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

Introduction

Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality worldwide,¹ representing a significant health and economic burden on society.² Timely reperfusion and revascularization therapies, including thrombolytic agents, percutaneous coronary intervention (PCI), and coronary artery bypass grafting, are effective in restoring blood flow to the ischemic myocardium.³ However, reperfusion itself can lead to further myocardial injury, a phenomenon known as myocardial ischemia/reperfusion injury (MI/RI).⁴ The underlying mechanisms of MI/RI are multifaceted and involve calcium overload, apoptosis, oxidative damage, mitochondrial dysfunction, and inflammation.⁵ The inflammatory response is vital in MI/RI, with miR-24 mitigating this by blocking the S100A8/TLR4/MyD88/NF-κB pathway,⁶ thus enhancing heart function. Additionally, apoptosis and ferroptosis are crucial in MI/RI.⁷ Uncoupling Protein 2 (UCP2) reduces MI/RI by inhibiting ferroptosis, possibly via the p53/TfR1 pathway.⁷ Honokiol protects the heart by activating the PI3K/AKT pathway and preventing mitochondrial apoptosis.⁸ Despite ongoing research, there is currently no effective treatment to prevent or manage MI/RI.⁹ In recent years, the focus of medical research has shifted toward finding effective strategies to prevent and reduce MI/RI, with growing evidence supporting traditional Chinese medicine (TCM) offer a promising complementary approach to MI/RI management.^10–12

Buyang Huanwu Decoction (BYHWD), a formula comprising Radix Astragali, Angelicae Sinensis Radix, Radix Paeoniae Rubra, Chuanxiong Rhizoma, Carthami Flos, Persicae Semen, and Pheretima, has been widely used for its vascular protective properties.¹³ Research has demonstrated that BYHWD offers cerebral protection in both humans and animals.^14,15 BYHWD has demonstrated potential in improving myocardial perfusion, reducing infarct size, and alleviating angina.^16,17 Furthermore, studies suggest that BYHWD exerts its therapeutic effects by modulating angiogenesis through pathways such as Cav-1/VEGF.¹⁸ Importantly, BYHWD demonstrates substantial clinical efficacy in the management of myocardial ischemia-reperfusion injury. A plethora of studies have indicated that BYHWD significantly attenuates the expression of inflammatory mediators,¹⁹ reduces cardiomyocyte apoptosis,²⁰ enhances myocardial energy metabolism,²¹ thereby augmenting myocardial repair capacity and improving cardiac function. Studies indicate that Radix Astragali protects cardiomyocytes from MI/RI by reducing oxidative stress and inflammation.²² Angelicae Sinensis Radix enhances blood circulation and reduces ischemia-reperfusion injury by improving vascular endothelial function.²⁰ Chuanxiong Rhizoma inhibits platelet aggregation and improves microcirculation, while Carthami Flos offers protection through its antioxidant and anti-inflammatory properties.¹⁴ Additionally, components like Pheretima and Persicae Semen support cardiomyocyte survival and regeneration by modulating signaling pathways such as PI3K/Akt and HIF-1.²⁰ Consequently, BYHWD exhibits multiple protective mechanisms in the treatment of myocardial ischemia-reperfusion injury, positioning it as a promising candidate within traditional Chinese medicine for MI/RI therapy.

Despite its long-standing clinical use, the precise molecular mechanisms underlying BYHWD’s protective effects on MI/RI remain poorly understood. Given the complexity of MI/RI pathogenesis and the multi-component nature of TCM formulations, targeting a single pathway or gene is insufficient to fully elucidate its therapeutic effects. Network pharmacology, a systems biology approach that integrates multi-target pharmacology with bioinformatics, provides a powerful tool to unravel the intricate mechanisms of TCM.^23,24 This method allows us to examine the complex interactions between BYHWD’s compounds, their targets, and the relevant biological pathways, providing insights that cannot be achieved by traditional pharmacological approaches. This study combines network pharmacology with experimental validation to systematically explore the molecular mechanisms by which BYHWD exerts its protective effects in MI/RI. Our findings offer a scientific basis for the clinical use of BYHWD in ischemic heart disease and provide insights into its multi-target therapeutic potential. A comprehensive workflow of the study is presented in Figure 1.

