Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2016, Article ID 3439521, 10 pages http://dx.doi.org/10.1155/2016/3439521

Research Article Significant Modules and Biological Processes between Active Components of Salvia miltiorrhiza Depside Salt and Aspirin

Yuan Li,1 Yanming Xie,1 Lianxin Wang,1 Yingying Zhang,1 Hao Gu,1 and Yan Chai2

1 Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, No. 16 Nanxiao Street, Dongzhimennei, Dongcheng District, Beijing 100700, China 2Department of Epidemiology, University of California, Los Angeles, 405 Hilgard Avenue, CA 90095, USA

Correspondence should be addressed to Yanming Xie; [email protected] and Yan Chai; [email protected]

Received 16 October 2015; Revised 8 December 2015; Accepted 12 January 2016

Academic Editor: Helmut Hugel

Copyright © 2016 Yuan Li et al. This 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.

The aim of this study is to examine and compare the similarities and differences between active components of S. miltiorrhiza depside salt and aspirin using perspective of pharmacological molecular networks. Active components of S. miltiorrhiza depside salt and aspirin’s related genes were identified via the STITCH4.0 and GeneCards Database. A text search engine (Agilent Literature Search 2.71) and MCODE software were applied to construct network and divide modules, respectively. Finally, 32, 2, and28 overlapping genes, modules, and pathways were identified between active components of S. miltiorrhiza depside salt and aspirin. A multidimensional framework of drug network showed that two networks reflected commonly in human aortic endothelial cells and atherosclerosis process. Aspirin plays a more important role in metabolism, such as the well-known AA metabolism pathway and other lipid or carbohydrate metabolism pathways. S. miltiorrhiza depside salt still plays a regulatory role in type II diabetes mellitus, insulin resistance, and adipocytokine signaling pathway. Therefore, this study suggests that aspirin combined with S. miltiorrhiza depside salt may be more efficient in treatment of CHD patients, especially those with diabetes mellitus or hyperlipidemia. Further clinical trials to confirm this hypothesis are still needed.

1. Introduction overdamagedwallsofbloodvessels.Duetothefactthatit inhibits formation of blood clot in people with high risk [3], Antithrombotic Therapy and Prevention of Thrombosis (Ver- aspirinisalsousedinthelongterm,atlowdoses,tohelp sion 9) [1], announced by the American College of Chest prevent heart attacks [4]. Salvia miltiorrhiza, a Chinese herbal Physicians (ACCP) Evidence-Based Clinical Practice Guide- medicine which promotes blood circulation to remove blood lines, proposed lasting low-dose aspirin therapy to be used stasis drugs [5], has been widely used to treat cardiovascular astheprimarypreventiontechniqueforpatientsabove50 diseases such as CHD. In 2005, S. miltiorrhiza depside salt years old or patients diagnosed with coronary heart disease passed the certification of new drug application for chronic (CHD). For patients with acute coronary syndrome (ACS) angina treatment at the State Food and Drug Administra- or undergoing stent implantation with PCI, dual antiplatelet tion (SFDA). 80% of its active components are magnesium therapy for up to one year is required. Antithrombotic lithospermate B (MLB) and its analogs (salvianolic acid B and therapy plays a crucial role in prevention and treatment lithospermic acid B) [6] extracted from Salvia miltiorrhiza of CHD, in which aspirin undoubtedly is the most widely major water-soluble active ingredients. The other 20% are used conventional drug. Aspirin irreversibly inhibits COX- mainly rosmarinic acid (RA) and lithospermic acid (LA). 1 and modifies the enzymatic activity of COX-2, which nor- A clinical noninferiority study showed that S. miltiorrhiza mally produces prostanoids [2]. Antiplatelet effect of aspirin depside salt had definite therapeutic effect in patients with inhibits the prostaglandin production which downregulates CHD angina pectoris, with no evidence of adverse drug thromboxane A2 (TXA2) levels. TXA2 is bound by platelet reaction (ADR) [7]. Aspirin and S. miltiorrhiza depside molecules under the normal circumstances to create a patch saltarethuscommonlyusedinCHDtreatment,butthe 2 Evidence-Based Complementary and Alternative Medicine molecular relationships between the two drugs are still highly interconnected regions clusters in the network by under study. Current evidence shows that both similar and MCODE (http://baderlab.org/Software/MCODE). MCODE different molecular therapeutic patterns exist between these is a clustering algorithm tool, which divides network module two drugs, but the exact pattern of whether they are the and provides detailed algorithm [15] (parameters: degree same, different, or partially overlapping remains unknown. cutoff=2;K-core=2;nodescorecutoff=0.2).Networkcan Therefore, a network pharmacology approach seems to be of be divided into several modules. Results can be visualized by interest as it would reveal the potential overlapping or unique Cytoscape. modules that are affected by either treatment. “Network pharmacology” [8], which combines systems 2.4. GO Biological Process and KEGG Pathway Enrichment. biology and biological networks, balances the perspective of DAVID software was used (http://david.abcc.ncifcrf.gov/) to development process to explain the disease [9]. It improves analyze the function of modules. DAVID software provides or restores biological networks from a balanced perspective hypergeometric distribution tests and mainly includes typical and explains the interaction between the drug and the human batch annotation and gene-GO term enrichment analysis to body. It emphasizes the discovery of drugs’ signaling pathway highlight the most relevant GO terms associated with a given and provides a reference to improve the therapeutic effect gene list [16] (parameters: Count = 2; EASE = 0.01; species and and reduce side effects. According to the characteristics of the background = Homo sapiens). DAVID functional annotation multicomponent and multitarget of Chinese herbal medicine, clustering uses an algorithm to measure relationships among network pharmacology may be a new and well-documented the annotation terms based on the degrees of their coassoci- method to find some meaningful information. Therefore, ation genes to group similar, redundant, and heterogeneous related genes were used to construct the molecular network, annotation contents from the same or different resources combined with module division with modular analysis to into annotation groups. It can display genes from a user’s excavate the associations of aspirin and active components list on pathway maps to facilitate biological interpretation of S.miltiorrhizadepside salt in this study. We believe that in a network context by taking full advantage of the well- exploring the internal connection of potential molecular known KEGG pathways. The biological processes and KEGG interactions between the two drugs can provide a clue for pathway corresponding to the modules were identified and combination therapy of CHD. ranked by 𝑃 values (𝑃 < 0.05; reliability is higher).

