Molecular Targets and Pathways Contributing to the Effects of Wenxin Keli on Atrial Fibrillation Based on a Network Pharmacology Approach

Background Atrial fibrillation (AF) is the most common sustained arrhythmia and is associated with high rates of mortality and morbidity. The traditional Chinese medicine Wenxin Keli (WXKL) can effectively improve clinical symptoms and is safe for the treatment of AF. However, the active substances in WXKL and the molecular mechanisms underlying its effects on AF remain unclear. In this study, the bioactive compounds in WXKL, as well as their molecular targets and associated pathways, were evaluated by systems pharmacology. Materials and Methods Chemical constituents and potential targets of WXKL were obtained via the Traditional Chinese Medicine Systems Pharmacology (TCMSP). The TTD, DrugBank, DisGeNET, and GeneCards databases were used to collect AF-related target genes. Based on common targets related to both AF and WXKL, a protein interaction network was generated using the STRING database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGGs) pathway enrichment analyses were performed. Network diagrams of the active component-target and protein-protein interactions (PPIs) were constructed using Cytoscape. Results A total of 30 active ingredients in WXKL and 219 putative target genes were screened, including 83 genes identified as therapeutic targets in AF; these overlapping genes were considered candidate targets for subsequent analyses. The effect of treating AF was mainly correlated with the regulation of target proteins, such as IL-6, TNF, AKT1, VEGFA, CXCL8, TP53, CCL2, MMP9, CASP3, and NOS3. GO and KEGG analyses revealed that these targets are associated with the inflammatory response, oxidative stress reaction, immune regulation, cardiac energy metabolism, serotonergic synapse, and other pathways. Conclusions This study demonstrated the multicomponent, multitarget, and multichannel characteristics of WXKL, providing a basis for further studies of the mechanism underlying the beneficial effects of WXKL in AF.


Introduction
Atrial fibrillation (AF) is one of the most common arrhythmias, and its prevalence is increasing [1]. e occurrence of AF can lead to heart failure, embolism, and stroke and significantly increases the rates of disability and death, presenting a serious health issue [2,3]. erefore, the prevention and treatment of AF are major concerns.
According to the American Heart Association practice guidelines, current strategies for AF management include rate control, rhythm control, anticoagulation, lifestyle, and risk factor management [4]. Antiarrhythmic drugs and catheter ablation are the main treatment options. However, available medications for AF are suboptimal based on the high rate of arrhythmia recurrence and the potential proarrhythmic effect [5,6]. Catheter ablation is also limited by a high rate of recurrence, and patients often require additional surgery [6,7]. erefore, researchers, clinicians, and patients seek new effective and safe treatment strategies for AF.
Traditional Chinese medicines (TCMs) have been used for the prevention and treatment of arrhythmias in China for over a thousand years [8]. Wenxin Keli (WXKL), a classical Chinese patent medicine with high efficacy and favorable safety, is useful for the management of patients with AF [9][10][11]. WXKL is composed of five Chinese herbal extracts: Radix (Dang Shen, DS), Polygonati Rhizoma (Huang Jing, HJ), Notoginseng Radix et Rhizoma (San Qi, SQ), Ambrum (Hu Po, HP), and Nardostachyos Radix et Rhizoma (Gan Song, GS). It is the first Chinese antiarrhythmic medicine approved by the China Food and Drug Administration [11][12][13].
Recent animal and cell studies [14][15][16] have demonstrated that WXKL inhibits and prevents atrial arrhythmias via complicated antiarrhythmic mechanisms, but the specific molecular mechanism remains unclear.
Similar to other TCM formulas, WXKL is a multicomponent and multitarget agent that achieves its specific therapeutic efficacy via the regulation of molecular networks by active components. In this study, we used a comprehensive network pharmacology-based approach to investigate the mechanisms by which WXKL exerts therapeutic effects in AF. A flowchart of the experimental procedure is shown in Figure 1.

Database Building and Active Compound Screening.
All compounds in the five herbal components of WXKL were retrieved from the TCM Systems Pharmacology (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php) database and analysis platform, which captures relationships among drugs, targets, and diseases. e database includes chemicals, targets, drug-target networks, and associated drug-targetdisease networks, as well as pharmacokinetic properties of natural compounds, such as oral bioavailability (OB), druglikeness (DL), and blood-brain barrier penetration [17,18]. Compounds were screened based on absorption, distribution, metabolism, and excretion (ADME), and pharmacokinetic information retrieval filters were used to obtain bioactive compounds for further analysis using the thresholds OB ≥ 30% and DL ≥ 0.18 [19,20]. A network was generated using Cytoscape to visualize the complex relationships between active compounds and potential targets [21]. Nodes represent compounds and targets, and edges indicate the intermolecular interactions.

