Understanding the Multitarget Pharmacological Mechanism of the Traditional Mongolian Common Herb Pair GuangZao-RouDouKou Acting on Coronary Heart Disease Based on a Bioinformatics Approach

GuangZao and RouDouKou (Fructus Choerospondiatis and Nutmeg, FCN) are one of the most common herb pairs in traditional Mongolian medicine for the treatment of coronary heart disease (CHD). However, evidence for the protective effect of FCN is limited, and its underlying mechanism of action remains unclear. The present study employed a network pharmacology approach to identify the potentially active ingredients and synergistic effects of the herb pair FCN as traditional Mongolian medicine. We predicted the targets of all available FCN ingredients with PharmMapper, SWISS, and SuperPred Server and clustered CHD-related targets from the DrugBank and the OMIM database. We also evaluated the links between herbal ingredients and pharmacological actions to explore the potential mechanism of action of FCN. We found that FCN targets a network of CHD-related key processes, including stress responses, cell adhesion and connections, angiogenesis, cell apoptosis and necrosis, the endocrine system, inflammatory and immune responses, and other biological processes. To confirm the predicted results, we investigated the protective effect of FCN on isoproterenol- (ISO-) induced myocardial ischemia in rats. Pathological assessment indicated that FCN inhibits apoptosis and inflammatory responses involving the myocardium. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analyses demonstrated the therapeutic effects of FCN on ISO-induced myocardial ischemia rats, possibly via regulating stress and inflammatory responses and inhibiting cardiomyocyte apoptosis. The findings of the present study indicate that bioinformatics combined with experimental verification provide a credible and objective method to elucidate the complex multitarget mechanism of action of FCN.


Introduction
Coronary heart disease (CHD) remains the leading cause of death in adults worldwide. CHD is a complex multifaceted condition. The main causes of CHD include myocardial ischemia, hypoxia, necrosis, and coronary atherosclerosis [1,2]. Because CHD is the final manifestation of various pathological insults, there is a strong demand to find safe and efficacious multitargets products to combat this health epidemic. Traditional Mongolian medicine has been used to prevent and treat cardiovascular disease for many years using various herbal formulas and has lately been the subject 2 Evidence-Based Complementary and Alternative Medicine of formal clinical studies. Herb pairs have been considered the halfway point between single herbs and herbal formulas, and typical herb pairs display basic synergistic effects of two kinds of herbs that have often been used in various herbal formulas with similar signs and symptoms. The GuangZao and RouDouKou (Fructus Choerospondiatis and Nutmeg, FCN) herb pair is commonly used in the treatment of CHD in traditional Mongolian medicine [3,4]. The present study aimed to evaluate this herb pair as a potential CHD treatment regimen.
In traditional medicine, single herbs, herb pairs, and herbal formulas are treated as single units, and these are composed of diverse, complex components. Their pharmacological effects are generally due to various active monomers that involve multiple pathways and targets. Numerous studies have focused on the mechanism of action of various active monomers in herbs; however, these active monomers apparently have significantly different characteristics. The advent of systems biology and network pharmacology offers a novel opportunity to assess traditional medicine from the perspective of holistic regulation [5,6].
The present study was designed to investigate the anti-CHD action of FCN based on network pharmacology, which encompasses identification of the components of FCN, CHD gene prediction-based data mining, drug target prediction, protein-protein interactions (PPI), drug-gene-disease comodule association, drug network analysis, and synergistic ingredients combination screening. Finally, an acute myocardial ischemia (AMI) experiment was performed on rats to confirm the mechanism of action of the FCN herb pair on AMI, including its targets and pathways. A schematic representation of our method is shown in Figure 1.  21st day. Model rats and herb-treated rats each received 0.3% CMC-Na solution/GuangZao/RouDouKou/FCN for a period of 21 days and 85 mg/kg of ISO hydrochloride (ISO (Shanghai, China), 1 mL/kg, s.c.) on the 20th and 21st days to induce myocardial injury, and the doses of three herb-treated groups were 650 mg/kg/day, i.g. All animals received standard diet and water ad libitum. The experimental protocols were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Inner Mongolia Medical University and approved by the Committee of Animal Ethics.