2. Materials and Methods 3. Result 2.1. Gene Obtaining. GeneCards (http://www.genecards 3.1. Genes Related to Active Components of S. miltiorrhiza .org/) is a comprehensive and authoritative database that DepsideSaltandAspirininSTITCHandGeneCardsDatabase. provides information of human genes [10] and is known as After searching the STITCH (Homo sapiens,score> 0.400, the most inclusive resource of gene-centered information medium confidence) and GeneCards Database (on March of the human genome [11]. STITCH (http://stitch.embl.de/) 16,2015),wefound55genesrelatedtoactivecomponents is a database of protein-chemical interactions integrating of S. miltiorrhiza depside salt and 498 genes related to massive literature information and various databases of aspirin. There were 32 overlapping genes shared by the biological pathways and drug-target relationships [12]. It is two drugs, and these genes accounted for 58.18% (32/55) used to create a network of interactions [13]. “MLB”, “RA”, of the identified genes related to active components of S. “LA” (active components of S. miltiorrhiza depside salt), and miltiorrhiza depside salt, 6.43% (32/498) of the identified “Aspirin” were entered into the integrated and searchable genes related to aspirin, and 5.79% (32/553) of all genes related database of GeneCards and STITCH4.0 to search and export to both drugs. After entering 32 overlapping genes’ list in related genes in Homo sapiens,respectively. GO functional annotation, it showed that the common roles included response to chemical stimulus, response to stress, 2.2. Network Construction. The genes related to active com- multiorganismprocess,responsetostimulus,andresponse ponents of S. miltiorrhiza depside salt (“MLB”, “RA”, and to external stimulus (Table 1). “LA”)and“Aspirin”weresubmittedtotheAgilentLiterature Search software v.3.1.1 (http://www.agilent.com/labs/research/ 3.2. Topological Analysis of Network. After submitting 55 and litsearch.html), which is a powerful automatic metasearch 498 genes related to active components of S. miltiorrhiza tool for querying multiple text-based PubMed and USPTO, depside salt and aspirin into the Agilent Literature Search for associations among genes of interest and construct- 3.1.1, two networks were created (Figures 1(a) and 1(b)). ing a network. Agilent Literature Search is a registered There were 528 nodes (genes) and 1506 edges (interactions) plugin and can be used in conjunction with Cytoscape identified from the active components of S. miltiorrhiza (http://www.cytoscape.org/) to realize the visualization and depside salt-related genes and 2120 nodes (genes) and 9064 analyzation of the network [14] (parameters: Max Engine edges (interactions) from the aspirin-related genes. Similar Matches = 10; Concept Lexicon = Homo sapiens;Interaction distributions of node degree that followed the power-law Lexicon = limited). distribution appeared in two networks (Figures 1(c) and 1(d)). The topological analysis of two networks such as clustering 2.3. Identification of Modules. The modular structures exist coefficient, centralization, density, diameter, and radius was in a complex biological systematic network, so we detect shown in Table 2. Evidence-Based Complementary and Alternative Medicine 3

Table 1: Top 10 overlapping biological functions of the 32 overlapping genes related to active components of S. miltiorrhiza depside salt and aspirin.

GO terms 𝑃 value Gene XDH, TNF, MCL1, IL8, PTGS2, RELA, EDN1, SOD1, MMP2, TGFB1, CCL11, MAPK1, GO:0042221: response to 9.5𝐸 − 19 CASP3, CCR3, JUN, BCL2, SERPINE1, IFNG, IL1B, NOS3, MAPK8, ALOX5, NOS2, and chemical stimulus IKBKB TNF, PTGER3, IL8, PTGS2, RELA, EDN1, NFKB1, SOD1, MMP2, TGFB1, CCL11, GO:0006950: response to 4.3𝐸 − 16 MAPK1, CASP3, CCR3, JUN, BCL2, AKR1B1, SERPINE1, IFNG, IL1B, NOS3, MAPK8, stress ALOX5, and NOS2 GO:0051704: 5.6𝐸 − 14 TNF, PTGS2, RELA, SOD1, MMP1, TGFB1, CCL11, VCAM1, MAPK1, APP, CCR3, JUN, multiorganism process BCL2, IFNG, IL1B, NOS3, and NOS2 XDH, TNF, MCL1, PTGS2, EDN1, NFKB1, MMP2, TGFB1, CASP3, APP, BCL2, GO:0050896: response to 7.5𝐸 − 14 SERPINE1, IFNG, IL1B, NOS3, NOS2, PTGER3, IL8, RELA, SOD1, CCL11, MAPK1, stimulus CCR3, JUN, AKR1B1, MAPK8, ALOX5, and IKBKB GO:0009605: response to 2.9𝐸 − 13 TNF, PTGER3, IL8, PTGS2, RELA, NFKB1, SOD1, TGFB1, CCL11, MAPK1, CASP3, external stimulus CCR3, JUN, BCL2, IFNG, SERPINE1, IL1B, and ALOX5 GO:0042127: regulation of 5.2𝐸 − 13 PTGER2, TNF, IL8, PTGS2, RELA, EDN1, TGFB1, VCAM1, MAPK1, CASP3, JUN, BCL2, cell proliferation IFNG, SERPINE1, IL1B, NOS3, and NOS2 GO:0042981: regulation of 7.3𝐸 − 13 TNF, PTGS2, MCL1, RELA, NFKB1, SOD1, TGFB1, MAPK1, APP, CASP3, JUN, BCL2, apoptosis IFNG,IL1B,NOS3,MAPK8,andIKBKB GO:0043067: regulation of 8.5𝐸 − 13 TNF, PTGS2, MCL1, RELA, NFKB1, SOD1, TGFB1, MAPK1, APP, CASP3, JUN, BCL2, programmed cell death IFNG,IL1B,NOS3,MAPK8,andIKBKB GO:0010941: regulation of 9𝐸 − 13 TNF, PTGS2, MCL1, RELA, NFKB1, SOD1, TGFB1, MAPK1, APP, CASP3, JUN, BCL2, cell death IFNG,IL1B,NOS3,MAPK8,andIKBKB GO:0065008: regulation of 3.4𝐸 − 12 XDH, TNF, PTGER3, PTGS2, MCL1, EDN1, SOD1, TGFB1, CCL11, CASP3, APP, CCR3, biological quality JUN, BCL2, IFNG, SERPINE1, IL1B, NOS3, NOS2, and IKBKB

