Mechanisms of Cinnamomi Ramulus for coronary heart disease treatment:prediction based on network pharmacology

Shi-Quan Chang,Yi Lin,Di Zhang,Jian-Xin Sun,Bing Yang,Guo-Ping Zhao＊

1College of Traditional Chinese Medicine,Jinan University,Guangzhou,510630,China.

Abstract

Objective: Guizhi (Cinnamomi Ramulus) has been widely applied in treating coronary heart disease in traditional Chinese medicine.However, little is known about the potential mechanism.Elucidating the effective components and mechanism based on network pharmacology was our purpose. Methods: Traditional Chinese Medicine Systems Pharmacology was screened to collect the possible active ingredients and their CAS and SMILES was searched in Pubchem, and the related potential targets were further predicted in Swiss Target Prediction database.Coronary heart disease molecular members were gained from GeenCards database, and the predicted targets of Cinnamomi Ramulus for coronary heart disease’s treatment were selected by Wayne diagram.As for mechanism analysis,String was used to construct the protein-protein interactions,and DAVID was used to conduct the GO and KEGG analysis. Results: Through GO and KEGG analysis, we found that cAMP signaling pathway, estrogen signaling pathway, calcium signaling pathway was related with coronary heart disease.Using network-based systems biology, we predicted that 10 active ingredients in Cinnamomi Ramulus have the treating effects with 78 potential targets.PIK3CG, MAPK8, BCL-2, BAX, PRKACA, CASP3, CALM1, CALM2, CALM3, NOS2, NOS3 were mainly involved in the treating effects of Cinnamomi Ramulus. Conclusion: Cinnamomi Ramulus may treat coronary heart disease through cAMP signaling pathway, estrogen signaling pathway, calcium signaling pathway.PIK3CG,MAPK8,BCL-2,BAX,PRKACA,CASP3, CALM1,CALM2,CALM3,NOS2,NOS3 were supposed to be considerable targets for treating coronary heart disease.

Key words:Cinnamomi Ramulus,Coronary heart disease,Network pharmacology,Target,Pathway

Background

Coronary heart disease (CHD) is one of the chronic diseases with high morbidity and mortality worldwide[1].A narrowing of the blood vessels that transfer blood and transport oxygen to the heart is the characteristic of this group of diseases.The basic pathological mechanism involves the dysfunction of both blood vessels and myocardium, which are induced and exacerbated by hypoxia,inflammation and apoptosis.It is well-known that atherosclerosis is the most general pathological process in the development of cardiovascular diseases.Various activated immune cells and smooth muscle cells can cause a chronic inflammation in the blood vessel wall, which results in atherosclerosis [2].Atherosclerosis is characterized by abnormal apoptosis of vascular endothelial cell,macrophage or vascular smooth muscle cell.There are evidences demonstrated that this kind of apoptosis could possess either detrimental or protective effects on coronary atherosclerosis, contributing to the formation or instability of atherosclerotic plaques.[3].

A recommendation about treating with aspirin and clopidogrel is popular,which is called dual antiplatelet therapy.Since atherosclerosis is acknowledged of vascular wall’s chronic inflammatory condition, there have been several anti-inflammatory schemes to prevent atherosclerosis complications.Indeed, Aspirin is more likely to to achieve cardioprotection through antiplatelet action rather than direct anti-inflammatory effect [4].Statins also exert anti-inflammatory effects except their cholesterol-lowering effects [5].Successful resolution and restoration of tissue damage are preferred results with the acute inflammatory response, instead of standing of inflammation, which can bring about scarring and loss of organ function[6].Modern pharmacological studies have shown that Guizhi (CinnamomiRamulus) has various physiological activities such as strengthening the heart,anti-inflammatory, and anti-platelet aggregation.Three classic prescriptions, namely Xuefu Zhuyu Decoction,Zhishi Xiebai Guizhi Decoction, and Gualou Xiebai Banxia Decoction, have been extensively applied in treating CHD.A comparative pharmacogenomic analysis based on molecular network modeling was made to systematically determine the drug-targeting spectrum of the three prescriptions at molecular level.The three decoctions featured at covering wide-area targets of CHD, demonstrating their therapeutic functions[7].

In this study, the main components and targets ofCinnamomi Ramuluswere screened through network pharmacology methods.Related targets of CHD were discovered, and the biological process of target Gene Ontology (GO) was analyzed through the biological information annotation database (DAVID), and the metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes(KEGG)for analysis.

Materials and methods

Screening of active components of Cinnamomi Ramulus

The main components ofCinnamomi Ramuluswere identified by searching the pharmacological database and analysis platform of Traditional Chinese Medicine Systems Pharmacology (TCMSP:http://lsp.nwu.edu.cn/index.ph).Moreover, the CAS obtained from the TCMSP, while the drug "SMILES"was found in PubChem (PubChem:http://www.ncbi.nlm.nih.gov/pccompound).

