# Network Pharmacology-Based Investigation into the Mechanisms of Quyushengxin Formula for the Treatment of Ulcerative Colitis

Authors: Haojie Yang; Ying Li; Sichen Shen; Dan Gan; Changpeng Han; Jiong Wu; Zhenyi Wang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2019)

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## Abstract

Objective. Ulcerative colitis (UC) is a chronic idiopathic inflammatory bowel disease whose treatment strategies remain unsatisfactory. This study aims to investigate the mechanisms of Quyushengxin formula acting on UC based on network pharmacology. Methods. Ingredients of the main herbs in Quyushengxin formula were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Absorption, distribution, metabolism, and excretion properties of all ingredients were evaluated for screening out candidate bioactive compounds in Quyushengxin formula. Weighted ensemble similarity algorithm was applied for predicting direct targets of bioactive ingredients. Functional enrichment analyses were performed for the targets. In addition, compound-target network, target-disease network, and target-pathway network were established via Cytoscape 3.6.0 software. Results. A total of 41 bioactive compounds in Quyushengxin formula were selected out from the TCMSP database. These bioactive compounds were predicted to target 94 potential proteins by weighted ensemble similarity algorithm. Functional analysis suggested these targets were closely related with inflammatory- and immune-related biological progresses. Furthermore, the results of compound-target network, target-disease network, and target-pathway network indicated that the therapeutic effects of Quyushengxin on UC may be achieved through the synergistic and additive effects. Conclusion. Quyushengxin may act on immune and inflammation-related targets to suppress UC progression in a synergistic and additive manner.


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## Body

## 1. Introduction

Ulcerative colitis (UC) is a chronic and progressive immunologically mediated disease causing consecutive mucosal inflammation of the colon [1, 2]. The onset of UC is most often during young adulthood, which is well characterized by homogeneous and continuous lesions [3]. Although the incidence of UC is increasing in Asia, it is highly diagnosed in the developed countries, especially in Western Europe and North America. Previous reports showed that the overall incidence and prevalence of UC are nearly 1.2/20.3 cases and 7.6/245 per 100,000 persons per year, respectively [4, 5].UC therapy is aimed to reduce the recurrent rate, as well as improve the life quality and minimize drug-related adverse events. Basic therapies for UC are determined based on the severity of symptoms, which are often thought as step-up approaches. To date, 5-aminosalycilates (5-ASAs) have been the mainstay for treatment of mild-to-moderate UC [6]. Though 5-ASAs are safe and have no dose-related toxicity in short-term use with a dose-response efficacy, long-term use of them might induces adverse events, such as headache, diarrhea, nausea, interstitial nephritis, and hepatitis. In addition, patients with more moderate-to-severe UC after 5-ASAs therapy are typically treated with corticosteroids, and these patients are often followed by transition to a steroid-sparing agent with a thiopurine, adhesion molecule inhibitor, or anti-tumor necrosis factor (TNF) agent [6]. However, these corticosteroid-based therapies also accompany with side effects, such as cataracts, osteopenia, avascular necrosis, insomnia, mood changes, delirium, glaucoma, and adrenal insufficiency [7, 8]. Besides, despite improved medical therapies, it is estimated that about 15% of UC patients still require proctocolectomy [9]. Therefore, it is of great significance to develop more optimized and integrated therapies for UC patients.To date, an increasing number of traditional Chinese herbal compounds are successfully used for treating UC with less side effects, such as Gegen Qinlian decoction [10], Jianpi Qingchang decoction [11, 12], Zhikang capsule [13], Huangkui Lianchang decoction [14], and Qingchang Wenzhong decoction [15, 16]. Quyushengxin formula is mainly composed of four herbs, Panax ginseng C.A. Mey. (Araliaceae), Astragalus membranaceus (Fisch) Bunge, Pulsatilla chinensis (Bge.) Regel, and Coptis chinensis Franch. Our clinical practice demonstrated Quyushengxin formula could relieve the clinical symptoms in active stage and suppress the inflammatory reaction of UC patients and could be used for treating mild-to-moderate UC [17]. Although the therapeutic effects of Quyushengxin on UC are attractive, molecular mechanisms of its action remain to be further elucidated.Traditional Chinese medicine- (TCM-) oriented network pharmacology provides us a novel way to unveil the molecular mechanisms of TCM through pharmacokinetic evaluation, network/pathway analysis, and target prediction [18, 19]. In this study, we tried to unveil the molecular mechanisms of Quyushengxin formula acting on UC based on network pharmacology.


