Network pharmacology based method for mechanistic investigation of the Compound Xintahua in the treatment of atherosclerosis

Yu-Che Wu,Yan-Ming Wang,Na-Bi Xinhua*

1College of pharmacy,Xinjiang Medical University,Urumqi,Xinjiang,China.

Abstract

Key words:Network pharmacology,Compound Xintahua(XTH),Atherosclerosis,Pharmacological mechanism,Targets

Background

Atherosclerosis(AS)is an arterial vascular lesion that is based on endothelial cell damage and is mediated by various pathological factors.There are many hypotheses about the pathogenesis of AS,such as lipid metabolism disorders,proliferation and migration of vascular smooth muscle cells,oxidative stress,inflammation,and platelet aggregation[1,2].Numerous studies have shown that a Chinese herbal compound has achieved good results in treatment of AS[3].Further,various drugs are compatible with each other and can inhibit the occurrence of AS through different mechanisms[4,5].

Traditional Chinese medicine(TCM)has been continually practiced since 2000 years.Due to the unique regional climate and dietary habits of its local residents,Xinjiang has a high incidences of cardiovascular disease[6,7].Compound Xintahua(XTH)is one of the most representative traditional Chinese medical prescriptions,which has good curative effect for clinical prevention and treatment of AS[12].It consists of three herbs:Xintahua(Ziziphora bungeana Juz.)is a dried flower of a grass of the genusZiziphora bungeana Juz.Its main active ingredients are flavonoids and volatile oils,steroids,as well as alkaloids[8,9],studies have shown that it has the pharmacological activity of increasing myocardial antioxidant capacity and myocardial protection[10-11].Hyssopuscuspidatus Boriss.Shenxiangcao(Hyssopus officinalis L.)is the aerial part of the genusHyssopus cuspidatus Boriss.Main active ingredients are volatile oil,flavonoids and other active ingredients[13,14].Modern pharmacological studies have shown that its volatile oil has antibacterial and antispasmodic activity[15],while its non-volatile extracts have strong anti-oxidant activity[16].It not only inhibits the secretion of interleukin-17,but also corrects the imbalance of Th1/Th2 type cytokines,thereby exerting anti-inflammatory effects [17,18].Origanum vulgare L.Niuzhi(Origanum vulgare L.)is the dry aerial part of the orchardOriganum vulgare L.orOriganum tyttanthum Gontsch.Main active ingredients include flavonoids,triterpenoids,organic acids,volatile oils and other chemical components,including rosmarinic acid.It is capable of free radicals,has anti-inflammatory and immune regulation,anti-thrombosis and anti-platelet aggregation[19,20].

A network pharmacology(NP)has recently emerged as a technique to decipher complex pharmacological problems[21].Recently,the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)was developed as a digital repository of traditional medicines.In addition,it can predict pharmacological targets and specific maladies of every dynamic compound,and is a major analytical tool in network pharmacology that helps determine the complex interactions between drugs and targets[22].Network pharmacology has helped elucidate the mechanism of several TCM formulations so far[23,24].

The Chinese herbal compound treats the disease through the synergy of multi-flavor Chinese medicine of Jun-Chen-Zuo-Shi,emphasizing systemicity and integrity.The theory of disease treatment by traditional Chinese medicine is similar to that of network pharmacology.The anti-AS effect of XTH is in line with both Chinese medicine theory and modern pharmacology.Although there are many reports on the pharmacological studies of three traditional Chinese medicine in the compound,research on the mechanism of multi-component,multi-target and multi-path treatment of AS is still not comprehensive.Therefore,it is important to screen the active components of XTH and its target by means of network pharmacology.

Methods

Screening for active XTH compounds

TCMSP(http://lsp.nwu.edu.cn/tcmsp.php)is a Chinese medicine pharmacology database containing information about the herbs used in TCM,and absorption,distribution,metabolism and excretion(ADME)characteristics of the individual compounds,their targets,related diseases,and pathways.CHEM-TCM(http://www.chemtcm.com)is the digital database of individual molecules,constituents of plants used in the traditional Chinese herbal medicine.The database consists of four major parts:chemical identification,botanical information,predicted activity againstcommon Western therapeutic targets,and estimated molecular activity according to traditional Chinese herbal medicine categories.PubChem(http://pubchem.ncbi.nlm.nih.gov)is an open chemistry database at the National Institutes of Health(NIH).It collect information on chemical structures,identifiers,chemical and physical properties,biological activities,patents,health,safety,toxicity data,and many others.