Prediction of potential targets for active components

Log on to Swiss Target Prediction(http://www.swisstargetprediction.ch) and enter"SMILES" of monomer drugs with species limited to"Homo sapiens".The obtained protein targets and corresponding Uniprot ID were imported into Excel.

Screening of CHD targets

By inserting the keywords "coronary heart disease"into the GeneCards database(https://www.genecards.org), we searched for the reported CHD genes,removing the false positive genes.In addition, the common targets ofCinnamomi Ramuluswith CHD were screened by Wayne diagram.

Acquisition of uniprot ID

According to the 1.3 Wayne diagram, common targets were obtained and their corresponding Uniprot IDs were searched.Uniprot KB (https://www.uniprot.org/)was used to identify the gene target ID.

Protein-protein interaction (PPI) network construction

String database (https://string-db.org/) is a database containing known and predicted protein-protein interactions.The 1.3 Wayne diagram was input into String to obtain common targets, and the species was defined as "Homo sapiens" to obtain a protein-protein interaction relationship.

Biological process and pathway analysis

DAVID (https://david.ncifcrf.gov/) can provide large-scale functional annotations of genes or proteins,and can identify the most significant enrichment of biological annotations.The common target obtained was imported into DAVID database, and the species was limited to "Homo sapiens".GO analysis and KEGG pathway analysis were carried out on the target of CHD.

Results

The active components of Cinnamomi Ramulus

Using TCMSP platform, 220 main components ofCinnamomi Ramuluswere screened.There were seven monomers meeting the criteria of OB (＞30%) and DL(＞0.18).According to the mass spectrometry results obtained in the literature, considering thatCinnamomi Ramuluscontains volatile oil, we also included its high-concentration components cinnamic acid,cinnamaldehyde and coumarin as active ingredients.They were selected for further study(Table 1).

Drug target prediction

The SMILES of these active ingredients were both input into Swiss Target Prediction and STITCH.178 targets were obtained by removing repetitive targets.Uniprot database was used to identify Uniprot IDs of part of the 178 target proteins.

Screening of CHD targets

By inserting the keywords “CORONARY HEART DISEASE” into the GeneCards database, we searched 7100 genes.We screened 1,300 targets based on the relevance score greater than 20.We listed the top 100 genes in the table below (Table 2).Moreover, we selected the 1300 genes and imported them into Web gestalt for KEGG enrichment(http://www.webgestalt.org/).We selected the top 10 channels to make a volcano map (Table 3, Figure 1).Among them PI3K-Akt signaling pathway, MAPK signaling pathway, Apelin signaling pathway, HIF-1 signaling pathway, Cholesterol metabolism are especially significant.

Table 2 Top 100 genes of coronary heart disease

Table 3 Top 10 KEGG pathways of coronary heart disease

Figure 1 Volcano plot for coronary heart disease KEGG pathway

Figure 2 Common targets A:Targets of coronary heart disease;B:Targets of Cinnamomi Ramulus

Screening of CHD targets of Cinnamomi Ramulus

The targets ofCinnamomi Ramulusand CHD related targets were imported into Venny 2.1 to obtain the therapeutic target ofCinnamomi Ramulus(http://bioinfogp.cnb.csic.es/tools/venny/index.html).Target No.1–78 was the related target ofCinnamomi Ramulusfor CHD treatment, while target No.79–178 was not related to treatment(Figure 2).

Construction of protein-protein interaction network and target analysis

Targets ofCinnamomi Ramulusfor CHD were input into String database with species limited to "Homo sapiens".Among them,the top 20 points in the score of the relationship between nodes are as follows (Table 4,Figure 3).

Figure 3 Protein interaction network construction for common targets

Table 4 Top 20 high score nodes in the protein interaction network construction

GO classification and enrichment analysis

The common targets were imported into DAVID database with species limited to "Homo sapiens".The target ofCinnamomi Ramulusfor CHD treatment was used to conduct GO analysis, which found that 306 biological processes, 47 cellular components and 90 kinds of molecular function were involved in the mechanism of effects ofCinnamomi Ramulusfor treating CHD.We select the first ten items from three aspects to make a visual bar graph and visual bubble plots (Figure 4, Figure 6, Figure 7 and Figure 8).The pathway enrichment analysis revealed that the significant bioprocess networks ofCinnamomi Ramuluson CHD were response to hypoxia,inflammatory response, and negative regulation of apoptotic process.

