Identification of Potential Bioactive Ingredients and Mechanisms of the Guanxin Suhe Pill on Angina Pectoris by Integrating Network Pharmacology ...
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Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2021, Article ID 4280482, 13 pages
https://doi.org/10.1155/2021/4280482
Research Article
Identification of Potential Bioactive Ingredients and
Mechanisms of the Guanxin Suhe Pill on Angina Pectoris by
Integrating Network Pharmacology and Molecular Docking
Mingmin Wang ,1 Shuangjie Yang,2 Mingyan Shao,3 Qian Zhang,2 Xiaoping Wang,2
Linghui Lu,2 Sheng Gao,4 Yong Wang ,2,3 and Wei Wang 1,2
1
Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China
2
School of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
3
School of Life Sciences, Beijing University of Chinese Medicine, Beijing 100029, China
4
Department of Gastroenterology, The Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200092, China
Correspondence should be addressed to Yong Wang; doctor_wangyong@163.com and Wei Wang; wangwei26960@126.com
Received 12 April 2021; Revised 15 July 2021; Accepted 26 July 2021; Published 11 August 2021
Academic Editor: Amir Syahir
Copyright © 2021 Mingmin Wang 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 Guanxin Suhe pill (GSP), a traditional Chinese medicine, has been widely used to treat angina pectoris (AP) in Chinese
clinical practice. However, research on the bioactive ingredients and underlying mechanisms of GSP in AP remains scarce.
In this study, a system pharmacology approach integrating gastrointestinal absorption (GA) evaluation, drug-likeness (DL)
evaluation, target exploration, protein-protein-interaction analysis, Gene Ontology (GO) enrichment analysis, network
construction, and molecular docking was adopted to explore its potential mechanisms. A total of 481 ingredients from five
herbs were collected, and 242 were qualified based on GA and DL evaluation. Target exploration identified 107 shared targets
between GSP and AP. Protein-protein interaction identified VEGFA (vascular endothelial growth factor A), TNF (tumor
necrosis factor), CCL2 (C-C motif chemokine ligand 2), FN1 (fibronectin 1), MMP9 (matrix metallopeptidase 9), PTGS2
(prostaglandin-endoperoxide synthase 2), IL10 (interleukin 10), CXCL8 (C-X-C motif chemokine ligand 8), IL6 (inter-
leukin 6), and INS (insulin) as hub targets for GSP, which were involved in the inflammatory process, ECM proteolysis,
glucose metabolism, and lipid metabolism. GO enrichment identified top pathways in the biological processes, molecular
functions, and cell components, explaining GSP’s potential AP treatment mechanism. Positive regulation of the nitric oxide
biosynthetic process and the response to hypoxia ranked highest of the biological processes; core targets that GSP can
regulate in these two pathways were PTGS2 and NOS2, respectively. Molecular docking verified the interactions between the
core genes in the pathway and the active ingredients. The study lays a foundation for further experimental research and
clinical application.
1. Introduction control symptoms of myocardial ischemia by reducing
myocardial oxygen demand and increasing coronary blood
Angina pectoris (AP) is chest pain or discomfort which often flow. However, despite these antianginal drug therapies
occurs with stimulating factors, like physical activity or being able to effectively control chest pain symptoms and
emotional stress. There is accumulating evidence to suggest improve physical exercise tolerance, these drugs have also
that AP is associated with myocardial ischemia and coronary been reported to induce receptor tolerance because of their
atherosclerosis [1]. Frequently used antianginal drug therapy regular prescription [3].
includes organic nitrates, β-blockers, calcium channel Traditional Chinese medicine has been reported to ef-
blockers, and nicorandil [2]. These medications mainly fectively control AP. For instance, Panax notoginseng can
2 Evidence-Based Complementary and Alternative Medicine
reduce cardiovascular events, alleviate AP symptoms, and 2. Materials and Methods
reduce the attack frequency of AP [4]. Acupuncture can also
safely and effectively improve physical restrictions, emo- 2.1. Identification of Chemical Ingredients in GSP.
tional distress, and attack frequency in patients with stable According to the 2015 edition of Chinese Pharmacopoeia,
AP [5]. In addition, Suxiao Jiuxin Wan is effective in treating GSP included five herbs: Styrax (Storax, Suhexiang), Bor-
AP with no severe side effects identified to date [6]. neolum Syntheticum (Borneol, Bingpian), Resi oliani
The Guanxin Suhe pill (GSP) is a traditional Chinese (Frankincense, Ruxiang), Lignum Santali Albi (Sandalwood,
medicine formula for AP in Chinese Pharmacopoeia. It Tanxiang), and Inula helenium (Elecampane Inula,
includes five herb components: Styrax (Storax, Suhexiang), Tumuxiang) (Table 1). We searched the HERB database (a
Borneolum Syntheticum (Borneol, Bingpian), Resi oliani high-throughput experiment- and reference-guided data-
(Frankincense, Ruxiang), Lignum Santali Albi (Sandalwood, base of TCM) (http://herb.ac.cn.) for the ingredients of the
Tanxiang), and Inula helenium (Elecampane Inula, five herbs in GSP. HERB integrates multiple TCM databases
Tumuxiang). The GSP and its components have been proved to construct a list of TCM herbs and ingredients [15]. Several
to have an antianginal effect [7]. A clinical trial on 120 widely used TCM databases such as SymMap, TCMID 2.0,
patients with cold obstruction causing “qi stagnation” TCMSP 2.3, and HIT were included [16–19]. To date, the
syndrome was conducted to determine whether GSP in- HERB database is the most comprehensive database for
creases the effect of isosorbide mononitrate. The result found Chinese medicine ingredients. This database was interro-
that GSP can significantly relieve the symptoms of AP, gated to get a complete view of the known chemical in-
including the frequency and duration of angina attacks [8]. gredients of GSP. Chemical features of the ingredients were
However, the mechanism of GSP on AP has not been downloaded for the next step in the analysis.
thoroughly investigated.