Identification of Drug Targets.
Protein targets of the compounds were retrieved from the TCMSP. Gene names were extracted from UniProtKB (http://www.uniprot.org).

Screening of Potential
Targets for AF. AF-associated target genes were obtained from various databases, such as TTD, DrugBank, DisGeNET, and GeneCards. e Drug-Bank database (https://www.drugbank.ca/) is a unique bioinformatics and cheminformatics resource that combines detailed drug data with comprehensive drug target information [22,23].
e DisGeNET database (https://www. disgenet.org/) includes one of the largest publicly available collections of genes and variants associated with human diseases [24]. e GeneCards database (https://www. genecards.org/) is a searchable, integrative database that provides comprehensive, user-friendly information for all annotated and predicted human genes [25]. e term "atrial fibrillation" was used for searches against these four databases to screen targets related to AF.

Collection of Common Compound-Disease Targets.
e screened chemical targets and disease targets were imported into the ImageGP (http://www.ehbio.com/ ImageGP/index.php) platform, and common compounddisease targets were obtained as candidates for further analysis [26].

PPI Network of Compound-Disease Targets.
A proteinprotein interaction (PPI) network was generated using the STRING database (https://string-db.org/), which covered almost all functional interactions between the expressed proteins [27]. Species was set to "Homo sapiens," and the target interaction information was obtained. e results were imported into Cytoscape (version 3.6.1; https://www. cytoscape.org/) to draw and analyze the interaction network.  e node size reflected the number of combined edges (degree), and nodes with a degree greater than twice the median degree of all nodes were selected as hubs.

Gene Ontology (GO) and KEGG Pathway Enrichment
Analyses. GO analyses based on the three main categories, i.e., biological process (BP), molecular function (MF), and cell component (CC), and a Kyoto Encyclopedia of Genes and Genomes (KEGGs) (https://www.kegg.jp/) pathway enrichment analysis were performed using the Metascape system (http://metascape.org/gp/index.html) [28] and the ImageGP. A P value less than 0.05 indicated significance.

Pathway Constructions and Analysis.
In order to explore the biological effects of cellular targets affecting the diseases through modulating specific pathways, an incorporated "AF pathway" was integrated based on the current understanding of AF pathology [29]. In brief, the obtained target proteins were firstly mapped to KEGG to distribute them to several pathways. Next, pathways closely related to AF were picked out and consolidated into an "AF pathway" under the pathological and clinical data.  (Table 1).

WXKL Target Identification.
In total, 30 compounds in WXKL were associated with 645 target proteins. After eliminating overlapping proteins, 219 proteins were obtained. To further understand the interactions between compounds and targets, we constructed a compound-target network, as shown in Figure 2, by mapping 30 compounds to 219 potential targets associated with inflammation, antioxidant stress, nuclear factor-kappa B, immune regulation, and so on. e network had 253 nodes and 531 edges. Compounds with the most targets in WXKL were quercetin, luteolin, 7-methoxy-2-methyl isoflavone, beta-sitosterol, baicalein, and stigmasterol, with 141, 55, 40, 35, 31, and 28 targets, respectively. ese results suggested that the six components are probably related to the therapeutic effects in AF.

Retrieval of Potential Disease
Targets. TTD, DrugBank, DisGeNET, and GeneCards retrieval results were integrated to obtain AF-related disease targets. As shown in Figure 3, the potential targets of WXKL were mapped to the disease targets using the ImageGP platform, and a Venn diagram was drawn. A total of 83 potential targets were obtained based on the intersection of the two sets of targets. ese targets are summarized in Supplementary Table S1.

Analyses of Candidate Target Proteins.
A total of 83 potential genes associated with AF were uploaded to the STRING database for analysis. e systematically selected protein targets with a median confidence score of 0.400 were plotted as a PPI network using the STRING database. A total of 83 nodes and 1253 edges were acquired, and the average node degree was 30.2. Furthermore, using Cytoscape, we constructed a network (Figure 4), in which the edges represent associations between a pair of targets, nodes represent the target, and the degree value represents the intensity. e top ten targets IL-6, TNF, AKT1, VEGFA, CXCL8, TP53, CCL2, MMP9, CASP3, and NOS3 had high degrees in this process, explaining their significance in the network.