Electrocardiography.
At the end of the experimental period, ECG were recorded by biological data acquisition and analysis system (BL-420F, Techman Soft, China). Under chloral hydrate (400 mg/kg, i.p.) anesthesia, blood was collected via the abdominal aorta. After clotting the blood, the serum was separated by centrifugation at 3,000 rpm, 10 min. The serum and heart tissues were stored at −80 ∘ C until further analysis.

Assay of Serum Myocardial Injury Markers.
Activity of serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) was measured using commercial kits (Biosino Bio-Technology and Science, Inc., Beijing, China) with an automated chemical analyzer (Biobase-Sapphire, Ireland).

Infarct Size Determination.
The hearts of rats that had been pretreated in different ways were sliced into six sections along the long axis, incubated in 2% TTC [2,3,5-triphenyltetrazolium chloride dissolved in PBS (0.1 mol⋅L-1)] for 20 min at 37 ∘ C. The infarct area (IA) was determined and calculated as described by Zhang [28]. Briefly, images were captured and the area was adjusted for weight. IA/LV% were calculated according to the formulas: where Wn is the weight of each heart section; WT is the weight of the entire heart; and In is the percentage of infarct area (IA) that was white in color in each section.

Histopathological Analysis.
Myocardial tissue was fixed in 10% buffered formalin at least 48 h. Subsequently, the fixed tissues were embedded, sectioned, and stained (H&E). The specimens were examined under a light microscope (DM4000, Leica Germany) by experienced pathologists.

Statistical
Analysis. Data are presented as mean ± SD. One-way ANOVA was used to determined statistical differences, and Tukey's test was performed for statistical comparisons among the groups. The S-N-K test was used when there was homogeneity of variance, and the chi-square test was employed to assess incidence. P< 0.05 was considered statistically significant.

Establishment of the Network of Anti-CHD Targets of FCN and Topological
Analysis. GuangZao (F. Choerospondiatis), a widely known Mongolian herb, derived from the dried fruit of C. axillaris (Roxb.) Burtt et Hill. Its preparation is described in traditional Mongolian medicinal theory. It has been used extensively as a remedy for ischemic heart disease and has been shown to have good clinical efficacy. It contains of several kinds of useful components, including organic acids, phenolic acids, and flavonoids [8][9][10][11][12]. RouDouKou (Nutmeg) is also a valued kitchen spice, but it has been often used for remedies for the stomach, heart, and nerve disorders in traditional Mongolian medicine. RouDouKou is a rich source of fixed and essential oils, triterpenes, and various types of phenolic compounds [13][14][15][16][17][18][19][20][21][22][23][24][25][26]. The essential compounds of GuangZao and RouDouKou are shown in Supplementary  Table S1. Statistical analysis of Mongolian medicine prescriptions for the treatment of cardiovascular disease in "Mongolian Medicine Prescription" and "Medicine Standard (1998 edition)" was conducted using binary quantization (expressed by 1 and 0), and SPSS22.0 was used for cluster analysis. Cluster analysis (Supplementary Figure S1) indicated that, at a distance of 25, the prescription ingredients can be divided into two categories, one for GuangZao (Fructus Choerospondiatis), RouDouKou (Nutmeg), Syzygium aromaticum (Clove), Radix aucklandiae, and Aquilaria agallocha Roxb. (Agilawood) and another for other herbs. The highest degree of association was observed between the GuangZao and RouDouKou.

Extracting Active Components in FCN.
The present study identified 25 components in GuangZao and 38 in RouDouKou, including those by filtering with DL ⩾0.18 and OB⩾30%; constituents migrating to blood, high-content compounds, and compounds listed as active in previous reports were included for further investigation. Altogether, these compounds accounted for 55.3% of the essential compounds are shown in Supplementary Table S2.

