To Investigate the Clinical Efficacy and Potential Mechanism of Tongxinluo Capsules in Preventing Coronary Restenosis Based on Meta-Analysis and Network Pharmacology Analysis

Objective The aim of this study is to evaluate the clinical efficacy of Tongxinluo capsules in preventing coronary restenosis based on meta-analysis and network pharmacology research methods and to preliminarily explore its intervention mechanism. Methods First, through meta-analysis, we comprehensively searched databases such as CNKI, Wanfang, PubMed, the Cochrane Library, and Web of Science to find out the randomized controlled trials of Tongxinluo capsules in the treatment of coronary restenosis until February, 2022. According to the Cochrane Library, risk bias assessment tools were used to evaluate the included literature and Review Manager 5.2 software was used to conduct statistical analysis of the included studies. Then, based on network pharmacology, through TCMSP database and BATMAN-TCM database screening, the chemical components of Tongxinluo capsules and their related effects, symptom, and common targets were analyzed. String net was used to construct protein-protein interaction (PPI) network, and R3.6.1 software was used to carry out GO biological process enrichment analysis and KEGG signaling pathway enrichment analysis to clarify key pathways. Results The meta-analysis finally included 10 RCTs with a total of 1318 subjects. Meta-analysis results showed that Tongxinluo capsules combined with conventional cardiovascular drugs could significantly improve the clinical efficacy of preventing in-stent restenosis and the clinical efficacy of preventing angina pectoris. There was no significant difference in the clinical efficacy of preventing myocardial infarction. Network pharmacology obtained a total of 101 chemical components and 149 targets through the online database. The results of network analysis showed that the targets were mainly involved in receptor ligand activity, carboxylic acid binding, steroid hormone receptor activity, and other related action pathways and were also involved in AGE-RAGE signaling pathway, cell senescence signaling pathway, and other related pathways. Conclusion Tongxinluo capsules combined with conventional cardiovascular drugs can improve the clinical efficacy of preventing in-stent restenosis and angina pectoris and have a significant effect on reducing inflammatory factors. The comprehensive result of the effect is mainly through the participation of receptor ligand activity, carboxylic acid binding, steroid hormone receptor activity, and other ways to achieve the purpose of treating coronary restenosis.


Introduction
With the improvement of Chinese people's material living standard and the change of lifestyle, the morbidity and mortality of coronary heart disease are increasing year by year, and it has become a major health hazard disease. Cardiovascular and cerebrovascular diseases (CVD) have caused serious health problems in China. According to relevant statistics, about 45 percent of patients died of CVD in 2019. About 2.5 million of them had coronary heart disease (CHD) and about 200,000 died [1]. It is estimated that there will be 11.39 million cases of coronary heart disease in China in 2022. Coronary heart disease is more common in the elderly, and coronary stenting percutaneous coronary intervention (PCI) is the main mode for the treatment of CHD [2,3], with the advantages of rapid recovery and small surgical trauma [4,5]. But studies have pointed out the existence of risk for stent restenosis after surgery [6]. Based on this, in recent years, combining traditional Chinese and Western medicine treatment for prevention and control of PCI in postoperative in-stent restenosis (ISR), thereby improving the quality of survival, has become the new exploration point.
Tongxinluo capsule is based on traditional Chinese medicine (TCM) collaterals epidemiology and under the guidance of the theory of ZangXiang doctrine developed by compound Chinese medicine, by ginseng, leech, scorpion, Peony root, cicada slough, terrapin, centipede, sandalwood, incense, frankincense, sour jujube kernel, and borneol, are composed of ten aftertaste Chinese herbs, which have the efects of nourishing blood, invigorating qi, promoting blood circulation, removing blood stasis, clearing collaterals, and relieving pain. Ginseng has the efect of replenishing qi; leeches, red peony root, terrapin, and frankincense have the efect of activating blood circulation; scorpions, cicada slough, and centipedes have the function of channeling channels and collaterals, sandalwood and incense have the function of qi. Jujube seed has the efect of nourishing blood; borneol has both the function of activating blood and channeling channels. Clinically, Tongxinluo capsules combined with conventional cardiovascular drugs have been proved to be efective in preventing coronary restenosis after PCI. Unfortunately, the mechanism is not clear.
Tis study investigated the clinical efcacy of Tongxinluo capsules combined with conventional cardiovascular drugs in preventing coronary restenosis after PCI through meta-analysis and preliminarily analyzed its target and possible pathway of action through network pharmacology to explore their intervention mechanisms, which provided a scientifc basis for Tongxinluo capsules in preventing coronary restenosis.

