Research on the Mechanism of Wuwei Yuganzi San in the Treatment of Coronary Heart Disease Based on Network Pharmacology

Background: Coronary heart disease (CHD) is a chronic cardiovascular disease across the world, which poses numerous threats to mankind. Wuwei Yuganzi San (WYS) is a famous traditional Tibetan medicine prescription. It has been conrmed effective in the treatment of CHD, but its specic mechanism remains still unclear. Objective: To elucidate the main pharmacological action of WYS in treating CHD and investigate the underlying multiple mechanisms of its multi-ingredient-multi-target by network pharmacology. Methods: Firstly, active ingredients of WYS and connected targets of ve herbs were retrieved by using traditional Chinese medicine systems pharmacology (TCMSP) and screening literature. Then, genome explanation databases (OMIM and GeneCards) were used to acquire targets related to CHD. The protein-protein interaction (PPI) network was built using the mutual targets ltering genes through protein interaction. Next, a network diagram could be established with the help of Cytoscape 3.8.2. And, STRING platform was used to construct a protein interaction network. Finally, GO and KEGG analyses were analyzed to further elucidate biological process enrichment. Results: After the screening, 36 active ingredients and 202 related targets in WYS in addition to 952 disease-related targets were acquired. A total of 37 key targets including AKT1, ESR1, and EGFR were screened in the PPI network. These targets were mostly concentrated on the transmembrane receptor protein tyrosine kinase signaling pathway, cellular response to growth factors stimulus, and response to growth factor. The KEGG enrichment demonstrated that the MAPK signaling pathway, P13K/AKT signaling pathway, Ras signaling pathway, and other corresponding signaling pathways were closely related to CHD. Conclusions. WYS plays a signicant role in the treatment of CHD. And this study provides a novel approach to disclose the therapeutic mechanisms of WYS on CHD.


Introduction
Over recent years, as a leading cause of death in the world, Coronary heart disease (CHD) remains one of the most epidemic health issues with a signi cant mortality and morbidity rate all over the world, which is caused by insu ciency in blood ow to cardiomyocyte because of coronary artery stenosis or myocardial infarction [1][2]. Consequently, percutaneous coronary intervention and drug therapy are very signi cant.
Otherwise, major adverse cardiovascular events will happen. Nitrates, beta-blockers, and calcium antagonists are both used for the intervention of anti-anginal agents [1]. As CHD has an intricate process, these drugs can not treat complicated pathology well. However, traditional Tibetan medicine (TTM) has distinctive curative e cacy on cardiovascular diseases during clinical practice, mostly because of its multi-targeted functions [3].
Wuwei Yuganzi San (WYS) comes from MaDiYiZhuXuanJi, a well-known book in TTM, and is widely used in the treatment of CHD for a thousand years [4]. WYS consists of Yuganzi (Phyllanthus Emblica L. YGZ), Zangjinjier (Caragana Jubata, ZJJE), Saibeizijin (Corydalis Impatiens, SBZJ), Dahuang (Rheum Palmatum L, DH), and Ganjiang (Zingiberis Rhizoma, GJ). Our research has demonstrated that WYS has signi cant effects in protecting cardiomyocytes from myocardial ischemia-reperfusion injury [5]. However, the underlying molecular mechanisms of WYS in the therapy of CHD are still not well understood, which has become an obstacle to its research and development.
The network pharmacology provides a novel method for revealing the effect mechanism of TTM prescriptions, which is a signi cant part of systematic biology. And it also emphasizes the function of multi-ingredient-multi-target-multi-pathway in deep sight [6]. Consequently, network pharmacology strategies are performed to explore the mechanisms of WYS in the treatment of CHD. The purpose of this study is to provide a convincing scienti c basis for WYS to treat CHD. Screening active ingredients in WYS and underlying targets in the treatment for CHD to construct a comprehensive network of multiingredient-multi-target-multi-pathway is an important method. What is more, KEGG and GO analysis also play a key role in analyzing this study. The owchart of this study is shown in Fig. 1.

Materials And Methods
Active ingredients in WYS collection and screening.
Firstly, traditional Chinese medicine systems pharmacology (TCMSP: https://sm.nwsuaf.edu.cn/lsp/tcmsp.php.) and literature were used to collect the main active ingredients in the prescription of WYS [7]. Then, the main active chemical components of WYS were selected by absorption, distribution, metabolism, and excretion (ADME) [7]. Another part of the active ingredients was screened by SwissADME [8]. And oral bioavailability (OB) value was set as greater than or equal to 30%.
Construction of multi-ingredient-multi-target network Cytoscape software 3.8.2 was used to construct a multi-ingredient-multi-target network to acquire the relationship between the active ingredients and underlying targets [15].