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. Abbreviation: ns, not significant.

Materials and Methods

Network Pharmacology Analysis

Collection of BYHWD-Related Targets and MI/RI-Related Targets

We collected BYHWD compounds from several databases, including the TCMSP,²⁵ ETCM,²⁶ BATMAN-TCM,²⁷ and SymMap database.²⁸ Next, active components of BYHWD were screened based on absorption, distribution, metabolism, and excretion (ADME) properties, adhering to the criteria of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18.²⁹ Information on the corresponding target proteins for these active compounds was retrieved, and the target protein names were mapped to official gene symbols through the UniProt database.³⁰ The target gene data were then consolidated and de-duplicated to generate a list of BYHWD-associated targets.

For MI/RI-related targets, we extracted data from seven publicly available databases: DisGeNET,³¹ GeneCard,³² OMIM,³³ Comparative Toxicogenomics Database (CTD),³⁴ TTD,³⁵ DrugBank,³⁶ and MalaCards.³⁷ Using the keyword “myocardial ischemia reperfusion injury”, the targets identified were aggregated, unified, and filtered for duplicates to produce a comprehensive list of MI/RI-associated genes.

Collection of Potential Common Targets for BYHWD Against MI/RI

The potential intersection targets of BYHWD and MI/RI were obtained and drawn using Venn (Figure 2).

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

Protein-Protein Interaction (PPI) Network Construction and Hub Gene Analysis

The intersection targets of BYHWD and MI/RI were analyzed using String 11.5³⁸ to construct a PPI network and a minimum confidence score of 0.7 was applied to ensure high reliability of the interactions. The PPI network result was visualized using Cytoscape software. Hub genes were identified using the CytoHubba plugin within Cytoscape, based on the maximum clique centrality (MCC) algorithm.

Gene Ontology (GO) and Pathway Enrichment

We used the DAVID database³⁹ for annotation of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The pathway maps were further visualized through an online tool⁴⁰ to enhance interpretability.

Network Construction

Data on BYHWD-MI/RI targets, active compounds, key signaling pathways and herbs were integrated into Cytoscape to construct and refine two networks: (1) the compound-target network of treatment with BYHWD for MI/RI, and (2) the herb-target pathway network of treatment with BYHWD for MI/RI.

Molecular Docking Verification

To confirm compound-target interactions, molecular docking was conducted using AutoDock Vina.⁴¹ The selected active compounds were used as ligands, while the top 10 hub genes served as receptors. Three-dimensional crystal structures of the hub genes were retrieved from the RCSB Protein Data Bank,⁴² and the MOL2 files of the active compounds were obtained from PubChem.⁴³ Protein preparations involved removing water molecules, adding polar hydrogens, and merging nonpolar hydrogens, followed by conversion to PDBQT format. The docking results, visualized with PyMOL,⁴⁴ evaluated binding affinity, with a binding energy below −5 kcal/mol indicating strong interactions.⁴⁵

In vivo Experiment Validation

Reagent

BYHWD, consisting of Radix Astragali, Angelicae Sinensis Radix, Radix Paeoniae Rubra, Chuanxiong Rhizoma, Carthami Flos, Persicae Semen, and Pheretima at a dispensing ratio of 120:10:10:10:10:10:4.5 (dry weight), was purchased from the affiliated hospital of Guizhou Medical University. The preparation and quality control of BYHWD were performed according to previously established methods.⁴⁶ For Western blot analysis, the following primary antibodies were used: anti-MMP-9, anti-AKT1, anti-ICAM1, anti-VCAM1, and anti-phospho-AKT. GAPDH was used as a loading control. Secondary antibodies were matched appropriately with the host species of the primary antibodies and were used at the dilutions recommended by the manufacturers (Antibody information is provided in the Supplementary Table).