Meta-Analysis
2.1.1. Literature Inclusion Criteria. Te literature inclusion criteria were as follows: (1) Randomized controlled trials (RCTs) published in China and abroad; (2) the language is limited to Chinese and English; and (3) the subjects were patients after PCI, that is, patients with angina symptoms confrmed by coronary angiography (CAG) as single or multiple vessel lesions (target vessel stenosis I > 70%) with coronary heart disease or myocardial infarction.

Literature Exclusion Criteria.
Te literature exclusion criteria were as follows: (1) Inclusion criteria were not met; (2) animal experiments; (3) literature review; (4) literature with poor design and incomplete data; (5) repeated publications; and (6) patients with other complications (such as acute myocardial infarction).

Literature Retrieval Strategy Databases Retrieved
Include. China National Knowledge Infrastructure (CNKI, https://www.cnki.net/), Wanfang Journal Database (Wanfang, https://www.wanfangdata.com.cn), PubMed (https:// pubmed.ncbi.nlm.nih.gov), the Cochrane Library (https:// www.cochranelibrary.com/), and Web of Science (https:// www.webofscience.com/). Te search period was from the establishment of the database to February, 2022. Chinese keywords mainly included "coronary artery," "restenosis," "clinical medicine," and "Tongxinluo capsule." English search words mainly include "Tongxinluo capsule," and "instent restenosis." 2.1.5. Data Extraction and Quality Assessment. All literature were screened according to inclusion and exclusion criteria, and relevant baseline information including title, author, year, intervention measures, and observation indicators were summarized for those literature that met the criteria. Te quality of the included literature was evaluated according to the cochrane risk bias assessment tool, including: (1) generation of random sequences; (2) allocation hidden; (3) blind subjects or researchers; (4) data integrity; (5) whether there is selective reporting of results; and (6) other biases. According to the literature content, three grades of "low risk," "high risk," and "unbelievable risk" were carried out for the literature.

Data Statistics and Analysis
Review. Manager 5.2 software was used for data analysis, and heterogeneity assessment was made for the included literature according to the P value and I 2 index of heterogeneity test. If there was homogeneity among studies (i.e., P ≥ 0.1 and I 2 ≤ 50%), a fxed-efect model was used for analysis; otherwise, random efect model was used. Odds ratio (OR) and 95% confdence intervals (CI) were selected to describe the results. Te control group was divided into "conventional treatment" and "conventional treatment + one drug" for subgroup analysis according to diferent intervention measures.

Chemical Constituents of Tongxinluo Capsules.
Chemical constituents of Tongxinluo capsules were collected through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https:// tcmspw.com/tcmsp.php); the Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine database (BATMAN-TCM, https://bionet.ncpsb.org.cn/ batman-tcm/) was used to search the chemical constituents of Tongxinluo capsules. Te TCMSP database is screened based on oral bioavailability (OB) ≥ 30% and druglike (DL) ≥ 0.18 in the absorption, distribution, metabolism, and excretion (ADME) parameters [7]. Te BATMAN-TCM database is screened by predicting candidate targets (including known targets) with a score of no less than 20 points for each component, P ≥ 0.5 as the standard [8]. Cytoscape

Construction and Analysis of the Protein Interaction
Network. Te intersection targets were imported into STRING database to obtain the protein interaction network and save it in TSV format. Te Node1, Node2, and combined score information in the fle are imported to Cytoscape V3.6.1 software, and the "Network Analyser" function in Cytoscape software is used for topology analysis of proteinprotein interaction (PPI) Network, taking degree value as reference. We calculate the "connectivity," "closeness centrality (CC)," and "betweenness centrality (BC)" of all nodes to determine the key targets in the network.

GO Biological Analysis and KEGG Pathway Analysis.
R3.6.1 software was used for enrichment analysis of GO biological process and KEGG signal pathway, and histogram, bubble diagram, and pathway diagram were drawn for visualization. GO bioanalysis was explained and annotated from three aspects: biological process, molecular function, and cellular location.

Literature Retrieval and Inclusion
Results. Based on the above retrieval strategy, a total of 86 Chinese literature and 10 English literature were retrieved, and 36 literature were obtained after removing duplicates. After further screening, 10 Chinese literature were included, with a total of 1318 patients. Te literature screening process and results are shown in Figure 1.

Basic Features of the Included Literature.
A total of 1318 patients were included in 10 studies, including 668 in the experimental group and 650 in the control group. Te included basic characteristics are shown in Table 1.