Table 2: The topological attributes of the gene interaction networks. functional annotations and 2 pathways (Figure 2(c)). Bio- logical annotations included immune effector processes (4) Active components of (regulation of immune response, regulation of immune effec- Parameters S. miltiorrhiza Aspirin tor process, positive regulation of immune system process, depside salt and regulation of immune system process), lipid biosynthetic Clustering coefficient 0.618 0.586 and metabolic processes (5) (lipid biosynthetic process, lipid Nodes 528 2120 metabolic processes, fatty acid metabolic process, cellular Edges 1506 9064 lipid metabolic process, and fatty acid metabolic process), Network centralization 0.081 0.101 other biosynthetic and metabolic processes (5) (carboxylic Network density 0.011 0.004 acid biosynthetic process, organic acid biosynthetic process, cellular biosynthetic process, monocarboxylic acid metabolic Network diameter 10 9 process, and biosynthetic process), and regulation of response Network radius 1 1 to stimulus (1). So the main biological functions of the 2 overlapping modules were lipid biosynthetic and metabolic and immune effector. The 2 pathways from overlapping 3.3. Module Identification. After submitting two networks modules were cell adhesion molecules (CAMs) pathway and into MCODE software, 38 modules were identified from intestinal immune network for IgA production pathway. active components of S. miltiorrhiza depside salt network. The maximum module was composed of 46 nodes while the 3.5. Functional Enrichment of Unique Modules for 2 Drugs. minimum was composed of 3 nodes. There were 122 modules GO functional enrichment analysis was implemented on inaspirinnetwork.Themaximummoduleiscomposedof acquired top 10 nonoverlapping modules of the 2 drugs sorted 112 nodes while the minimum is composed of 3 nodes. Two by MCODE score, respectively (Figures 3(a) and 4(a)). 762 networks shared 2 overlapping functional modules including GO biological functions and 63 KEGG pathways were found the same genes: M(s3a10) and M(s33a100) (Figure 2(a)). M(s3a10) in modules of active components of S. miltiorrhiza depside contains 7 nodes (CD86, TNFSF9, EMR2, CD37, ICOSLG, salt’s network, and 1391 GO biological functions and 80 SPN, and CD97) and 21 edges. M(s33a100) contains 3 nodes KEGG pathways were found in modules of aspirin’s network. (FASN, SCD, and DGAT2) and 3 edges (Figure 2(b)). The 63 pathways of Salvianolate network (Figure 3(b)) are asfollows:23humandiseasespathways(cancers(pancreatic 3.4. Functional Enrichment Analysis of 2 Overlapping Mod- cancer, bladder cancer, and another 17 cancers), immune dis- ules. The two overlapping modules contained 15 biological eases (primary immunodeficiency and autoimmune thyroid 4 Evidence-Based Complementary and Alternative Medicine

apcs plcg1

irgm

sh3glb1 tnfsf10

tspyl2 tpr trna ubc cyp2b6 nqo1 sgms2 relb mapk7 il17d tyrp1 ppig s100b pik3c3

sptlc2

calml3 ifng nf-kappab grap2 pgk1 plp1 sgca rfx5 lilrb4 ikzf1 il1r2 trpc6 zbtb16 cd5l tat agxt clec10a eef1a1 sptlc1 ifna14 actb lat il13 vav1 3.1.4.3 ptx3 sgta il2 hla-e il1rn hsr lcp2 ifna6 cr1 blk slamf7 hsf1 btk ifna5 ahr ciita trpm4 gja1 alox5ap rps6ka3 zbtb32 chuk ndufb6 lat2 il1b acp1 ifna8 blnk pcna ikbkg ptger4 tyr uts2r pla2g2a mcm8 atf1 tnf camk2g pkc kcna3 dnai1 ptgfrn dapk3 plxna2 sp1 ptpn22 lta esr2 pcsk1 syk zap70 ctd hspa4 pkb itpr1 fcn2 tec abl1 map3k7ip2 rfc3 serpina1 il10 ptpn11 pip ncam2 ervk2 txk cxcl3 lyn cdc7 adam33 gadd45gip1 prkaa2 pml zhx2 angpt1 mmp3 ptger2 tymp serpina3 usp10 gc becn1 crtc1 traf6 rorc par1 timp3 mirn23a hsp90aa1 myc mirn200b cdk9 timp2 mmp12 creb1 irak1 atg12 il8 alox5 pmaip1 irf6 mc3r arg2 gnrh1 hif1a pde5a ly96 tnfa brd4 cdk5 mmp1 ifi27 crtc2 tlr2 pten itk tnfaip6 sod1 stx2 cdc25a stk11 rps6kb1 tank chek1 thy1 snf1lk ldlr hcc cxcr4 il1r1 mmp2 il17a fos cdk2 cdc25cklf5 hdac4 cxcl2 erbb2 park7 timp1 mcl1 akt1s1 gapdh crtc3 tlr4 ptges parp1 slc2a4 crmp1 nfatc1 mmp9 mmp7 bak1 igfals cish src eif4e hspa5 insr usp9x pold3 bcl2l11 zc3h12a mmrn1 hsp90b1 cdkn2a mink1 brms1 flt4bax bcl2l1 f2rl1 csf2 cdc2 cxcr5 cdkn1a ryr1 hdac5 tnfsf11 ccnh slc16a1 bad trib3 snf1lk2 adc ceacam5 socs3 pin1 sh2d2a bcl2 ppp2r4 cd4 kdr ros1 nr2e3 atf6 tp53 vegfa fyn dynamin cma1 itga2b cnr1 ppme1 twist1 2.1.1.71 ddit3 plod2 apaf1 slc16a8 slc16a7 muc3a spry1 wnt3a akt1 ca2 il17ra bcr ephb2 srpk1 arhgef2 mmp13 camk1 cd2 pvrl1 nod1 edem1 stam2 hgf atf4 1.13.11.17 plau jun cdkn2b ebp tnfsf13 spry2 mef2c mlc1 mt1jp muc1 hdac6 rela vegfc pik3ca crebbp atf2 3.6.1.8 slc16a4 nr0b2 irs2 nod2 setd2 nampt slc16a3 ptpn6 mapk8 map2k1 plod1 lck nos2 il6 vcp klf4 aprt acvr1b cd69 ptgs2 app stat3 mapk3 fgf21 nppa mapk9 fam20c kras prkaa1 cd8a ripk1 shc1 olr1 dok1 eif2ak3 ecd ep300 pex13 tslp lgals3 sirt1 eif4g2 alkfgfr3 mapk14 mirn100 mcs icam1 vcam1 irs1 p2rx7 cxcl13 grb2 frap1 prdx5 fgf2 serpine1 ccl24 rage apc2 lch ibsp acvrl1 wdtc1 ednrb raf1 atp8a2 abca1 stat1 dpep1 egfr hmgb1 osm fpr2 sp7 pln lcn2 cx3cl1 crh ccl11 ccl2 apom p2ry2 f9 pgr edn1 2.6.1.77 slc3a2 p2rx4 sirt7 smpd2 egr1 nova2 braf cox1 igf1r tnc preb cga smad2 gsg2 runx2 eif4ebp1 aaas apc smad3 cxcl11 bsg klf2 csf1 slc2a3 adam9 cxcl10 mid1 lrp1 sirt2 sgsm3 rgs2 pc-3 trpv1 ccl5 fosl2 csf3 cemp1 ccl3 thbs1 ezh2 ece1 st3gal4 gabpa ppia mirn433 nfat5 pik3r3 nos3 slc17a5 igf1 mirn34a icoslg ifna1 ctbs ctnnd1 ccl4 arpc2 ptgir mapk1 tspan2 igfbp1 stat5a ptpn1 cd68 spn emr2 smad1 irf3 fgfr2 alpp cd97 trim69 actin apex1 map3k7 bdnf cd37 nos1 fap cd86 tbk1 ntrk2 visa cdc25b h2afz tnfsf9 snai2 tmem173 irf7