Lipinski's rule of five also known as the Pfizer's rule of five or simply the rule of five(RO5)is a rule of thumb to evaluate drug likeness or determine if a chemical compound with a certain pharmacological or biological activity has chemical properties and physical properties that would make it a likely orally active drug in humans.The rule was formulated by Christopher A.Lipinski in 1997,based on the observation that mostorally administered drugs are relatively small and moderately lipophilic molecules [25,26].The rule describes molecular properties important for a drug's phar macokinetics in the human body,including their absorption,distribution,metabolism,and excretion(ADME).The rule is important to keep in mind during drug discovery when a pharmacologically active lead structure is optimized step-wise to increase the activity and selectivity of the compound as well as to ensure drug-like physicochemical properties are maintained as described by Lipinski's rule[27](Table 1).Candidate drugs that conform to the RO5 tend to have lower attrition rates during clinical trials and hence have an increased chance of reaching the market[28].

Drug-like evaluation(DL)refers to physicoche mical properties (such as solubility,stability,etc.) and biological properties associated with good clinical efficacy,i.e.absorption,distribution,metabolism,excretion,toxicity(ADME-Tox)[29].Oral availability(OB)refers to the relative amount and rate at which a drug is absorbed into the blood circulation system after oral administration via an extravascular route.TCMSP provides molecular OB/DL values for reference.Oral availability and drug-like evaluation were used to screen the oral bioavailability of the drug molecules and other key parameters required for drug-making[30].

Table 1.Lipinski's rule of five

Selection of drug component targets

Swiss Target Prediction(http://swisstargetprediction.ch/)allows you to predict the targets of a small molecule.Using a combination of 2D and 3D similarity measures,it compares the query molecule to a library of 280'000 compounds active on more than 2000 targets of 5 different organisms.The parameters set were as follows:Choose an organism:Homo sapiens;Provide a SMILES:Paste a SMILES in this box;click “submit”Download CSV files,create Excel documents,consolidate CSV files,screen out duplicates.

Selection of AS targets

DisGeNET(http://www.disgenet.org/) is a discovery platform designed to address a variety of questions concerning the genetic underpinning of human diseases[31].In the pursuit to gather different aspects of the current knowledge on the genetic basis of human diseases,DisGeNET covers information on all disease areas(Mendelian,complex and environmental diseases).With more than 400000 genotype-phenotype relationships from different origins integrated and annotated with explicit provenance and evidence information,DisGeNET is a valuable knowledge and evidence-based discovery resource for Translational Research.These valuable set of tools allows investigating the molecular mechanisms underlying diseases of genetic origin [32],and are designed to support the data exploitation from different perspectives.The DisGeNET server was started,in the“search” button, “diseases” option was selected,“atherosclerosis”was entered and “search”initiated.The“Top 10 gene associations for this disease”option was selected in the results bar,and “Browse details”clicked.The AS target excel document was downloaded,the duplicates were screened out.

Analysis of protein interaction relationship

The GeneMANIA (http://genemania.org/)finds other genes that are related to a set of input genes,using a very large set of functional association data.Association data include protein and genetic interactions,pathways,co-expression,co-localization and protein domain similarity.Server was started, “Homo sapiens”option was selected and the gene names of targets entered,finally the “submit”button was clicked.This analyzed the interaction between protein and protein,to give the protein interaction network map,organized the network layout,saved the Image,gave the protein-protein association network(PPI),and the report was download.Enrichment analysis and path annotation

FunRich (http://www.funrich.org/) is a stand-alone software tool used mainly for functional enrichment and interaction network analysis of genes and proteins.By importing the acquired targetin formation into the FunRich server,the relevant path information of the target were obtained.The parameters were set as follows,Add Data Set;Chart Type:Bar Chart;NO.of displayed terms:10-15;Size:Auto;Resolution:Extra High;Export Excel.In addition,it contains a database of pathways that store information about gene function,and graphical representations of cellular components,molecular functions,and biological processes within the cell.

