Using Network Pharmacology to Explore Potential Treatment

Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2019, Article ID 7631365, 15 pages https://doi.org/10.1155/2019/7631365

Research Article Using Network Pharmacology to Explore Potential Treatment Mechanism for Coronary Heart Disease Using Chuanxiong and Jiangxiang Essential Oils in Jingzhi Guanxin Prescriptions

Jia Tai, Junbo Zou , Xiaofei Zhang, Yu Wang, Yulin Liang, Dongyan Guo, Mei Wang, Chunli Cui, Jing Wang, Jiangxue Cheng, and Yajun Shi

Shaanxi Province Key Laboratory of New Drugs and Chinese Medicine Foundation Research, Pharmacy College, Shaaxi University of Chinese Medicine, Xianyang 712046, China

Correspondence should be addressed to Yajun Shi; [email protected]

Received 3 July 2019; Revised 30 August 2019; Accepted 14 September 2019; Published 20 October 2019

Academic Editor: Gioacchino Calapai

Copyright © 2019 Jia Tai et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. To predict the active components and potential targets of traditional Chinese medicine and to determine the mechanism behind the curative effect of traditional Chinese medicine, a multitargeted method was used. Jingzhi Guanxin prescriptions expressed a high efficacy for coronary heart disease (CHD) patients of which essential oils from Chuanxiong and Jiangxiang were confirmed to be the most important effective substance. However, the interaction between the active components and the targets for the treatment of CHD has not been clearly explained in previous studies. Materials and Methods. Genes associated with the disease and the treatment strategy were searched from the electronic database and analyzed by Cytoscape (version 3.2.1). Protein-protein interaction network diagram of CHD with Jiangxiang and Chuanxiong essential oils was constructed by Cytoscape. Pathway functional enrichment analysis was executed by clusterProfiler package in R platform. Results. 121 ingredients of Chuanxiong and Jiangxiang essential oils were analyzed, and 393 target genes of the compositions and 912 CHD-related genes were retrieved. 15 coexpression genes were selected, including UGT1A1, DPP4, RXRA, ADH1A, RXRG, UGT1A3, PPARA, TRPC3, CYP1A1, ABCC2, AHR, and ADRA2A. *e crucial pathways of occurrence and treatment molecular mechanism of CHD were analyzed, including retinoic acid metabolic process, flavonoid metabolic process, response to xenobiotic stimulus, cellular response to xenobiotic stimulus, cellular response to steroid hormone stimulus, retinoid binding, retinoic acid binding, and monocarboxylic acid binding. Finally, we elucidate the underlying role and mechanism behind these genes in the pathogenesis and treatment of CHD. Conclusions. Generally speaking, the nodes in subnetwork affect the pathological process of CHD, thus indicating the mechanism of Jingzhi Guanxin prescriptions containing Chuanxiong and Jiangxiang essential oils in the treatment of CHD.