tcdd pah

ddx58

itgam rb1 tcf4 dgat2 spi1 mpo th1l s100a9 spib ccnd1 lin28b scd ddx41 s100a12 cd34

ptprc

eng spic tcf7l2 s100a6 fasn ikbkb nfkb1 cd28 s100a8

mcam

lilra1 fcgr3a araf asah1 ptger3 card9 il23a xdh 2.3.1.16 f2 cd79b epo akr1b10 kat5 cd79a

camp tlr9 mirn19a erbb3 bok cd22 myd88 il22 brca2 hadha par4 epor akr1b1

(a) (b)

100 400

100

10 10 Number of nodes of Number Number of nodes of Number

1 1 1 10 50 1 10 100 300 (deg.) (deg.) (c) (d)

Figure 1: Characteristics of two gene interaction networks. (a) Active components of S. miltiorrhiza depside salt-related genes interaction network. (b) Aspirin-related genes interaction network. Yellow diamonds denote known active components of S. miltiorrhiza depside salt and aspirin-related genes, and red and green denote genes obtained from text mining. (c) The degree distribution of active components of S. miltiorrhiza depside salt network. (d) The degree distribution of aspirin network.

disease), endocrine and metabolic diseases (type II diabetes pathways (cancers (prostate cancer, prostate cancer, and mellitus), and infectious diseases (epithelial cell signaling in another 9 cancers), immune diseases (5), viral myocarditis Helicobacter pylori infection)), 17 immune system pathways, (1), and Alzheimer’s disease (1)), 7 signaling molecules and 8 cellular processes pathways (cell growth and death (6), interaction pathways, 5 cellular processes (cell growth and regulation of autophagy, and focal adhesion), 5 endocrine death (3) and cellular community (2)), 5 signal transduction system pathways, and so forth. The 80 pathways (Figure 4(b)) pathways, and so forth. of aspirin network are as follows: 21 metabolism pathways After eliminating repeated pathways, active components (carbohydrate metabolism (6), xenobiotics biodegradation of S. miltiorrhiza depside salt had 44 pathways while aspirin (5), cofactors and vitamins metabolism (3), lipid metabolism had 53 pathways, including 26 common pathways. Common (3), and others), 21 immune system pathways (hematopoietic pathways were as follows: immune systems (11) (chemokine cell lineage, natural killer cell mediated cytotoxicity, Toll- signaling pathway, NOD-like receptor signaling pathway, like receptor signaling pathway, etc.), 18 human diseases hematopoietic cell lineage pathway, T cell receptor signaling Evidence-Based Complementary and Alternative Medicine 5

Aspirin’s modules Active components of S. miltiorrhiza depside salt’s modules

36 2 120

(a)

M(s3a10) M icoslg (s33a100) dgat2 2 spn emr scd

cd97

cd86 cd37 fasn tnfsf9

(b)

The 15 overlapping biological processes from 2 overlapping modules Regulation of response to stimulus Two pathways from 6% 2 overlapping modules

Immune effector processes Intestinal Biological 27% processes immune Cell adhesion 40% network for IgA molecules Metabolic production (CAMs) processes pathway pathway 27%

(c) (d) Figure 2: Number of two-drug modules, overlapping modules, and biological functions. (a) Number of modules identified from two networks. (b) Blue circles indicate the genes in the 2 overlapping modules. (c) GO biological processes of 2 overlapping modules. (d) Pathways of 2 overlapping modules.

pathway, Toll-like receptor signaling pathway, RIG-I-like signal transduction (3) (ErbB signaling pathway, Jak-STAT receptor signaling pathway, and 5 other related immune sys- signaling pathway, and VEGF signaling pathway), signaling tem pathways), human diseases (5) (cancers (bladder cancer, molecules and interactions (2) (CAMs and cytokine-cytokine small cell lung cancer, and colorectal cancer and pathways receptor interaction), neurotrophin signaling pathway (1), in cancer) autoimmune thyroid disease), cellular processes and progesterone-mediated oocyte maturation. Unique path- (3) (apoptosis, p53 signaling pathway, and focal adhesion), ways of active components of S. miltiorrhiza depside salt 6 Evidence-Based Complementary and Alternative Medicine