Construction of PPI Network Construction.
The related targets of WYS in the intervention of CHD were input into STRING (http://string-db.org/) online platform [16]. Firstly, "multiple proteins" were selected. Then, the mutual target genes of WYS and CHD were input into the blank, and the organism was set as Homo sapien. Finally, the interaction score was set as 0.7, and the PPI network was constructed.
Go and KEGG Analysis of Core Genes.
To further investigate the mechanism of WYS in the treatment of CHD. Go and KEGG analysis of potential targets were analyzed by Metascape (https://metascape.org/gp/index.html) [17]. THE Metascape online platform was used to elucidate the biological processes, molecular function, cellular components, and signaling pathways in the treatment of CHD.

Construction of drugs-targets-pathways-disease network
Cytoscape software 3.8.2 was used to construct a drugs-targets-pathways-disease network to elucidate speci c mechanisms and targets of the function of WYS.

Results
Retrieving Results of Effective Ingredients of WYS 92 ingredients for YGZ, 92 ingredients for DH, and 148 ingredients for GJ were obtained from TCMSP online platform. 31 ingredients for SBZJ and 33 ingredients for ZJJE were acquired from the literature [18][19][20][21][22]. When ADME was set as OB≥ 30% and DL ≥ 0.18 in TCMSP online platform, the probability ≥ 0.88 in Swiss Target Prediction online platform, and the norm t ≥ 0.88 in PharmMapper online platform, 13 ingredients and 370 targets for YGZ, 12 ingredients, and 353 targets for DH, 1 ingredient and 1 target for GJ, 1 ingredient and 2 targets for SBZJ, 9 ingredients and 34 targets for ZJJR. Finally, 36 active ingredients and 202 related targets in WYS (Table. 1). There are 37 core genes between WYS and CHD (Table. 2).  Potential Therapeutic Targets in CHD Firstly, genes were retrieved from GeneCards, and the Inferred Functionality Score ≥30.34 was selected in GeneCards, which ltered genes in this database. 445 known therapeutic targets for CHD were acquired from the GeneCards database. Secondly, 548 known therapeutic targets for CHD were acquired from the OMIM database. In total, there were 951 therapeutic targets from CHD Finally, the target identi cation was performed by the Venny2.1.0 online system. And the results are displayed in Fig. 2.
Multi-ingredient-multi-target Interaction Network 36 ingredients of WYS and 202 interactive genes were connected to construct a complex PPI network using that included 243 nodes and 795 edges (Fig. 3).

PPI Network Diagram Construction
At the beginning, the 37 intersection targets of WYS in the treatment of CHD were input into the STRING database. And a protein interaction network diagram was acquired, with 37 nodes, 93 edges, and an average node degree of 5.03 (Fig. 4).

Results of Go Enrichment Analysis
Metascape online platform was used to perform KEGG analysis and GO analysis. There are three parts in GO enrichment analysis, including biological process, cellular component, and molecular function (Fig.   5).
From the results of biological process, it was signi cantly related to transmembrane receptor protein tyrosine kinase signaling pathway (GO: 0007169), cellular response to growth factor stimulus (GO: 0071363), and response to growth factor (GO: 0070848).
From the results of cellular component, it was closely correlated to membrane raft (GO: 0045121), membrane microdomain (GO: 0098857), and membrane region (GO: 0098589).
From the results of molecular function, it was closely correlated to protein kinase activity (GO: 0004672), phosphotransferase activity, alcohol group as acceptor (GO: 0016773), and kinase activity (GO: 0016301).

Drugs-targets-pathways-disease network
The Drugs-targets-pathways-disease network was shown in Fig. 7, which included 3 drugs, 22 core targets, 20 signaling pathways, 144 nodes. The light blue circle node is WYS; red node is CHD; green octagon node is signi cant signaling pathway; dark triangle node is core targets; purple diamond node is the drug, including YGZ, DH, and ZJJE. This network analysis demonstrated the characteristics of multiple drugs and multiple targets of WYS in the treatment of CHD.