1. Background were often accompanied by a high prevalence of coronary atherosclerosis [6, 7]. Despite the decline in mortality from Cardiovascular diseases (CVD) cause more than 17.3 million heart disease in recent years, the social burden of coronary deaths per year with an estimated mortality increase to 23.6 heart disease remains worrisome, particularly in developing million by 2030 [1, 2]. Coronary heart disease (CHD) is the countries. However, the potential molecular mechanism of leading cause of cardiovascular disease, usually caused by CHD is unclear. *erefore, there is an urgent need for in- coronary artery occlusion [3], and is the cause of the highest depth research and improvement of the treatment of CHD, in morbidity and mortality in the world [4, 5]. Myocardial in- order to achieve the purpose of reducing the health and farction (MI), palpitation, and angina pectoris are the main economic burden of patients with coronary heart disease. clinical manifestations of CHD [2]. Necropsy analyses of Traditional Chinese medicine (TCM) plays a systemic patients who suffered a fatal cerebral stroke indicated that they role with multiple targets and multiple ways in treating 2 Evidence-Based Complementary and Alternative Medicine diseases. Jingzhi Guanxin prescription is a standardized 5.1.1, released 03 July 2018, https://www.drugbank.ca/), cardiovascular herb medicine from Chinese Pharmacopoeia which is a unique bioinformatics and chemical informatics 2015 editions [8]. Jingzhi Guanxin prescriptions can pro- database, containing 11,628 drugs and related chemical mote blood circulation and remove blood stasis, which is information, drug targets, protein data, and so on [20]. In used to treat angina pectoris and coronary heart disease [8]. the study, genes were included from the DisGeNETdatabase *ese prescriptions contain five herbs, i.e., Salvia miltior- with DSI scores above the median, as well as all CHD-related rhiza Bge. (Danshen), Ligusticum chuanxiong Hort. genes from DrugBank and TTD. *rough the retrieval of (Chuanxiong), Paeonia lactiflora Pall. (Chishao), Dalbergia Universal Protein Resource (UniProt, http://www.uniprot. odorifera T. (Jiangxiang), and Carthamus tinctorius L. org/), all the genes were normalized into consistent symbols, (Honghua), all of which are recorded in Chinese Pharma- and the unified information contained UniProt number and copoeia 2015 edition [8]. Several studies have indicated that gene abbreviation. Jingzhi Guanxin prescriptions have been highly effective for patients with CHD [9, 10], but the specific mechanism is still unclear. Network pharmacology is a field in which network 3.2. Compositions of Essential Oil from Chuanxiong and biology and multipharmacology are combined [11]. Prin- Jiangxiang. *e essential oil from Chuanxiong was cipally, the methods are focused on identifying and ranking extracted by steam distillation, and the compositions were the targets in biological networks [12]. *e network analyses analyzed by gas chromatography-mass spectrometer (GC- of biological pathways and interactions have revealed that MS). *e constituents of Jiangxiang essential oil were ob- the robustness of biological systems can be obtained from tained from the literature by searching CNKI, Wanfang, and the network structure to a large extent [11, 13, 14]. Coex- PubMed databases with the keywords of “Jiangxiang volatile pressed genes were enriched for searching functionally re- oil” and “dalbergiae odoriferae volatile oil”. Studies were lated genes, and its network showed mutual investigation referenced which reported compositions of essential oil from and mutual relationship [15]. Further, functional enrich- Jiangxiang analyzed by GC-MS. Because the chemical ment analysis could determine the mechanisms of the pu- composition is usually represented by multiple chemical tative targets. names, we converted the chemical name to Chemical Ab- In Jingzhi Guanxin prescriptions, Chuanxiong and stracts Service (CAS Number) so that TCMSP or PubChem Jiangxiang essential oils are the most important material can be used to identify these chemical compounds. *e basis for promoting blood circulation and removing blood composition name, name code, and CAS Number of stasis [16, 17]. Previously, our research group had carried out compositions are arranged in the supplementary documents. a massive systemic research of the essential oils. On this basis, the related genes of CHD and essential oils were re- 3.3. Collection of Compositions Associated with the Gene. trieved, and their biological functions were analyzed in order *e main source of composition-targets was obtained from to further clarify the molecular mechanism of the essential TCMSP (version 2.3, update to 31 May 2014, http://lsp.nwu. oils in the treatment of CHD and to provide a reference for edu.cn/tcmsp.php) database [21]. TCMSP database has the clinical application of these essential oils and for further specific informatics methods to infer drug-disease connec- drug development. tion, which were collected from 499 herbs which were all registered in the Chinese Pharmacopoeia (2010) with a total 2. Materials and Methods of 12,144 chemicals [21]. Another major database for ac- quiring composition-targets is STITCH (http://stitch.embl. *e technical strategy of this research is shown in Figure 1. de/) by searching SMILES structure; STITCH is a database of *e research strategy was based on network pharmacology known and predicted interactions between chemicals and of deciphering key pharmacological pathways involved in proteins currently containing 9,643,763 proteins from 2,031 Chuanxiong and Jiangxiang essential oils acting on CHD. organisms [22]. *e SMILES structure of compositions obtained from PubChem (https://pubchem.ncbi.nlm.nih. 3. Data Collections gov/) which is an open chemistry database with 96,502,248 compositions of which 3,151,393 have been 3.1. Collection of Coronary Heart Disease- (CHD-) Related tested. Genes. Significant genes associated with CHD were obtained from DisGeNET (version 5.0, http://www.disgenet. org/web/DisGeNET/menu/home). DisGeNET is a discovery 3.4. Network Construction and Analysis. Many common platform containing collections of 561,119 genes associated diseases such as cancer and CHD are often caused by with human diseases [18]. In order to collect comprehensive multiple molecular abnormalities [23]. In the “network retrieval results, *erapeutic Target Database (TTD, last target” theory, the establishment of molecular connections update by 15 September 2017, https://db.idrblab.org/ttd/) between drug/herbal formulae and diseases/TCM syn- was also used to retrieve genes related to coronary heart dromes is crucial [24]. *ese molecular connections are disease, which is a database that provides known and ex- derived from a disease-specific network, which can be plored therapeutic proteins, targeted diseases, correspond- formed due to the interactions of genes or gene products ing drugs for these targets, and so on [19]. In addition, CHD- [24]. Meanwhile, the introduction of a “network” in drug related genes were also collected from DrugBank (version discovery incorporates the assessment of network topology, Evidence-Based Complementary and Alternative Medicine 3

Database construction

TCMSP DisGeNET TCMSP DrugBank Therapeutic target STITCH DisGeNET DrugBank

Chuanxiong essential oil Jiangxiang essential oil Coronary heart disease targets targets targets

Network construction

Functional enrichment analysis

Figure 1: Process overview. as well as dynamics, and thus offers a quantifiable de- network was constructed by using the Top-Go package of R scription of the complex biological system and its response platform [33]. to various drug/herbal treatments [24]. *e nodes with high centrality (e.g., network degree) can be viewed as key nodes 4. Results in a network [24]. *e concept attempts to comprehensively describe all of the possible vulnerable targets for clarifying 4.1. Collection of 487 Genes Associated with CHD from Dis- the efficiency of drug treatment [24]. Composition-target- GeNET, TTD, and DrugBank. A total of 912 CHD-related CHD regulatory network was constructed by using Cyto- genes were retrieved from DisGeNET and 457 genes with scape (version 3.2.1) software; all node degrees of the net- a DSI score higher than the median (0.55) were selected, work were calculated at the same time. Different expression 28 CHD target genes were found in DrugBank, and one profiles of genes in relation to the compositions and CHD gene was obtained from TTD. Further, 487 genes were were filtered by Venny2.1.0 [25]. All nodes whose degrees identified and an analytical network was established. were more than twice the median were considered as key Details of these genes are provided in Supplementary nodes, which are filtered by the Cytoscape plug-in MCODE Table S1. [26–28].

4.2. Compositions and Targets of Chuanxiong and Jiangxiang. 3.5.GOEnrichmentandKEGGPathwayAnalysis. In order to As shown in the GC-MS analysis results, 83 ingredients were better understand the potential biological process of pre- detected from Chuanxiong essential oil. And 32 composi- dictive genes, KEGG (Kyoto Encyclopedia of Genes and tions of Jiangxiang essential oil were obtained from the Genomes, Release 88.0, October 1, 2018) and GO (Gene literature [34, 35]. An overview of ingredients of Chuan- Ontology, last updated on March 9, 2018) were analyzed for xiong and Jiangxiang essential oils is shown in Supple- pathway functional enrichment using clusterProﬁler soft- mentary Table S2. A total of 315 essential oil-related targets ware package on R platform [29–32]. *e interaction of Chuanxiong essential oil were retrieved from TCMSP and 4 Evidence-Based Complementary and Alternative Medicine

