Effect of Xuefu Zhuyu Capsule on Myocardial Infarction: Network Pharmacology and Experimental Verification

Background Myocardial infarction (MI) is the most severe manifestation of cardiovascular disease. Xuefu Zhuyu Capsule (XFC), a proprietary Chinese medicine, is widely used in various cardiovascular diseases. At present, the molecular mechanism of XFC remains unclear. Objective To explore the mechanism of anti-MI effects of XFC by combining network pharmacology and experiments. Methods TCMSP, GeneCards, and DisGeNET databases were used to find the target of XFC. PPI analysis was performed by the STRING database. KEGG and GO analyses were performed by Metascape Database. Molecular docking was performed by Autodock Vina. HE staining, echocardiography, immunofluorescence, and TUNEL were performed to verify the prediction results. Results Network pharmacology showed that quercetin, kaempferol, β-sitosterol, luteolin, and baicalein were the main active ingredients of XFC. TNF, IL6, TP53, VEGFA, JUN, CASP3, and SIRT1 were the main targets of XFC. KEGG results showed that key genes were mainly enriched in lipid and atherosclerosis, PI3K-Akt signaling pathway, MAPK signaling pathway, and NF-κB signaling pathway. HE staining showed that XFC could improve the morphology of myocardial tissue. Echocardiography showed that XFC could improve cardiac function. TUNEL showed that XFC could reduce cardiomyocyte apoptosis. Immunofluorescence showed that XFC could reduce the expression of α-smooth muscle actin (α-SMA) and increase the expression of CD31. In addition, we found that XFC may exert its therapeutic effects through SIRT1. Conclusion This study demonstrated that SIRT1 may be the target of XFC in the treatment of MI. The active ingredients of XFC and SIRT1 can be stably bound. XFC could inhibit apoptosis, promote angiogenesis, and improve myocardial fibrosis through SIRT1.


Introduction
Myocardial infarction (MI) is the most severe manifestation of cardiovascular disease (CVD), which afects more than 7 million individuals worldwide each year [1]. Te usual initiating mechanism for MI is the rupture or erosion of a vulnerable, lipid-laden, atherosclerotic coronary plaque, resulting in exposure of circulating blood to a highly thrombogenic core and matrix materials in the plaque [2]. Te method of saving ischemic myocardium from MI is timely reperfusion, including drug thrombolysis and percutaneous coronary intervention; however, the process of reperfusion may lead to arrhythmia, myocardial stunning, microvascular obstruction, and lethal myocardial reperfusion injury [3,4].
Network pharmacology is a new subject based on systems biology and bioinformatics, which can elucidate the mechanism of drug action at the molecular level [13,14]. Te comprehensive and systematic concept of network pharmacology accords with the characteristics of multicomponent, multitarget, and multipathway of TCM. Terefore, in this study, we aimed to explore the active ingredients, therapeutic targets, and signaling pathways of XFC in the treatment of MI through network pharmacology and in vivo. Our study may provide evidence for the pharmacological efects of Chinese herbal compounds on MI. A fowchart of the study approach is shown in Figure 1.  [15], the TCMSP parameter was set as bioavailability (OB) ≥30% and drug-like properties (DL) ≥0.18, as suggested by the TCMSP. Disease targets of MI were collected by the GeneCards database (https://www.genecards.org/) [16] and the DisGeNET database (https://www.disgenet.org/) [17]. Te verifed and predictive targets of XFC to treat MI were obtained by overlapping these targets.

Protein-Protein Interaction (PPI) and Gene Enrichment
Analysis. PPI analysis of overlapping targets was performed using the STRING platform [18], and the calculation results were imported into Cytoscape 3.9.0 software [19] for network topology analysis. Screen for key targets using the CytoHubba plugin. Gene enrichment analysis of overlapping targets was performed through the Metascape database [20]. It mainly includes molecular function (MF), cellular components (CC), and biological process (BP), KEGG. Te parameters of the Metascape database were set as follows: min overlap: 3, P value cutof: 0.01, and min enrichment: 1.5. Hiplot (https://hiplot-academic.com/) [21] is used to visualize the results.

