Network Pharmacology Analyses of the Pharmacological Targets and Therapeutic Mechanisms of Salvianolic Acid A in Myocardial Infarction

Objective Salvianolic acid A, a natural polyphenolic ingredient extracted from traditional Chinese medicine, possesses an excellent pharmacological activity against cardiovascular diseases. Herein, therapeutic mechanisms of salvianolic acid A in myocardial infarction were explored through systematic and comprehensive network pharmacology analyses. Methods The chemical structure of salvianolic acid A was retrieved from PubChem database. Targets of salvianolic acid A were estimated through SwissTargetPrediction, HERB, and TargetNet databases. Additionally, by GeneCards, OMIM, DisGeNET, and TTD online tools, myocardial infarction-relevant targets were predicted. Following intersection, therapeutic targets were determined. The interaction of their products was evaluated with STRING database, and hub therapeutic targets were selected. GO and KEGG enrichment analyses of therapeutic targets were then implemented. H9C2 cells were exposed to oxygen‐glucose deprivation/reoxygenation (OGD/R) to mimic myocardial infarction and administrated with salvianolic acid A. Cellular proliferation was assayed via CCK‐8 assay, and hub therapeutic targets were verified with RT-qPCR. Results In total, 120 therapeutic targets of salvianolic acid A in myocardial infarction were identified. There were close interactions between their products. Ten hub therapeutic targets were determined, covering SRC, CTNNB1, PIK3CA, AKT1, RELA, EGFR, FYN, ITGB1, MAPK8, and NFKB1. Therapeutic targets were significantly correlated to myocardial infarction-relevant pathways, especially PI3K-Akt signaling pathway. Salvianolic acid A administration remarkably ameliorated the viability of OGD/R-induced H9C2 cells, and altered the expression of hub therapeutic targets. Conclusion Our work uncovers therapeutic mechanisms of salvianolic acid A for the treatment of myocardial infarction, providing a new insight into further research on salvianolic acid A.


Introduction
Cardiovascular disease is a general term for diseases that damage the heart and blood vessels, characterized by rapid onset and high morbidity [1]. In accordance with the WHO, approximately 18 million individuals died from cardiovascular disease in 2019, which represented 32% of overall global deaths as the dominating cause of deaths in all diseases [2]. Myocardial infarction, often triggered by blood clot blocking artery or bypass graft, has the features of a sudden decrease in blood flow to myocardium, eventually resulting in heart failure even death [3]. Among cardiovascular diseases, myocardial infarction has become a primary international health issue [4].
rombolysis, percutaneous coronary intervention as well as coronary artery bypass graft remain the most commonly applied approaches in treating myocardial infarction. Nevertheless, patients often present complications such as bleeding, ischemia-reperfusion damage as well as coronary restenosis. erefore, more effective therapeutic approaches to alleviate apoptosis of cardiomyocytes and facilitate local angiogenesis are urgently required for preventing the expansion of irreversible myocardial damage [5].
Salvianolic acids are extracted from Salvia miltiorrhiza Bunge (Danshen). Salvianolic acid A is the strongest antioxidant among salvianolic acids, which is an effective free radical scavenger because of the polyphenolic structure [6]. It has been suggested that salvianolic acid A exerts diverse pharmacological properties, especially for cardiovascular diseases [7]. Preclinical evidence has demonstrated the cardioprotective property of salvianolic acid A against myocardial infarction. Salvianolic acid A attenuates myocardial infarction-triggered apoptosis and inflammation through activation of thioredoxin [8]. Also, it exhibits the antiapoptotic and cardioprotective effects on rat cardiomyocytes under ischemia/reperfusion via DUSP-induced modulation of ERK1/2/JNK signaling [9]. Experimental evidence also demonstrates that salvianolic acid A possesses antioxidant activity and exerts a remarkable protective function against isoproterenol-triggered myocardial infarction [10]. Additionally, salvianolic acid A exhibits cardioprotective effects by facilitating angiogenesis [11] as well as lowering plasma uric acid levels [12] and plasma and tissue dimethylarginine levels [13] in acute myocardial infarction animal models. Despite this, there is a lack of systematic and comprehensive analysis of the therapeutic mechanisms of salvianolic acid A in myocardial infarction.
Network pharmacology is an effective approach to establish a "compound-protein/gene-disease" network, which reveals the regulation principle of small molecule compounds with a high-throughput manner, thus providing a broader selection of pharmacologically relevant targets [14]. Herein, we applied network pharmacology analyses to dissect the therapeutic mechanisms of salvianolic acid A systematically and comprehensively in myocardial infarction. Additionally, the effects and therapeutic targets of salvianolic acid A in myocardial infarction were verified in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced H9C2 cells that mimicked myocardial infarction.