Risk Assessment of Bias in the Included Studies.
Te Cochrane Risk Assessment Scale was used to assess the risk of bias and the quality of the 10 included articles was evaluated. In this study, 1 literature [7] was grouped according to the order of medical treatment and evaluated as "high risk," while the other 9 literature mentioned random grouping and were all random number table and evaluated as "low risk." Eight studies did not mention distribution hiding and blindness, which was evaluated as "unbelievable risk." Two literature [10,16] mentioned distribution hiding and blindness, which was evaluated as "low risk." Five literature [7,8,10,12,13] did not clearly explain the data integrity and were evaluated as "unclear risk," while the other fve literature all had clear outcome indicators and were evaluated as "low risk." No repeated publication or published bias was found in all studies, and the evaluation was "low risk." Other bias is unknown and evaluated as "unclear risk." Te overall risk of bias for included studies is shown in Figure 2.

Meta-Analysis Results
(1) Evaluation and Analysis of Prevention of ISR. A total of 10 literature [7][8][9][10][11][12][13][14][15][16] reported the efective rate of clinical treatment for the prevention of in-stent restenosis, and the heterogeneity test result was (Chi 2 � 3.50, DF � 9 (P � 0.94), I 2 � 0%). Due to high experimental homogeneity and zero experimental heterogeneity, the fxed-efect model was adopted. According to the results of meta-analysis (OR � 0.37, 95% CI (0.26, 0.51), Z � 5.97, P < 0.00001), the efective rate of the experimental group was signifcantly higher than that of the control group, and the frequency of ISR in the experimental group was signifcantly lower than that of the control group, with a statistical signifcance. Terefore, it can be considered that Tongxinluo capsules combined with conventional cardiovascular drugs can improve the clinical treatment efciency of preventing ISR (See Figures 3 and 4).
(2) Prevention of AP Analysis. A total of 8 literature [7,8,[10][11][12][13][14]16] reported the clinical efectiveness of prevention of angina pectoris, and the heterogeneity test results were (Chi 2 � 20.17, DF � 7 (P � 0.005), I 2 � 65%). Due to high heterogeneity, the random-efect model was adopted. According to the results of meta-analysis (OR � 0.44, 95% CI (0.30, 0.64), Z � 4.23, P < 0.0001), the efective rate of the experimental group was signifcantly higher than that of the control group, while the number of angina cases in the experimental group was signifcantly lower than that in the control group with a statistical diference. Terefore, it can be considered that Tongxinluo capsules combined with conventional cardiovascular drugs group can improve the clinical efective rate of prevention of angina pectoris (See Figure 4).

Evidence-Based Complementary and Alternative Medicine
(3) Analysis of Prevention of MI. A total of 6 literature [8, 10-12, 14, 16] reported the clinical efectiveness of prevention of angina pectoris, and the heterogeneity test result was (Chi 2 � 3.84, DF � 5 (P � 0.57); I 2 � 0%). Due to homogeneity, the fxed-efect model was adopted. According to the results of meta-analysis (OR � 0.73, 95 %CI (0.37, 1.44), Z � 0.91, P < 0.36), the results showed that the efective rate of the experimental group was not signifcantly higher than that of the control group, which was not statistically signifcant. Terefore, it is not considered that Tongxinluo capsules combined with conventional cardiovascular drugs can improve the clinical treatment efciency of preventing myocardial infarction (See Figure 5).
(4) IL-6 Level. A total of 3 literature [9,14,15] reported the level of infammatory factor IL-6, and the heterogeneity test result was ( Chi 2 � 9.69, DF � 2 (P � 0.008), I 2 � 79%), indicating large heterogeneity, so the random efect model was adopted. According to the meta-analysis results (MD � −2.85, 95% CI (−4.24, −1.45), Z � 4.00, P < 0.001), there was a statistical diference between the treatment group and the control group. Te level of IL-6 in the treatment group was signifcantly lower than that in the control group, which proved that Tongxinluo capsules had a good function of improving the IL-6 level (See Figure 6).

(5) Incidence of Total Adverse Cardiovascular Events (TACE).
A total of 3 literature [8,10,16] reported total adverse cardiovascular events, and the heterogeneity test result was (Chi 2 � 5.86, df � 2 (P � 0.05); I 2 � 66%). Due to high heterogeneity, the random efect model was adopted. According to the results of meta-analysis (MD � 0.21, 95% CI (0.09, 0.50), Z � 3.50, P � 0.0005), there was a statistical diference between the treatment group and the control group. Te number of patients having adverse cardiovascular events in the treatment group was signifcantly lower than that in the control group, so Tongxinluo capsules can be considered to have a good therapeutic efect in adverse cardiovascular events (See Figure 7).