Experimental Validation
2.2.1. Drug and Animals. XFC was purchased from Tianjin Hongrentang Pharmaceutical Co., Ltd. (Approval number: Z12020223). EX527 was purchased from Selleckchem, USA, Product No: S1541, male Wistar rats (weighing 190-210 g), purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China). All rats were housed in specifc pathogen-free animal rooms in the laboratory animal center of Guang'anmen hospital, China Academy of Chinese Medical Sciences. Te temperature of the animal room is 20-25°C, and the relative humidity is 55-60%; all rat feeding methods and animal experiment procedures strictly follow the relevant guidelines stipulated by the experimental animal ethics committee of Guang' anmen Hospital, China Academy of Chinese Medical Sciences.

Model of MI and Drug
Treatment. Te rats' model of MI was established by left anterior descending (LAD) ligation as previously described [24]. Rats were anesthetized with pentobarbital sodium (60 mg/kg, i.p.). An incision was made between the 4th and 5th intercostal space on the left side of the rat, the thoracic cavity was opened, the pericardium was torn, and the thorax was gently pressed to squeeze out the heart. Find the left atrial appendage and determine the ligation position (2 mm below the junction of the pulmonary conus and the left atrial appendage). Te LAD was ligated at the ligation site, the heart was immediately put back into the thoracic cavity, the incision was closed, and the skin was sutured. Animals in the sham group were only threaded without ligation. An electrocardiograph was performed immediately after modeling, and the ST-Segment elevation was the standard screening model and the rats without ECG changes were excluded. After the operation, the surviving rats were randomly divided into the following four groups: (1) sham-operated group (without LAD ligation); (2) model group (LAD ligation); (3) XFC treated group (LAD ligation and intragastrically administered XFC at 0.432 g/kg [25]); (4) EX527 (SIRT1 inhibitor) group (LAD ligation, intragastrically administered XFC at 0.432 g/kg and intraperitoneal injection of EX527 at 5 mg/ kg) [26]. Te sham group and model group were given equivalent distilled water. All groups began drug intervention within 12 hours after modeling. Te drugs were administered once a day for 4 weeks.

Echocardiographic Evaluation of Cardiac Function.
After 4 weeks of administration, the Vevo 3100 Imaging System (Fujiflm Visual Sonics Vevo 3100, Toronto, Canada) was used to observe and record the echocardiographic of rats. After the rats were anesthetized with isofurane, the rats were fxed in a supine position, and the skin was prepared in front of the left chest. Te RMV-716 high-frequency probe was used to take the short axis of the left ventricle, and the level of the mitral valve of the rat heart was detected. Te average value of 3 consecutive cardiac cycles was taken for each group. Ultrasound detection indexes: left ventricular short-axis ratio (FS) and left ventricular ejection fraction (EF).

Histological Analysis.
After echocardiography, the rats were euthanized, anesthetized by intraperitoneal injection of 1% pentobarbital, fxed in the supine position, quickly opened the chest, and the hearts were removed. Some samples were transferred to a refrigerator at −80°C. Another portion of the sample was fxed with 4% paraformaldehyde and used for subsequent studies. Hematoxylin-eosin (HE) staining was prepared according to standard procedures. Te rat heart tissue was fxed, dehydrated, embedded in parafn, and sliced along the longitudinal axis with a thickness of 4 μm. After HE staining (Beyotime, China), the morphology of cardiac tissue cells was observed under the microscope.

Immunofuorescence Staining.
Immunofuorescence staining of CD31, α-smooth muscle actin (α-SMA), and c-TnI was performed. Briefy, the procedure was as follows: sections were prepared, washed 3 times with PBS, repaired with citrate bufer, washed 3 times with PBS, blocked with sheep embryo serum at 37°C for 15 min, washed 3 times with PBS, incubated with primary antibody at 4°C for 17 h, washed 3 times with PBS, incubated with fuorescently labeled secondary antibody at 37°C for 30 min, washed 3 times with PBS, sealed with DAPI, and photographed with the confocal microscope. Ten felds were randomly selected.