Building a “Compound-Target-Path” Network

The network construction,analysis and visualization were carried out using Cystoscope 3.6.1(http://www.cytoscape.org/).Cytoscape is an open source bioin formatics software platform for visualizing molecular interaction networks and integrating with gene expression profiles and other state data.The interaction targets predicted by chemical components were used as nodes to establish the relationship,and the“Compound-Target-Pathway”network map was drawn.The “Compound-Target-Pathway”network diagram of XTH treatment AS was analyzed by Network Analyzer.In the network,the chemical composition,target,and pathway were represented by nodes.A connected edge indicated that a target is a potential compound and was associated with a pathway.

Results

Potential targets and major chemical constituents

A total of 316 XTH chemical constituents were identified from TCMSP,CHEM-TCM and literature mining.while 198 chemical components were screened in PubChem by the Lipinski's rule of five.Two conditions of drug-like(DL)≥30%and oral availability(OB)≥0.18 were set in the 198 compounds,16 major drug components were obtained.The corresponding SMILES format file were downloaded from the PubChem database and backed up(Table 2).

The 16 main drug components obtained in the previous stage were imported into Swiss Target Prediction server for targetanalysis and sortout 117 drug targets.Compared with the 1268 AS disease targets screened by DisGeNET server,39 common targets obtained were directly or indirectly related to AS(Figure 1,Table 3).

Figure 1.A total of 39 common targets were obtained from two target databases,including 117 from drug targets,1268 fromAS targets,of which 39 common targets were shared across two target databases.

Table 2.Pharmaceutical ingredients specific information

Table 3.Common targets(top 20 targets)

Among the predicted targets,AKRIBI,FABP4,HSD11BI,MPO,PLA2G1B,and MAPT were related to lipid metabolism;ADORA2A,CYP1B1,MMP1,MMP2,PTPN1,SRC,and XDH were related to angiogenesis;EGFR,ESR1,NOX4,PPARD,PTGS1,and MAOA were related to oxidation reaction;AR,MMP3,MMP9,ESR2,GLO1,and AHR were related to cell survival and apoptosis;while ALOX5,PPARA,ABCC1,PTGS1,PTGS2,and CYP19A1were related to inflammatory reaction.Therefore,XTH prevention and treatment of AS may be related to the regulation of lipid metabolism,oxidation,cell growth as well as angiogenesis to protect vascular endothelial cells,reduce inflammation and other effects.At the same time,the corresponding compounds with thehighestnumberoftargetswereluteolin,arachidonic acid,apigenin,kaempferol,quercetin,genistein,rosmarinic acid,spruce,and baicalein.XTH is composed offlavonoidsand phenoliccomponents,suggesting that its used in treatment of AS mainly depends on these two compounds.The corresponding targets of each compound were different and similar in the same way,which reflects XTH multi-component and multi-target mechanism in treatment ofAS(Table 4).

Table 4.Potential targets and numbers of major chemical constituents

Analysis of protein interaction relationship

The 39 common targetswere imported into the GENEMANIA database as standard gene names to obtain a network map of protein interactions(Figure 2).The interaction between proteins was mainly through physical correlation,protein co-expression,shared protein domains,prediction,co-localization and the pathway was manifested.Among them,protein co-expression,shared protein domain,pathway,physical correlation,co-localization,and predicted accounted for,42.40%,21.58%,13.17%,10.25%,9.56% and 3.03%,respectively.

GO enrichment analysis

A two-dimensional bar graph of GO enrichment analysis results was obtained through the FunRich server(Figure 3,Figure 4,and Figure 5).The results indicated that the predicted target was in the biological process,and Metabolism,Energy pathways were ranked high.Among the molecular functions,lipid kinase,growth factor,helper transport protein,receptor,catalytic,G protein coupled receptor,and transcription factor activities were consistent.Cytoplasm,extracellular,extracellular space,exosomes accounted for the largest proportion of cellular components.