Drug Target Prediction and Synergistic Action Analysis.
According to the filters previously described in the methodology, we obtained 453 candidate genes that were targeted by FCN. We found 126 common targets by overlap analysis Evidence-Based Complementary and Alternative Medicine 5 among GuangZao and RouDouKou to prove that this herb pair acts synergistically. By comparing the candidate targets of FCN to known CHD-related genes obtained from OMIM and DrugBank databases, we found 87 CHD-related genes in the candidate targets of FCN, in which 24 genes frequencies greater than 4 were shown in Supplementary Table S3. 3.1.3. Network of Compound-Target and Analysis. The compound-target network was constructed using Cytoscape 3.5.1 software, which included 2,343 protein interactions between candidate targets (Figure 2). After calculation of topological features, if the Degree, Betweenness, and Closeness of this node were greater than median of corresponding parameters of all nodes, then the nodes were designated as hub nodes. Our results showed that the hub nodes included 20 key compounds and 29 key targets.
These 29 key targets included 10 cotargets that are important in the pathogenesis of CHD: mitogen-activated protein kinase 14 (MAPK14/p38), cellular tumor antigen p53 (TP53/p53), serine/threonine-protein kinases 1 (AKT1), prothrombin (F2), and matrix metalloproteinase-9 (MMP9) ( Figure 3). The activation of MAPK14 in the course of myocardial ischemia injury aggravates cellular injury induces inflammation, apoptosis, and other cellular processes [30]. In response to ischemia reperfusion (I/R) stress, p53 has been known to become activated and accumulated in the mitochondrial matrix. This gave rise to ROS generation, subsequently causes transient mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome C, which triggers apoptosis by activating caspase 3 [31]. The action of Akt on its downstream targets determines its function in cardiovascular processes such as cell survival, growth, proliferation, angiogenesis, mediating eNOS phosphorylation, and vasorelaxation [32]. F2 is a common target of antithrombotic drugs, and MMP9 plays an essential role in local proteolysis of the extracellular matrix and in leukocyte migration. The top three in the Betweenness order are p53, ESR1, and p38 in the 10 cotargets of FCN (Betweenness, N = 0.042509, 0.024799, 0.019129). The detailed information of 10 cotargets is listed in Supplementary Table S4. The 20 key compounds included 15 GuangZao, 4 RouDouKou ingredients and 1 common composition, indicating that GuangZao plays significant roles against CHD in FCN. Among the ingredients, gallogen and citric acid found in GuangZao and safrole found in RouDouKou exhibited the highest degrees (N = 116, 102, 100) to CHD, followed by chebulic acid and gallic acid (Degree, N=87, 52) from GuangZao ( Figure 3, Supplementary Table S5).

Network of Compound-Target-Pathway and Analysis.
Then, based on the results of DAVID analysis, we conducted enrichment analysis using KEGG pathways and GO terms according to key targets of FCN. As is shown in Figure 3, the seven enriched pathways and four GO function terms are closely related to CHD, including the pathways of VEGF signaling pathway, PI3K-Akt signaling pathway, leukocyte transendothelial migration, FoxO signaling pathway, HIF-1 signaling pathway, regulation of lipolysis in adipocytes, MAPK signaling pathway, and the GO terms of negative regulation of apoptotic process, angiogenesis, positive regulation of nitric oxide biosynthetic process, and platelet activation. Acute cardiovascular events, on account of rupture or erosion of an atherosclerotic plaque, are the major cause of morbidity and mortality in patients. Intraplaque (IP) neovascularization and hemorrhage, hypoxia, and influx of inflammatory mediators are stimulating factor of plaque rupture. The molecular mechanism of regulating this pathological process involves the HIF-1 signaling pathway, VEGF signaling pathway, angiogenesis, inflammatory response, leukocyte transendothelial migration, and other processes [33]. Reperfusion therapy is believed to be the most effective method of treating acute myocardial infarction (AMI), but it may lead to further damage of ischemic myocardium, which is related to p38/MAPK activation, myocardial apoptosis, PI3K-Akt signaling cascade, endothelial nitric oxide synthase (eNOS) activity, and immune system activation [34,35]. These representative pathways indicate that FCN acts as an anti-CHD agent.