Animal Model and Treatment

Male Sprague-Dawley rats (200 ± 20 g, aged 5–6 weeks) were purchased from Changsha Tianqin Bioscience Co., Ltd. (certificate no. SCXK (Xiang) 2022–0011) and randomly assigned to sham, MI/RI, and MI/RI + BYHWD groups (n=10 per group). The study was approved by the Animal Ethics Committee of the Guizhou Medical University (Approval No. 2305219). The MI/RI model was established by occluding the left anterior descending (LAD) coronary artery for 30 minutes, followed by 2 hours of reperfusion. Briefly, rats were anesthetized with an intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg body weight). The chest cavity was exposed through a midline sternotomy, and the heart was carefully exteriorized. The LAD coronary artery was located, and a 6–0 silk suture was passed around the artery. The suture was tightened to achieve ischemia, confirmed by a change in the myocardial color to a pale gray or cyanotic appearance and a ST-segment elevation on electrocardiogram (ECG). After 30 minutes of ischemia, the suture was released to allow reperfusion for 2 hours. Rats in the BYHWD group were orally administered BYHWD (12.84 g/kg/day) for three weeks before ischemia induction.¹⁶ The sham group underwent the surgical procedure without LAD ligation. ECG monitoring was continuously conducted to assess changes in real time. LAD ligation resulted in myocardial discoloration (gray or cyanotic appearance) and ST-segment elevation on ECG, which confirmed successful ischemia. Reperfusion was indicated by more than 50% reduction in ST-segment elevation.

Following reperfusion, animals were euthanized by overdose of isoflurane anesthesia and cervical dislocation. The heart was immediately excised, placed in cold phosphate-buffered saline (PBS), and then snap frozen in liquid nitrogen for further analysis. The frozen heart tissues were stored at −80°C until further processing for protein expression.

Assessment of Myocardial Infarction Size

Myocardial infarction size was assessed via triphenyltetrazolium chloride (TTC) staining. Immediately after reperfusion and heart excision, the hearts were then frozen at −20°C for 10–15 min and transversely sectioned into 1–2 mm thick slices from the apex to the base. Frozen heart tissue slices were then incubated in TTC (1%, 5 mL) solution at 37°C for 30 minutes in the dark. The infarcted regions, which lacked TTC reduction, appeared white or pale, whereas the non-infarcted areas, which retained the TTC, appeared red. Myocardial infarct size was calculated using Image-Pro Plus software (Media Cybernetics), with the following formula: infarct size (%) = (infarct area/total heart area) × 100%.

Histopathology

For histopathological examination, the myocardial tissue was fixed in 4% paraformaldehyde, dehydrated in ethanol, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining was performed, and histopathological changes were examined under a light microscope at 200× magnification. The images were captured using the BA210 Digital Trinocular Camera Microscopy System from Motic Industrial Group Co., Ltd. (China).

ELISA

Serum concentrations of cytokines, including IL-6, IL-10, and TNF-α, were measured using quantitative ELISA kits according to the manufacturer’s instructions. Briefly, 50 µL of serum sample was added to pre-coated ELISA plates, followed by incubation at room temperature for 2 hours to allow the cytokines to bind to the capture antibody. After incubation, the plates were washed three times with washing buffer to remove unbound substances. HRP-conjugated secondary antibody was then added and incubated at room temperature for 1 hour. After another wash, the substrate solution was added, and a colorimetric reaction was allowed to develop. The optical density was measured at 450 nm using a microplate reader. Cytokine concentrations were determined using a standard curve constructed for each cytokine.

Blood was collected from the tail vein of each animal, and approximately 1–2 mL of blood was obtained per animal. The blood was allowed to clot at room temperature for 30 minutes and then centrifuged at 1500 rpm for 10 minutes. The serum was carefully separated and stored at −80°C until further analysis.

Western Blot

Proteins were extracted from myocardial tissue obtained from the apex of left ventricle using RIPA buffer (Thermo Scientific), and concentrations were measured with a BCA Protein Assay Kit (Thermo Fisher Scientific, P0010). Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% milk, and incubated with primary antibodies (AKT, phospho-AKT, ICAM1, VCAM1, MMP9, and GAPDH) overnight. After washing, membranes were treated with HRP-conjugated secondary antibodies, and protein expression was visualized using enhanced chemiluminescence (ECL). Protein expression levels were quantified using Image Lab software and normalized to GAPDH.