Figure 2.Protein interaction network diagram

Protein co-expression,shared protein domain,pathway,physical correlation,co-localization,and predicted accounted for,42.40%,21.58%,13.17%,10.25%,9.56%and 3.03%.

Figure 3.Biological process terms were Metabolism,Energy pathways,Protein metabolism,Cell communication,Transport,Regulation of nucleobase,Signal transduction etc.

Figure 4.Molecular function terms were lipid kinase,growth factor,helper transport protein,receptor,catalytic,G protein coupled receptor,transcription factor activities etc.

Figure 5.Cell component terms were cytoplasm,extracellular,extracellular space,exosomes,endoplasmic reticulum,nuclear envelope lumen,intracelluar organgelle,membrane,intracelluar membrane-bounded organgelle etc.

Pathway analysis

290 signal paths were obtained through the FunRich server,and 43 channels were indirectly or directly related to the AS(Table 5).There were 13 pathways related to cellproliferation,namely:Adherensjunction,TNF signaling pathway,VEGF signaling pathway,Epithelial cell signaling in Helicobacter pylori infection,EGFR tyrosine kinase inhibitorresistance,ErbB signaling pathway,Gap junction,Focal adhesion,Signaling events Mediated by PTP1B,Class I PI3K signaling events,mTOR signaling pathway,p38 MAPK signaling pathway,and Negative regulation of FGFR signaling.There were10 pathways related to platelet aggregation including:Arachidonic acid metabolism,Platelet activation,Calcium signaling pathway,Thrombin/protease-activated receptor(PAR)pathway,Nectin adhesion pathway,PDGF receptor signaling network,uPA and uPAR-mediated signaling,Integrins in angiogenesis,COX reactions,and Plateletaggregation.The five pathwaysrelated to vasoconstriction and relaxation included:Adherens junction,Vascular smooth muscle contraction,Endothelins,Prostanoid metabolism,and Metabolism of serotonin.Further,two pathwaysincluding EGFR tyrosine kinase inhibitor resistance and Metabolism of serotonin were related to angiogenesis.There were also10 pathways comprising of Arachidonic acid metabolism,Regulation of lipolysis in adipocytes,TNF signaling pathway,VEGF signaling pathway,Leukocyte transendothelial migration,Focal adhesion,S1P pathway,p38 MAPK signaling pathway,Osteopontin-mediated events and HIF-2-alpha transcription factor network which were related to inflammation.A total of 10 endocrine-related pathways included:PPAR signaling,Regulation of lipolysis in adipocytes,GnRH signaling,Drug metabolism-cytochrome P450,Metabolism of xenobiotics by cytochrome P450,Focal adhesion,Steroid hormones,Insulin,Metabolism of Steroid hormones and vitamins A and D,IGF Activity by IGFBPs.6 pathways namely:TNF signaling,ErbB signaling,Leukocyte transendothelial migration,S1P,Signaling events mediated by PTP1B,and Osteopontin-mediate were related to immunity events.There were also 4 pathways related to lipid metabolism:Linoleic acid metabolism,Regulation oflipolysis in adipocytes,Glycerolipid metabolism,and Metabolism of lipids and lipoproteins.

Table 5.Pathway information

Network Construction

The “Compound-Target-Pathway”network map of XTH was established using Cytoscape 3.6.1(Figure 6).The relationship between 15 compounds(in which Danshensu and salvianolic acid correspond to the same target,so that salvianolic acid was selected),39 common targets,and 43 action pathways and AS were presented.The blue diamond represents the signal pathway,the red circle represents the XTH target,the pink triangle represents the compound,the yellow represents theZiziphora,the green represents theHyssopus,and the orange represents theOriganum.Analysis of the results showed that,the access nodes 1-14 of the network were ranked first(the number of targets acting on the pathway are≥6),and were the central pivot node of the network,suggesting that they may be the core action pathway of treatment AS by XTH.The corresponding targets of the compounds were the same which reflects the multi-component and multi-target treatment mechanism of AS using XTH.This mechanism could be related to cell proliferation,lipid metabolism,inflammatory reaction,and oxidation reaction while various mechanisms such as immune regulation and endocrine hormone metabolism are closely related.This network map reveals the characteristics of XTH in treatment ofAS from multiple perspectives.