Network pharmacology is becoming a cutting-edge re-
2.2. ADME Screened Ingredients with Gastrointestinal Ab-
search field in drug discovery and development [9]. By
sorption (GA) and Drug-Likeness (DL) Prediction.
integrating reductionist and systems approaches and com-
Pharmacokinetic parameters like bioavailability and DL are
putational and experimental methods, network pharma-
as crucial in a small molecule as an effective ingredient. Since
cology studies emphasize the paradigm shift from “one
GSP is orally administered, we considered GA a necessary
target, one drug” to “network target, multicomponent
pharmacokinetic behavior for active ingredient evaluation.
therapeutics” [10]. The multi-ingredient and multitarget
The BOILED-Egg model (the Brain or Intestinal Estimate D
nature of Chinese medicine makes it an ideal field for
permeation method) was applied to determine GA [20].
network pharmacology [11].
DL, which is established from structural or physico-
Molecular docking can predict ligand-target interaction
chemical inspections of oral drug-candidate compounds,
at a molecular level [12]. As an established structure-based
assesses qualitatively the chance for an ingredient to become
in silico simulation assay, molecular docking has been
an oral drug according to bioavailability [21]. Five methods
widely used in the drug discovery field [13]. The experi-
were applied as filters for DL evaluation, that is, the Lipinski
mental screening of large libraries of compounds against
(Pfizer), Ghose (Amgen), Veber (GSK), Egan (Pharmacia),
molecular target panels, that is, high-throughput screening
and Muegge (Bayer) methods [22–26]. All the above
(HTS), has been recognized as the gold standard in biology
methods are integrated into an online tool named Swis-
discovery. However, the high cost of the experimental
sADME (http://www.swissadme.ch/) [27]. According to the
screening remains a drawback. Docking enables re-
PubChem identification of ingredients provided by HERB,
searchers to virtually screen databases of approved drugs,
we searched the simplified molecular-input line-entry sys-
natural products, or synthesized compounds into a group
tem (SMILES) in PubChem (https://pubchem.ncbi.nlm.nih.
of biological targets of interest within a reasonable time
gov/) and uploaded the SMILES onto the SwissADME
[14]. Virtual screening and target profiling have made
website. Chemicals were screened by the following criteria: if
molecular docking a novel approach for active ingredient
the prediction results of the component were both “high”
screening and mechanism deciphering in Chinese medi-
GA and “yes” in more than two of the five filters in the DL
cine research.
prediction, it met our inclusion criteria and progressed to
Here, we took advantage of the most comprehensive
the next screening step.
traditional Chinese medicine (TCM) database to date, the
HERB database (a high-throughput experiment- and ref-
erence-guided database of TCM), to explore the core in- 2.3. Common Targets of GSP Ingredients in AP
gredients and targets of GSP in treating AP. Next, a
systematic pharmacological method integrating ADME 2.3.1. Potential Therapeutic Targets of GSP Active Ingredients.
(absorption, distribution, metabolism, and excretion) The HERB database was used to acquire potential thera-
screening, network pharmacology, and molecular docking peutic targets of GSP active ingredients. Apart from da-
was used to elucidate the underlying mechanism of the active tabase mining targets in the TCM databases such as
ingredients in GSP for AP treatment. It is anticipated that TCMSP, SymMap, HIT, and TCMID, as mentioned above,
the study will promote future studies in TCM, with the HERB also integrates the functional module named “ref-
concomitant development of more effective therapeutic erence mining,” which contains target information from
remedies for AP. manually curated reference data from 17886 references.
Evidence-Based Complementary and Alternative Medicine 3
Table 1: Information of herbs in GSP.
Name Number
No. Chinese Use part Properties Candidate Abbreviation
Latin English Components
pinyin compounds
Balsam from
1 Styrax Storax Suhexiang Warm; pungent 123 52 SHX
trunk
Borneolum Minor cold;
2 Borneol Bingpian Resin 75 30 BP
Syntheticum pungent; bitter
Warm; pungent;
3 Resi oliani Frankincense Ruxiang Balsam 170 85 RX
bitter
Lignum Santali
4 Sandalwood Tanxiang Heartwood Warm; pungent 134 80 TX
Albi
Elecampane Warm; pungent;
5 Inula helenium Tumuxiang Root 14 7 TMX
Inula bitter
HERB identities of active ingredients were uploaded to the annotation of the herbs’ targets [34]. In addition, GO
database to retrieve them according to potential thera- pathway enrichment was performed for the biological
peutic targets. processes, cell components, and molecular functions.