Statistical Analysis

Statistical analysis was performed using SPSS software (Version 25, IBM Corp., USA). Data are expressed as mean ± standard deviation. For normally distributed data, one-way ANOVA followed by Bonferroni post-hoc tests was used. Statistical significance was set at p < 0.05.

Results

Targets of BYHWD and MI/RI

A total of 794 compounds were initially identified across the seven herbal components of Buyang Huanwu Decoction (BYHWD): Radix Astragali (87 compounds), Angelicae Sinensis Radix (125 compounds), Chuanxiong Rhizoma (189 compounds), Radix Paeoniae Rubra (119 compounds), Persicae Semen (66 compounds), Carthami Flos (189 compounds), and Pheretima (19 compounds). Applying the ADME criteria (OB ≥ 30%, DL ≥ 0.18), 106 potential compounds from the seven herbal medicines in BYHWD were screened. Of these, 95 were identified as potential active compounds, which is provided in Table 1. Subsequently, the UniProt database was used to identify and verify the target protein names of these compounds and their corresponding gene names. This process resulted in the identification of 285 unique BYHWD-related targets after integration and deduplication. Additionally, 455 targets associated with myocardial ischemia/reperfusion injury (MI/RI) were identified using seven databases: DisGeNET, OMIM, CTD, TTD, GeneCards, DrugBank, and MalaCards. A Venn diagram revealed 75 overlapping genes between the BYHWD and MI/RI target datasets. These common targets are listed in Table 2 and visually represented in Figure 2.

Table 1 Active Compounds of BYHWD and Their Parameters

Table 2 Determined Target Information of BYHWD Related to MI/RI

BYHWD-MI/RI PPI Network and Hub Genes Analysis

The 75 intersecting targets were analyzed using the STRING database, and their interaction network was visualized in Cytoscape 3.7.2. The network consists of 72 nodes and 342 edges (Figure 3A and B), with pink nodes indicating higher connectivity. The top 10 genes based on MCC algorithm are shown in Table 3, with IL6 having the highest score. Using the CytoHubba plugin and MCC algorithm, the top hub genes associated with BYHWD’s effects in MI/RI were identified: IL6, AKT1, TNF, ICAM1, MMP9, IL10, VCAM1, NOS3, ALB, and CRP (Figure 3C).

Table 3 The Top 10 Genes Based on MCC Method

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.

Gene Ontology and Pathway Enrichment Analysis

GO analysis was conducted to classify the biological processes (BP), cellular components (CC), and molecular functions (MF) associated with the 75 overlapping targets. A total of 349 GO terms were enriched, including 276 BP, 29 CC, and 44 MF terms, all with p < 0.05. The top 10 terms in each category are summarized in Figure 4 and Table 4. KEGG pathway analysis identified 80 enriched pathways, of which 25 were closely linked to MI/RI based on a PubMed literature review. The top pathways include TNF, apoptosis, p53, NOD-like receptor, HIF-1, and VEGF signaling pathways (Figure 5 and Table 5).

Table 4 Top 10 Items of Gene Ontology (GO) Enrichment Analysis

Table 5 The Enriched 25 Potential Related Pathways for MI/RI

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.

Compound-Target Network Analysis

A compound-target interaction network was constructed using Cytoscape, comprising 327 nodes and 1336 edges. The compounds with the highest degree centrality included quercetin, kaempferol, baicalein, stigmasterol, baicalin, beta-sitosterol, luteolin, hederagenin, 3,9-di-O-methylnissolin, and oleic acid (Figure 6).

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.