RXRG ADH1A

RXRA UGT1A3

SEMA3D PPARA ADRA2A RPA1 GRM8 PLA2G10 PHACTR1 FUT3 DDAH1 GP6 CTSS CA1 UGT1A10 KCNK3 CA2 TRPV3 NR1H3 TRPC3 AHR UGT1A7 ARSD HOMEZ CHRNA2 GPR162 UGT1A8 LRP6 CHDS1 CHRM3 LTC4S HDAC2 TAB2 ABCG1 CYP1A1 ABCC2 MAOA NREP LGALS14 CHRM2 OLR1 ALX4 OR2A25 TRAF5 MMP9 SENP2 ESYT3 KIAA1462 DDAH2 CHRM1 MIR2909 PRDM10 GUK1 MAPK1 BCL2A1 CFB SLC2A9 LGALS2 PNN PLA2G5 ANGPTL4 IGHG1 MIR23A IL1F10 GMCL1 SERPINA9 JUN SRPX HSPA8 IL15RA APOC4 TRIB1 SCARB1 PTPRT GABRA6 DLEU7 PLTP LIPE MRAS MYBPC3 SELPLG ADH1B KCNE2 TMEM57 GPD1L LPAL2 CYP2D6 PPIA GABRA5 IL36RN SMAD1 VAMP8 FABP2 CHDH C5AR2 NR2F2 FADS3 PROC GMCL1P1 ATP4B ADH1C ADAM11 EIF3M BHMT2 NDST1 FEN1 BHMTANGPTL3 CKM TAS2R50 GABRA3 CD93 ZNF132 USP9Y CYP7A1 CFDP1 SREBF2 PLGLB2 GREM1 CHDS4 TBX20 MAOB CAPN10 FRA1H CYP2C8 CHDS3 PPBP PALLD SNX19 MYLIP PLGLB1 HAP1 KDSR P2RY1 SENP1 GABRA2 PLA2G2D SLCO1B1 LTBP1 NEUROD1 C2 PEAR1 TMED1 IL1RL1 STN1 NCOA2 ADTRP SLC26A8 HAND1 PSMA6 MIR19B1 PON3 TIMP4 HDL3 MMP26 CYP2B6 PTGS1 CIMT ABCG5 EPRS KLF2 CYP2J2 NLN GPER1 MIRLET7I SLC2A6 JPH3 MIR365A GDF1 FCRLB ALDH1B1 KLF5 TBX3 PTGS2 PF4V1 HEATR6 TFAP2BHPS1 EPHX2 TRPM3 ANP32B SLC25A20 ITGAE IRF8 KLF14 FOLH1 TLR4 FADS2 ACKR2 ACVR1 LMAN1 LIPA PRCP RBL2 ITGAL THBS4 PRDX5 ST8SIA4 DLC1 GOT2 OR13G1 JX4 CYP3A5 LIPG SLC6A2 ABCG8 UCP3 TNFSF4 GCA JX5 JX3 BRAP PCLAF ELMO1 MCFD2 NOX5 SOAT2 FSHMD1A TLR2 PLA2G7 FGF21 MIA3 MADD CACNA1C DAB2IP CPY1A2 CYP2C9 TOX HNRNPUL1 HSPA1L CCHCR1 HAL CPE CYBRD1 CUBN SAA4 JX6 JX2 GABRA1 RPLP0P4 UBL5 NEXN AQP5 S1PR1 ABCA7 BCAR1 PALD1 LEFTY1 OPN4 ABCC4 KRT74 APOA4 P2RX6 STRN SERTAD3 GNGT2 CYP4A11 TBX5 MYH15 AMPD1 MEF2A PRKCE MIR224 SUMO1 FRZB WDR12 APOM MRPS6 PLPP3 TRNA JX7 JX1 CHRNA7 MKL1 GCLM CNDP1 CAMTA1 TMPRSS11B LRP8 ADAM33 IL18RAP PGLYRP2 RASGRF2 WRB SCAR2 TES GLP1R POU2F3 ROBO3 SLCO2B1 KAT2B CCL19 APOL1 PDZK1 HEATR5B DPP4 APOC1 APOA5 CHD TMEM170A PRMT8 ADRA1A CHDS2 AVSD1 F2RL3 HAS2 HCAR2 USF1 JX8 JX16 APOD SCAP AQP10 RPN1 TAS2R38 TRIB3 HIRA PRRC2ACSNK1D WDHD1 AHSG LCAT MESP1 GSTM5 ADORA3 FLAD1 RIPK4 SERPINA12 CITED2 NODAL VKORC1 SUMO4 NNMT ADRA1B CYSLTR1 ARL15 CORT ISL1 SGIP1 MIR486−1 ALOX5AP ZIC3 SH3BGR NPPC HLP TNNT1 GHS PGR ADD1 RFC1 SELENBP1 ZNF627 GRK4 WDR31 OAZ1 IRX4 JX9 JX15 INSIG1 CDKN2B−AS1 PARL CYP3A7 ADRA1D 5−HT1B SREBF1 PCDH8 AS3MT ZNF202 GALNT2 PROCR CHDS9 SRSF2 WDR55 TAF3 INSIG2 LEFTY2 LPL SP6 MLF1 NECTIN2 NPFFR2 CES1 ASD1 ZBTB21 THRA1/BTR KIDINS220 ACAT2 RN7SL263P JX10 JX14 MYH6 GSK3A ADRA2B MIR361 UTY OSR1 DTNA APLNR ABCC1 EHD3 CHIT1 KALRN TBX18 BMP1 KIF28P CES1P1 IVL LDB2 JX11 JX13 FSTL4 NANOS3 ADIPOR1 SRSF1 TP53COR1 COG2 IL1RAP JX12 SLC7A13 MPZL2 CBSL ADRA2C LY6G5B ADH7 PGPEP1 WNT16 ARMS2 DNAH11 ADIPOR2 OR2F2 NAXE CYP3A4 NDST4 IL6 KIF6 MTHFD1L GSTM4 IL18BP ZBTB7C SVEP1 ACTC1 MIA NTN1 SORT1 RYR3 CASQ2 ALOX5 SLC30A8 HPSE2 FAM175B QKI DMXL2 ABCB11 F12 UGT2B7 OR10A4 APOA2 NDUFS2 HEY2 MIR206 IGHD POC1A UGT1A1 CFC1 MIR499A QRSL1 ANGPTL2 KLK1 TCF21 FCGR2B GJA4 NTRK3 PGR−AS1 ADRB1 IMPACTSLC2A3 ASGR1 SLCO1A2 ADAMTS7 SOD3 CFD FABP4 CRELD1 PCSK1 P2RY12 MYOM3 GLS2 CHDS8 FCAR SLC5A3 ADRB2 PRKY EARS2 KCTD12 MT1B APOC2 ST2 MCM10 SLC15A1 KIF2C CREG1 ZNF568 ST6GALNAC5 CASP3 EHMT1 VTI1A GRIK4 HMGCR PCSK9 ATP5J ALOX12B ATXN2L NEUROG3 KIF9 ATP2C1 ZGLP1 ZPR1 FADS1 FCGR2C LRPAP1 CELSR2 NR1H4 CYP2C19 PSRC1 RNLS CTRB1 ADAMTS1 RENBP CXCL16 BUD13 IRF2BP2 SERPINB8 DEFB4A NOS1AP NPC1L1 NAMPT LOC107987506 CYP1B1 SELENOS PCSK6 UTS2 SLC39A7 BAK1 DRD1 HHEX DSCAM PLAU APOF CPS1 ENOX2 MIR197 TBL1Y RELA LTA4H MIR4513 CD86 MUC1 SLC6A3 NOS3 SCN5A CRYZ EXTL3 PPP3CA