Retrieval of the Chemical Structure of Salvianolic Acid A.
PubChem (https://pubchem.ncbi.nlm.nih.gov) is an important chemical information resource, which comprises over 293 million depositor-provided substance descriptions, 111 million unique chemical structures as well as 271 million biological activity data points from 1.2 million bioassay experiments [15]. e chemical structure of salvianolic acid A was accessed from PubChem.

Analyses of Salvianolic Acid A Targets.
e Swis-sTargetPrediction web tool (http://www.swisstargetprediction. ch) allows users to predict the most possible macromolecular targets of a specific small molecule compound on the basis of 2D and 3D similarity with a library of 370000 known actives on over 3000 proteins from three distinct species [16] O" of salvianolic acid A was uploaded to Swis-sTargetPrediction, and potential molecular targets of salvianolic acid A were downloaded. HERB (http://herb.ac. cn/) is a high-throughput experiment and reference guide database of traditional Chinese medicine [17]. Salvianolic acid A ingredient was input into HERB database, and relevant gene targets were gathered from curated references. TargetNet (http://targetnet.scbdd.com) is a web service to estimate potential drug-target interactions through multitarget SAR models [18]. Potential targets of salvianolic acid A were screened on the basis of ECFP2 molecular fingerprint in accordance with the area under the receiver operating characteristic curve � 1. On the basis of SwissTargetPrediction, HERB, and TargetNet databases, potential molecular targets of salvianolic acid A were merged and deduplicated. rough the Uniprot database (https://www.uniprot.org/) [19], gene name of targets was corrected and matched.

Acquirement of Targets of Myocardial Infarction.
GeneCards (http://www.genecards.org/) [20] and Online Mendelian Inheritance in Man (OMIM; http://omim.org) [21] are comprehensive, and authoritative research resources of annotative information of human genes. DisGeNET (http://www.disgenet.org/) is a knowledge management platform that integrates and standardizes data about diseaserelevant genes and variants from a variety of sources, covering over 24,000 diseases and phenotypes, 17,000 genes as well as 117,000 genomic variants [22]. erapeutic Target Database (TTD; http://db.idrblab.net/ttd/) is a popular information resource of the known therapeutic protein and nucleic acid targets, the targeted diseases, the pathway information as well as the matched drugs or ligands [23]. Potential targets of myocardial infarction were searched from above databases, followed by merging, deduplication, and correction through the Uniprot database.

Identification of Targets Shared by Salvianolic Acid A and
Myocardial Infarction. Targets of salvianolic acid A and myocardial infarction were intersected, and imported into Venny 2.1 online tool (http://bioinfogp.cnb.csic.es/tools/ venny/index.html).
e Venn diagram of targets shared by salvianolic acid A and myocardial infarction was drawn.

Protein-Protein Interaction (PPI) Analyses.
e PPIs of myocardial infarction targets of salvianolic acid A were estimated with the STRING online tool (https://string-db. org/) that integrates all known and predicted PPIs comprising physical and functional interactions [24]. e criteria included organism, Homo sapiens; settings, highest confidence (0.900). e PPI network was drawn via Cytoscape 3.7.2 (https://cytoscape.org/) [25]. e degree of targets was computed with Count package. rough cytoHubba plugin, hub genes were selected.

