Gui-Ping Cao ,Ling Xu ,Yu-Long Wang ,Fei-Xiang Ma ,Hua Yuan ,Rong-Fang Tang

1Traditional Chinese Medicine Hospital of Yancheng City attached to the Nanjing University of Chinese Medicine,Yancheng 224001,China.2Yancheng Third People's Hospital Affiliated to Southeast University,Yancheng 224005,China.3The First Clinical Medical College of Nanjing University of Traditional Chinese Medicine,Nanjing 210049,China.

Abstract Objective:To collect the main components and targets of Jiang-zhi-dai-pao-cha (JZDPC)and investigate the mechanism of JZDPC for the treatment of hyperlipidemia by network pharmacology.Methods:The components and targets of JZDPC were searched from ETCM databases,the targets related to hyperlipidemia were searched from DisGeNET and GeneCards databases,and then the intersection targets and corresponding key components were obtained.Cytoscape 3.8.2 software was used to construct and analyze networks,and then Metascape online database was applied for gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of core putative targets.Results:There were 99 overlapping targets between JZDPC and hyperlipidemia,among which NR3C1,ESR1,NR1I2,NFKB1,ESR2,ALOX5,PTGS1,PPARA,RXRA,LPL,PLA2G1B,PYGM,CYP2C9 were the core putative targets,and many members of nuclear receptor 1 (NR1) subfamily were included.The core components of JZDPC,such as Ursolic Acid,β-Sitosterol,Resveratrol,Arirubic Acid,Alisol A,Oleanolic Acid,Rhein,Chrysophanol and Emodin,can regulate blood lipid by regulating a series of signaling pathways including the above core potential targets,such as non-alcoholic fatty liver disease(NAFLD) signaling pathway,pathways in cancer,arachidonic acid (AA) metabolism signaling pathway and peroxisome proliferator activated receptor (PPAR) signaling pathway,Starch and sucrose metabolism signaling pathway,etc.They play many roles in the treatment of hyperlipidemia by participating in lipid synthesis and metabolism,anti inflammation,anti oxidative stress,regulating hormone levels and carbohydrate metabolism.Conclusion:Network pharmacology provides a theoretical basis for investigating the mechanism of action of JZDPC,and the NAFLD signaling pathway is one of the most valuable pathways.

Keywords:Hyperlipidemia;Jiang-zhi-dai-pao-cha;network pharmacology;nuclear receptor 1 subfamily;hosphatidylinositol 3-kinase complex,class IA;non-alcoholic fatty liver disease signal pathway;arachidonic acid metabolism signal pathway;peroxisome proliferator activated receptor signal pathway

Background

Hyperlipidemia usually refers to the increase of total cholesterol (TC)and triglyceride (TG) in plasma,and also generally refers to various dyslipidemia such as the increase of low-density lipoprotein cholesterol (LDL-C) and the decrease of high-density lipoprotein cholesterol (HDL-C).Lipids combine with proteins to form lipoproteins to increase their solubility and facilitate their circulation in blood.Therefore,hyperlipoproteinemia is also known as hyperlipidemia [1].Hyperlipidemia is an important risk factor for coronary heart disease,stroke,fatty liver and many other diseases,which needs active intervention.At present,the commonly used drugs for regulating blood lipid mainly include statins,fibrates,cholic acid chelators and ezetimibe.There are many adverse reactions such as rhabdomyolysis,elevated aminotransferase and diarrhea.The new pre drug protein invertase subtilisin/kexin9 (PCSK9) inhibitors elouximab and aliciumab are administered non orally and are expensive,Most of them are selected for patients with statin resistance and homozygous familial hypercholesterolemia [2-3].Shanzha (Crataegus pinnatifida Bge.),Zexie (Alisma orientalis (Sam.) Juzep.),Jiaogulan (Gynostemma pentaphyllum (Thunb.) Makino) and other traditional Chinese medicines have been proved to have definite lipid-lowering effect,mild effect and less adverse reactions[4-6].They can be used alone or in combination for the prevention and treatment of hyperlipidemia.Network pharmacology is systematic,integrated and comprehensive by establishing drug component target disease data relationship and analyzing network topology relationship,studying drug action mechanism and developing new drugs [7],which is consistent with the characteristics of multi-component,multi-target,multi-channel and overall function of traditional Chinese medicine [8],and can be used to predict the material basis and potential targets of traditional Chinese medicine,Clarify the mechanism of action and develop new uses.