ABCC5 Figure 2: *e composition-target-disease networks of Jiangxiang and CHD. *e triangle node represents CHD; the diamond nodes represent related genes of CHD; the circular blue nodes represent ingredients of Jiangxiang essential oil; the red V-shape nodes represent related genes of Jiangxiang essential oil; the circular purple nodes represent coexpression of CHD and Jiangxiang essential oil.

Figure 3: *e composition-target-disease networks of Chuanxiong and CHD. *e rectangle yellow node represents CHD; the diamond purple nodes represent related genes of CHD; the purple V-shape nodes represent ingredients of Chuanxiong essential oil; the blue oval nodes represent related genes of Chuanxiong essential oil; the red triangle nodes represent coexpression of CHD and Chuanxiong essential oil.

STITCH databases. And 78 related targets of Jiangxiang 4.3. Network Analyses. In order to explain the potential essential oil were searched from TCMSP, DrugBank, and pharmacological eﬀects of Chuanxiong and Jiangxiang es- DisGeNET. Details of all component-related genes can be sential oils in the treatment of CHD, a composition-target- found in Supplementary Table S3. disease network was established (Figures 2 and 3, layout type Evidence-Based Complementary and Alternative Medicine 5

CHRM1

JX13

CHRM2

JX8

Figure 4: *e cnetplot of Jiangxiang composition-target-disease network. *e circular blue nodes represent ingredients of Jiangxiang essential oil; the circular red nodes represent related genes of Jiangxiang essential oil.

GNAI2

CHX63

CHRM1

CHX77

Figure 5: *e cnetplot of Chuanxiong composition-target-disease network. *e purple V-shape nodes represent ingredients of Chuanxiong essential oil; the blue oval nodes represent related genes of Chuanxiong essential oil. is “circular layout”). In the network, hub genes with degrees Jiangxiang Chuanxiong greater than twofold of the median were considered key nodes which were filtered by Cytoscape plug-in MCODE [36, 37]. Key nodes in the pathway were selected by the R language, including CHRM1, CHRM2, JX13, JX8, GNAI2, CHRM1, 11 10 104 CHX77, and CHX63, CNET networks as shown in Figures 4 (1.8%) (1.6%) (17.1%) and 5. 3 Apart from these, 12 coexpression genes were selected by (0.5%) Venny2.1.0, including UGT1A1, DPP4, RXRA, ADH1A, 7 2 (1.2%) (0.3%) RXRG, UGT1A3, PPARA, TRPC3, CYP1A1, ABCC2, AHR, and ADRA2A. *e Venny plot is shown in Figure 6. 470 (77.4%) 4.4. Functional Enrichment Analyses. In order to determine the mechanism of predicting the target, KEGG and GO were executed by the clusterProfiler software package in the R language. A total of 944 pathways were enriched and are CHD arranged in Supplementary Table S4. Among them, 111 Figure 6: Twelve coexpression genes of CHD and ingredients. *e pathways were enriched from KEGG; the first 20 pathways blue represents related genes of Jiangxiang essential oil; the yellow are displayed in Table 1, and Figure 7 shows the top 10 represents related genes of Chuanxiong essential oil; the green pathways with the highest p value. As shown in Figure 7, represents related genes of CHD. 6 Evidence-Based Complementary and Alternative Medicine