M(s1) M(s2) M(s3)

serpina1 nampt pln tnfa gc prkaa1

ros1 frap1 osm il8 akt1 pik3ca vav1 sirt1 stat1 serpina3 lat2 f9 ephb2 blk bcl2 mmp2 kdr timp3 zap70 lyn mapk14 smad2 mapk8 blnk ccl2 il10 mmp12 mmp7 ccl5 lat rela sod1 bax btk vegfa il17ra syk lcp2 jun igf1r timp2 src tp53 cdkn2a mmp9 ccl11 il17a csf1 myc spry2 shc1 il2 mmp3 lta ifna6

grb2 irs2 ifna14 ifna8 timp1 dok1 igf1 pold3 ifna5 ccl3 mmp1 adam33 sh2d2a csf3 il13 spry1 stam2 ifng Signaling molecules M(s4) M(s5) M(s6) Signal and interaction slc16a3 6% slc16a4 il1b cd34 transduction Cellular processes slc16a7 8% 13% mcam il1rn ptprc pgk1 Nervous system 2%

slc16a8 Endocrine system 8%

slc16a1 eng actb itgam cma1 il1r2 Immune system 27% M(s7) M(s8) M(s9) M(s10) Human diseases 36 mirn200b % gja1 cd68 cdc25c

cdk2 esr2 itk

actin

fap

cxcr4 cdkn1a ptger4 ifi27 snai2 par1 ptgfrn

(a) (b)

Figure 3: Top 10 unique modules and their pathways of active components of S. miltiorrhiza depside salt. (a) The top 10 unique modules for active components of S. miltiorrhiza depside salt network. (b) The 63 KEGG pathways identified from top 10 unique modules that divided into 7 categories.

M(a1) M(a2) M(a3)

jmjd3 ptger4

dnmt3b

jmjd2a hdac1 dnmt3a dnmt1

aof2 sin3a mbd1 ep300

hdac2

mecp2 hdac3 ezh2

ptger2 rcor1

suv39h1 cbx5

Signaling M( ) M( ) M( ) a4 a5 a6 Transport and molecules Signal and transduction vhl c3ar1 catabolism 6 fgd5 1 interaction % iqsec1 sco1 % slc3a2 9%

bcl6 Membrane Metabolism gnai2 3 fcgr3b transport % lrrc25 26%

aldh1l1

ctdspl rad21 slc39a8

foxp1 Cellular processes lrrn1 retn Immune system 6 26% % Human diseases M(a7) M(a8) M(a9) M(a10) 23%

cbs ppp2r1b icoslg lpl plec1 tnfsf9

flt1 cgref1 cd86 cdkn2b basp1 cd37 psrc1 lama5 spn

phactr1 pa2g4 emr2 cnnm2 cd97 itgb4

(a) (b)

Figure 4: Top 10 unique modules and their pathways of aspirin. (a) The top 10 unique module networks for aspirin network. (b) The80 KEGG pathways identified from top 10 unique modules that divided into 9 categories. wereasfollows:cancers(6),immunediseasesorsystems mellitus, adipocytokine signaling pathway, GnRH signaling (4) (autoimmune thyroid disease, primary immunodefi- pathway, and progesterone-mediated oocyte maturation), ciency, B cell receptor signaling pathway, and Fc gamma cellular processes (3) (cell cycle, regulation of autophagy, and R-mediated phagocytosis pathway), endocrine diseases or oocyte meiosis), and mTOR signaling pathway (1). Unique systems (4) (insulin signaling pathway, type II diabetes pathways of aspirin were as follows: metabolism pathways Evidence-Based Complementary and Alternative Medicine 7