2.3.2. Prediction of Known Therapeutic Targets for AP. 2.5. Network Construction. The possible protein-protein
To establish a known therapeutic target dataset of AP, interactions (PPIs) were acquired from the STRING data-
several disease target databases were utilized, including base (https://string-db.org/), which covered almost all the
DisGeNET (https://www.disgenet.org/), GeneCards known functional interactions between the expressed pro-
(https://www.genecards.org/), OMIM (https://www. teins [35]. Moreover, PPI network pairs with overall com-
omim.org/), PharmGKB (https://www.pharmgkb.org/), bined scores above 0.4 were included. The combined results
and Therapeutic Target Database (http://db.idrblab.net/ of PPIs were imported to CytoSpace software (version 3.7.1,
ttd/) [28–32]. The keyword “angina pectoris” was used to Boston, MA, USA), and the PPI network was reconstructed
search the candidate targets. Since GeneCards is a data- with the “CytoHubba” plugin. The degrees of freedom in a
base with enormous web-based, deep-linked cards for topology network reflect the strength of a node’s connection
each of the >73000 human gene entries, its disease target with other nodes in the network. A high degree value in the
prediction result contains thousands of low-relevance PPI network indicates a high number of node edges. Thus,
targets. To eliminate the native effect of low-relevance high targets with high degree values are more likely to play
targets, targets from GeneCards with relevance scores
4 Evidence-Based Complementary and Alternative Medicine
docking conformation. Docking conformation with the 3.5. PPIs and Pathway Functional Enrichment
lowest binding energy was used for analysis. The top-five
ingredients with strong binding to each target were selected. 3.5.1. Possible PPIs in Intersection Targets. The PPI results of
PyMol was used to draw the binding graphs [37]. 107 intersection targets derived from STRING were im-
ported to CytoSpace to construct a topology network with
the “CytoHubba” plugin (Figure 2(b)). The intersection
3. Results targets were ranked according to the degree value of the
3.1. Workflow. The specific workflow is depicted in Figure 1. topology network. Among them, ten targets including
Firstly, the chemical ingredients of the five components in VEGFA (vascular endothelial growth factor A), TNF (tumor
GSP were searched for in the database, and the active in- necrosis factor), CCL2 (C-C motif chemokine ligand 2), FN1
gredients were screened out by pharmacokinetics, including (fibronectin 1), MMP9 (matrix metallopeptidase 9), PTGS2
GA and DL. Next, the targets of active ingredients from GSP (prostaglandin-endoperoxide synthase 2), IL10 (interleukin
and the targets of AP were retrieved from multiple databases. 10), CXCL8 (C-X-C motif chemokine ligand 8), IL6 (in-
Overlap targets were acquired from Gene Venn diagrams. terleukin 6), and INS (insulin) were ranked highest using
Subsequently, common targets of ingredient and disease Maximal Clique Centrality. These ten targets were recog-
targets were uploaded for possible PPIs and GO analyses. nized as hub targets. CytoSpace software was used to con-
PPI network and herb-ingredient-target networks were struct the PPI networks and herb-ingredient-target networks
constructed. Core targets of top pathways were applied for (Figures 2(c) and 2(d)).
molecular docking screens with interacted ingredients.
PyMol software was used to conduct interaction simulations 3.5.2. GO Pathway Enrichment. The GO enrichment anal-
of core targets and high-ranked ingredients. ysis of the 107 common targets was analyzed with DAVID.
All the biological processes (BP), molecular functions (MF),
3.2. Candidate Active Ingredients in GSP. After searching and cell component (CC) pathways obtained by GO en-
HERB, a total of 481 compounds were collected, including richment were ranked using −LogP (Figure 4). In the bio-
123, 75, 170, 134, and 14 compounds in Styrax (Storax, logical process GO enrichment, several AP and coronary
Suhexiang), Borneolum Syntheticum (Borneol, Bingpian), atherosclerosis-related pathways, such as the positive reg-
Resi oliani (Frankincense, Ruxiang), Lignum Santali Albi ulation of nitric oxide biosynthetic process, ranked top
(Sandalwood, Tanxiang), and Inula helenium (Elecampane according to the −LogP value. The herb-ingredient-target
Inula, Tumuxiang), respectively (Supplementary Table 1). network of the two pathways ranked highest in the biological
According to the PubChem identification provided by the process as shown in Figure 5. The positive regulation of gene
HERB database, the SMILE structure of every component expression ranked second in the biological processes. Since
was collected from PubChem. this process is very general and does not clearly provide an
insight into the mechanism of the disease, this pathway was
omitted in further analyses.
3.3. Ingredient Screening for High GA and Good DL. The
SMILE structure of ingredients was then uploaded to
SwissADME, and the ingredients were screened according to 3.6. Molecular Docking of the Most Targeted Proteins in the Top
the GA and DL by the criteria mentioned in the methods. Pathways. According to the GO pathway enrichment, the
After deduplication, 242 components were qualified, in- positive regulation of nitric oxide biosynthetic process and
cluding 52, 30, 85, 80, and 7 in Styrax (Storax, Suhexiang), the response to hypoxia ranked highest in the biological
Borneolum Syntheticum (Borneol, Bingpian), Resi oliani process pathways. PTGS2 in NO and NOS2 in hypoxia
(Frankincense, Ruxiang), Lignum Santali Albi (Sandalwood, demonstrated 54 and 11 compound interactions, respec-
Tanxiang), and Inula helenium (Elecampane Inula, tively, ranking the highest in these two pathways (Supple-
Tumuxiang), respectively (Supplementary Table 1). Several mentary Table 2). Thus, PTGS2 and NOS2 were identified as
qualified components were presented in more than one herb. core targets in these two pathways. We then conducted
All ingredients qualified by GA and DL were adopted to molecular docking between PTGS2, NOS2, and the corre-
screen intersection targets with AP. sponding compounds. Interaction scores are shown in
Supplementary Tables 3 and 4. The molecular interaction
graphs for the top-five ingredients of each component were
3.4. Intersection Targets of GSP Ingredients and AP. After then constructed using PyMol (Figures 6 and 7). All ten
deduplication, 699 related targets of GSP were collected ingredients interacted with corresponding targets mainly
from HERB. Meanwhile, 393 AP-related targets were col- through a hydrogen bond. Residual interaction information
lected from OMIM, DisGeNET, GeneCards, OMIM, is shown in Tables 2 and 3.
PharmGKB, and TTD. Among the intersection of GSP and
AP targets, there were 107 shared targets adopted for PPI 4. Discussion
analysis in STRING (Figure 2(a)). To provide a general view
of interactions between herbs, ingredients, and AP-related According to the topology network’s degree value, the top-
targets, an herb-ingredient-target network was constructed 10 hub genes in the PPI network were identified. They were
for these 107 intersection targets (Figure 3). VEGFA, TNF, CCL2, FN1, MMP9, PTGS2, IL10, CXCL8,
Evidence-Based Complementary and Alternative Medicine 5
Step 1
Target prediction
HERB database
PharmGKB, GeneCards, OMIM,
Identify chemical ingredients DisGeNET, and Therapeutic target
database
PubChem, SwissADME
Pharmacokinetic screen Disease-related
target prediction
HERB database
Theraputic target prediction
Intersection target
Step 2
Network analysis and
pathway enrichment
STRING DAVID
Protein-protein-interaction analysis, and
Pathway enrichment
hub genes identifiaction
Biology process Molecular function Cell component
Step 3
Molecular docking
Core target in pathways
AutoDock PyMol
Binding strength ranking Molecular interaction simulation
Figure 1: Workflow diagram of the present study.