Table 1: Top 20 enriched KEGG pathways and top 10 enriched GO pathways. Description p value p.adjust q value Count Source Retinol metabolism 2.77E – 06 8.12E – 05 3.51E – 05 4 KEGG Metabolism of xenobiotics by cytochrome P450 4.60E – 06 8.12E – 05 3.51E – 05 4 KEGG Chemical carcinogenesis 6.25E – 06 8.12E – 05 3.51E – 05 4 KEGG Steroid hormone biosynthesis 9.79E – 05 9.54E – 04 4.12E – 04 3 KEGG Adipocytokine signaling pathway 1.56E – 04 1.07E – 03 4.63E – 04 3 KEGG Drug metabolism—cytochrome P450 1.77E – 04 1.07E – 03 4.63E – 04 3 KEGG PPAR signaling pathway 1.93E – 04 1.07E – 03 4.63E – 04 3 KEGG *17 cell differentiation 5.72E – 04 2.79E – 03 1.20E – 03 3 KEGG Ascorbate and aldarate metabolism 8.12E – 04 3.52E – 03 1.52E – 03 2 KEGG Pentose and glucuronate interconversions 1.29E – 03 5.03E – 03 2.17E – 03 2 KEGG *yroid cancer 1.53E – 03 5.41E – 03 2.34E – 03 2 KEGG Porphyrin and chlorophyll metabolism 1.97E – 03 6.39E – 03 2.76E – 03 2 KEGG Non-small-cell lung cancer 4.79E – 03 1.44E – 02 6.21E – 03 2 KEGG Bile secretion 5.53E – 03 1.54E – 02 6.65E – 03 2 KEGG Drug metabolism—other enzymes 6.81E – 03 1.77E – 02 7.64E – 03 2 KEGG Small cell lung cancer 9.33E – 03 2.27E – 02 9.82E – 03 2 KEGG Parathyroid hormone synthesis, secretion, and action 1.20E – 02 2.75E – 02 1.19E – 02 2 KEGG *yroid hormone signaling pathway 1.43E – 02 3.09E – 02 1.33E – 02 2 KEGG Nonalcoholic fatty liver disease (NAFLD) 2.29E – 02 4.25E – 02 1.83E – 02 2 KEGG Hepatitis C 1.80E – 02 3.69E – 02 1.59E – 02 2 KEGG Cellular response to xenobiotic stimulus 6.58E – 08 3.75E – 05 1.97E – 05 5 GO-BP Flavonoid metabolic process 1.09E – 07 3.75E – 05 1.97E – 05 3 GO-BP Retinoic acid metabolic process 4.82E – 07 8.37E – 05 4.41E – 05 3 GO-BP Cellular response to steroid hormone stimulus 4.85E – 07 8.37E – 05 4.41E – 05 5 GO-BP Response to xenobiotic stimulus 6.64E – 07 9.16E – 05 4.82E – 05 5 GO-BP Xenobiotic metabolic process 9.64E – 07 1.11E – 04 5.83E – 05 4 GO-BP Antibiotic metabolic process 1.74E – 06 1.71E – 04 9.00E – 05 4 GO-BP Regulation of lipid metabolic process 2.62E – 06 2.26E – 04 1.19E – 04 5 GO-BP Response to steroid hormone 3.62E – 06 2.78E – 04 1.46E – 04 5 GO-BP Xenobiotic catabolic process 2.32E – 05 1.46E – 03 7.67E – 04 2 GO-BP Transcription factor complex 1.21E – 03 2.37E – 02 1.45E – 02 3 GO-CC Receptor complex 1.53E – 03 2.37E – 02 1.45E – 02 3 GO-CC RNA polymerase II transcription factor complex 4.38E – 03 3.97E – 02 2.43E – 02 2 GO-CC Nuclear transcription factor complex 6.15E – 03 3.97E – 02 2.43E – 02 2 GO-CC Endoplasmic reticulum chaperone complex 6.40E – 03 3.97E – 02 2.43E – 02 1 GO-CC Invadopodium 1.02E – 02 5.28E – 02 3.23E – 02 1 GO-CC Lamellipodium membrane 1.40E – 02 5.90E – 02 3.61E – 02 1 GO-CC Apical plasma membrane 1.52E – 02 5.90E – 02 3.61E – 02 2 GO-CC Cell projection membrane 1.84E – 02 6.24E – 02 3.82E – 02 2 GO-CC Cytochrome complex 2.10E – 02 6.24E – 02 3.82E – 02 1 GO-CC Nuclear receptor activity 2.61E – 08 1.45E – 06 7.15E – 07 4 GO-MF Transcription factor activity, direct ligand-regulated 2.61E – 08 1.45E – 06 7.15E – 07 4 GO-MF sequence-specific DNA binding Monocarboxylic acid binding 6.41E – 08 2.37E – 06 1.17E – 06 4 GO-MF Retinoic acid binding 1.98E – 07 5.50E – 06 2.71E – 06 3 GO-MF Retinoid binding 1.44E – 06 2.92E – 05 1.44E – 05 3 GO-MF Isoprenoid binding 1.58E – 06 2.92E – 05 1.44E – 05 3 GO-MF Carboxylic acid binding 6.03E – 06 8.72E – 05 4.30E – 05 4 GO-MF Organic acid binding 6.29E – 06 8.72E – 05 4.30E – 05 4 GO-MF Steroid hormone receptor activity 7.77E – 06 9.59E – 05 4.73E – 05 3 GO-MF Protein heterodimerization activity 1.24E – 05 1.37E – 04 6.76E – 05 5 GO-MF retinol metabolism, metabolism of xenobiotics by cyto- small cell lung cancer, parathyroid hormone synthesis, se- chrome P450, chemical carcinogenesis, steroid hormone cretion and action, thyroid hormone signaling pathway, biosynthesis, adipocytokine signaling pathway, drug hepatitis C, nonalcoholic fatty liver disease (NAFLD) were metabolism—cytochrome P450, PPAR signaling pathway, involved in the pathological development of CHD. *e *17 cell differentiation, ascorbate and aldarate metabolism, execution of GO pathways was done using the clusterProfiler pentose and glucuronate interconversions, thyroid cancer, software package of R language, including BP (biological porphyrin and chlorophyll metabolism, non-small-cell lung process), CC (cellular component), and MF (molecular cancer, bile secretion, drug metabolism—other enzymes, function) analysis, a total of 833 pathways were enriched Evidence-Based Complementary and Alternative Medicine 7

p.adjust

Retinol metabolism

Metabolism of xenobiotics by cytochrome P450 0.001 Chemical carcinogenesis

Steroid hormone biosynthesis 0.002 Adipocytokine signaling pathway

Drug metabolism-cytochrome P450 0.003 PPAR signaling pathway

Th17 cell differentiation 0.004

Ascorbate and aldarate metabolism

Pentose and glucuronate interconversions 0.005

0.20 0.25 0.30 Gene ratio

Count 2.0 3.5 2.5 4.0 3.0 Figure 7: Top 10 enriched KEGG pathways with p value.