Functional Enrichment Analyses.
Utilizing clusterProfiler package [26], functional enrichment analyses of myocardial infarction targets of salvianolic acid A were implemented. Gene ontology (GO) analyses were utilized for describing biological functions of gene products, comprising biological processes (BPs), cellular components (CCs), along with molecular functions (MFs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were applied for probing out signaling pathways enriched by myocardial infarction targets of salvianolic acid A. A P value < 0.05 was regarded as significant enrichment. e map of KEGG pathways was drawn via pathview package [27].
H9C2 cells were pretreated with 50 μm salvianolic acid A with 98.15% purity (MedChemExpress, China) for 24 h, as previously described [8]. For establishing an in vitro model of myocardial infarction, H9C2 cells were washed with PBS and maintained in DMEM. Afterwards, they were grown lasting 3 h in an incubator flushed with a gas mixture (1% O 2 , 5% CO 2 as well as 94% N 2 ). Following OGD, they were grown in complete culture medium with 5% CO 2 and 95% air lasting 6 h. Control cells were grown in DMEM with normoxia.

Cell Viability Assay.
Cell viability was assayed with cell counting kit-8 (CCK-8) kit (MedChemExpress, China). H9C2 cells were grown in a 96-well plate (1 × 104 cells/well). 10 μL CCK-8 reagent was added to each well with H9C2 cells. e plate was cultured at 37°C for 2 h away from light. e optical density was monitored with microplate reader at 450 nm.

Reverse Transcription-Quantitative PCR (RT-qPCR).
Total RNA extraction from H9C2 cells was implemented via TRIzol reagent (Solarbio, China). e RNA content was measured with UV spectrophotometry. Extracted RNA was utilized for reverse transcription and cDNA was synthesized. e qPCR assays were implemented utilizing SYBR Premix Ex Taq II and RT-qPCR detection system. GAPDH was utilized for normalizing mRNA expressions. Relative expressions were computed with 2 −ΔΔCt . Sequences of primers (SRC, CTNNB1, PIK3CA, AKT1, RELA, EGFR, FYN, ITGB1, MAPK8, and NFKB1) utilized for RT-qPCR are listed in Table 1.

Statistical
Analyses. Data were analyzed through appropriate R packages and GraphPad Prism 8 software. ANOVA was applied for comparisons between groups, and the difference was statistically significant when P < 0.05.

e Chemical Structure and Potential Targets of Salvianolic Acid A.
e chemical structure of salvianolic acid A was retrieved from the PubChem database, as illustrated in Figure 1. To determine potential molecular targets of salvianolic acid A, we employed three web tools, comprising SwissTargetPrediction, HERB, and TargetNet databases. As a result, 100 (Table 2), 33 (Table 3), and 79 ( Table 4) potential targets of salvianolic acid A were predicted on the basis of SwissTargetPrediction, HERB, and TargetNet databases, respectively. After merging and deduplication, we finally retrieved 180 potential targets of salvianolic acid A (Figure 2(a)). SwissTargetPrediction-, HERB-, and Target-Net-predicted targets of salvianolic acid A occupied 47%, 16%, and 37% of all targets, respectively.
rough Swis-sTargetPrediction web tool, target classes of the top 15 potential targets of salvianolic acid A were analyzed. In Figure 2(b), 40.0% belonged to protease, with 26.7% for lyase, 13.3% for membrane receptor, and with 6.7% for secreted protein, enzyme, or kinase.

Estimation of Targets of Myocardial Infarction.
To estimate the potential targets of myocardial infarction, this study integrated four comprehensive databases comprising

Identification of Targets Shared by Salvianolic Acid A and Myocardial Infarction.
To determine myocardial infarction targets of salvianolic acid A, we took the intersection between targets of salvianolic acid A and myocardial infarction. As illustrated in Figure 4,120 targets shared by salvianolic acid A and myocardial infarction were eventually obtained.