The Effect of FCN on ECG.
To verify the effect of FCN on myocardial ischemia, we established the rat model of myocardial ischemia. Figures 4(a)-4(e) show the ECG patterns of model rats induced with 85 mg/kg ISO (model group, Figure 4(b)) revealed ST segments upward; the incidence of abnormal ECG showed a significant difference between normal and ISO model group ( 2 = 5.495, P = 0.019, P < 0.05). The incidence of abnormal ECG was 50.0% in the GaoZao group and 70% in RouDouKou group (650 mg/kg body weight) (GaoZao and RouDouKou group; Figures 4(c) and 4(d)). The ECG of the major ISO rats pretreated with FCN was generally normal, and the rate of abnormal ECG was 30% (FCN group; Figure 4(e)). Chi-square testing indicated that the difference was statistically significant, with a difference of 2 =9.295, P= 0.002, P<0.05.

TTC Macroscopic Enzyme Mapping Assay.
To determine the extent of myocardial ischemia, the cardiac tissues were sectioned and stained with TTC. Figures 5(a)-5(e) exhibited that the hearts of model rats depicted a large unstained area, with several necrotic patches ( Figure 5(b));

Histological Changes.
We explored further the effect of FCN in ISO-mediated histological changes of hearts. Figures 6(a)-6(e) show that while the heart tissues of control rats showed normal myocardial fibers, extensive disruption and fragmentation of heart myofibrils with visible necrosis, loss of striations, hyperemia, and inflammatory infiltration were observed in the model rats. Administration of RouDouKou, GuangZao, or FCN all reduced the ISO-induced myocardial necrosis in different degrees. The heart tissue of RouDouKoutreated rats showed a small amount of vacuolar degeneration, and the myocardial fibers showed wider gaps. GuangZao group showed evidence of inflammatory infiltration and relatively narrow gaps among myocardial fibers; meanwhile, FCN group merely displayed a small amount of hemorrhage. These results suggested that FCN imparts a therapeutic effect on myocardial ischemia.  Table S4). To verify the results of network analysis, the gene expression of p38 and p53, the downstream proinflammatory cytokines TNF-and IL-1 , and apoptosis-related gene Bcl2 and Bax in each group were detected by qRT-RCR. The levels of p38, p53, TNF-, IL-1 , and Bax dramatically increased in the model rat heart tissues compared to the control (P<0.01). In addition, Bcl-2 expression significantly decreased (P<0.01). Treatment with RouDouKou or GuangZao resulted in the downregulation of p38, p53, TNF-, IL-1 , and Bax in different degrees compared with model group. The p38 levels of the RouDouKou group were lower than those of the GuangZao group (P<0.05), whereas the p53, TNF-, IL-1 , and Bax levels of the GuangZao group were lower than the RouDouKou group. However, Bcl-2 expression and Bcl-2/Bax ratio showed the opposite profiles. The above targets in the FCN group showed similar profiles to those of the RouDouKou or GuangZao groups, and FCN induced more pronounced effects (Figures 7(a)-7(g)).