Jiang-zhi-dai-pao-cha (JZDPC) is a self-made preparation of Traditional Chinese Medicine Hospital of Yancheng City,with approval No.:z0400997.The prescription is composed of four traditional Chinese medicines:Danshen (Salvia miltiorrhiza Bge.),Zexie(Alisma orientalis (Sam.) Juzep.),Shanzha (Crataegus pinnatifida Bge.)and Heshouwu (Polygonum multiflorum Thunb.).Salvia miltiorrhiza Bge.,Alisma orientalis (Sam.) Juzep.are sovereign drugs as they can romote blood circulation and remove blood stasis,promote dampness and resolve phlegm.Crataegus pinnatifida Bge.is the minister herb as it can eliminate food,strengthen the stomach,promote qi and dissipate blood stasis,and reduce blood lipid.Polygonum multiflorum Thunb.is the supplementary herb as it can detoxify,moisten intestines and defecate,reduce blood lipid and help phlegm turbidity.The preparation has been used clinically for 40 years.It has definite curative effect,mild drug properties,dispelling evil without harming the health.Hyperlipidemia is one of its main symptoms,but the relevant mechanism of action is not clear.In this study,the active components,action targets and signal pathways of JZDPC in the treatment of hyperlipidemia were analyzed by network pharmacological method,so as to preliminarily clarify the action mechanism of JZDPC in hyperlipidemia in theory.It provides a reference for subsequent experimental research.

Method

Collection of JZDPC components and targets

Taking the Chinese phonetic name of four traditional Chinese medicines in JZDPC as the keyword,query the compounds and corresponding potential targets (gene symbols) in the Encyclopedia of traditional Chinese medicine database (ETCM,http://www.tcmip.cn/ETCM/index.php/Home/) [9].All compounds are evaluated by the compound class proposed by Bickerton team[10],predicted by medchemstudio (version 3.0),and the target with the reliability score Tanimoto ＞0.8 is retained,as a potential target of compounds.

Screening disease targets

Take "hyperlipidemia" as the keyword,obtain the disease targets related to hyperlipidemia in the disease-related gene and mutation site database (DisGeNET,http://www.disgenet.org/) [11] and gene business card database (GeneCards,https://www.genecards.org/),take the union set and delete duplicates.

Construct the network diagram of herbs -key compounds -intersection targets

Upload potential targets of compounds and disease targets contained in JZDPC to gideo bioinformation cloud platform,draw Wayne diagram and sort out intersection targets.According to the intersection target,the key compounds are inversely screened,that is,the compounds acting on the intersection target are selected.The network diagram of "herbs-key compounds-intersection target" is constructed by Cytoscape 3.8.2 software.

Construct the protein-protein interaction network diagram of intersecting targets and screen the core potential targets

Input the screened intersection target into the Search Tool for the Retrieval of Interaction Gene/Proteins (STRING,https://string-db.org/) developed by peer Bork team of European Molecular Biology Laboratory (EMBL) [12],select Homo sapiens,construct the functional and physical interaction network of intersection target protein (full network),set the minimum required interaction score to 0.7 (highly trusted),remove isolated nodes and save the corresponding interaction map,and export the corresponding TSV file.Import the first two columns of TSV file into Cytoscape 3.8.2 software,analyze the network topology with tools network analyzer function in the software,and then analyze with mcode clustering function.Collect targets with degree and betweenness Centrality greater than the median in network analyzer analysis and targets in densely connected network modules obtained in mcode analysis,merge these targets,delete duplicates and then are the core potential targets.

Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis

Metascape [13] (http://metascape.org/gp/index.html#/main/step1)is an online gene research tool website integrating gene ID conversion,gene annotation and function enrichment analysis.It has the advantages of fast data update,wide coverage and simple operation.In order to further understand the functional characteristics of the core potential targets and their main signal pathways,the screened core potential targets are input into the metascape website for go enrichment analysis and KEGG pathway enrichment analysis.The species is set as H.sapien,and the analysis method is custom analysis.In the next step,the core potential targets are enriched and analyzed by GO Biological Processes,GO Molecular Functions,GO Cellular Components and KEGG Pathway respectively.Save the corresponding results,and draw bubble charts and bar charts with the top items of P value significance to visualize the results.

Results

Composition and corresponding targets of JZDPC

A total of 159 compounds with different names were obtained from the four traditional Chinese medicines in JZDPC in ETCM database,including 19 fromPolygonum multiflorum Thunb.,30 fromCrataegus pinnatifida Bge.,30 fromAlisma orientalis (Sam.) Juzep.and 83 fromSalvia miltiorrhiza Bge..Among the 159 compounds,99 compounds with different names have a reliability score of Tanimoto ＞0.8 in ETCM,corresponding to 554 different targets.Table 1 shows the number of compounds and targets contained in each traditional Chinese medicine in JZDPC.

Table 1 Numbers of compounds contained in herbs of JZDPC and corresponding putative targets

Disease targets and the network diagram of herbs-key compounds-intersection targets

472 and 835 disease-related targets were screened from disgenet database and genecards database respectively.The disease targets were obtained by merging and deleting duplicates.A total of 99 intersection targets between disease targets and potential targets of compounds screened in "composition and corresponding targets of JZDPC" are obtained through online Wayne diagram,as shown in Figure 1.Based on the intersection target,89 key compounds with different names were inversely screened,including 28 fromCrataegus pinnatifida Bge.,18 fromPolygonum multiflorum Thunb.,17 fromAlisma orientalis (Sam.) Juzep.and 29 fromSalvia miltiorrhiza Bge..Each compound is given a number,and herb,key compound and intersection target is mapped one by one.Figure 2 is the network diagram of herbs-key compounds-intersection targets (gene symbol)drawn by Cytoscape 3.8.2 software.

Protein protein interaction (PPI) and mcode cluster analysis of intersection targets

Input the 99 intersection targets obtained under "2.2" into the string database to obtain a highly reliable (interaction score ≥ 0.7)protein-protein interaction network diagram of the intersection targets,as shown in Figure 3.The network diagram contains 85 nodes and 264 edges.The average node degree of network nodes is 6.2.And 41 targets with node connectivity and median centrality greater than the median are obtained.The core gene module obtained by mcode clustering function analysis in Cytoscape 3.8.2 software contains 42 targets,as shown in Figure 4.After the two are combined and the duplicates are deleted,53 core potential targets are obtained,which are sorted from large to small according to the target regulated score[14] (Number of herb-compound targeting specific targets),including AR,NR3C1,ESR1,NR1I2,HSD11B1,NFKB1,ESR2,ALOX5,AHR,PTGS1,PPARA,RXRA,NR1H4,LPL,PLA2G1B,TNF,PYGM,CYP2C9,CYP2E1,CYP3A4,NR1H2,NR1H3,PIK3CG,etc.The compounds related to 53 core potential targets are Ursolic Acid,β-Sitosterol,Resveratrol,Resveratrol Glycoside,Rhein,Rhrysophanol,Emodin,Emodin Methyl Ether,Cauliflower Acetylben Glycoside,2,3,5,4′-Tetrahydroxystilbene-2-o-β-D-Glucoside,Linoleic Acid,Perforating Acid,Dehydrogenated Perforating Acid,Alimaric Acid A,Alimaric Acid B,Alimaric Acid D,Alimaric Acid E,Sulfur Porous Bacterial Acid,Alisol A,Alisol A Monoacetate,11-Deoxyalisol A,23-Acetylalisol E,24-Acetylalisol E,Alismatine Diterpenoids,Neoalisol,Oleanolic Acid,Baicalin,Stigmasterol,Taxol,Carotene Salvianol and other compounds.These compounds and targets are of great significance for JZDPC in the treatment of hyperlipidemia.

Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis

Input 53 core potential targets into metascape website for go and KEGG enrichment analysis.The pathways conforming toP＜0.01(-logP＞2) are arranged in descending order according to -logP,and count the number of core potential targets falling on each enrichment pathway.The GO biological process,go molecular function and go cell component enrichment analysis results of the top 15 items are shown in bubble chart,as shown in Figure 5-7.All items less than 15 items are listed;The enrichment analysis results of the top 20 KEGG pathways are displayed in a bar chart,as shown in Figure 8.

As can be seen from Figure 5,GO biological process mainly involves biological processes such as lipid biosynthetic process,steroid metabolic process,monocarboxylic acid metabolic process,cellular response to lipid,cellular response to hormone stimulus,regulation of inflammatory response,regulation of protein secretion,response to oxidative stress,glycerolipid biosynthetic process,carbohydrate metabolic process,etc.As can be seen from Figue 6,go molecular functions mainly involve nuclear receptor activity,lipid binding,cofactor binding,glucose binding,monocarboxylic acid binding,nitric-oxide synthase regulator activity,phosphatase binding,antioxidant activity,hormone binding,oxidoreductase activity,etc.As can be seen from Figue 7,GO cellular components mainly involve hosphatidylinositol 3-kinase complex,class IA (PI3K IA),secretory granule lumen,perinuclear region of cytoplasm,blood microparticle,RNA polymerase II transcription factor complex,membrane raft,etc.As can be seen from Figure 8,KEGG pathway mainly involves signal pathways such as non-alcoholic fatty liver disease,pathways in cancer,arachidonic acid metabolism,PPAR signaling pathway,linoleic acid metabolism,starch and sucrose metabolism,IL-17 signaling pathway,ovarian steroidogenesis,bile secretion and pancreatic secretion.Among these pathways,the most relevant is the signal pathway of non-alcoholic fatty liver disease,core potential targets such as AKT1,CYP2E1,IL1B,INS,NFKB1,NR1H3,PIK3CA,PIK3R1,PPARA,PTGS1,PTGS2,RXRA,SREBF1 and TNF are enriched in this pathway,as shown in Figure 9.

Figue 1 Wayne diagram of the intersection targets of compounds in four herbs of JZDPC and hyperlipidemia

Figue 2 Herbs-key compounds-intersection targets network diagram of JZDPC

Figue 3 Protein-protein interaction network diagram of JZDPC targets in the treatment of hyperlipidemia (interaction score ≥0.7)

Figue 4 Mcode cluster analysis of protein-protein interaction of JZDPC targets in the treatment of hyperlipidemia

Figue 5 GO biological process enrichment analysis of core putative targets of JZDPC in the treatment of hyperlipidemia

Figue 6 GO molecular functions enrichment analysis of core putative targets of JZDPC in the treatment of hyperlipidemia

Figue 7 GO cellular components enrichment analysis of core putative targets of JZDPC in the treatment of hyperlipidemia

Figue 8 KEGG pathway enrichment analysis of core putative targets of JZDPC in the treatment of hyperlipidemia

Figue 9 Non-alcoholic fatty liver disease signaling pathway

Discussion

According to the network diagram of herbs-key compounds-intersection targets,89 key compounds in JZDPC act on 99 intersection targets of drugs and diseases,which is consistent with the theory that traditional Chinese medicine has multi-component and multi-target in the treatment of diseases.Through further screening by mcode clustering function analysis in String database and Cytoscape software,it is predicted that the core components of JZDPC in the treatment of hyperlipidemia are mainly Ursolic Acid,β-Sitosterol,Resveratrol,Alimaric Acid,Alisol A,Oleanolic Acid,Rhein,Chrysophanol,Emodin,etc,and the core potential targets are NR3C1,ESR1,NR1I2,NFKB1,ESR2,ALOX5,PTGS1,PPARA,RXRA,LPL,PLA2G1B,PYGM,CYP2C9,CYP2E1,NR1H2,NR1H3,NR1H4,AKT1,AMY2A,GCK,HK1,PYGL,PTGS2,etc.