Evidence-Based Complementary and Alternative Medicine
Image J software was used to measure the integrated option density (IOD) value of the stained positive part in the captured Image, and the arithmetic mean was taken as the IOD value of the section.

TUNEL Assay.
Cardiomyocyte apoptosis was detected by the In Situ Cell Death Detection Kit (11684817910, Roche, Switzerland) according to the manufacturer's protocol. Briefy, the procedure was as follows: parafn section, dewaxing, hydration, cell permeation, adding TUNEL reaction solution, adding converter-POD, reaction with substrate DAB, counting, and photographing (FV1000, Olympus, Japan). Finally, use Image J software to analyze the pictures, calculate the number of positive cells and total cells in each picture, calculate the percentage of apoptosis, and make a statistical analysis.

Statistical Analysis.
Statistical analysis was performed using GraphPad Prism 8. All data are presented as the mean-± standard deviation (SD). One-way analysis of variance followed by Tukey's test was used for multiple comparisons among groups. P < 0.05 was considered statistically signifcant.

Analysis of Drug and Disease Targets.
A total of 152 active ingredients and 253 targets corresponding to the active ingredients were collected and 2345 disease targets were collected. Te list of the top 20 active ingredients was drawn according to OB values (Table 1). Te intersection of drug targets and disease targets was calculated, and 147 overlapping targets were obtained (Figure 2(a)). Te ingredients-target network fgure (Figure 2(b)) was drawn by active ingredients and targets. From this fgure, it can be seen that quercetin, kaempferol, β-sitosterol, luteolin, baicalein, stigmasterol, naringenin, isorhamnetin, wogonin, and nobiletin correspond to many targets with large degree values. It is speculated that XFC treatment of MI is mainly these active ingredients with large degree values.

Gene Enrichment Analysis of XFC against MI.
PPI processing was conducted on the overlapping targets through the STRING platform, and Cytoscape was used for network topology analysis (Figure 3). Te key targets were TNF, IL6, TP53, VEGFA, JUN, CASP3, PTGS2, MAPK3, EGFR, STAT3, and SIRT1. Gene enrichment analysis was conducted on the overlapping targets through the Metascape database. KEGG results showed that key genes were mainly enriched in lipid and atherosclerosis, PI3K-Akt signaling pathway, MAPK signaling pathway, NF-κB signaling pathway, cAMP signaling pathway, and calcium signaling pathway (Figure 4(a)); CC results showed that key genes were mainly enriched in membrane raft, transcription regulator complex, vesicle lumen, receptor complex, and integral component of presynaptic membrane (Figure 4(b)). BP results showed that key genes were mainly enriched in the cellular response to nitrogen compound, response to an inorganic substance, response to the hormone, response to xenobiotic stimulus, and cellular response to lipid (Figure 4(c)). MF results showed that key genes were mainly enriched in DNA-binding transcription factor binding, oxidoreductase activity, kinase binding, protein homodimerization activity, and phosphatase binding (Figure 4(d)). Based on our previous studies and network pharmacology results [12,27], we selected SIRT1 as the validation gene among these targets.

Molecular Docking Verifcation.
Quercetin, kaempferol, β-sitosterol, luteolin, and baicalein were selected as binding ligands, and SIRT1 was selected as the target protein. Molecular docking was performed by Autodock Vina. Te results showed that the docking energy was ≤−7 kcal·mol −1 . It can be seen from Figure 5 that the ligands are stably combined in the binding pocket to form a stable structure. For example, quercetin could bind to the SIRT1 via putative hydrogen bonds to the Arg303, Ser370, Leu372, Lys375, Lys377, and Lys408 residues of SIRT1.