6 Evidence-Based Complementary and Alternative Medicine
LPL OLR1PLAT
PLB1NR1I2 HMGCR
HFE LCAT
LDLR LTA
PPARA APOB
ADRB1 PON1
SLC6A2 TLR4 PPARG CETP
EDN1 APP
ADRB2 SCN5A
EGFR ICAM1
SLC2A4 ABCA1
BDNF CRP
ADRB3 CXCL8 IGF1 HBA1
HMOX1 LEP NOS3 APOE
NOS1 GLB1
GCG IL6 TNNT2
MMP2 MPO
592 107 286 INSR
MMP9 IL1B
KCNH2
TGFB1 INS PECAM1
PRKCD AKT1 SOD2
IL10 CCL2
THBD SERPINE1 ESR1 HIF1A
TNF
ECE1 VEGFA CEBPA
IL2 SRC PTGS2 PTEN
TTN HBB
IFNG TP53 FN1 SELE
TNFRSF1A CASP3 CYP19A1
IL1A IL4 MMP1 G6PD
GSR CAT PLG AR
Herb target KDR CYCS
PPBP PLAU
FGF2 CSF2 MMP3
CCND1 CD40
Disease target
MAPK8IP1 PTPN1
KRAS CD40LG
Intersection target NOTCH33 NR3C2 Degree
PIK3CA
A F3
SOD1 IL18 TERTHSPA5NOS2
0 100
(a) (b)
HBIN030821
H HBIN031753
H
HBB 7553
5
HBIN022577 HBIN041495
HBIN020921 HBIN047613
H
IL6 HBIN020789
HBIN032605
HBIN033803
HBIN033890
HBIN031378
HBIN019208 HBIN038026 HBIN046359
TNF HBIN028546
HBIN015704 HBIN039150HBIN048744
FN1 HBIN038627
HBIN028125 IL6 FN1
HBIN048757
HBIN040811
MMP9 HBIN033267 HBIN028076 MMP9
Tanxiang INS HBIN048952
HBIN043061
INS HBIN031211HBIN024535 HBIN015037
Ruxiang Tumuxiang
VEGFA PTGS2 HBIN0327544
PTGS2 HBIN030497 HBIN009471
HBIN020782
Suhexiang
HBIN025445 Bingpian H
7 BIN017771
CCL2 HBIN032807
HBIN017908 IL10 CXCL8
HBIN019918 HBIN018781
VEGFA HBIN032812
HBIN018729 HBIN016408 CCL2 TNF HBIN020653
IL10 HBIN037713
HBIN017508 HBIN010477 HBIN022965
HBIN038058
CXCL8 HBIN017057
HBIN047744
HBIN040358
HBIN042501 HBIN030840
MCC HBIN047751
HBIN0477477 HBIN035142
HBIN032583
HBIN045031
0033311 HBIN0386800
HBIN042339
HBIN040854
339
33
339 8855544
Low High
Herb
Target
Ingredient
(c) (d)
Figure 2: Intersection target genes and protein-protein interactions (PPIs) network of top-10 hub targets. (a) Intersecting target genes
between targets of GSP and AP; (b) PPIs network of intersecting targets; (c) PPI network of top-10 hub targets; (d) herb-ingredient-target
network of top-10 targets.
IL6, and INS. VEGFA is a member of the PDGF/VEGF hormone that plays a vital role in the regulation of carbo-
growth factor family. It induces proliferation and migration hydrate and lipid metabolism [46].
of vascular endothelial cells [39]. VEGFA has also been The function of hub targets mainly focuses on the
proved to be essential for both physiological and patho- regulation of inflammation, ECM regulation, and regulation
logical angiogenesis [40]. TNF, CCL2, IL10, and IL6 belong of metabolism. These are vital processes in the pathology of
to the cytokine family and are involved in inflammatory coronary atherosclerosis. Inflammation is an essential
processes [41]. FN1 and MMP9 are proteins in the extra- pathological process of coronary atherosclerosis, which is
cellular matrix and are involved in ECM proteolysis [42]. the major underlying cause of AP. A critical aspect in
Research has proved that AP shows an imbalanced collagen managing patients with stable angina is treating underlying
turnover even without significant obstructive coronary ar- coronary atherosclerotic disease and reducing the overall
tery disease [43]. PTGS2 is an inducible isozyme responsible risk burden to prevent future cardiac events and progression
for prostanoid biosynthesis and involved in inflammation of coronary disease. GSP might exert a protective effect on
and mitogenesis [44]. CXCL8, also known as IL-8, is a AP through these processes.