Some compounds have the effect of regulating blood lipid and its mechanism have been reported.Ursolic acid and oleanolic acid are isomers,belonging to pentacyclic triterpenoid acids,and they both can regulate the absorption,synthesis and metabolism of TG and TC[15].Liu et al.[16] found that ursolic acid inhibits lipid synthesis by competitively inhibiting the activities of fatty acid synthase such as acetyltransferase and malonyltransferase.Li et al.[17] confirmed that ursolic acid can also up regulate the expression of triglyceridase,the rate limiting enzyme of lipolysis,and promote lipolysis.Oleanolic acid can not only increase the expression of low density lipoprotein receptor (LDLR),but also reduce the expression of LDLR degrading enzyme PCSK9 protein,thus reducing the level of LDL-C [18].β-Sitosterol has structural similarity with cholesterol.It can competitively inhibit the intestinal absorption of cholesterol and promote the excretion of cholesterol in feces by up regulating the expression of ABCA1 [19-20],and inhibit the chronic inflammation related to hyperlipidemia by down regulating the expression of inflammatory factors in adipose tissue,such as IL-6,TNF-α,MCP-1,etc.[21].Resveratrol can up regulate the expression of pathway proteins such as CYP7A1,ABCG5 and PPARA [22-23],and down regulate the expression of pathway proteins such as PI3K,Akt and mTOR [24] to improve lipid metabolism.ALISOL A is a protoalkane triterpenoid component of Alisma orientalis,which up regulates the expression of CYP7A1 by activating farnesol X receptor (FXR) and regulates bile salt efflux pump [25].Anthraquinone compounds such as rhein,chrysophanol and emodin have good effects on regulating blood lipid.They can inhibit the activity of SREBPs in human hepatoma Huh7 cells and regulate the expression of lipolytic genes,such as PPARG,FAS,C/EBPα,etc,so as to inhibit the induced differentiation of preadipocytes 3T3-L1 into adipocytes,improve insulin sensitivity,reduce the contents of TC and TG,and reduce the particle size of white adipocytes and brown adipocytes [26-27].

Among the predicted core potential targets,NR1I2,NFKB1,PPARA,RXRA,LPL,NR1H2,NR1H3,NR1H4,AKT1,GCK and HK1 are mainly involved in the absorption,transport,synthesis and metabolism of lipids,cholesterol and triglycerides,and can regulate the levels of insulin and glucagon.Cholesterol in human cells cannot be directly catabolized.It needs to be transported to cholesterol carrier protein in plasma such as HDL through a series of transporters such as ABCA1 and ABCG1,and then reversed and transported back to the liver,and then transformed into bile acids under the action of invertase and discharged out of the body.Nuclear receptor 1 (NR1) subfamily is one of the members of nuclear receptor superfamily,which is closely related to glucose and lipid metabolism in vivo,mainly including NR1I2 (PXR),NR1C1-3 (PPARA,PPARB,PPARG),NR1H4(FXRA),etc.[28-29].Activation of PXR can reduce the expression of cholesterol reverse transporter ABCA1,inhibit the reverse transport of extrahepatic cholesterol back to the liver [30],inhibit the metabolism of cholesterol to bile acids by reducing the expression of CYP7A1,the rate limiting enzyme for the conversion of cholesterol to bile acids[31],and reduce the catabolism of cholesterol.Activation of PPARA accelerates the of fatty acids β Oxidation,improve fat utilization and regulate glycogen synthesis,and activation of PPARG can improve insulin sensitivity [32].LXRA And LXRB are two subtypes of liver X receptor.The former is mainly expressed in liver,small intestine,adipose tissue and macrophages,and the latter is widely expressed throughout the body [33].Activating LXR can promote the reverse transport of cholesterol,promote the metabolism of cholesterol in hepatocytes into bile acids,and reduce the reabsorption of cholesterol in bile [34-35].FXRA negatively regulate the synthesis and reabsorption of bile acids,regulate the efflux of bile acids,and activate the key enzyme lipoprotein esterase (LPL) of lipolysis and promote TG metabolism by up regulating the expression of apolipoprotein C-II (ApoC-II) and apolipoprotein V (ApoV) [36].The experiment showed that [37] the levels of TC and TG increased in the plasma and liver of mice with FXR gene knockout.ALOX5,CYP2C9,CYP2E1,PLA2G1B,PTGS1,PTGS2 and others mainly regulate the synthesis of prostaglandins and prostacyclin through arachidonic acid metabolic pathway and participate in inflammatory response [38-39].PYGM,PYGL,AMY2A and others are mainly involved in the metabolism of starch and sucrose.