Interactions between Products of Myocardial Infarction
Targets of Salvianolic Acid A. rough the STRING online Evidence-Based Complementary and Alternative Medicine tool, we evaluated the interactions between products of myocardial infarction targets of salvianolic acid A. Figure 5 illustrates their interactions. We computed the degree of each target.

Biological Functions and Pathways of Myocardial Infarction Targets of Salvianolic Acid A.
GO enrichment analyses were implemented for probing out biological functions of 120 myocardial infarction targets of salvianolic acid A. In Figure 7(a), the myocardial infarction targets of salvianolic acid A were remarkably linked to biological processes of response to molecule of bacterial origin, response to lipopolysaccharide, cellular response to chemical  Evidence-Based Complementary and Alternative Medicine stress, reactive oxygen species metabolic process, response to oxidative stress, regulation of reactive oxygen species metabolic process, response to reactive oxygen species, peptidylserine phosphorylation, cellular response to biotic stimulus, and peptidyl-serine modification. Additionally, cellular components of membrane raft, membrane microdomain, membrane region, vesicle lumen, secretory granule lumen, cytoplasmic vesicle lumen, integrin complex, platelet alpha granule, protein complex involved in cell adhesion, and cell projection membrane were significantly enriched by the myocardial infarction targets of salvianolic acid A. We also found the significant enrichment of molecular functions of protease binding, endopeptidase activity, metallopeptidase activity, metalloendopeptidase activity, serine-type peptidase activity, serine hydrolase activity, serine-type endopeptidase activity, protein tyrosine kinase activity, 1phosphatidylinositol-3-kinase activity, and integrin binding by the myocardial infarction targets of salvianolic acid A. On the basis of them, the myocardial infarction targets of salvianolic acid A exerted key functions in myocardial infarction. KEGG pathway enrichment analyses were conducted for unveiling the pathways involved in the myocardial infarction targets of salvianolic acid A. As illustrated in Figure 7(b), myocardial infarction-relevant pathways (PI3K-Akt signaling pathway, lipid and atherosclerosis, fluid shear stress and atherosclerosis, focal adhesion, AGE-RAGE signaling pathway in diabetic complications, sphingolipid signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, IL-17 signaling pathway, etc.) were significantly correlated to the myocardial infarction targets of salvianolic acid A. Especially, the details of PI3K-Akt signaling pathway were visualized, as illustrated in Figure 7(c).