Cardiac Function.
Te results showed that compared with the sham group, the EF and FS in the model group were signifcantly decreased (P < 0.01). Compared with the model group, the EF and FS in the XFC group were signifcantly increased (P < 0.01), and the EF and FS in the EX527 group showed an upward trend, but there was no statistical signifcance. Compared with the XFC group, the EF and FS in the EX527 group were signifcantly decreased (P < 0.01) ( Figure 6).

Pathological Changes in Heart
Tissue. Cardiac pathology was compared by HE staining. Te results showed that in the sham group, the myocardial fbers were striated, the Evidence-Based Complementary and Alternative Medicine nucleus of the myocardial cells was in the middle, and the interstitial blood vessels of the myocardial fbers were normal, without interstitial edema and infammatory cell infltration. In the model group, the myocardial space of the ischemic heart was enlarged, the fber arrangement was disordered, and a large number of infammatory cells infltrated. Te changes in the XFC group and the EX527 group were between the sham group and the model group, and the degree increased in turn (Figure 7(a)).

Changes in Cardiomyocyte
Apoptosis. TUNEL assay detects cardiomyocyte apoptosis. Te results showed that compared with the sham group, the apoptotic rate of cardiomyocytes in the model group was signifcantly increased (P < 0.01); compared with the model group, the apoptosis rate of cardiomyocytes in the XFC group was signifcantly decreased (P < 0.01). Compared with the XFC group, the apoptosis rate of cardiomyocytes in the EX527 group was signifcantly increased (P < 0.01) (Figure 7(b)).

Changes in Myocardial Fibers and Angiogenesis.
Cardiac fbrosis is the main pathological feature of MI, and fbrosis is usually caused by excessive accumulation of extracellular matrix. Myofbroblasts are the main source of extracellular matrix, and they express the high-contractile protein α-SMA [28,29]. Te immunofuorescence of α-SMA can be  Evidence-Based Complementary and Alternative Medicine used to evaluate the degree of myocardial fbrosis. Te results showed that compared with the model group, α-SMA in the XFC group and EX527 group was signifcantly decreased (P < 0.01). Compared with the XFC group, α-SMA in the EX527 group was signifcantly increased (P < 0.01) (Figure 8(a)). Angiogenesis plays an important role in the recovery process of MI. As a marker of endothelial cells, CD31 represents the change in capillary density [30]. Compared with the sham group, CD31 in the model group was signifcantly increased (P < 0.01). Compared with the model group, the CD31 in the XFC group and the EX527 group was signifcantly increased (P < 0.01   Evidence-Based Complementary and Alternative Medicine in the EX527 group was signifcantly decreased (P < 0.01) (Figure 8(b)). cTnI is a common method and index for clinical diagnosis of MI. AMI is usually accompanied by an increase in cTnI [31]. Compared with the sham group, the proportion obligation as previously described of cTnI in the model group was signifcantly increased (P < 0.01). Compared with the model group, the proportion of cTnI in the XFC group and the EX527 group was signifcantly decreased (P < 0.01). Compared with the XFC group, the proportion of cTnI in the EX527 group was signifcantly increased (P < 0.01) (Figure 8(c)).