Gene Ontology Enrichment Analysis.
We imported the 83 candidate target genes into the Metascape system for a GO analysis.
Based on this analysis, the potential targets were related to many biological processes, molecular functions, and cellular components, including functions that maybe important for the occurrence and development of AF. e top 20 highly enriched BP terms were retained for analysis and included negative regulation of cytokine, reactive oxygen species, and cell migration ( Figure 5(c)). Previous study showed that AF targets were the diversity of molecular pathways affecting mechanical stress, sarcomere disarray, chronic inflammation, reactive oxygen species, and activation of atrial fibroblasts, which have collectively emerged from translational research and genome-wide association studies (GWASs) [30].
ese results indicated the therapeutic effects of WXKL in AF maybe mediated by its effects on these biological processes. Accordingly, these processes are of great significance for our understanding of the mechanism by which WXKL influences AF.
A total of 119 GO terms in the molecular function category ( Figure 5(b)) were enriched, and the top 20 entries were selected for analysis. ese terms mainly included cytokine receptor binding, kinase binding, adrenergic receptor activity, phosphatase binding, and antioxidant activity.
In total, 58 GO terms in the cell components category ( Figure 5(a)) were enriched, and the top 19 entries were selected for analysis based on P < 0.01. e targets were closely related to membrane raft extracellular matrix, RNA polymerase II transcription factor complex, endoplasmic reticulum lumen, neuron projection cytoplasm, mitochondrial outer membrane, perinuclear region of cytoplasm, focal adhesion, and nuclear membrane. Previous study showed interconnectivity of numerous targets functioning in the cardiomyocyte cell membrane, sarcoplasmic reticulum, nucleus, sarcomere, and inflammasome with reported roles in human or preclinical models of AF [31,32]. us, it can be seen that WXKL plays a role in the treatment of AF through these cell components category.
Evidence-Based Complementary and Alternative Medicine 3.6. KEGG Pathway Enrichment Analysis. To further reveal the mechanisms underlying the effect of WXKL on AF, we conducted a KEGG pathway enrichment analysis of 83 targets and screened the top 20 pathways based on a threshold value of P < 0.01 ( Figure 6). Many pathways involving potential target genes were identified, such as the VEGF signaling pathway, HIF-1 signaling pathway, and PI3K-Akt signaling pathway, which are associated with signal transduction. Hepatitis B, TNF signaling pathways, NF-kappa B signaling, cytokine-cytokine receptor interaction, and hepatitis C pathways are related to inflammatory reactions. Moreover, amyotrophic lateral sclerosis, allograft rejection, and allograft rejection are closely related to immunological processes. e calcium signaling pathway modulates intracellular Ca 2+ levels and a number of Ca 2+dependent intracellular signaling processes. Serotonergic synapses and tryptophan modulate 5-hydroxytryptamine, which is a hormonal trigger for AF. In addition, complement and coagulation cascades revealed that WXKL has a potential role in other related diseases. e AGE-RAGE signaling pathway is highly important in diabetic complications; it elicits the activation of multiple intracellular signaling pathways involving NADPH oxidase, protein kinase C, and MAPKs, thereby resulting in NF-kappa B activity.
Previous study showed that the mechanisms of AF were related to atrial cardiomyopathy, mechanical stress, activation of atrial fibroblasts, fibrofatty infiltrations, inflammation, activation of inflammasomes and macrophages, hypercoagulability and autonomic nervous system dysregulation, sarcoplasmic reticulum Ca 2+ leak, and so on [30]. e results verified that WXKL alleviates AF by regulating inflammatory, immunity, antioxidant stress, serotonergic synapses, Ca 2+ levels, and a number of Ca 2+ -dependent intracellular signaling processes (Figures 7 and 8).

Discussion
AF, the most common sustained arrhythmia, currently affects over 33 million individuals worldwide, and its prevalence is expected to be more than double over the next 40 years. AF is associated with a twofold increase in premature mortality and important major adverse cardiovascular events, such as heart failure, severe stroke, and myocardial infarction [33]. AF is not just an atrial disease, with documented associations with systemic inflammation, endothelial dysfunction, cardiometabolic disturbance, and wider abnormalities in myocardial structure and function [34,35]. In this study, we identified the main active ingredients of WXKL as quercetin, luteolin, 7-methoxy-2- Evidence-Based Complementary and Alternative Medicine methyl isoflavone, beta-sitosterol, baicalein, and stigmasterol and used network and functional analyses to show that they are potentially valuable for the treatment of AF. Some compounds detected in this study have reported effects other than anti-inflammatory, antiimmune, and antistress activity. Quercetin, a very common flavonoid found in plant foods, prevents arrhythmias and dramatically improves heart muscle function and repair following heart attack. Quercetin has a protective effect against AF, probably by inhibiting platelet aggregation and TXA2 formation, as well as increasing PGI2 generation [35]. Previous studies have suggested that luteolin, a flavonoid, possesses antioxidative, antitumor, and anti-inflammatory properties. e cardiac protective effects of luteolin have recently been demonstrated in vitro and in vivo [36].
As illustrated in the compound-target network, many targets could be altered by multiple compounds, such as TNF, IL-6, JUN, and PTGS1. Others, including CRP, CXCL2, and COL3A1, could only be regulated by quercetin.