member of the CXC chemokine family and is a significant In the biological process GO enrichment, several AP and
mediator of the inflammatory response [45]. INS is a peptide coronary atherosclerotic related pathways ranked top
Evidence-Based Complementary and Alternative Medicine 7
HBIN037796
HBIN037713
HBIN037630HBIN038058
HBIN036188 HBIN039096
HBIN034630 HBIN040296
HBIN033277 HBIN040358
HBIN041495
HBIN032812
HBIN042501
HBIN032807
HBIN042679
HBIN032754
HBIN044636
HBIN031753
HBIN046008
HBIN030821
HBIN047613
HBIN027030
HBIN003176
HBIN026019 Ruxiang
HBIN003303 HBIN031211
HBIN025813 HBIN038604
HBIN030497
HBIN004074
HBIN024321 HBIN038627
HBIN030387 HBIN007722
HBIN022943 HBIN024976
HBIN030386 HBIN010592
HBIN022577 Bingpian HBIN015702
HBIN015036 HBIN010894
HBIN021904 HBIN019918
Tumuxiang HBIN011919
HBIN020921 HBIN012772
HBIN020789 HBIN013503 HBIN017057
HBIN021599 HBIN020643
HBIN019208 HBIN015704 HBIN018729
HBIN015037 HBIN015804
HBIN018743
HBIN018688 HBIN017508
HBIN018727
HBIN019502
Bing-Su
Ru-Su HBIN042339 Ru-Tan HBIN033218
AADACL2 SLPI CDC25A HTR3A IGHG1 PRKCA LHFPL6 Api5-ps NOS3 GCLM AOC2 Cox20b NOX4 SYP DGKD AMY2A IL13 STAT3 MAPK14 HSPA1B BAX MAPK1 RASA1 SLC5A5 MAPK12 MAPK6 IL18 AHR PRKCB RTP1
ALDH2 APOD PPARG TFEB APOB PLAU GUCY2F CASP1 Ppp1r18os MARK4 ABCG2 FXYD2 PLK1 SUFU PGD Ptges3-ps RIPK1 HSD17B8 SLC6A2 NOTCH3 HSD11B1 SLC6A15 ALOX5 NFATC1 CAV1 CT45A2 SLC26A7 Dpml OPRM1 COL1A1
MAPK11 PM20D2 TRPV4 CYP7A1 CTH Ctsm-ps BCHE ZNF442 TGFB1 MSTN PRSS1 CD86 AKR1C1 CNOT2 FGF2 PLAT EDN1 SULT1A2 VIM MGAM MAP2K2 MT1X LRP12 Salpa3 TNFRSF1A SLC8A1 IL7R SLC2A2 BCAP29 CALCA
RASGRF2 PPBP ABAT PIK3CB ACCSL MAPK15 FASN BIRC5 PDE3B TOP1 TPO Polr2k-ps HSDL2 FTH1 CYP1A2 ENPEP GSTM1 MAFF DNAJB4 C2orf81 ERICH4 ANTXR2 AKR1C2 PPARGC1B IL16 ICAM1 MAPK9 SMAD2 HTATIP2 HSD17B14
FCER2 DDIT3 MCL1 BMP2 CA2 ADAM23 ABCA1 ADM G6PD TUG1 G6PC BTK RBFOX2 CLK2 SLC1A2 B4GALT4 EAF2 TERT HBB AADACL3 TAT NFE2L2 UCP2 LCAT NFKBIA SYNE2 GPR21 CYP19A1 GPX3 P4HB
ADRB3 IGF1 DRD5 KDR GH1 AR PTGER3 CD40LG LITAF IFI27 UGT1A6 SLC6A20 CCL2 CHUK TNF GRIN3A PIR CYP3A4 RBM5 RUNX2 RELA H3P16 HBA1 AFMID PXDN CEBPA ESR1 SOD1 IL1B CSF2
NQO2 PRDM5 ABCG1 RUNX1T1 ODC1 VEGFA PLG WDR12 UCP3 PTPN6 CLEC4A CDC25B CREG1 Egfros PTGS1 SGCD CCNB1 ERBB3 Kcnmb4os2 HSF1 ADCY2 GSTM2 NR3C1 INSR CDC37 E2F2 DHRS12 TP53COR1 ALAS1 HAS2
BDNF DHRSX Sod1m LIPE DECR1 DHTKD1 RDH13 TGFB1I1 SLC35D2 NOSTRIN IGF1R MAPK8 SQSTM1 ALOX12 NCF1 BCL2 EBAG9 PRKAA2 KEAP1 SERPINE1 HSPA6 VEZF1 MS4A2 BBC3 E2F1 ATP2C1 EGLN1 TRIM16L GNRH1 TXNRD1
DHRS7C PIK3CG HSP90AA1 MGST1 PHLDA2 TOP2A AKT1 CD80 BDH2 MAPK8IP2 MET PARP1 MAP2K1 CDH1 GABBR1 ACAA2 SELE VCAM1 FTL DHRS13 CHRNA4 TNFRSF21 TRP53 NFKB1 Oaz2-ps CDK12 EIF6 CASP7 ELK1 SEL1L3
DHRS2 SCN5A NCOA1 GLS2 CASP8 CITED1 STAT1 HSD17B11 SRXN1 FECH MAP2K7 SLC6A3 PRKCG RBM45 PCNA UGP2 COL7A1 Ccpg1os CETP CLDN4 Akr1c12 IRAK1 CAMK2G GRIN1 ATAD2B MMP2 TRPM8 HPGD FASLG ATMIN
EGF DCAF5 STC2 FGFR2 CYP1B1 IGFBP3 NUF2 GOT1 PLK2 IL4 DDIT4 AKR1B1 PDX1 CSK DHRS7B SRSF11 PPP1R10 CFLAR CHEK2 SNX24 HSD3B2 RDH10 NKX3-1 PTGR1 EGFR Hsdr1 HFE CD40 IGF2 OXA1L
HERC5 SOD2 BARD1 GADD45A MPO CASP9 PTGS2 PECR DNMT1 GHR BIRC2 DHRS4L2 Appbp2os GLB1 TP53 IL6 AAGAB EGR1 STAU2 CDKN1A CHRM1 GJA1 DMTN GCH1 PXDNL TRPA1 Hmgb1-ps9 Ifi206 FOS SPOCK1