[38], and top 10 significant pathways are listed in Table 1. In Jiangxiang and Chuanxiong are important components of order to reflect the internal relationship between these GO decoctions and Chinese patent medicine for the treatment of terms, the clusterProfiler software package was used to re- CHD and angina pectoris [8]. Wei et al. [41] indicated that construct the interactive network (Figures 8–10). Jingzhi Guanxin prescription can reduce the range of *e pathway network of hub genes was filtered, and the myocardial infarction in rats with acute myocardial ische- cnetplot is shown in Figures 11 and 12. *e cnetplot in- mia. Jiangxiang essential oil and water extract can protect dicated that the subnetwork could participate in the path- rats from ischemia/reperfusion injury, which may play a role ological development processes of CHD retinoic acid by regulating sugar metabolism, lipid metabolism, and metabolic process, flavonoid metabolic process, response to amino acid metabolic pathways [42]. Meanwhile, Jiangxiang xenobiotic stimulus, cellular response to xenobiotic stimu- has been observed to reduce the degree of atherosclerosis lus, cellular response to steroid hormone stimulus, retinoid and erythrocyte deformability in experimental atheroscle- binding, retinoic acid binding, monocarboxylic acid bind- rotic rabbits [43]. A large number of experimental results ing, transcription factor activity, and direct ligand-regulated showed that Jiangxiang volatile oil could inhibit thrombosis sequence-specific DNA binding. *e target genes partici- and increase platelet cAPM in incubated rabbits [44, 45]. In pating in the core network include AHR, CYP1A1, UGT1A3, addition, the flavonoids in Jiangxiang have the effects of UGT1A1, ABCC2, RXRA, RXRG, and PPARA. antioxidation, anticancer, anti-inflammation, analgesia, and antiplatelet aggregation [46–48]. *e volatile oil of Ligu- 5. Discussion sticum chuanxiong can significantly slow down the heart rate and weaken the myocardial contractility [17]. *ese effects *e overall and local burden of CHD is enormous. On an may be attributed to the mechanism of volatile oil in the annual basis, about 785,000 Americans have new coronary treatment of CHD. attacks and 470,000 have a recurrence [5]. Based on the Networks may provide a scaffold for the integration of theoretical principles of TCM, CHD belongs to the category omics data [15]. Network target can provide predictive and of chest arthralgia and heartache [39]. Jingzhi Guanxin quantitative measures to the mechanistic role of drugs or prescription is a classical prescription for the treatment of herbal formulae in the treatment of diseases [24]. With the angina pectoris of CHD [40]. It is worth mentioning that rapid advancement in bioinformatics, systems biology, and 8 Evidence-Based Complementary and Alternative Medicine

p.adjust

0.001

0.002

0.003

0.004

0.005

0.006

Size 2 4 3 5 Figure 8: *e GO interaction network of coexpression genes (the emapplot of coexpression genes from BP enriched pathway). polypharmacology, “network pharmacology,” there is a shift Studies found that expression of DPP4 in the heart was from “one target, one drug” paradigm to the “network target, coordinated with a set of gene expression signature char- multicomponent” strategy, as it can not only reveal the acteristic for whole blood proliferation, which is enriched for underlying complex interactions between a herbal formula genes involved in cell cycle control and DNA replication, and cellular proteins, but also detect the influence of their potentially impacting peripheral stem cell mobilization [53]. interactions on the function and behavior of the human Ku et al. [54] found that DPP4 can protect the heart from system. *is key idea is in line with the holistic theory of ischemia/reperfusion through GLP-1 receptor-dependent TCM [36]. In order to study the relationship between drugs and receptor-independent mechanisms. Methylation of and disease targets and to clarify the targets of volatile oils in RXRA gene promoter may be one of the reasons for the the treatment of CHD, a component-target-disease network downregulation of the expression of right subventricular was constructed, which provided a basis for further un- bundle myocardium in patients with tetralogy of Fallot [55]. derstanding the changes of disease tissues and revealed the Research shows that drugs can improve lipid metabolism in molecular mechanism of CHD. *e coexpressed genes in the ischemic heart model by regulating transcriptional factors target network represent the potential target of Jiangxiang such as RXRA and PPARs [56]. And ADH1A makes an and Chuanxiong volatile oils in the treatment of coronary important impact on the omega oxidation pathway [57]. heart disease. In the results, twelve coexpression genes were Further, studies have identified a human-specific sub- selected by Venny2.1.0, including UGT1A1, DPP4, RXRA, network regulated by RXRG, which has been validated to ADH1A, RXRG, UGT1A3, PPARA, TRPC3, CYP1A1, play a different role in hyperlipidemia and type 2 diabetes ABCC2, AHR, and ADRA2A. Many studies have shown a between human and mouse [58]. Familial combined hy- significant association between low serum bilirubin levels perlipidemia (FCHL) is the most common atherogenic and CVD [49]. UGT1A1 is the only enzyme that contributes disorder of lipid metabolism [59]. Variation in the RXRG substantially to bilirubin glucuronidation and thus enhances gene may contribute to genetic dyslipidemia in FCHL bilirubin elimination (catalyzed by UGT1A1 enzyme) subjects [59]. UGT1A3 serves as potential therapeutic targets [50, 51]. Further, TA repeat polymorphism may be a key for CHD risk and has an effect on the function of high- characteristic in the gene controlling bilirubin level [52]. density lipoprotein [60]. PPARA is an important gene that Evidence-Based Complementary and Alternative Medicine 9

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Size 1 4 2 5 3 Figure 9: *e GO interaction network of coexpression genes (the emapplot of coexpression genes from MF enriched pathway). 10 Evidence-Based Complementary and Alternative Medicine

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Receptor complex 0.024

RNA polymerase II transcription factor complex

0.028

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0.036 Nuclear transcription factor complex

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Size 1.0 2.5 1.5 3.0 2.0 Figure 10: *e GO interaction network of coexpression genes (the emapplot of coexpression genes from CC enriched pathway).

