 hub genes for BYHWD anti-MI/RI; (C) Heatmap of molecular docking scores; (D) Herb-compound-target network; (E) Herb-target-pathway network; (F) Molecular docking between hub genes and key active compounds; (G) Gene Ontology (GO) enrichment analysis; (H) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis; (I) Effect of BYHWD on myocardial infarction size and hematoxylin-eosin (HE) staining; (J) Effect of BYHWD on protein expression of hub genes; (K) Effect of BYHWD on inflammatory cytokine expression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2. Venn diagram for BuYang HuanWu Decoction (BYHWD)-related targets and myocardial ischemia/reperfusion injury (MI/RI)-related targets.

Figure 3. Protein-protein interaction (PPI) network and top 10 hub genes for BuYang HuanWu Decoction (BYHWD) anti-myocardial ischemia/reperfusion injury (MI/RI). (A) PPI network constructed with STRING database. (B) PPI network constructed with Cytoscape software (the darker the node color, the higher the number of connected proteins). (C) Top 10 hub genes for BYHWD anti-MI/RI were obtained by using the CytoHubba plug-in.

Figure 4. Gene Ontology (GO) enrichment analysis of BYHWD anti-MI/RI targets. Top 10 enriched terms in biological processes (BP), cellular components (CC), and molecular functions (MF).

Figure 5. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of myocardial ischemia/reperfusion injury (MI/RI)-related targets. Top 10 pathways ranked by −log10 (P value) and number of involved genes.

Figure 6. The red triangle represents the herbs contained in BuYang HuanWu Decoction (BYHWD); the blue diamond represents the potential therapeutic targets for BYHWD anti-myocardial ischemia/reperfusion injury (MI/RI); the yellow octagon represents the active compounds contained in Chi shao (RPR); the light blue octagon represents the active compounds contained in Chuang xiong (CR); the pink octagon represents the active compounds contained in Hong hua (CF); the purple octagon represents the active compounds contained in Huang qi (RA); the green octagon represents the active compounds contained in Tao ren (PS); the ellipse represents the common active compounds of the herb.

Figure 7. Herb-target-pathway network. The purple circles stand for 25 myocardial ischemia/reperfusion injury (MI/RI)-related pathways and the red, yellow, green, dark green, light blue, pink, and Orange diamond stand for compounds of Angelicae Sinensis Radix, Radix Astragali, Chuanxiong Rhizoma, Persicae Semen, Pheretima, Radix Paeoniae Rubra, and Carthami Flos, respectively. Besides, blue circles stand for genes related to BuYang HuanWu Decoction (BYHWD).

Figure 8. Heatmap of the scores of molecular docking in this study.

Figure 9. Results of molecular docking between hub genes and key active compounds. (A) Protein Kinase B alpha (AKT1) with quercetin, (B) AKT1 with kaempferol, (C) Interleukin-10 (IL-10) with beta-sitosterol, (D) IL-10 with stigmasterol, (E) Matrix metalloproteinase-9 (MMP9) with baicalein, (F) MMP9 with baicalin. Yellow lines represent hydrogen bonds between the protein and the ligand. Green lines indicate hydrophobic interactions. Red lines show electrostatic interactions or ionic bonds. The stick model represents the ligand, while the ribbon model represents the protein structure. The residue labels indicate key amino acids involved in the interactions.

Figure 10. Effect of BuYang HuanWu Decoction (BYHWD) on myocardial infarction size and myocardial tissue pathological morphology. (A and B) Representative images of myocardial infarct size in each group, assessed using triphenyltetrazolium chloride (TTC) staining. Recombinant Human Glutathione Peroxidase 4 (rhGPx4) prevents pathological damage of myocardial ischemia. The black arrow indicates the location of myocardial ischemia/reperfusion injury (MI/RI). (C) Representative hematoxylin-eosin (HE) staining images showing myocardial tissue morphology in each group. The black arrows indicate cardiomyocyte necrosis, edema, and inflammatory cell infiltration. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; sample size, n = 4.

Figure 11. Effect of BuYang HuanWu Decoction (BYHWD) on protein expression of hub genes. (A–G) Western blot analysis for the protein expression levels of Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), Matrix Metalloproteinase-9 (MMP-9), and Protein Kinase B alpha (AKT1) / Phosphorylated AKT1 (p-AKT1) in each group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns = not significant, sample size, n = 6.

Figure 12. Effect of BuYang HuanWu Decoction (BYHWD) on inflammatory cytokine expression (A–C) Enzyme-Linked Immunosorbent Assay (ELISA) analysis showing the expression levels of inflammatory cytokines Tumor Necrosis Factor-alpha (TNF-α), Interleukin-6 (IL-6), and Interleukin-10 (IL-10) in each group. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n=6.