Construction and Analysis of the Protein Interaction
Network. Te intersection targets were imported into STRING database to obtain the protein interaction network and save it in TSV format. Te Node1, Node2, and combined score information in the fle are imported to Cytoscape V3.6.1 software, and the "Network Analyser" function in Cytoscape software is used for topology analysis of PPI Network, taking Degree value as reference. We calculate the "connectivity," "closeness centrality (CC)," and "betweenness centrality (BC)" of all nodes to determine the key targets in the network.2.2.4 GO biological analysis, and KEGG Pathway Analysis R3.6.1 software was used for enrichment analysis of GO biological process and KEGG signal pathway, and histogram, bubble diagram, and pathway diagram were drawn for visualization. GO bioanalysis was explained and annotated from three aspects: biological process, molecular function, and cellular location.

Ingredient Screening of Tongxinluo Capsules.
Te ADME and prediction target component scores of Tongxinluo capsules were searched by TCMSP and BAT-MAN-TCM databases, and OB ≥ 30% and DL ≥ 0.18 were obtained. Score ≥ 20 and P ≥ 0.5 were the screening conditions. A total of 101 chemical components were retrieved after weight removal. See Table 2 for details. Drug component-target network diagram is shown in Figure 9.

Retrieval Results of Chemical Component Targets and
Coronary Artery Restenosis Targets. Te chemical targets of Tongxinluo capsules were obtained by searching TCMSP and BATMAN-TCM databases, and 678 targets were obtained after weight removal. A total of 763 disease targets were identifed in GeneCards and OMIM databases using "coronary restenosis" as the key word. Te drug action targets and disease targets were mapped and compared, and 149 intersection targets were obtained. Te intersection results are shown in Figure 9.

Protein Interaction Network Construction and Analysis
Results. Te common targets were input into STRING database, the species were defned as human, the protein interaction relationship was obtained, and the TSV format fle was saved and imported into Cytoscape software for visualization. Te results are shown in Figure 10, and the topology parameter analysis is shown in Table 3 (the frst 30). R3.6.1 software was used to conduct GO enrichment analysis on the intersection target of Tongxinluo capsules and coronary artery restenosis. "Receptor ligand activity," "carboxylic acid binding," "receptor ligand activity," and 148 items including "steroid hormone receptor activity" (P < 0.05, Figure 11). KEGG pathway analysis found "Lipid and atherosclerosis," "MAPK signaling pathway," and "nonalcoholic fatty liver" disease and 180 related pathways. Te result is shown in Figure 12

Signifcance of Tis Study. PCI is currently the main means for the treatment of acute coronary syndrome (ACS).
Although PCI can efectively and quickly relieve the symptoms of patients, the incidence of postoperative coronary artery restenosis is still high, between 20% and 30%, accompanied by the occurrence of AP and MI, mentioned adverse cardiac and cerebrovascular events (MACCE), etc.
Te Tongxinluo capsule is widely used in the treatment of cardiovascular diseases. Its main components are ginseng, borneol, leeches, centipede, turtle worm, peony root, etc., which have the efects of antiplatelet aggregation and antithrombosis and lowers blood lipids. Combination drug therapy can efectively reduce the incidence of restenosis after PCI.
Network pharmacology partial coronary restenosis ISR is a serious problem in patients undergoing PCI. In this study, the ADME and predictive target component scores of single drug in Tongxinluo capsules were searched through the advantages of network pharmacology, which is broad, multiaspect, and multitarget, to explore the possible mechanisms of active components, key targets, and potential pathways in the treatment process, so as to provide scientifc basis for the treatment of coronary artery restenosis by Tongxinluo capsules.