CYP1A1 Retinoic acid metabolic process UGT1A3 Flavonoid metabolic process Response to xenobiotic stimulus AHR Cellular response to xenobiotic stimulus UGT1A1

ABCC2

Cellular response to steroid hormone stimulus PPARA

RXRA RXRG

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Figure 11: *e GO interaction network of coexpression genes (the cnetplot of coexpression genes from BP enriched pathway). controls lipid metabolism. Studies have found that reduced plays an important role in the process of myocardial fibrosis PPARA expression during heart failure leads to reduced fatty [63]. *e members of CYP1 family (1A1, 1A2, and 1B1) play acid oxidation and myocardial energy deficiency [61, 62]. a major role in the bioactivation of PAHs to genotoxic Volatile oil may affect the lipid metabolism in the heart by metabolites which lead to DNA adducts, atherosclerosis, and acting on the expression of PPARA in the heart tissue. carcinogenesis [64]. Ko and Shin [65] demonstrated that TRPC3 is highly expressed in the heart and participates in cardio-sulfa caused aberrant heart development in zebrafish the pathogenesis of cardiac hypertrophy and heart failure as and was activated through the AhR signaling pathway in a a pathological response to chronic mechanical stress [38]. CYP1A-independent manner. Research results indicate that Meanwhile, TRPC3 channel is an indispensable regulator of variations in the ABCC2 gene might influence the left fibrosis development, by promoting fibroblasts to transition ventricular parameters [66]. Additionally, AHR is highly into myofibroblasts via intracellular Ca2+ overload, and it correlated with heart defects. Studies have shown that when Evidence-Based Complementary and Alternative Medicine 11

UGT1A3 UGT1A1

Retinoid binding Retinoic acid binding Monocarboxylic acid binding

RXRA

PPARA

Nuclear receptor activity Transcription factor activity, direct ligand regulated sequence−specific DNA binding

AHR

RXRG Size 3 4

Figure 12: *e GO interaction network of coexpression genes (the cnetplot of coexpression genes from MF enriched pathway).

AHR is activated, it can lead to heart malformations in drugs for the treatment of coronary heart disease and can zebrafish embryos [67]. Furthermore, a study indicated that increase the survival rate of patients with acute myocardial the cardiac developmental toxicity of PM2.5 might be infarction [1]. G (i) protein can affect the response of cyclase prevented by targeting AHR or wnt/β-catenin signaling [67]. to β-adrenergic stimulation by participating in the hormone *is study suggested that fetal ADRA2A may be important regulation of adenylate cyclase [71]. *e activation of β1- for normal heart development. However, it has been sug- adrenergic receptor can produce positive myocardial effect, gested that adult cardiac myocytes are virtually devoid of which leads to the aggravation of contraction, the acceler- postsynaptic ADRA2A [68]. *ese results provide an im- ation of cardiac ejection velocity, and the increase of heart portant reference for the further study of the pathogenesis of rate, and β2-adrenergic receptor can cause smooth muscle CHD and the pathway mechanism for the treatment of relaxation [72]. GNAI2 is guanine nucleotide-binding pro- CHD. Overall, these coexpressed genes participate in the tein G (i) subunit alpha-2, and it may affect the role of process of lipid metabolism, myocardial fibrosis, ischemia/ epinephrine in the heart by inhibiting the stimulation of reperfusion, and other physiological changes in the heart to β-epinephrine by cyclase. K+ plays an important role in varying degrees, thus affecting the development of CHD. maintaining the normal operation of cardiac electrophysi- Jiangxiang and Chuanxiong volatile oils may play a role in ology, and the abnormal change in the K+ channel is an the treatment of coronary heart disease by acting on these important factor leading to various heart diseases [73]. targets and giving full play to the corresponding biological Muscarinic acetylcholine receptors play a role in regulating effects. cardiac function and smooth muscle contraction [74]. GO analysis provides the most comprehensive resource Studies have shown that CHRM2 is closely related to the which is currently available for computable knowledge re- expression of human cardiac function and plays an im- garding the functions of genes and gene products, used to portant role in the regulation of cardiovascular functions recognize shared associations between proteins and anno- [75, 76]. CHRM1 plays an important role in the regulation of tations to GO [69]. KEGG is a database resource for the IK, Ach atrial repolarization [77]. CHRM1 and CHRM2 can understanding of high-level functions and utilities of the regulate K+ channels through the action of G protein. biological system. We used KEGG and GO to enrich 944 Meanwhile, CHRM1 can increase heart rate [78, 79] and pathways, which revealed the molecular mechanism of CHD contractile force [80], CHRM2 can modulate pacemaker and better explained the variation between healthy and activity, atrioventricular conduction, and force of contrac- diseased tissues. Hence, this can be used to develop effective tility [81] and sympathetic neurotransmitter release in atria treatment strategies. Key nodes in the pathway were selected [82]. It is worth mentioning that isobornyl acetate (CHX63) by R language; the results suggest CHRM2, GNAI2, CHRM1, has a clear analgesic and anti-inflammatory effect, which JX8, JX13, CHX63, and CHX77 may play an important role may be an important role in the treatment of coronary heart in the pathological and therapeutic mechanism of CHD. disease. JX13 and JX8 are positive and negative isomers of Adrenaline can restore heartbeat by increasing coronary and each other and are the main components of volatile oil. *e cerebral perfusion pressure [70]. β-Blockers are first-line CHX77, CHX63, JX13, and JX8 components act on GNAI2, 12 Evidence-Based Complementary and Alternative Medicine and CHRM1 and CHRM2 and thus activate the important Discipline of Shaanxi Province (grant number: 303061107); molecular mechanisms for the treatment of CHD with Key Research and Development Plan of Shaanxi Province volatile oils. Further, its main function may be in regulating (grant number: 2018SF-314); and Discipline Innovation Team heart rate, myocardial contraction, and ion channels. *ese Project of Shaanxi University of Chinese Medicine (2019- studies provide indirect evidence to support our predictions. YL11). *e authors would like to acknowledge the Shaanxi Province Key Subject of Pharmacy Engineering of Shaanxi 6. Conclusions Provincial Traditional Chinese Medicine Administration.