Herb-Target-Pathway Network

By inputting 75 targets that intersected with MI/RI genes into the DAVID database, we identified 80 pathways associated with MI/RI. After searching the PubMed database and comparing these results with the 80 enriched pathways, 25 pathways were confirmed to be significantly enriched in relation to MI/RI. The herb-target-pathway network analysis identified Radix Astragali and Carthami Flos as the primary contributors to BYHWD’s anti-MI/RI effects, with the TNF signaling pathway displaying the highest centrality, followed by the HIF-1, PI3K/AKT, p53, Toll-like receptor, FoxO, and MAPK signaling pathways (Figure 7).

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).

Molecular Docking Verification

Among the 95 active compounds, six (quercetin, kaempferol, baicalein, stigmasterol, baicalin, and beta-sitosterol) exhibited the highest degree centralities, prompting us to perform molecular docking with the top 10 hub proteins. The heatmap in Figure 8 displays the binding affinities between these compounds and the hub proteins. The binding affinities range from −5.0 to −10 kcal/mol, with darker colors indicating stronger binding. The results show that quercetin and kaempferol exhibited the strongest binding affinities with key hub proteins such as AKT1 and TNF, followed by baicalein, stigmasterol, baicalin, and beta-sitosterol. These findings suggest that the active compounds in BYHWD have potential interactions with the identified hub proteins, which could contribute to the therapeutic effects of BYHWD in MI/RI.

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

In Figure 9, molecular docking reveals key interactions between BYHWD compounds and target proteins. Quercetin binds strongly to AKT1, with hydrogen bonds to THR-211 and LYS-179, and hydrophobic interactions with TYR-229 and MET-227. Kaempferol also interacts with AKT1 through hydrogen bonds with GLU-228 and LYS-179, and hydrophobic interactions with ALA-177 and LYS-179. Beta-sitosterol binds moderately to IL10, interacting with LEU-94, LEU-98, and MET-77 via hydrophobic forces, suggesting its role in inflammation regulation. Stigmasterol interacts with IL10 via both hydrophobic interactions with VAL-124 and PHE-128, and hydrogen bonds with GLU-142. Baicalein and Baicalin both show strong binding to MMP9, forming hydrogen bonds with key residues (PRO-415, GLU-397, PRO-421, GLY-186, THR-423) and hydrophobic interactions with PHE-145, VAL-398, and MET-422. These interactions suggest that these compounds may modulate their respective pathways to exert therapeutic effects in MI/RI.

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.

BYHWD Reduces Myocardial Infarction Size and Restores Tissue Morphology

TTC staining demonstrated that myocardial tissue in the sham group exhibited consistent red coloration, with minimal evidence of infarction. In contrast, in the MI/RI group, significant infarct size increase was observed, with pale and infarcted walls. Notably, BYHWD treatment reduced infarct size compared to the MI/RI group (Figure 10). HE staining showed that myocardial fibers in the sham group were regularly arranged with uniform staining and no obvious signs of tissue damage. In contrast, the MI/RI group showed disordered myocardial fibers, with marked swelling, necrosis, degeneration, and extensive inflammatory cell infiltration in the interstitial spaces. However, BYHWD treatment improved myocardial structure, showing more organized fibers, reduced swelling, and less inflammatory infiltration (Figure 10).

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.

Regulates Protein Expression in MI/RI

Western blot analysis showed significant reductions in ICAM1, VCAM1, and MMP9 expression in BYHWD-treated groups compared to the MI/RI model group. Moreover, BYHWD upregulated phosphorylated AKT1 (p-AKT1) levels (Figure 11).

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.

Anti-Inflammatory Effects of BYHWD

ELISA results indicated that BYHWD significantly decreased pro-inflammatory cytokines IL-6 and TNF-α, while increasing anti-inflammatory IL-10 levels (Figure 12), highlighting its anti-inflammatory potential in MI/RI.

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.

Discussion

MI/RI is one of the primary causes of mortality in patients with ischemic heart disease and significantly impacts the outcomes of those with this condition. As such, while the pathogenesis of MI/RI is well-understood, developing safe and effective treatments remains a major challenge due to the limited efficacy of current therapeutic options. These treatments fail to adequately address the underlying mechanisms of ischemic and reperfusion injury, highlighting the need for more effective therapies. This study elucidated the molecular mechanisms by which BYHWD provides cardioprotection against MI/RI using a network pharmacology approach. Specifically, we identified 95 active compounds in BYHWD that influence various biological processes associated with MI/RI, modulating myocardial ischemia outcomes through pathways such as TNF signaling pathway, apoptosis, and p53 signaling pathway.