Evidence-Based
It is generally believed that oxidative stress, inflammation, and related processes are important factors for the promotion of apoptosis or myocardial fibrosis, leading to atrial electrical remodeling and structural remodeling and eventually to the occurrence and persistence of AF [37,38]. e identification of significant leukocyte infiltration in atrial myocardium in AF patients and observed correlation between elevated circulating proinflammatory cytokines and AF severity support this contention [39]. Established inflammatory factors and biomarkers related to AF include tumor necrosis factor α, C-reactive protein, interleukin-2, interleukin-6, interleukin-8, matrix metalloproteinase, endothelin, and myeloperoxidase [34]. Cardiac fibroblasts which were a highly plastic population of resident cells play an important role in the development of an AF substrate [40]. Activated fibroblasts secrete extracellular matrix proteins and recruit immune cells, leading to dispersion and blockade of conduction in the myocardium. Combined with an already heterogeneous and anisotropic fiber bundle arrangement, focal fibrosis renders the left atrium extremely susceptible to reentrant circuits [40,41]. VEGF, a pivotal activator of angiogenesis and calcium (Ca 2+ ) signaling in endothelial cells, increases collagen production in atrial fibroblasts [42]. In addition, after treatment, procollagen type I, procollagen type III production, myofibroblast differentiation, and the migratory ability of fibroblasts are reduced in AF. ese results further confirm that the targets identified in this study are consistent with previous reports, indicating supporting the therapeutic effects of WXKL.
Obesity is associated with an increased amount of epicardial fat, which is a major mediator of the relationship between obesity and AF [43]. A probable mechanism by which epicardial fat promotes AF is direct adipocyte infiltration into the atrial myocardium, resulting in slow or anisotropic conduction. A second probable mechanism is atrial fibrosis caused by adipokines secreted from epicardial fat. ird, it is possible that epicardial fat contributes to AF by the secretion of proinflammatory factors, such as IL-6, IL-8, and TNF-α. By a KEGG analysis, we found that WXKL can regulate lipolysis in adipocytes (Figures 7 and 8   Evidence-Based Complementary and Alternative Medicine   Evidence-Based Complementary and Alternative Medicine implying that it is a good treatment strategy for patients with obesity and AF. In 2019, Wang et al. [44] proposed that the relationship between AF and DM is complex and is mediated by structural, electrical, electromechanical, and autonomic changes in the atria, triggered by oxidative stress, inflammation, and glycemic fluctuations. Glucose-lowering therapies may affect the development of AF. Accordingly, it is important to elucidate the mechanism underlying DM-related AF and to evaluate the best treatment strategy for patients with DM and AF. Our KEGG analysis indicated that the most important pathway by which WXKL effects AF is the AGE-RAGE signaling pathway (Figures 7 and 8), involved in diabetic complications, which elicits the activation of multiple intracellular signaling pathways involving NADPH oxidase, protein kinase C, and MAPKs, resulting in NF-kappa B activity. Combining our KEGG analysis, WXKL maybe is a good treatment strategy for patients with DM and AF.
Serotonin, 5-hydroxytryptamine, is a hormonal trigger for AF [45,46]. By a KEGG analysis, we found that pathways are related to the role of WXKL in AF including the regulation of serotonergic synapses and tryptophan metabolism.
Accordingly, the targets identified in our study are consistent with previous findings, further supporting the use of WXKL for the treatment of AF by regulating the inflammatory response, oxidative stress reaction, immune regulation, cardiac energy metabolism, tryptophan metabolism, and other pathways.

Conclusions
In summary, WXKL is beneficial for the treatment of AF, consistent with previous studies. We further characterized the biological functions of active ingredients in WXKL and their corresponding targets by a network pharmacological approach, improving our understanding of the molecular biological mechanism underlying the effects of WXKL in AF. ese findings provide an important theoretical basis for the clinical treatment of AF.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.