Discussion
Salvianolic acid A is extracted from traditional Chinese medicine Salvia miltiorrhiza, which is a major water-soluble as well as a biologically active ingredient [28]. e present study employed network pharmacology analyses to uncover the pharmacological targets and therapeutic mechanisms of salvianolic acid A in myocardial infarction. Further, H9C2 cells were administrated with OGD/R to mimic myocardial infarction, and the effects and hub therapeutic targets were confirmed. us, our findings unveiled the possible functional mechanisms and pharmacological targets of salvianolic acid A as an antimyocardial infarction therapy.
rough intersecting the targets of salvianolic acid A and myocardial infarction, 120 pharmacological targets of salvianolic acid A in myocardial infarction were determined. Among them, ten hub therapeutic targets were identified, covering SRC, CTNNB1, PIK3CA, AKT1, RELA, EGFR, FYN, ITGB1, MAPK8, and NFKB1. Evidence has demonstrated the functions of the hub therapeutic targets in myocardial infarction. Blockage of SRC can stabilize Flk/ cadherin complexing, reduce edema as well as tissue damage after myocardial infarction [29]. Additionally, SRC suppression reverses Cx43 remodeling and improves heart function following myocardial infarction [30]. CTNNB1 protein product β-catenin is a key integral part of the canonical Wnt/β-catenin pathway. Wnt/β-catenin damage response enables to activate the epicardium as well as cardiac fibroblasts for promoting cardiac repair [31]. e blockage of the Wnt/β-catenin pathway improves the cardiac function of myocardial infarction [32]. Activated RELA/p65 results in myocardial infarction [33], and activation of EGFR-dependent pathway strengthens cardiac fibrosis and exacerbates cardiac dysfunction in myocardial infarction [34]. e basic fibroblast growth factor activates Nrf2-triggered  [35]. Endothelial ITGB1 (β1 integrin) is essential for the heart to adapt cardiac ischemia and protects from myocardial infarction [36]. ITGB1 upregulation is capable of increasing cardiac function and clinical outcome after myocardial infarction [37,38]. NFKB1 gene rs28362491 ins/ del variation correlates to increased susceptibility to myocardial infarction among Chinese Han patients [39]. erapeutic targets were significantly linked to myocardial infarction-relevant pathways, such as PI3K-Akt signaling pathway. Evidence demonstrates that salvianolic acid A is regarded as a potential PI3K/Akt inhibitor. For instance, salvianolic acid A enables to attenuate CCl4triggered liver fibrosis through inactivation of the PI3K-Akt pathway [40]. Moreover, it hinders vasculogenic mimicry formation in human non-small cell lung carcinoma through the PI3K-Akt pathway [41]. Additionally, through suppressing PI3K-Akt pathway, salvianolic acid A triggers cellular apoptosis as well as blocks tumor growth in acute myeloid leukemia [42]. e PI3K-Akt pathway participates in myocardial ischemia/reperfusion damage in diabetic murine models, which is blocked by salvianolic acid A [43]. Salvianolic acid A hinders malignant development of glioma as well as strengthens temozolomide sensitivity through weakening PI3K-Akt pathway [44]. Because the PI3K-Akt pathway plays a key role in administering the process of myocardial infarction, targeting this aberrant signaling pathway as well as improving the pathological manifestation of myocardial infarction remains indispensable [5].
We further verified the effects and therapeutic targets of salvianolic acid A in myocardial infarction in OGD/R-    APP  MTOR  KDR  ITGAV  ITGB3  IL6  ERBB2  MAPK14  PLG  ESR1  NFKB1  MAPK8  ITGB1  FYN  EGFR  RELA  AKT1  PIK3CA  CTNNB1  SRC   8  9  9  9  10  10  11  12  13  13  14  14  14  14  14  15  19  20  23   Evidence-Based Complementary and Alternative Medicine GO Results of ree Ontologies response to molecule of bacterial origin response to lipopolysaccharide cellular response to chemical stress reactive oxygen species metabolic process response to oxidative stress regulation of reactive oxygen species metabolic process response to reactive oxygen species peptidyl-serine phosphorylation cellular response to biotic stimulus peptidyl-serine modification membrane ra membrane microdomain membrane region vesicle lumen secretory granule lumen cytoplasmic vesicle lumen integrin complex platelet alpha granule protein complex involved in cell adhesion cell projection membrane protease binding endopeptidase activity metallopeptidase activity metalloendopeptidase activity serine-type peptidase activity serine hydrolase activity serine type endopeptidase activity protein tyrosine kinase activity 1-phosphatidylinositol-3-kinase activity integrin binding   Evidence-Based Complementary and Alternative Medicine induced H9C2 cells. As expected, salvianolic acid A administration enabled to ameliorate the viability of OGD/Rinduced H9C2 cells as well as alter the expression of the hub therapeutic targets. Nevertheless, several limitations should be pointed out. First, the hub therapeutic targets of salvianolic acid A should be verified in myocardial infarction animal models. In our future research, we will further investigate the therapeutic effects as well as pharmacologically relevant targets of salvianolic acid A in myocardial infarction animal models. Second, further clinical trials are urgently needed to verify the therapeutic effects of salvianolic acid A against myocardial infarction.

Conclusion
e present study unveiled the pharmacological targets and therapeutic mechanisms of salvianolic acid A in myocardial infarction utilizing network pharmacology analyses and in vitro OGD/R H9C2 cellular models of myocardial infarction, which paved the way for further clinical trials.
Data Availability e datasets analyzed during the current study are available from the corresponding author on reasonable request.