Quercetin, a flavonoid found in several herbs, is one of the primary cardioprotective components in the compound-target network. Research has demonstrated that quercetin exerts cardioprotective effects on isolated heart tissues affected by MI/RI injury via the HMGB1/TLR/NF-κB pathway.⁴⁷ Additionally, Zhang et al reported that quercetin reduces oxidative stress damage by blocking NADPH oxidase. Its anti-inflammatory and anti-apoptotic properties mitigate inflammatory responses and prevent myocardial cell apoptosis.⁴⁸ Furthermore, Tang et al showed that quercetin enhances cardiomyocyte survival during MI/RI by regulating the SIRT1/PGC-1α signaling pathway.⁴⁹

Kaempferol, another prominent flavonoid, is well-recognized for its antioxidant and anti-inflammatory properties.⁵⁰ Dong et al demonstrated that kaempferol alleviates oxidative stress and inflammatory responses, reducing myocardial infarct size in MI/RI by inhibiting the Nrf2 and cleaved caspase-3 signaling pathways.⁵¹ Additionally, Zhou et al found that kaempferol provides cardiac protection during MI/RI by inhibiting the GSK-3β pathway.⁵² These findings suggest that kaempferol holds significant potential as a treatment for MI/RI.

Baicalein and baicalin, major constituents of Carthami Flos and Radix Paeoniae Rubra, exhibit a range of beneficial properties, including antiviral, antioxidant, anticancer, anti-apoptotic, and anti-inflammatory effects.^53,54 Song et al showed that baicalein reduces myocardial infarction size and alleviates MI/RI by activating the ERK1/2 and AKT pathways.⁵⁵ Similarly, Luan et al reported that baicalin can alleviate inflammation reaction and apoptosis during MI/RI by regulating the Akt/NF-κB pathway.⁵⁶

Luteolin, another component of Carthami Flos, has been shown to reduce LDH and CK-MB levels, as well as diminish leukocyte infiltration and cytokine levels in MI/RI rats by modulating the Sirt1/NF-κB pathway.⁵⁷ In vivo studies further demonstrated that luteolin decreases infarct size, suggesting that luteolin may protect the heart by upregulating SERCA2a and Sp1.⁵⁸

Persicae Semen, Radix Paeoniae Rubra, Carthami Flos, and Angelicae Sinensis Radix all contain beta-sitosterol and stigmasterol, which are increasingly recognized for their beneficial roles in cardiovascular diseases. Lin et al showed that beta-sitosterol can reduce myocardial MI/RI injury in vivo by influencing the PPARγ/NF-κB signaling pathway.⁵⁹ These findings suggest that BYHWD may provide cardioprotection through various components, targets, and pathways.

PPI analysis identified IL6, TNF, AKT1, ICAM1, IL10, MMP-9, VCAM1, NOS3, ALB, and CRP as the top ten targets with the highest MCC scores. Additionally, pathway enrichment analysis revealed that the primary pathways involved were TNF signaling, apoptosis, p53 signaling, and VEGF signaling. IL6, TNF, and CRP are well-known inflammatory cytokines that trigger inflammatory responses, exacerbating myocardial damage during ischemia/reperfusion injury (MI/RI). In contrast, IL-10, a significant anti-inflammatory cytokine, is associated with reduced myocardial damage.⁶⁰ In this study, BYHWD treatment significantly reduced pro-inflammatory cytokines IL-6 and TNF-α, while increasing anti-inflammatory IL-10 levels. These findings suggest that BYHWD may counteract inflammation and mitigate MI/RI through its modulation of key inflammatory cytokines.