Taken together, bioinformatics data show that the positive Supplementary Materials effect of Jiangxiang and Chuanxiong volatile oils on CHD may be predominantly due to its effect on ischemia/reper- Supplementary Table 1: genes of CHD. Supplementary fusion, lipid metabolism, and myocardial fibrosis and may Table 2: ingredients of Jiangxiang and Chuanxiong essential be related to the regulation of ion channels, myocardial oils. Supplementary Table 3: related genes of essential oil contraction, and heart rate. *ese results highlight that the compounds. Supplementary Table 4: pathways from KEGG predicted therapeutic target may be a potential biomarker and GO. (Supplementary Materials) for the treatment of CHD with Jiangxiang and Chuanxiong volatile oils. However, systematic and rigorous experiments References are needed to verify our findings. [1] J. E. Dalen, J. S. Alpert, R. J. Goldberg, and R. S. Weinstein, Abbreviations “*e epidemic of the 20th century: coronary heart disease,” @e American Journal of Medicine, vol. 127, no. 9, pp. 807–812, CVD: Cardiovascular diseases 2014. CHD: Coronary heart disease [2] N. D. Wong, “Epidemiological studies of CHD and the MI: Myocardial infarction evolution of preventive cardiology,” Nature Reviews Cardi- TCM: Traditional Chinese medicine ology, vol. 11, no. 5, pp. 276–289, 2014. UniProt: Universal Protein Resource [3] V. L. Lazaro, “2014 PHA clinical practice guidelines for the diagnosis and management of patients with coronary heart GC-MS: Gas chromatography-mass spectrometer disease,” ASEAN Heart Journal, vol. 24, no. 1, 2016. CAS Number: Chemical Abstracts Service [4] C. Berry, D. Corcoran, B. Hennigan, S. Watkins, J. Layland, KEGG: Kyoto Encyclopedia of Genes and Genomes and K. G. Oldroyd, “Fractional flow reserve-guided man- GO: Gene Ontology agement in stable coronary disease and acute myocardial BP: biological process infarction: recent developments,” European Heart Journal, CC: cellular component vol. 36, no. 45, pp. 3155–3164, 2015. MF: molecular function [5] R. Kones, “Primary prevention of coronary heart disease: NAFLD: Nonalcoholic fatty liver disease. integration of new data, evolving views, revised goals, and role of rosuvastatin in management. A comprehensive survey,” Drug Design, Development and @erapy, vol. 5, pp. 325–380, Data Availability 2011. *e data used to support the findings of this study are in- [6] F. Gongora-Rivera, J. Labreuche, A. Jaramillo, P. G. Steg, J.-J. Hauw, and P. Amarenco, “Autopsy prevalence of coro- cluded within the article. nary atherosclerosis in patients with fatal stroke,” Stroke, vol. 38, no. 4, pp. 1203–1210, 2007. Conflicts of Interest [7] Z. Chen, P. Venkat, D. Seyfried, M. Chopp, T. Yan, and J. Chen, “Brain-heart interaction,” Circulation Research, *e authors declare that they have no conflicts of interest. vol. 121, no. 4, pp. 451–468, 2017. [8] L. Ji, Pharmacology of Traditional Chinese Medical Formulae, Authors’ Contributions Traditional Chinese Medicine Publishing House, Beijing, China, 2012. Jia Tai, Junbo Zou, and Yu Wang searched articles in [9] Q. Shujun, “*erapeutic effect of purified Guanxin soft electronic databases and wrote the manuscript. Yulin Liang, capsule in the treatment of angina of coronary heart disease,” Xiaofei Zhang, Dongyan Guo, and Mei Wang analyzed the Journal of Practical Medical Techniques, vol. 14, pp. 1425-1426, data. Chunli Cui, Jing Wang, and Jiangxue Cheng performed 2007. the data extraction. Yajun Shi designed the study and [10] L. Yuanxiang and D. Shenchun, “Application of Jingzhi amended the paper. Jia Tai, Junbo Zou, and Xiaofei Zhang Guanxin pills in the treatment of angina pectoris,” Chinese and Foreign Health Digests, vol. 22, pp. 248-249, 2009. contributed equally to this work and are co-first authors. [11] A. L. Hopkins, “Network pharmacology: the next paradigm in drug discovery,” Nature Chemical Biology, vol. 4, no. 11, Acknowledgments pp. 682–690, 2008. [12] V. Bezhentsev, S. Ivanov, S. Kumar, R. Goel, and V. Poroikov, *is work was supported by the Natural Science Foundation of “Identification of potential drug targets for treatment of re- China (grant number: 81703720); Key Research and Devel- fractory epilepsy using network pharmacology,” Journal of opment Program of Shaanxi Province of China (grant number: Bioinformatics and Computational Biology, vol. 16, no. 1, 2017ZDXM–SF–008); Chinese Medicine Pharmaceutical Key Article ID 1840002, 2018. Evidence-Based Complementary and Alternative Medicine 13

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