(17) (arachidonic acid metabolism, linoleic acid metabolism, only activate a variety of factors for apoptosis [26], but also other carbohydrate metabolisms, and another 14 related participate in the process of platelet formation [27]. MMP1 pathways), immune diseases and system (4) (Huntington’s and MMP2 belong to the metalloproteinase family. They disease, allograft rejection, amyotrophic lateral sclerosis areinvolvedindiversefunctionssuchasremodelingofthe (ALS) pathway, and complement and coagulation cascades vasculature, angiogenesis, inflammation, blood coagulation, pathway), human diseases (3) (viral myocarditis, Alzheimer’s and atherosclerotic plaque rupture. MMPs have a role in disease, and Parkinson’sdisease), PPAR signaling pathway (1), myocardial cell death pathways and vascular remodeling ABC transporters pathway (1), and ECM-receptor interaction [28], such as vascular smooth muscle cell migration into pathway (1). the intima [29]. Regulation of cell proliferation, apoptosis, programmed cell death, and cell death are common biological 4. Discussion functions of the 32 overlapping genes, which are considered remarkable aspects of CHD [30]. Thus, these overlapping Aspirin and S. miltiorrhiza depside salt are commonly used genes’ biological functions are mainly reflected in HAECs and drugs for the treatment of CHD, but there is lack of rela- atherosclerosis process. tionship studies between the two drugs from large-scale There are 28 common pathways in overlapping and molecular perspective. Two overlapping modules and many top 10 unique modules between active components of S. common biological processes and pathways between the two miltiorrhiza depside salt and aspirin. Most of these pathways drugs were known in this investigation. These results are are related to the antitumor and inflammatory immune likely to reveal the overlap of potential therapy mechanism response (p53 signaling pathway, ErbB signaling pathway, T and internal connection between the two drugs. Except for cell receptor signaling pathway, etc.). But there are also some the overlap, the two drugs also have some unique functions, pathways participating in the process of antiatherosclerosis. whichhaveanexcellentadvantageindifferentranges. CAMs pathway participates in cell growth and death, which There are 32 overlapping genes between aspirin and active are associated with cardiovascular diseases [31]. CAMs are components of S. miltiorrhiza depside salt, such as JUN, proteins expressed on the cell surface and function in HAECs VCAM-1, TGFB1, TGFB1, IL8, IL1B, NOS2, NOS3, MAPK1, adhesion. CAMs play a critical role in a wide array of bio- MAPK8, CASP3, MMP1, and MMP2 that have been associ- logical processes that include hemostasis, immune response, ated with the cardiovascular diseases (coronary heart disease, inflammation, embryogenesis, and development of neuronal angina pectoris, atherosclerosis, and thrombosis which are tissue, which establish strong adhesion on the endothelium all within the scope of CHD) as biomarkers or therapeutic of arteries. VEGF signaling pathway can regulate vascular targets from the Comparative Toxicogenomics Database. endothelial cell proliferation and migration. It also leads to Vascular endothelial cell proliferation and apoptosis are an change of vascular permeability and control of angiogenesis. early marker for atherosclerosis. Therefore, prevention of There is now much evidence that VEGFR-2 is the major smooth muscle cell and endothelial cell proliferation and mediator of VEGF-driven responses in endothelial cells and removal of superoxide radicals have a positive effect for treat- it is considered to be a crucial signal transducer in both ment of CHD. JUN participates in the biological process of physiologic and pathologic angiogenesis [32]. JAK-STAT angiogenesis [17] and increases endothelial cell proliferation pathway is the principal signaling mechanism for a wide array and smooth muscle cell hyperplasia [18]. IL8, IL1B, TGFB1, of cytokines and growth factors, closely related to the main and VCAM-1 participate in the inflammatory response [19]. factor VEGF which mediates proliferation and migration TGFB1 positively regulates cell migration, apoptosis, and of vascular endothelial cells [33]. In IFN-𝛾-treated HAECs, blood vessel endothelial cell migration [20]. VCAM-1, which MLB inhibited IFN-𝛾-induced JAK-STAT signaling pathways belongs to immunoglobulin superfamily (IgSF), is widely and consequently suppressed IFN-𝛾-induced expression of expressed in human aortic endothelial cells (HAECs). It also chemokines, IP-10 promoter activity, IP-10 protein release, plays an important role in cell adhesion. Overexpression of and monocyte adhesion to HAECs [34]. VCAM-1 causes increased endothelial adhesion [21], which Antiplatelet treatment with aspirin is widely considered further leads to the formation of atherosclerotic plaque as a cornerstone of atherosclerotic vascular disease’s primary rupture NOS2 and NOS3 belonging to NO (produces nitric [35]andsecondarypreventionandalsousedforacute oxide) [22], which is implicated in vascular smooth muscle treatment [36]. In this study, we found that aspirin may play a relaxation through a cGMP-mediated signal transduction moreimportantroleinmetabolism,suchasthewell-known pathway.NOS2andNOS3positivelyregulatevasodilation AA metabolism pathway and other lipid or carbohydrate and mediate vascular endothelial growth factor- (VEGF-) metabolism pathways. Moreover, we found that aspirin par- induced angiogenesis in coronary vessels [23] and promote ticipates in the PPAR signaling pathway, and PPAR-𝛾 has blood coagulation through the activation of platelets [24]. played a pivotal role in anti-inflammation, atherosclerosis, MAPK1, MAPK8, and CASP3 play an irreplaceable role insulinresistance,andantitumor[37].Activecomponentsof in biological process of apoptosis. MAPK is a key signal S. miltiorrhiza depside salt may play a more important role in transduction receptor from the surface to the nucleus, which endocrine system, such as type II diabetes mellitus, insulin is involved in cell proliferation, differentiation, migration, signaling pathway, and adipocytokine