KEGG analysis

Common targets were imported into DAVID database with species limited to "Homo sapiens".KEGG enrichment analysis was carried out forCinnamomi Ramulus, involving 88 pathways.The first 10 pathways are selected and plotted (Figure 5).The possible treating mechanisms ofCinnamomi Ramulusinclude cAMP signaling pathway, estrogen-signaling pathway,calcium signaling pathway.We can find these in the pathways above, which are PIK3CG, MAPK8,BCL-2, BAX, PRKACA, CASP3, CASP8, CALM1,CALM2,CALM3,NOS2,and NOS3.

Figure 4 GO enrichment analysis of Cinnamomi Ramulus related targets on coronary heart disease

Figure 5 KEGG pathway visual bar graph

Figure 6 GO Analysis Biological process visual bubble plot

Figure 7 GO analysis cellular component visual bubble plot

Figure 8 GO analysis molecular function visual bubble plot

Discussion

Network pharmacology can predict the targets and pathways of drugs to treat diseases at the molecular level, and has gradually gained more and more widespread recognition and application.Zhishi Xiebai Guizhi decoction and Xiaoxianxiong decoction are both commonly used in clinical treatment of CHD in traditional Chinese medicine whileCinnamomi Ramulusis part of the prescription.Therefore, this study uses this method to study the mechanism ofCinnamomi Ramulus's treatment of CHD.

The results of the study show thatCinnamomi Ramulushas 10 effective compounds with 78 main targets acting on CHD, mainly involving the physiological and pathological processes of blood coagulation, angiogenesis, vasodilation, inflammation,and antioxidant.

Through KEGG analysis of 1300 CHD related targets, we found PI3K-Akt signaling pathway, MAPK signaling pathway, Apelin signaling pathway, HIF-1 signaling pathway, Cholesterol metabolism especially significant.Class I phosphatidylinositol 3-kinases(PI3Ks), one of the most considerable relevant targets for novel antithrombotic therapies, have been recognized to play a crucial role in multiple steps of platelet activation [8].The activation of PI3Ks promotes PIP3 then initiates the recruitment,phosphorylation and activation of the Ser/Thr kinase Akt, which mediates corresponding cellular responses[9].MAPKs are involved in a variety of cellular processes, such as proliferation, migration,differentiation and apoptosis [10].They have been confirmed in blood platelets, which are the further targets of PI3Ks.However, damage to the vascular endothelium can cause platelets to being activated and aggregated,leading to the formation of hemostasis and thrombosis.Activating the apelin receptor by its cognate peptide ligand generates quite a few physiological effects, including vasodilation, increased myocardial contractility, angiogenesis, and balanced energy metabolism and fluid homeostasis [11].The apelin/apelin receptor pathway is a promising therapeutic target resulting from its implications in atherosclerosis, hypertension, coronary artery disease,heart failure, diabetes and obesity [12].Chen SM conducted a clinical experiment which resulting in that patients with CHD expressed proteins of heme oxygenase-1, hypoxia inducible factor-1α, and ubiquitin more than controls, and the expression levels is proportional to the severity of the disease.Heme oxygenase-1 is positively correlated with hypoxia-inducible factor-1α and ubiquitin.Antioxidative therapy with adrenergic receptor blocker,angiotensin-converting enzyme inhibitor or statins up-regulated the expression of heme oxygenase-1 and hypoxia inducible factor-1α [13].A substantial number of data indicate that cholesterol in the atherosclerotic process in charge of coronary disease.Cholesterol influences similarly to blood pressure on the occurrence of CHD.Other cardiovascular risk factors such as glucose,fibrinogen,cigarette smoking, and left ventricular hypertrophy markedly have effects on the risk associated with the serum cholesterol level.When it comes to correcting hypertension or diabetes, blood lipid values are a key consideration in determining the urgency,type,and efficacy of treatment[14].

The GO enrichment analysis revealed that the significant bioprocess networks ofCinnamomi Ramuluson CHD were response to hypoxia,inflammatory response, and negative regulation of apoptotic process.Evidence can be found that response to hypoxia is related to CHD.Hypoxia-inducible factor prolyl 4-hydroxylase 2 inhibition in endothelial cells activates the hypoxia response, which seems to be a determining factor of ischemic cardioprotective effects[15].It has been demonstrated that atherosclerosis would develop due to a persistent chronic inflammatory state of the arterial wall.The inflammatory stimulation, recruitment of inflammatory cells and production of pro-inflammatory cytokines enhances vascular inflammation [16].Wright B Lauten demonstrated that after the low-density lipoprotein cholesterol and blood pressure reached the required levels in patients with coronary artery disease,simultaneous treatment with quinapril or irbesartan reduced the inflammatory cytokine IL-6 in the vasculature and serum binding of monocytes.RAS inhibitors can be acknowledged to show powerful additive anti-inflammatory effects on vascular endothelium and smooth muscle cell layers, regardless of blood pressure or cholesterol reduction [17].Apoptotic macrophage accumulation also took place in the process of atherosclerotic plaque stability and plaque rupture, apart from significant effects of metalloproteases secreted by macrophages [18, 19].Evidences have shown that statins can modulate the apoptosis of macrophage both in vitro and in vivo,which is a core cell type involved in multiple processes of atherosclerosis[20].