Active Component Analysis.
A total of 101 efective components related to Tongxinluo capsule in the treatment of coronary artery restenosis were identifed. However, due to the complex composition of the Tongxinluo capsule, the core components in the treatment of coronary artery restenosis cannot be determined temporarily.      Evidence-Based Complementary and Alternative Medicine restenosis. Trough KEGG pathway analysis of common targets, it was found that the age-rage signaling pathway may indirectly improve diseases such as angiogenesis, infammation, and atherosclerosis through a series of signal transduction by acting on AGEs molecules. It can promote the treatment of coronary artery restenosis with Tongxinluo capsules. Among them, the age-rage signaling pathway has an obvious mechanism of action and ranks high in the signaling pathway. Te Tongxinluo capsule may ameliorate coronary restenosis through the age-rage signaling pathway. Te specifc infuence mechanism is as follows: aging, infammation, oxidative stress, ischemia reperfusion, and other diseases can indirectly afect AGEs molecules, activate and promote RAGE genes, and indirectly afect PLC genes, thereby promoting DAG and IP3 molecules. DAG directly promotes PKC gene, while IP3 indirectly promotes calcium ion, and calcium directly promotes PKC gene. PKC gene indirectly acts on ERK1/2, P38, and JNK genes in three pathways, respectively. Erk1/2 and P38 indirectly act on AP-1, while P38 and JNK indirectly act on NF-κB. It directly acts on VEGF, McP-1, TNF-α, IL-6, IL-1, and IL-8 genes, and ultimately indirectly improves angiogenesis, infammation, and atherosclerosis (shown in Figure 13).
Te improvement of coronary restenosis by Tongxinluo capsules may be related to the cellular senescence signaling pathway. When afected by external oxidative stress, ionizing radiation, telomere shortening, and loss of tumor inhibition, SIRT1 gene is indirectly inhibited, and FOXO3 gene is directly inhibited, p21 gene is directly promoted through ionization, CDK2 gene and CycE gene are directly inhibited, RB gene is directly inhibited by phosphorylation, and fnally E2F gene is directly inhibited. Finally, cell cycle arrest, senescence related chromatin lesion, and lysosome content increase were indirectly afected, that is, cell senescence was ultimately afected (shown in Figure 14).
In addition, studies have found that cell senescence signals are also associated with coronary artery restenosis. When stimulated by external adverse factors, cell cycle arrest is indirectly afected, and senescence-related chromatin lesions and lysosome content are increased, that is, cell senescence is ultimately afected and coronary artery restenosis is promoted.
In summary, through this study, the main medicinal components of Tongxinluo capsules as well as its possible targets and related pathways were preliminarily clarifed. Te method is relatively complete and the screening results are supported by relevant literature. Subsequent cell or animal experiments on the predicted results are required, which provides ideas and directions for more subsequent studies.

Clinical Efcacy Analysis.
Te 10 studies included in this study analyzed the clinical efcacy of Tongxinluo capsules combined with conventional chemical therapy compared with conventional combined therapy in preventing the occurrence of coronary artery restenosis. Compared with conventional chemotherapy, Tongxinluo capsules combined with conventional chemotherapy has a better preventive efect and can efectively reduce the postoperative incidence of ISR, AP, IL-6, and total adverse cardiovascular events but has no signifcant efect on the postoperative prevention of MI.

Recommendations for Clinical Studies.
Tis study has a certain reference value for the clinical treatment of coronary artery restenosis. Te results of this study showed that Tongxinluo capsules combined with conventional chemical drugs had better efect than conventional combined drugs and no signifcant adverse reactions were observed. Terefore, in the future clinical treatment of coronary artery restenosis, Tongxinluo capsules can be used on the basis of conventional combined drugs according to patients' conditions to achieve a better therapeutic efect. In addition, it is suggested that researchers should clearly record the specifc methods of blind uncovering and allocation of conceals in future clinical studies and carefully record various indicators and clinical trial reports to minimize the risk of deviation.     (1) Only two of the literature included in this study MACCE, so the sample size was insufcient to conduct efectiveness analysis; (2) the quality of the literature included in this study was uneven, only two literature clearly pointed out that the blind method was applied, so various biases might be generated; (3) a small number of research literature were included, resulting in a small sample size, which may produce bias; (4) the course of treatment and dose selected in the included literature are inconsistent, which may cause bias; (5) most of the included literature have few indicators and cannot be efectively analyzed; (6) network pharmacology is based on the results of network data and computational simulation and has not been verifed by in vitro and in vivo experiments.

. Conclusion
Based on the meta-analysis and network pharmacology research techniques, this study frst evaluated the clinical efcacy of Tongxinluo capsules in treating ISR and analyzed its potential mechanism of action. Te results showed that in 10 studies, the experimental group was signifcantly better than the control group in the treatment of ISR, AP, infammatory cytokine IL-6, and the incidence of total adverse cardiovascular events. However, there was no signifcant diference in the treatment of MI between the control and treatment groups. A total of 101 efective components related to Tongxinluo capsules in the treatment of coronary artery restenosis were identifed. Tere were 148 related pathways, mainly involving the age-rage signaling pathway and cell senescence signaling pathway, etc. Tongxinluo capsules may improve coronary artery restenosis through the age-rage signaling pathway and cell senescence signaling pathway.

Conflicts of Interest
Te authors declare that they have no conficts of interest.