signaling pathway. transformation, and apoptosis in the whole process. MAPKs Cardiovascular disease, especially CHD, is the primary cause play an important role in regulating the expression of proin- of mortality among diabetes mellitus patients. Entirely due flammatory molecules in many cells [25]. CASP3 can not to more extensive coronary atherosclerosis, more than 50% 8 Evidence-Based Complementary and Alternative Medicine of diabetes mellitus patients will die from a cardiovascular Acknowledgments event. Meanwhile, diabetes mellitus is a risk factor for coronary heart disease [38]. Type II diabetes mellitus is The authors are very grateful to Professor Dr. Rob Verpoorte a chronic low-grade inflammatory disease. Inflammatory for all the guidance and valuable suggestions. The study was factor and adipose cytokines are involved in the development financially supported by Beijing Technology Development of of type II diabetes mellitus and its complications [39]. The Chinese Medicine Special (JJ2014-53); Method Study of Eval- metabolic syndrome can promote the development of type II uation and Mechanism on Chinese and Western Medicine diabetes and CHD. Insulin resistance plays a pivotal role in Combination (no. Z0417); the ninth-Science Foundation the progression of this syndrome and cardiovascular diseases. of Institute of Basic Research in Clinical Medicine, China Improvement of insulin resistance, therefore, is most likely to Academy of Chinese Medical Sciences (no. Z0406); Scientific reduce the high cardiovascular event rate in type II diabetes Research Innovation Team Project of China Academy of [40]. Control of blood glucose and blood lipid has a positive Chinese Medical Sciences (no. PY1303). role to prevent and delay the development of atherosclerosis. Previous studies have demonstrated that Salvianolate has References positive effect on attenuating atherosclerosis [41], scavenging free radical [42], preventing endothelial dysfunction [43], [1] A. Holbrook, S. Schulman, D. M. Witt et al., “Evidence-based regulating matrix metalloproteinases expression, activity, management of anticoagulant therapy: antithrombotic therapy and anti-inflammation [44], modulating lipid profiles [45], and prevention of thrombosis, 9th ed: American College of and protecting against myocardial ischemia and reperfusion Chest Physicians Evidence-Based Clinical Practice Guidelines,” (MI/R) injury [46]. Chest, vol. 141, no. 2, supplement, pp. e152S–e184S, 2012. Finally, through analyzing the molecular networks mod- [2] I. Morita, “Distinct functions of COX-1 and COX-2,” ule, these two drugs were found not only sharing certain Prostaglandins and Other Lipid Mediators,vol.68-69,pp. biological processes and pathways, but also having unique 165–175, 2002. character. So using aspirin combined with S. miltiorrhiza dep- [3]H.D.LewisJr.,J.W.Davis,D.G.Archibaldetal.,“Protective side salt may be more efficient in treatment of CHD patients, effects of aspirin against acute myocardial infarction and death especially those with diabetes mellitus or hyperlipidemia. in mean with unstable angina. Results of a veterans administra- tion cooperative study,” New England Journal of Medicine,vol. 309,no.7,pp.396–403,1983. 5. Conclusion [4] D. G. Julian, D. A. Chamberlain, and S. J. Pocock, “A com- parison of aspirin and anticoagulation following thrombolysis We adopted network-based approach to investigate the sim- for myocardial infarction (the AFTER study): a multicentre ilarities and differences between S. miltiorrhiza depside salt unblinded randomised clinical trial,” British Medical Journal, and aspirin in great detail. A multidimensional framework of vol. 313, no. 7070, pp. 1429–1431, 1996. drug network showed that the two drugs reflected commonly [5] L.Zhou,Z.Zuo,andM.S.S.Chow,“Danshen:anoverviewofits in human aortic endothelial cells and atherosclerosis process. chemistry, pharmacology, pharmacokinetics, and clinical use,” Aspirin plays a more important role in metabolism, such as Journal of Clinical Pharmacology,vol.45,no.12,pp.1345–1359, the well-known AA metabolism pathway and other lipid or 2005. carbohydrate metabolism pathways. S. miltiorrhiza depside [6]A.Watzke,S.J.O’Malley,R.G.Bergman,andJ.A.Ellman, saltstillplaysaregulatoryroleintypeIIdiabetesmellitus, “Reassignment of the configuration of salvianolic acid B and insulinresistance,andadipocytokinesignalingpathway. establishment of its identity with lithospermic acid B,” Journal Therefore, this study suggests that aspirin combined with S. of Natural Products,vol.69,no.8,pp.1231–1233,2006. miltiorrhiza depside salt may be more efficient in treatment [7] Q. Zhang, A.-D. Liu, and Y.-S. Huang, “Clinical non-inferiority of CHD patients, especially those with diabetes mellitus or trial on treatment of coronary heart disease angina pectoris hyperlipidemia, but should be confirmed by further clinical of Xin-blood stasis syndrome type with lyophilized Salvia salt trials. of lithospermic acid powder for injection,” Chinese Journal of Integrative Medicine,vol.12,no.1,pp.12–18,2006. [8]A.L.Hopkins,“Networkpharmacology,”Nature Biotechnology, Competing Interests vol. 25, no. 10, pp. 1110–1111, 2007. The authors declare that there are no competing interests. [9] A. L. Hopkins, “Network pharmacology: the next paradigm in drug discovery,” Nature Chemical Biology,vol.4,no.11,pp.682– 690, 2008. Authors’ Contributions [10] M. Safran, I. Dalah, J. Alexander et al., “GeneCards Version 3: the human gene integrator,” Database,vol.2010,ArticleID Yuan Li designed the study and drafted the paper. Yan baq020, 2010. Chai amended English writing of this review. Lianxin Wang, [11] M. Shklar, L. Strichman-Almashanu, O. Shmueli, M. Shmoish, Yingying Zhang, and Hao Gu participated in the design and M. Safran, and D. Lancet, “GeneTide—Terra Incognita Dis- analysis of the study. Yanming Xie arbitrated disagreements. covery Endeavor: a new transcriptome focused member of All authors have read and approved the final version of the the GeneCards/GeneNote suite of databases,” Nucleic Acids paper. Research, vol. 33, supplement 1, pp. D556–D561, 2005. Evidence-Based Complementary and Alternative Medicine 9