Common targets were input into DAVID to perform KEGG analysis.Possible pathways to treat CHD are chosen.Intracellular cAMP is an ubiquitous intracellular second messenger that regulates important cellular functions, whose levels are regulated by the enzymes adenylyl cyclase and phosphodiesterase.It is reported that cAMP plays an important role in modulating atherosclerotic plaque because it removes excess free cholesterol from macrophages as the inducer of the ABCA1 secretory pathway and by means of reducing cholesterol-circulating level [21].Predication may be that cAMP may be a feasible target for preventing and treating atherosclerosis.The female reproductive hormone estrogen is well known to affect cardiovascular functions, such as maintenance of lipid profile, anti-atherosclerotic and anti-inflammatory action,increasing vasodilation, enhancing high-density lipoprotein levels, attenuation of cardiac cell apoptosis[22].The underlying physiological actions of estrogen are important determinants of cardiovascular physiology and development of vascular disease [23].The beneficial effects of estrogen on lipid metabolism may be partial reason to protect premenopausal female from atherosclerosis since the vascular calcification process involved in CAD is lipid sensitive.Regulation of lipoprotein activity and structure during atherosclerosis is also in the charge of estrogens.Evidence shows that estrogen signaling can hinder the formation of initial fatty streaks germane to atherosclerosis by improving lipid profiles and dampening inflammation through the ER subtypes[24].Oxidation of low density lipoprotein in vessel wall leads to an inflammatory cascade that activates atherogenic pathway leading to foam cell formation.The accumulation of foam cells leads to fatty streak formation, which is the earliest visible atherosclerotic lesion.In contrast, the cardiac sarco/endoplasmic reticulum Ca2+-ATPase and hepatic apolipoprotein E expression can improve cardiovascular function [25].Ca2+is an important cytoplasmic signaling molecule in most cellular responses, mainly distributed in extracellular and intracellular organelles, such as sarcoplasmic reticulum and mitochondria [26].Myocardial ischemia/reperfusion (MI/R) is an inevitable process in the treatment of cardiovascular diseases such as acute myocardial infarction,thrombolysis, coronary angioplasty, and cardiac arrest[27].Few therapeutic methods are carried out for MI/R injury so far mainly through ischemic post-conditioning and drugs of anti-free radicals,dilating blood vessels, and reducing calcium overload.Clinically MI/R injury is suggested to be intervened by maintaining calcium homeostasis, a preferred target that is particularly important for myocardial cell structure and function [28].No hesitation should be with concentrating on some cardioprotective reagents relevant to calcium overload in treatment.

PIK3CG, part of the PI3K family of enzymes, is a protein encoding PI3Kγ or p110γ.Cellular functions such as cell proliferation, differentiation, and intracellular trafficking need its participation.PI3Kγ is suggested to have essential influences on the development of cardiovascular diseases such as atherosclerosis, thrombosis and myocardial infarction[29].Detection of its expression has also been completed in several cells including platelets, vascular endothelial cells, cardiomyocytes, and smooth muscle cells, resulting in establishment of connections between its expression and different diseases [30].Same as MAPKs.PKA, whose expression is high in platelets as serine/threonine kinase, is related to suppressing platelet adhesion and shape change [31].The best described to suppress platelet activities is the channels induced through cyclic adenosine monophosphate/protein kinase A signaling pathway[32].Protein kinase A is reported to be activated by cAMP, which induces platelet inhibition.Cell apoptosis is a type of programmed cell death regulated by genes, which is induced by initiation of the apoptosis signaling pathway and the expression of related genes.BCL-2 is an anti-apoptosis gene while BAX is the pro apoptotic gene [33].The role Caspase-3 plays in signaling pathways related to apoptosis is conflux.Evidences have shown that polymorphisms in the NOS genes are associated with CHD and hypertension, replicating results from earlier studies, especially NOS2 and NOS3, which appeared in the HIF-1 signaling pathway [34].Ca2+flux across cell membranes achieves rapid cellular responses.Calmodulin is aware of fractional adjustments in Ca2+concentration and transports the message to various interacting partners.The fact that there are three independent CaM genes in the human genome highlights the essential role of accurate Ca2+signaling for cell function[35].

Conclusion

Cinnamomi Ramulusmay treat CHD through cAMP signaling pathway, estrogen-signaling pathway,calcium signaling pathway.PIK3CG, MAPK8,BCL-2,BAX,PRKACA, CASP3, CALM1,CALM2, CALM3,NOS3, NOS2 are supposed to be considerable targets for treating CHD.