Figure 6.Compound-target-pathway network diagram

Blue diamond represent pathways,red circle represent XTH targets,pink triangle represent compounds,yellow node represent Xintahua,green node represent Shenxiangcao,orange represent Niuzhi,and the edges represent inter-nodal relationships.

Discussion

Network pharmacology is a discipline that builds the “drug- target-disease” interaction network based on high-throughput histological data analysis,computer virtual computing and network database retrieval,and further uses the holistic view of network balance to analyze the interaction between drugs and the body.It also represents a conformity of the overall characteristics of traditional Chinese medicine new research ideas,new methods and new technology.It also effectively links the overall characteristics of traditional Chinese medicine system and modern cutting-edge technology for the innovation and development of traditional Chinese medicine from experience level towards the mechanism level.Further,it provides a new methodology to support the molecular and system level to promote synergistic communication between TCM and western medicine[33].It also provides a new idea and method for the study of the pharmacology of traditional Chinese medicine and gives quantifiable data for the multi-target and multi-path of traditional Chinese medicine and national medicine.Moreover,it realizes the integrated and systematic study of the mechanism of action of multi-component or compounds of single medicine[34].

This study found that flavonoids and phenolic components play a pharmacodynamic role in XTH.Among the 16 compounds screened in this work,there were 11 flavonoids,4 phenolic components,and one Iridoids.Flavonoids have a wide range of pharmacological effects,including anti-oxidation,anti-inflammatory,vasodilation,inhibition of platelet aggregation and regulation of blood lipids.Their mechanism of action includes inhibition of enzymes involved in the production of oxygen free radicals,inhibition of lipid peroxidation,down-regulation of inflammation factor,cyclooxygenase activity,activation of AMPK andNF-κB signalingpathways[35,36].Phenolic compounds on the other hand have significant antioxidant effects,protective effects on cerebral ischemic injury and endothelial cells as well as inhibition of glycocataracts.Manyin vitroexperiments have shown that phenolic acids have many antioxidant effects such as scavenging oxygen free radicals and inhibiting liposome peroxidation[37,38].

Luteolin in XTH induced the effects of CYP1A2,CYP2B1 and CYP3A1 in rats.In addition,luteolin was synchronized with the induction of enzyme activity and gene level of CYP450 in rats,indicating the regulation of luteolin on enzyme activity.This may have been achieved by interfering with mRNA expression levels[39].Apigenin inhibits lipopolysaccharide(LPS)-induced NO production in macrophages in a dose-dependent manner.In addition,apigenin could significantly reduce the adhesion of monocytes induced by TNF-α to HUVEC,and significantly inhibit the upregulation of VCAM-1,ICAM-1 and ET-1 to exert anti-inflammatory effects[40].Kaempferol inhibits ox-LDL-induced decrease in HUVEC activity,reduces apoptosis,down-regulates the expression of inflammatory factors and adhesion molecules,and alleviates oxidative stress,which is associated with activation oftheMAPK signaling pathway[41].Quercetin effectively inhibits VSMCs,collagen synthesis and secretionin vitro.The mechanism may be related to quercetin activation of PI3K/Akt/NF-κB signaling pathway,up-regulation of phosphorylated PI3K and Akt expression,and inhibition nuclearrelated transportof NF-κB [42].Animal experiments confirmed that salvianolic acid A can delay the occurrence and development of AS by lowering blood lipids,ET-1,reducing IL-6,TNF-α,inhibiting the formation of AS plaques and down-regulating LDL expression.The cells confirmed that ox-LDL can inhibit the proliferation of HUVEC and promote its apoptosis,which could induce the inflammatory secretion by HUVEC.Its noteworthy that,Salvianolic acid A can exert anti-AS effectby increasing HUVEC proliferation,reducing HUVEC apoptosis and inflammatory factor secretion,and itsmechanism could be related to inhibition of MAPKs signaling pathway[43].