[12] M. Kuhn, D. Szklarczyk, A. Franceschini et al., “STITCH 2: an smooth muscle cells,” TheJournalofBiologicalChemistry,vol. interaction network database for small molecules and proteins,” 277, no. 12, pp. 9800–9805, 2002. Nucleic Acids Research, vol. 38, pp. D552–D556, 2009. [29] N. V.O. Sweeney, P.M. Cummins, Y. A. Birney, E. M. Redmond, [13] M. Kuhn, D. Szklarczyk, S. Pletscher-Frankild et al., “STITCH and P. A. Cahill, “Cyclic strain-induced endothelial MMP-2: 4: integration of protein-chemical interactions with user data,” role in vascular smooth muscle cell migration,” Biochemical and Nucleic Acids Research,vol.42,no.1,pp.D401–D407,2014. Biophysical Research Communications,vol.320,no.2,pp.325– [14]G.Su,J.H.Morris,B.Demchak,andG.D.Bader,“Biological 333, 2004. network exploration with cytoscape 3,” Current Protocols in [30] M. Levy,´ C. Maurey, D. S. Celermajer et al., “Impaired apop- Bioinformatics,vol.47,pp.8–13,2014. tosis of pulmonary endothelial cells is associated with intimal [15] C. G. Rivera, R. Vakil, and J. S. Bader, “NeMo: network proliferation and irreversibility of pulmonary hypertension in module identification in cytoscape,” BMC Bioinformatics,vol. congenital heart disease,” Journal of the American College of 11, supplement 1, article S61, 2010. Cardiology,vol.49,no.7,pp.803–810,2007. ´ [16] T. R. Burkard, U. Rix, F. P. Breitwieser, G. Superti-Furga, and J. [31] U. Catalan,´ S. Fernandez-Castillejo,´ L. Pons et al., “Alpha- Colinge, “A computational approach to analyze the mechanism tocopherol and BAY 11-7082 reduce vascular cell adhesion of action of the kinase inhibitor bafetinib,” PLoS Computational molecule in human aortic endothelial cells,” Journal of Vascular Biology, vol. 6, no. 11, Article ID e1001001, 2010. Research,vol.49,no.4,pp.319–328,2012. [17] J. Folkman, “Angiogenesis and c-Jun,” Journal of the National [32]T.Eleftheriadis,G.Antoniadi,V.Liakopoulos,G.Pissas,I. Cancer Institute,vol.96,no.9,p.644,2004. Stefanidis, and G. Galaktidou, “Plasma angiogenin and vascular endothelial growth factor a among hemodialysis patients,” [18] A. Pedram, M. Razandi, and E. R. Levin, “Extracellular signal- Iranian Journal of Kidney Diseases,vol.6,no.3,pp.209–215, regulated protein kinase/jun kinase cross-talk underlies vas- 2012. cular endothelial cell growth factor-induced endothelial cell [33] S. Cagnin, M. Biscuola, C. Patuzzo et al., “Reconstruction and proliferation,” The Journal of Biological Chemistry,vol.273,no. functional analysis of altered molecular pathways in human 41, pp. 26722–26728, 1998. atherosclerotic arteries,” BMC Genomics,vol.10,articleno.13, [19] J.-C. Tille and M. S. Pepper, “Mesenchymal cells potentiate 2009. vascular endothelial growth factor-induced angiogenesis in [34] S. C. Chen, Y. L. Lin, B. Huang, D. L. Wang, and J. J. Cheng, vitro,” Experimental Cell Research,vol.280,no.2,pp.179–191, “Salvianolic acid B suppresses IFN-𝛾-induced JAK/STAT1 acti- 2002. vation in endothelial cells,” Thrombosis Research,vol.128,no.6, [20]C.Benckert,S.Jonas,T.Crameretal.,“Transforminggrowth pp.560–564,2011. factor 𝛽1 stimulates vascular endothelial growth factor gene [35] S. J. H. Bredie, H. Wollersheim, F. W.A. Verheugt, and T. Thien, transcription in human cholangiocellular carcinoma cells,” “Low-dose aspirin for primary prevention of cardiovascular Cancer Research,vol.63,no.5,pp.1083–1092,2003. disease,” Seminars in Vascular Medicine,vol.3,no.2,pp.177– [21] E. Galkina and K. Ley, “Vascular adhesion molecules in 184, 2003. atherosclerosis,” Arteriosclerosis, Thrombosis, and Vascular Biol- [36] R. Wilson, J. Gazzala, and J. House, “Aspirin in primary and ogy,vol.27,no.11,pp.2292–2301,2007. secondary prevention in elderly adults revisited,” Southern [22]K.D.O’Brien,T.O.McDonald,A.Chait,M.D.Allen,andC. Medical Journal, vol. 105, no. 2, pp. 82–86, 2012. E. Alpers, “Neovascular expression of E-selectin, intercellular [37]S.A.Khan,A.Ali,S.A.Khanetal.,“Unravelingthecomplex adhesion molecule-1, and vascular cell adhesion molecule-1 in relationship triad between lipids, obesity, and inflammation,” human atherosclerosis and their relation to intimal leukocyte Mediators of Inflammation,vol.2014,ArticleID502749,16 content,” Circulation, vol. 93, no. 4, pp. 672–682, 1996. pages, 2014. [23] L. S. A. Capettini, S. F. Cortes, J. F. Silva, J. I. Alvarez-Leite, and [38] M. Laakso and J. Kuusisto, “Insulin resistance and hypergly- V. S. Lemos, “Decreased production of neuronal NOS-derived caemia in cardiovascular disease development,” Nature Reviews hydrogen peroxide contributes to endothelial dysfunction in Endocrinology, vol. 10, no. 5, pp. 293–302, 2014. atherosclerosis,” British Journal of Pharmacology,vol.164,no. [39] G. S. Hotamisligil, “Endoplasmic reticulum stress and the 6, pp. 1738–1748, 2011. inflammatory basis of metabolic disease,” Cell,vol.140,no.6, [24] H. Li and U. Forstermann,¨ “Prevention of atherosclerosis by pp.900–917,2010. interference with the vascular nitric oxide system,” Current [40] A. Tjokroprawiro, “New approach in the treatment of T2DM Pharmaceutical Design,vol.15,no.27,pp.3133–3145,2009. and metabolic syndrome (focus on a novel insulin sensitizer),” [25]S.Zhang,J.Yuan,M.Yuetal.,“IL-17Afacilitatesplatelet Acta Medica Indonesiana, vol. 38, no. 3, pp. 160–166, 2006. function through the ERK2 signaling pathway in patients with [41] K. Y. Hur, H. J. Seo, E. S. Kang et al., “Therapeutic effect of mag- acute coronary syndrome,” PLoS ONE,vol.7,no.7,ArticleID nesium lithospermate B on neointimal formation after balloon- e40641, 2012. induced vascular injury,” European Journal of Pharmacology, [26]Y.E.Nassef,M.A.Hamed,andH.F.Aly,“Inflammatory vol. 586, no. 1–3, pp. 226–233, 2008. cytokines, apoptotic, tissue injury and remodeling biomarkers [42] X.-J. Wu, Y.-P. Wang, W. Wang, W.-K. Sun, Y.-M. Xu, and L.-J. in children with congenital heart disease,” Indian Journal of Xuan, “Free radical scavenging and inhibition of lipid perox- Clinical Biochemistry, vol. 29, no. 2, pp. 145–149, 2014. idation by magnesium lithospermate B,” Acta Pharmacologica [27] M. Wang, G.-B. Sun, X. Sun et al., “Cardioprotective effect of Sinica,vol.21,no.9,pp.855–858,2000. salvianolic acid B against arsenic trioxide-induced injury in [43] J. Ba, H. Peng, Y. Chen, and Y. Gao, “Effects and mechanism cardiacH9c2cellsviathePI3K/Aktsignalpathway,”Toxicology analysis of vascular endothelial growth factor and salvianolic Letters, vol. 216, no. 2-3, pp. 100–107, 2013. acid B on 125I-low density lipoprotein permeability of the rabbit [28] W.-H. Fan and M. J. Karnovsky, “Increased MMP-2 expression aortary endothelial cells,” Cell Biochemistry and Biophysics,vol. in connective tissue growth factor over-expression vascular 70,no.3,pp.1533–1538,2014. 10 Evidence-Based Complementary and Alternative Medicine

[44] Y. Joe, M. Zheng, H. J. Kim et al., “Salvianolic acid B exerts vasoprotective effects through the modulation of heme oxygenase-1 and arginase activities,” Journal of Pharmacology and Experimental Therapeutics,vol.341,no.3,pp.850–858, 2012. [45] T.-L. Yang, F.-Y. Lin, Y.-H. Chen et al., “Salvianolic acid B inhibits low-density lipoprotein oxidation and neointimal hyperplasia in endothelium-denuded hypercholesterolaemic rabbits,” Journal of the Science of Food and Agriculture,vol.91, no. 1, pp. 134–141, 2011. [46] Y.-H. Chen, G.-H. Du, and J.-T. Zhang, “Salvianolic acid B protects brain against injuries caused by ischemia-reperfusion in rats,” Acta Pharmacologica Sinica,vol.21,no.5,pp.463–466, 2000. M EDIATORSof INFLAMMATION

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