INSIG1 CBR1 Idd6.3 ALAS2 MAOB APP Atp5k-ps2 GNMT HMGCR CTNNB1 MT1A FOSL1 GLS COLQ LRRK2 INS COMT PNLIP ALOXE3 ECE1 PRKCD RBP2 RASSF1 PECAM1 PPARA DUOX2 ZDHHC21 SLC2A13 PAM MAFG
EIF2AK3 MAPK13 PGR SPP1 APOE PSMD3 MMP1 SRC GATM AKR1C3 TCEAL7 SULT1A1 NPEPPS TYW1 MTRR SLC22A5 IRF3 EPAS1 SNAI1 DUSP6 CASP3 LTA SMAD3 MMP10 HIF1A RRN3 HES1 CXCL8 DHRS4 CCND2
NQO1 UNG NOS1 JUND CALM3 KSR2 TXN LEP CREM THBD EZR MAPK7 ME1 NCEH1 IFI44L ARNTL2 ITGA11 TTN KRAS TDRD7 EPHX1 ANKRD37 HSPH1 STK24 RDH8 NTRK2 Gja6 OPCML CCR12P BRAF
EEF1E1 COQ3 ALPI KCNH2 Gnf4 IKBKB MMP13 FABP1 COL11A2 UBE2D1 PCYT1A SCD PLB1 TRPC5OS CRIM1 GOT1L1 IFNB1 MAFK CBR3 CRP ENPP7 ADRB1 MDM2 FBXO30 CYP21A2 GPX2 MAPK4 PKLR GSR FN1
ACHE Mod2 CES1 MAPK3 GCG TRPM2 ABHD12B TCF4 CDK6 TRIM16 HSD17B1 NOS2 GPT2 WWP2 IL1A KYAT1 CCND1 HMOX1 RHOA HSD3B1 ADRB2 FOSL2 PSME3 SULT1E1 B4GALT7 HK2 SARDH SIRT1 NR3C2 MMP3
IL2 CPT1A GRIA1 CD5L ATF2 SLC7A11 RXRA NDOR1 GSK3B POLD1 EPX BAD AADAC MKI67 OLR1 INPPL1 SOAT1 F3 TYR RDH12 ZNF639 CYP1A1 MMP9 PANX2 ERBB2 RAF1 AKR1B10 CDKN2A PTPRU CAPN2
DYNLRB1 AHSA1 GLRX PIK3CD CCK PTEN ATF3 PLIN1 Tlr12 MTOR GCK AARS1 Tstap198-15 SLC6A4 PIK3CA TEP1 LPL MYC RDH11 HSPA5 GRIA2 IRF1 RTN4RL1 LPO IRAK4 MTTP MMP8 IKBKG ACP4 PRSS50
CYCLIN
SOD3 TKT NR1I2 MAOA PCOLCE ACACA POR KIF20B PMAIP1 NR1I3 PPM1G TRPV1 BIRC3 PTPN1 TRIM63 CSNK2A2 TNNT2 DHRS1 NOX5 CXCL10 DNMT3B KYAT3 LDLR XIAP ESR2 GSTP1 RB1 CCDC34 CXCL2
B1
PON1 PYGL CYP17A1 SLC2A4 MT2A GPT HSD17B13 F8A1 PPARD GSK3A BCL2L1 BAP1 Topbp1-ps1 CAT GOT2 WEE1 LRP10 C5AR1 CCNA2 ABCC3 EP300 IFNG AADACL4 TFF1 PTGES ACP3 COX10 CTSD SGK3 APC
RAD51 NOP2 FABP5 FCGR2B RUNX3 Ciaaq1 AMPD3 PYY JUN GUSBP1 MAPK8IP1 NINL SDR16C5 ACMSD IL10 FOXO3 HSD17B3 TJP1 MX1 IGFBP1 FMO5 SLC14A1 ADTRP TXNRD2 HSPB1 ADK THNSL2 ALG5 DRP1 DNPEP
RDH14 LMCD1 Gssos2 CYCS TLR4 PPFIBP1 TXN2 DUT DIO1 ADRA1A LARS1 FZD2 Mapk6-ps1 GCLC Mipepos CBR4
HBIN040811
HBIN041350
HBIN039150
HBIN043061
HBIN038026 HBIN043075
HBIN036159 HBIN045254
HBIN035811 HBIN045942
HBIN035574 HBIN046359
HBIN020657
HBIN020831
HBIN020655 HBIN025445 HBIN033267 HBIN038680 HBIN047744 HBIN034297 HBIN047546
HBIN021604 HBIN020653
HBIN022965 HBIN018781
HBIN030840 HBIN017853 HBIN033890 HBIN048744
HBIN031031 HBIN017771 HBIN033803 HBIN048757
HBIN032583 HBIN014423 HBIN033609 HBIN048952
HBIN034284 HBIN011564 Ru-Su-Tan
HBIN033432 Tanxiang HBIN006084
HBIN035142 HBIN010919 Bing-Ru
HBIN032992 HBIN010477
HBIN036022 HBIN009471
HBIN032850 HBIN014419
HBIN036185 Suhexiang HBIN047756 HBIN032605 HBIN015668
HBIN039584 HBIN047755 HBIN031378 HBIN016408
HBIN040854 HBIN047754 HBIN031114 HBIN017056
HBIN029443 HBIN017908
HBIN045014 HBIN047753 HBIN028546 HBIN020782
HBIN045031 HBIN047752 HBIN028125
HBIN028076 HBIN020785
HBIN022584
HBIN024875
HBIN024535
HBIN045972 HBIN047751
HBIN047738 HBIN047750
HBIN047739 HBIN047749
HBIN047745 HBIN047748
HBIN047747
HBIN048365
HBIN047746
Herb
Ingredient
Target
Figure 3: Herb-ingredient-target network of GSP for angina pectoris.
according to the P value and gene counts. Positive regulation inhibition of overproduced NO also can alleviate the in-
of the nitric oxide biosynthetic process, response to hypoxia, flammation induced by NO.