ICAM1 is an adhesion molecule that mediates adhesion reactions and plays an important role in vascular endothelial dysfunction. ICAM-1 can induce circulating leukocytes to adhere to the endothelium and migrate to the vascular intima, aggravating the inflammatory response and leading to myocardial injury during MI/RI.⁶¹ ICAM-1 and VCAM-1, as key adhesion molecules, play a critical role in the migration of leukocytes and the amplification of inflammatory responses during MIRI, contributing to endothelial dysfunction and tissue damage.⁶² MMP-9 is the most important enzyme of matrix metalloproteinase (MMPs). Increased MMP-9 expression is associated with inflammation, microvascular synthesis, extracellular matrix degradation and synthesis, and cardiac dysfunction during MI/RI.⁶³ AKT1 is a serine/threonine-protein kinase (AKT1, AKT2, and AKT3) that plays a crucial role in major cellular functions, including cell growth, the cell cycle, glucose metabolism, transcription, protein synthesis, and angiogenesis.⁶⁴ AKT1 plays a key role in angiogenesis, particularly the activation of phosphorylated AKT1 (p-AKT1), is well established as a key mediator of cellular survival and anti-inflammatory responses. Activation of p-AKT1 can mitigate apoptotic pathways, reduce oxidative stress, and enhance endothelial barrier function, collectively protecting cardiac tissues from reperfusion injury.⁶⁵ Our study found that BYHWD significantly downregulated the expression of ICAM-1, VCAM-1, and MMP-9, while upregulating the expression of p-AKT1 compared to the MI/RI group. Furthermore, significant binding affinities were observed between hub genes and key compounds of BYHWD, indicating potential interactions. These findings underscore the therapeutic potential of BYHWD in attenuating MIRI through multi-target and multi-pathway effects.

However, this study had several limitations. First, the potential components of BYHWD identified through database mining rely heavily on previously published data. It is plausible that this formula contains additional, as-yet-undiscovered compounds, which could be identified using more advanced network pharmacology methodologies, such as multilevel network analysis, GWAS-based network pharmacology, and global network reconstruction and drug repurposing. These methodologies combine various layers of data and offer a more comprehensive approach to identifying critical compounds and their underlying mechanisms. Second, the predicted targets and pathways were derived from existing databases and literature, which may limit the discovery of novel mechanisms underlying MIRI pathogenesis and the pharmacological actions of BYHWD. Third, future research should consider including standardized drugs or interventions as comparisons in in vivo preclinical studies to provide a more robust evaluation of BYHWD’s therapeutic effects. Finally, we did not evaluate the dose-dependent effects of BYHWD on MI/RI, which could further elucidate the relationship between dosage and therapeutic efficacy. We will further elaborate on the dose-dependent effects of BYHWD, as well as the potential long-term impacts of its usage, based on the current data and future directions for research. This will provide a more comprehensive understanding of the therapeutic potential and safety profile of BYHWD. Future research should focus on comprehensive in vivo studies and well-designed clinical trials to validate the protective effects and optimal dosing of BYHWD in MI/RI.

Conclusions

We explored the mechanisms underlying the cardioprotective effects of BYHWD in MI/RI through a network pharmacology approach and in vivo validation. Key therapeutic targets identified in this study include AKT1, TNF, IL6, MMP9, IL10, ICAM1, ALB, NOS3, CASP3, and CRP. The main bioactive compounds in BYHWD, quercetin, kaempferol, baicalein, stigmasterol, baicalin, and beta-sitosterol were shown to play key roles in combating MI/RI. Enrichment analysis revealed several key pathways involved, including TNF signaling, apoptosis, and p53 signaling. Molecular docking studies demonstrated that these active compounds effectively bind to the identified key targets. Furthermore, in vivo experiments demonstrated that BYHWD significantly reduced myocardial infarct size, suppressed the expression of pro-inflammatory cytokines IL-6 and TNF-α, decreased the levels of ICAM1, VCAM1, and MMP9 proteins, and enhanced anti-inflammatory cytokine IL-10 and phosphorylated AKT1 (p-AKT1) protein expression. These findings reveal the multi-component, multi-target, and multi-pathway mechanisms by which BYHWD exerts cardioprotective effects against MI/RI.