Discussion
Cardiac repair after MI is accomplished through a series of complex events. Tese events begin with infammation that helps digest and removes damaged cells and extracellular   matrix tissues, followed by a repair phase with infammation subsiding, fbroblast proliferation, scarring, and new angiogenesis in the following days [32]. Herbal formula contains hundreds of chemical compounds, which makes it complicated and challenging to understand the mechanisms of action and bioactive ingredients. Network pharmacology integrates data, and carries out experimental and clinical studies to provide a powerful tool for exploring the mechanisms of TCM [33]. In this work, we integrated network pharmacology, molecular docking, and experimental verifcation methods to clarify the molecular biological mechanism of XFC in the treatment of MI.
In this study, we frst identifed the active ingredients and targets of action of XFC, while obtaining disease targets for MI. We found that quercetin, kaempferol, β-sitosterol, luteolin, baicalein, stigmasterol, naringenin, and isorhamnetin were likely to be the main active ingredients of XFC to exert anti-MI. Most of these active ingredients are favonoids; favonoids can improve CVD, such as reducing the brittleness of blood vessels, improving the permeability of blood vessels, reducing lipids and cholesterol, and preventing hypertension [34][35][36]. For example, Albadrani et al. found that quercetin ameliorates MI in rats by decreasing TGF-β1/Smad3 signaling [37]. Wang et al. demonstrated that kaempferol ameliorates oxidative stress and infammation by activating the PI3K/Akt/GSK-3β pathway, thereby reducing myocardial ischemia/reperfusion injury [38]. We next performed a PPI analysis on 147 overlapping targets to obtain the key targets among them. Tese key targets are mainly involved in apoptosis (TP53 and CASP3), immune infammation (TNF, IL6), and angiogenesis (VEGFA), which are closely related to the molecular mechanisms of MI. MI evokes activation of the innate immune system, resulting in increased levels of proinfammatory cytokines (IL-1β, IL6, and TNF-α) in the heart and circulation. Te TNF-mediated process is complex. TNF can show toxicity through TNFR1 and protection through TNFR2, showing bidirectional efects in MI [39]. VEGFA is a highly specifc vascular endothelial growth factor that promotes angiogenesis and increases vascular permeability. VEGFA plays a key role in triggering the cardiac angiogenic response following MI. VEGFA can inhibit cardiomyocyte apoptosis, promote cardiac repair, and promote vasodilation [40,41].
Combining our previous study and this bioinformatics analysis, we chose SIRT1 as an entry point among these key targets. First, we molecularly docked SIRT1 to the main active ingredients and found that SIRT1 can and stably bind,  Evidence-Based Complementary and Alternative Medicine which confrmed that SIRT1 might be one of the targets of XFC. Ten, we verifed our hypothesis through experiments. Our data suggested that XFC can efectively improve cardiac function, myocardial fbrosis, and cardiomyocyte apoptosis in MI model rats. Interestingly, SIRT1 inhibitors could inhibit the efects of XFC, which confrms that SIRT1 may be the target of XFC. SIRT1, an NAD+-dependent protein deacetylase, is capable of deacetylating histones and nonhistone substrates that have a powerful regulatory role and can regulate a variety of important physiological and pathological processes in cells, such as cell metabolism, gene repair, cell survival, cell senescence, apoptosis, infammatory response, oxidative stress, and vascular protection [42].
Previous studies have confrmed that activation of SIRT1 can alleviate MI injury [42,43]. Based on this, we hypothesized that the active component of XFC could activate SIRT1 to exert its anti-MI efect. Te efect of promoting blood circulation and eliminating blood stasis in Chinese medicine is similar to that of angiogenesis. Recanalization and angiogenesis in the infarcted region are essential for the survival of cells in the myocardium. CD31 is an endothelial cell-specifc marker that can be used to label vascular endothelial cells. α-SMA is a eukaryotic skeletal protein involved in the composition of microflaments and is a marker protein for mesenchymal cells [44][45][46]. Our data suggest that XFC improved the expression of CD31 and α-SMA and that SIRT1 inhibitors eliminate this efect, so we hypothesize that XFC can exert a neovascularizing efect through SIRT1. Indeed, SIRT1 has been shown to improve CD31 and a-SMA expression, and this evidence further supports the reliability of our conclusions [47,48].

Conclusion
In summary, we predicted by network pharmacology that SIRT1 might be a target of XFC against MI. Te in vivo study showed that XFC could inhibit apoptosis, promote angiogenesis and improve myocardial fbrosis through SIRT1.

Data Availability
All data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Tis study was reviewed and approved by the ethics committee of Guang'anmen Hospital, China Academy of Chinese Medical Sciences.

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