Western
Blot. MAPK14 (p38) plays an important role in the cascades of cellular responses evoked by cell stresses such as proinflammatory cytokines or physical stress, which is activated through phosphorylation on both Thr-180 and Tyr-182 and further phosphorylate additional targets including TP53(p53) [36]. p53 regulates cell-cycle checkpoint and induction of apoptosis in response to DNA damage. The available evidence suggests that promoter selectivity of p53 is regulated by its phosphorylation [37]. For instance, phosphorylation of Ser15 and Ser20 influences binding of p53 to promoters for cell-cycle arrest and DNA repair genes. An additional phosphorylation of Ser46 following severe DNA damage increases the affinity of p53 for promoters of proapoptotic genes. Gallogen (ellagic acid) and

RQ value
Bcl-2/Bax (g) Figure 7: The FCN alters the expression of genes that are related to stress reaction, apoptosis and necrosis of cardiomyocytes, the inflammatory response. (a-f) mRNA levels of the proinflammatory cytokines TNF-, IL-1 , stress cytokine p38, and the proapoptosis cytokines P53, Bax, and antiapoptosis cytokines Bcl-2, as well as Bcl-2/Bax ratio of the injured myocardium of each intervention group (n = 6 mice per group). Mean values ± SD. are represented. ## P < 0.01 versus the control group, # P < 0.05 versus the control group, * * P < 0.01 versus model, and * P < 0.05 versus model. quercetin in GuangZao and macelignan in RouDouKou are key compounds which exhibited the higher degrees (Degree, N=116,44,32). It has been reported that gallogen could exert its beneficial effects by regulating multiple genes, including several cell survival/cell-cycle genes such as p53, Bcl-2, Bax, and c-Myc and regulation of kinases, like MAPK, phosphoinositide 3-kinase (PI3-K), glycogen synthase kinase 3 beta (GSK-3), and so on [38]. Quercetin and macelignan were reported that the protective effects on hepatoprotective effect or anti-inflammatory may be associated with repression of P-p38 [22,39].
To evaluate the actions of FCN on stress reaction and cardiomyocyte apoptosis, the crucial predicted targets, including p38 and p53 protein levels in each group, were analyzed by western blotting. With different treatments, distinct changes in p38 and p53 expression were not observed among the five groups. Under the conditions of existing methods about network analysis and target prediction, whether small molecules affect protein expression or phosphorylation is not yet distinguishable. To further explore the involvement of p38 and p53, P-p38 and P-p53 protein levels in each group were analyzed. We found that FCN, GuangZao, and RouDouKou groups showed significantly lower levels of P-p38 (P< 0.01 (FCN, RouDouKou) or P< 0.05 (GuangZao), respectively, versus model) compared to the model rats (Figure 8(b)). Moreover, the levels of P-p53 in FCN, GuangZao rats were significantly lower (P < 0.05 (FCN), P < 0.01 (GuangZao), versus model) (Figures 8(a) and 8(b)). RouDouKou group showed lower levels of P-p38 expression than GuangZao (P < 0.05, RouDouKou versus GuangZao), whereas the RouDouKou group expressed lower levels of P-p53 than the RouDouKou (P =0.076, RouDouKou versus GuangZao). These findings suggest that FCN treatment effectively reduced stress reactions and cardiomyocyte apoptosis in ISO-induced rats possibly by interfering with phosphorylation levels of p38 or p53, which further affected downstream genes, rather than protein levels of p38 and p53. Overall, the results were consistent with systematic pharmacological analysis.

Conclusions
FCN, a traditional Mongolian herb pair for CHD treatment in China, can ameliorate ischemia in ISO-induced myocardial ischemia rats. In this study, we developed an integrative systems pharmacology approach to explore the pharmacological mechanisms of FCN against CHD by which a network pharmacology analysis and experimental validation. For the first time, we identified 25 in GuangZao and 38 in RouDou-Kou candidate compounds and they were predicted to bind to 89 CHD-related targets using multiple online platforms. We further defined the molecular mechanisms of FCN against CHD through multilevel data integration, including a compound-target-interacted proteins network, compoundtarget-pathway network, and topological analysis. The results confirm that the combination of GuangZao and RouDouKou represses myocardial ischemia in ISO rats by regulating reaction abilities, which in turn inhibits cardiomyocyte apoptosis and inflammatory responses. Results indicate that GuangZao and RouDouKou are effective for the treatment of CHD.

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

Conflicts of Interest
The authors declare that they have no conflicts of interest.