It was also found that the predicted targets in XTH were related to the regulation of lipid metabolism and angiogenesis,inflammatory response,and oxidative stress.Each targetand pathway play a synergistic role,suggesting that this may be the key to prevention and treatment of AS by XTH.Studies have shown that after arterial vascular injury,a large number of inflammatory factors adhere to endothelial cells,their interaction with macrophages causes formation of plaques on the surface of the cells.This weakens the endothelial cells,which in turn leads to their apoptosis in severe cases and eventual AS.On the other hand,lipid metabolism disorders can lead to increased LDL,TC,TG and cholesterol.A large amount of cholesterol enters monocytes,forming foam cells,which accumulate in the intima of the arteries to form fat streaks,leading to the formation of AS[44].Platelet aggregation,oxidative stress,and inflammatory reactionsfurtherdamageendothelialcells,promote thrombosis,and aggravate inflammatory responses,thereby accelerating AS pathological processes.The arachidonic acid pathway plays an important role in the whole anti-AS process,and its metabolites are involved in cholesterol metabolism,endothelial cell monocyte adhesion,platelet aggregation,and regulation of blood viscosity.SRC tyrosine kinase can activate of PLA2 which promotes the synthesis of arachidonic acid[45,46].

The results of protein interaction network show that AS target and drug target can interact through multiple pathways.Among them,39 targets such as PPARD,PPARG,MPO and AR exist in AS target and drug target.XTH may pass 39 targets to affect lipid metabolism,oxidative stress,protect angiogenesis,reduce inflammation,regulate the network and cell function,as well as exert anti-AS effects.

Based on the results of this study,the main mechanism of XTH prevention and treatment of AS may be that the target compound acts on VEGF signaling pathway,while EGFR tyrosine kinase inhibits resistance,with PI3K signaling events,p38 MAPK signaling pathway,Platelet activation and other signaling pathways to mediate endothelialcellproliferation and apoptosis,which regulates vascular permeability,protect angiogenesis,maintain tissue structure,and maintain cell stabilityin vivo.Signaling pathways such as Arachidonic acid metabolism,TNF signaling pathway,S1P pathway,and ErbB signaling pathway are involved in inducing the production of pro-inflammatory factorsaswellas up-regulating the costimulatory molecules to stimulate anti-inflammatory effects to improve the body's immune function.Signaling pathways such as Metabolism of lipids and lipoproteins,Linoleic acid metabolism,and regulation of lipolysis in adipocytes are involved in the regulation of lipid metabolism.It can be seen that the XTH active component group targets a multi-target AS,and exerts a pharmacological effect through a plurality of metabolic pathways in a lipid metabolism-angiogenesisimmuno-endocrine-cell process.

Conclusions

Based on the network pharmacology technology,this study took 16 main compounds of XTH as the research objects.The good oral bioavailability and drug-like properties of these compounds showed that they have good drug-forming properties,and they can represent XTH drugs well from drug metabolism perspective.Using TCMSP,PubChem,and Swiss Target Prediction as well as use of FunRich servers and Cytoscape software to analyze,predict the possible targets and pathways of the above components.A network was constructed to initially reveal synergistic effect of multi-component,multi-target and multi-path of XTH in prevention and treatment of AS.This provides a theoretical basis and method for further study on the basic research and mechanism of XTH in prevention and treatment ofAS.

Authors’contributions

WYC and XHNB conceived and designed the study.WYH and WYM collected the data.WYC performed the data analysis and wrote the manuscript.All authors are responsible for reviewing data.All authors read and approved the final manuscript.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.The datasets generated and/or analysed during the current study are available in the TCMSP repository,http://lsp.nwu.edu.cn/tcmsp.php. CHEM-TCM,http://www.chemtcm.com Pub Chem,http://pubchem.ncbi.nlm.nih.gov Swiss Target Prediction,http://swisstargetprediction.ch/ Dis Ge NET,http://www.disgenet.org/ Gene MANIA,http://genemania.org/ Fun Rich,http://www.funrich.org/ Cystoscope 3.6.1,http://www.cytoscape.org/