Discussion
The de nitions of "syndrome" and "disease" have been known in TTM. The recognition of the disease is based on the comprehensive condition of the disease. Consequently, the underlying occurrence and development of disease are known from a macro perspective. There is a comprehensive comprehension of the etiology, pathogenesis, and drug selection in the development of CHD [23]. However, the underlying mechanisms and pathways of TTM remain unclear. Recently, network pharmacology has been an optimized method to explore the "drug-component-target" of TTM or traditional Chinese medicine. In this study, our results showed that WYS has the effect of preventing CHD by regulating multi-ingredient-multitargets with multi-pathways. WYS consists of 5 herbs, including Yuganzi (Phyllanthus Emblica L. YGZ), Zangjinjier (Caragana Jubata, ZJJE), Saibeizijin (Corydalis Impatiens, SBZJ), Dahuang (Rheum Palmatum L, DH), and Ganjiang (Zingiberis Rhizoma, GJ). In this study, using the TCMSP and PubChem database at rst, 36 active components and 202 drug targets were testi ed in WYS by OB and DL. Then, a multiple network diagram was constructed, including a multi-ingredient-multi-target interaction network, a PPI network diagram, and a drug-targets-pathways-disease network. Finally, GO and KEGG enrichment analyses were performed to explain the underlying mechanisms.
To date, many studies had found that YGZ and its extract could protect RAW264.7 cell from H 2 O 2 -induced toxicity, reduce high cholesterol and narrow the area of atherosclerotic plaque, and inhibits the expression of ET-1 gene [24][25][26]. And it also exerted anti-in ammatory effects by reducing the expression of NO and pro-in ammatory cytokines [27]. Moreover, it could protect β cells, scavenge free radicals, reduce in ammation and reduce advanced glycation end-products [28]. DH could reduce oxidative stress by regulating blood lipid metabolism and improving antioxidant capacity, thereby regulating mitochondrial apoptosis. Besides, DH could also regulate Fas/FasL-mediated apoptosis and inhibit the signal transduction pathway of β cell apoptosis [29]. The thesis showed that network pharmacology and metabolomics methods were carried out and found that Aconiti Lateralis Radix Praeparata combined with Zingiberis Rhizoma could treat chronic heart failure through mitochondria-mediated energy metabolism [30][31]. PPARα/PGC-1α/Sirt3 signal pathway played a signi cant role in the treatment of chronic heart failure [32]. There was an exerting anti-in ammatory effect, which SBZJ could inhibit the production of TNF-α, IL-6, and NO by down-regulating the activation of NF-κB, the phosphorylation of ERK1/2, and MAPK signaling pathway [33]. ZJJE had the effects of inhibiting TNF-α, scavenging free radicals, inhibiting lipid peroxidation, and having anti-thrombosis effects to achieve cardioprotection [34][35][36].
Above all, WYS had been used to be a typical and effective prescription for CHD for a long time. And our research team found that WYS had a protective effect on myocardial ischemia-reperfusion injury in rats. The mechanism might reduce serum LDH and CK levels, increase the activity of SOD and GSH-Px in myocardial tissue, and reduce MDA. It promoted the expression of Bcl-2 protein, increased Bcl-2/Bax, and inhibited the expression of Bax protein [4][5].

Analysis of active ingredients
In the network of multi-ingredient-multi-target, key ingredients with a higher degree contain quercetin, sennoside D_qt, procyanidin B-5,3'-O-gallate, ellagic acid, and phyllaemblicin A. Among them, quercetin is a avonoid ingredient and exists widely in nature. Research showed that quercetin modulated AMPK/SIRT1/NF-κB signaling pathway [37] or JAK2/STAT3 pathway [38] to inhibit cell apoptosis and oxidative stress, reduce myocardial infarction size, improve ventricular remodeling and cardiac functionrelated biochemical indexes, and promote the recovery of cardiac blood ow. And a double-blind, placebocontrolled, randomized clinical trial demonstrated signi cantly elevated total antioxidant capacity [39]. Both two studies provided evidence for WYS treating CHD successfully.

Analysis of potential targets
The highest degree of the target was ADAM17. And the following targets were AKT1, ALB, ADH1C, AR in the network of multi-ingredient-multi-target. ADAM17, tumor necrosis factor-alpha converting enzyme, played a key role in cardiovascular. Increased shedding of ADAMs could induce various cardiovascular diseases, which are closely related to in ammation, tissue remodeling, and dysfunction. ADAMs may be promising therapeutic targets for hypertension and atherosclerosis [40].

GO analysis and KEGG analysis
Metascape online platform was used to perform KEGG analysis and GO analysis. From the results of GO analysis, the potential targets were enriched, which were connected with membrane raft(GO: 0045121), membrane microdomain(GO: 0098857), and membrane region (GO: 0098589) in biological process. Furthermore, from the results of KEGG analysis, 14 signaling pathways related to CHD as signi cant pathways from the 20 pathways of KEGG analysis were divided into three aspects: oxidative stress, metabolism, and immunity. As shown in Fig. 7: multi-targets and multi-pathways played a key role in the treatment of CHD. The pathways linked to oxidative stress contain MAPK signaling pathway, HIF-1 signaling pathway, Foxo signaling pathway, JAK/STAT signaling pathway, VEGF signaling pathway, Platelet activation. According to previous studies, CHD is a disease with complex mechanisms. When ischemia, blood oxygenation, and abnormal energy metabolism occurred, cardiomyocytes will deform or even die, which resulted in CHD. Metabolism included PI3K-Akt signaling pathway, Fluid shear stress and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, mTOR signaling pathway, Ras signaling pathway, Insulin resistance, Calcium signaling pathway. Abnormal changes in uid shear stress [41], calcium [42], and insulin resistance [43] might be more likely to develop into CHD. Both PI3K-Akt signaling pathway and mTOR signaling pathway were involved in autophagy, which played a signi cant role in myocardial ischemia-reperfusion injury [44]. Immunity included Chemokine signaling pathway.
Chemokines recruited in ammatory cells to the injured vascular endothelium and released in ammatory cytokines. Finally, the development of CHD is accelerating [45].

Conclusion
The mechanism of action of WYS in the treatment of CHD may be associated with multiple ingredients, targets, and signaling pathways. High-quality clinical research and basic research are needed to con rm these results.