positive regulation of smooth muscle proliferation, choles- Nitric oxide can cause vasodilation via its effect on
terol metabolic process, negative regulation of the apoptotic vascular smooth muscle cells. However, the overproduction
process, inflammatory response, and angiogenesis were of NO, which is proinflammatory, is detrimental to car-
marked as essential pathways involved in the therapeutic diovascular disease [50]. PTGS2 has been identified as the
mechanisms of GSP. core target gene involved in this pathway. PTGS2, also
Positive regulation of the nitric oxide biosynthetic known as COX2, is an important factor in the inflammatory
process ranked highest. Nitric oxide is a vasodilator pro- pathway. Research has proved that COX2 also influences the
duced by several cell types [47]. The function of nitric oxide positive regulation of the nitric oxide biosynthetic process
in vessel health is double-edged. On the one hand, endo- [51]. A low dose of COX2 inhibitor has been proved to have
thelial nitric oxide synthase (NOS) produces nitric oxide to a protective effect; the mechanism is based on what is known
maintain the physiological level of NO, which is crucial for about the complex biology of cyclooxygenase in different
vascular endothelial homeostasis, while on the other, dif- tissue compartments, including the vascular endothelium,
ferent stress-stimulating factors can induce the activation of myocardium, and atherosclerotic plaques. Ursolic acid,
inducible nitric oxide synthase (iNOS) [48]. NO overpro- apigenin, daidzein, baicalein, and dehydroabietic acid are
duction induced by the activation of iNOS can lead to the top-five ingredients according to the binding score.
endothelial dysfunction and the development of athero- Ursolic acid contacts with ARG120, which is a major
sclerosis in the late stages [49]. According to the target contributor to both the inhibition and catalysis of PTGS2.
prediction of GSP, ingredients in GSP can bind to iNOS and The PTGS2 inhibitory properties of other contact residues
COX2 (PTGS2) to inhibit the overproduction of NO. The have also been reported [52].
8 Evidence-Based Complementary and Alternative Medicine
Positive regulation of nitric oxide biosynthetic process
Positive regulation of gene expression
Response to hypoxia
Positive regulation of transcription from RNA polymerase II promoter
Positive regulation of smooth muscle proliferation
Cellular response to lipopolysaccharide
Cholesterol metabolic process
Response to drug
Response to ethanol
Negative regulation of the apoptotic process
Extracellular space
Extracellular region
External side of plasma membrane
Cell surface
Membrane raft
Term
Plasma membrane
Perinuclear region of cytoplasm
Platelet alpha granule lumen
Receptor complex
Caveola
Enzyme binding
Cytokine activity
Protein binding
Heme binding
Growth factor activity
Identical protein binding
NADP binding
Protease binding
Receptor binding
Heparin binding
0 10 20 30 40
–Lg P
Biological process
Cell component
Molecular function
Figure 4: Gene ontology pathway enrichment. Top-10 pathways in BP, MF, and CC according to −Log P.
HBIN028125
HBIN028076 HBIN028546
HBIN031378 HBIN028076
HBIN024535 HBIN032605 HBIN022584 HBIN031114
HBIN020782 HBIN033803
HBIN017908 HBIN016408 HBIN033803
HBIN033890
HBIN016408 NOS2
HBIN038026
HBIN010477 CCL2 NOS1
HBIN039150 HBIN015668 HBIN038026
HBIN047744
HBIN040811
HBIN047613 PPARA HMOX1
HBIN043061 HBIN014419 HBIN045942
HBIN042501
HBIN046359
HBIN041495 Tanxiang
INSR EGFR HBIN048744 CAT CASP3
HBIN040358 HBIN047613 HBIN021599
INS
IFNG HBIN048757 Tumuxiang
Ruxiang
HBIN038058 SOD2
ICAM1 HBIN048952
HBIN037713 Suhexiang HBIN041495 LEP HBIN020653
EDN1 HBIN015037 LTA
HBIN032812
AKT1 Bingpian Suhexiang
HBIN021599
Tanxiang Tumuxiang HBIN031753 HBIN020657
HBIN032807 TGFB1
PTGS2 VEGFA
HBIN009471
HBIN032754
IL6 HBIN011564 HBIN027030 HIF1A HBIN034284
HBIN031753 Ruxiang MMP2
Bingpian HBB
HBIN017771
HBIN030821 HBIN022577 PLAT VCAM1 HBIN036022
SOD2 HBIN018781 PLAU
HBIN022577 IL1B
HBIN020653 HBIN004074 HBIN045031
ESR1
HBIN020921 TLR4 HBIN019918 HBIN017057
TNF HBIN022965 HBIN017508
HBIN020789
Herb
HBIN030840
HBIN019208
HBIN032583 Target
HBIN015704
HBIN035142
HBIN038627 Ingredient
HBIN036022
HBIN033267
HBIN038680
HBIN031211
HBIN040854
HBIN030497
HBIN025445 HBIN042339
HBIN019918 HBIN045031
HBIN018729 HBIN047751
HBIN017057
HBIN017508
Herb
Target
Ingredient
(a) (b)
Figure 5: Herb-ingredient-target network of top pathways. (a) Herb-ingredient-target network of GO:0045429 (positive regulation of nitric
oxide biosynthetic process) related target; (b) herb-ingredient-target network of GO:0001666 (response to hypoxia) related target.
Evidence-Based Complementary and Alternative Medicine 9
(a) (b)
(c) (d)
(e) (f )
Figure 6: Molecular docking between NOS2 and top-five ingredients. (a) Protein structure of NOS2 (PDB ID:4cx7). (b) Molecular
interaction between MOL000006 luteolin and NOS2. (c) Molecular interaction between MOL000098 quercetin and NOS2. (d) Molecular
interaction between MOL000354 isorhamnetin and NOS2. (e) Molecular interaction between MOL000390 daidzein and NOS2.