The results of GO enrichment analysis show that the biological process is closely related to the synthesis and metabolism of lipids and steroids and the cellular response to hormones,inflammation and oxidative stress,molecular functions play a role by affecting nuclear receptor activity,lipid binding,cofactor binding and glucose binding,and the cellular components are mainly related to type PI3K IA,secretory granule lumen,perinuclear region of cytoplasm and so on.Nuclear receptors are a class of ligand activated transcription factor superfamily,which are located in the nucleus or transported from the cytoplasm to the nucleus after binding with ligands.Positive or negative regulation of nuclear receptors and their ligands is an important means to prevent and treat many diseases such as glucose and lipid metabolism diseases and cancer [40].PI3K IA can phosphorylate the hydroxyl group at the 3′ position of phosphatidylinositol to produce a second messenger,and then activate PI3K/ATP and other signal pathways,regulate cell proliferation,differentiation,metabolism and apoptosis,and participate in sugar,fat and protein metabolism [41].The results of KEGG pathway enrichment analysis show that the core pathways include non-alcoholic fatty liver disease cancer-related signal pathway,arachidonic acid metabolism signal pathway,peroxisome proliferator activated receptor signal pathway and starch and sucrose metabolism signal pathway.These pathways are closely related to hyperlipidemia and may be the reason for JZDPC to treat hyperlipidemia.The most relevant is the signal pathway of non-alcoholic fatty liver disease,which inhibits the synthesis of fatty acids,cholesterol and TG,promotes its catabolism,and participates in the processes of antioxidant stress,anti-inflammatory and anti fibrosis [42].From one side,it shows that JZDPC can also be used in the treatment of nonalcoholic fatty liver disease.

In conclusion,JZDPC plays a role in the treatment of hyperlipidemia,probably due to Ursolic Acid,β-Sitosterol,Resveratrol,Arirubic Acid,Alisol A,Oleanolic Acid,Rhein,Chrysophanol and Emodin,etc,act on signal pathways such as non-alcoholic fatty liver disease signaling pathway,pathways in cancer,arachidonic acid metabolism signaling pathway and peroxisome proliferator activated receptor signaling pathway,Starch and sucrose metabolism signaling pathway,etc,mediated by NR3C1,ESR1,NR1I2,NFKB1,ESR2,ALOX5,PTGS1,PPARα,RXRA,LPL,PLA2G1B,PYGM,CYP2C9,etc,and participate in lipid synthesis and metabolism,anti-inflammatory,antioxidant stress,regulating hormone level and carbohydrate metabolism.JZDPC is effective in the treatment of hyperlipidemia,and network pharmacology provides a certain theoretical basis for clarifying its mechanism.In the later stage,experimental studies such as content determination,proteomics and metabolomics need to be carried out to explore its exact mechanism.