(f ) Molecular interaction between MOL000422 kaempferol and NOS2.
(a) (b)
Figure 7: Continued.
10 Evidence-Based Complementary and Alternative Medicine
(c) (d)
(e) (f )
Figure 7: Molecular docking between PTGS2 and top-five ingredients. (a) Protein structure of PTGS2 (PDB ID: 5fdq). (b) Molecular
interaction between MOL008498 ursolic acid and PTGS2. (c) Molecular interaction between MOL000008 apigenin and PTGS2.
(d) Molecular interaction between MOL000390 daidzein and PTGS2. (e) Molecular interaction between MOL002714 baicalein and PTGS2.
(f ) Molecular interaction between MOL003782 dehydroabietic acid and PTGS2.
Table 2: Molecular binding information of PTGS2 and top-5 ingredients.
Target Compound TCMSP ID Contacting residue Binding distance Score
PTGS2 Ursolic acid MOL008498 ARG120 3.1 −9.6
TYR130 3
PTGS2 Apigenin MOL000008 −9
CYS47 2.1
GLN461 2.9
PTGS2 Daidzein MOL000390 −8.4
CYS47 3.2
THR212 3.2
PTGS2 Baicalein MOL002714 −7.5
ARG222 2.8, 3.1
SER121 2.7
PTGS2 Dehydroabietic acid MOL003782 LYS532 3.1 −7.5
GLN372 2.8
Table 3: Molecular binding information of NOS2 and top-5 ingredients.
Target Compound TCMSP ID Contacting residue Binding distance Score
ARG388 2.8
NOS2 Luteolin MOL000006 VAL352 3.1 −9.5
PHE369 2.8
ARG388 3
NOS2 Quercetin MOL000098 TYR347 2.3 −9.3
VAL352 3.2
VAL352 3.4
NOS2 Isorhamnetin MOL000354 ASP382 2.3 −8.9
ARG388 2.8
TYR373 3
NOS2 Daidzein MOL000390 ARG388 3.3, 3.3 −8.9
ARG266 2.9, 3.4
NOS2 Kaempferol MOL000422 ARG388 2.8 −8.9Evidence-Based Complementary and Alternative Medicine 11
The response to hypoxia ranked third. AP often involves and pharmacological mechanisms of the major bioactive
coronary artery spasm or stenosis, which leads to the dis- ingredients identified, more pharmacological experiments
ruption of oxygen and nutrient transportation via blood flow and in vitro molecular binding assays will be conducted in
[53]. This hemodynamic disorder can lead to heart tissue the near future.
hypoxia. Hypoxia signaling plays a vital role in cardiac and
vascular remodeling in the pathogenesis of cardiovascular Data Availability
diseases [54]. In a hypoxic environment, HIF-α protein is
stabilized. It binds to the hypoxia-responsive element All the relevant data of this article are available at https://
(HREs) of each target gene, including glucose metabolism github.com/MingminW/Guanxinsuhe-Pill.git.
(pyruvate dehydrogenase kinase (PDK)), angiogenesis
(vascular endothelial growth factor A (VEGFA)), and Conflicts of Interest
erythropoiesis (erythropoietin (EPO)) [55, 56]. Thus, the
ingredients of GSP may alleviate the cardiac remodeling The authors declare no conflicts of interest.
process through the tissue response to hypoxia.
Response to the hypoxia also ranked top in the biological Authors’ Contributions
process analysis. NOS2 is the core target in this pathway. The
exact mechanism of angina pain remains unclear, but it is Mingmin Wang, Shuangjie Yang, and Mingyan Shao have
related to a mismatch between myocardial oxygen demand contributed equally to this work. Mingmin Wang, Shuangjie
and supply [57]. It has been shown that, after tissue hypoxia, Yang, and Mingyan Shao performed data curation and
iNOS overexpression plays a vital role in tissue injury [58]. analysis. Qian Zhang, Xiaoping Wang, and Linghui Lu
Moreover, hypoxia alters the expression of several tran- conducted the network construction. Sheng Gao contributed
scription factors responsible for iNOS expression, and to the molecular docking. Yong Wang and Wei Wang
downregulation of iNOS can limit cell injury caused by designed and funded the research, revised the manuscript,
hypoxia [59]. Therefore, iNOS inhibition can be a novel and approved the submission of this manuscript. All authors
therapeutic mechanism for protection from hypoxia-in- have read and agreed to the final submission of the
duced injury and cell death. Luteolin, quercetin, iso- manuscript.
rhamnetin, daidzein, and kaempferol rank highest according
to the molecular docking study. Their contacting residue, Acknowledgments
mainly on the H4B binding pocket in the oxygenase domain,
This work was financially supported, in part, by grants from
is essential for H4B cofactor binding [60]. These ingredients
the National Natural Science Foundation of China (nos.
might contact this domain and thus influence the subse-
81930113, 81904169, and 81822049) and the National Major
quent dimerization and function of the enzyme.
Scientific and Technological Special Project for “Significant
Our results provide a potential binding model for the
New Drug Development” (no. 2019ZX09201004-001-011).
top-ranked ingredients of NOS2 and PTGS2. As mentioned
above, NOS2 inhibitors and PTGS2 inhibitors have been
identified for the treatment of cardiovascular disease. Supplementary Materials
However, the selective inhibitors have vast side effects. The
Supplementary Table 1: herb ingredient ID lists and qualified
multitarget and multi-ingredient nature of Chinese medi-
ingredient ID list. Supplementary Table 2: ingredient-target
cine provides another avenue for drug development from
interaction lists of top targets. Supplementary Table 3: in-
Chinese medicine.
teraction scores of PTGS2 and ingredients. Supplementary
Table 4: interaction scores of NOS2 and ingredients. (Sup-
5. Conclusions plementary Materials)
This study identifies the potential bioactive ingredients,
biological targets, and functional pathways of GSP for AP,
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