## 2. Materials and Methods

### 2.1. Screening of Potential Bioactive Compounds in Quyushengxin Formula

Traditional Chinese Medicine Systems Pharmacology Database (TCMSP,http://lsp.nwu.edu.cn) is a systems pharmacology platform of Chinese herbal medicines that captures the relationships between drugs, targets, and diseases [20]. Ingredients along with their molecular weight (MW), water partition coefficient (AlogP), number of hydrogen bond donors (Hodn), number of hydrogen acceptors (Hacc), oral bioavailability (OB), Caco-2 permeability (Caco-2), blood-brain barrier (BBB), drug-likeness (DL), fractional negative accessible surface area (FASA) ,and half-life (HL), of all four herbs in Quyushengxin formula were retrieved from TCMSP. Then, absorption, distribution, metabolism, and excretion (ADME) properties, including OB, DL, and HL, were evaluated for screening out bioactive compounds. The potential bioactive compounds in Quyushengxin were predicted and sifted out via an integrated model including PreOB (for prediction of OL), PreDL (for prediction of DL), and PreHL (for prediction of HL) [21, 22]. In detail, OB value was obtained by OBioavail 1.1, and the compounds with OB ≥ 30% were selected out for further analysis [20, 23]. PreDL was utilized to calculate the DL index of compounds, and compounds with DL ≥ 0.18 were included for further research. The DL evaluation approach was constructed via both Tanimoto coefficient and molecular descriptors, and the formula is listed as follows:(1)TX,Y=X⋅YX2+Y2−X⋅Y,where X was the molecular descriptors of herbal ingredients and Y showed the average molecular properties of all molecules in the DrugBank database (http://www.drugbank.ca/).Besides, PreHL was estimated by combining multivariable linear regression model and MLR (mixed logistic regression) algorithm [22], as follows:(2)Yt1/2=13.310±13.31+13.376±13.37×nArCO+7.092±nA7×H7m+0.053±0.007×D/Dr09+19.377±4.052×N−070−7.598±70−7.×C−033−347.423±33−347.×JGI6+32.752±JG2×nRC=N−0.100±nR0×Mor02e,R2=0.65,Q2=0.62,F=27.272,SEE=8.127,Ntraining=126,Ntest=43,where R2 was the correlation coefficient of training set and Q2 was the correlation coefficient of external test sets of the model. SEE was the estimated standard deviation of training set. F was the mean square ratio. Besides, Ntraining indicated the number of chemical compounds in the training set, and Ntest indicated the number of chemical compounds in the test set. It was evidenced that there were eight descriptors satisfying the linear regression as follows: nArCO, H7m, D/Dr09, N-070, C-032, JGI6, nRC=N, and Mor02e. Finally, 4 ≤ HL ≤ 8 was defined as appropriate selection criteria for drug HL evaluation.


### 2.2. Prediction of the Candidate Targets of Bioactive Compounds

Weighted ensemble similarity (WES) algorithm was applied for predicting direct targets of the bioactive compounds via a large scale of drug target relationships [24]. Those targets with likelihood score ≥7 were deemed as direct targets in this study. Thereafter, candidate targets were mapped to Uniprot (http://www.uniprot.org/) for annotation and normalization.


### 2.3. Functional Enrichment Analyses

Gene Ontology- (GO-) biological processes (BPs) and Kyoto Encyclopedia of Genes and Genomes (KEGG,http://www.genome.jp/kegg/) pathways of the candidate targets of bioactive compounds were predicted via the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database [25] with P<0.05 as the criterion for significance.


### 2.4. Prediction of Target-Related Disease

Target-related diseases were predicted by integrating multisource databases, including Comparative Toxicogenomics Database (CTD,http://ctdbase.org/) [26], Therapeutic Target Database (TTD, http://bidd.nus.edu.sg/group/cjttd/) [27], and PharmGKB database (https://www.pharmgkb.org/) [28].


### 2.5. Network Construction

Three kinds of networks in this study were established using Cytoscape 3.6.0 software [29]: compound-target network (C-T network), target-disease network (T-D network), and target-pathway network (T-P network). C-T network was composed of bioactive compounds and their potential targets, which was built to reveal the drug-target interactions. T-D network was built based on the potential targets and their related diseases. The pathway information of targets was selected from the results for KEGG pathway enrichment analysis by excluding those pathways with no relevance to UC based the latest pathological information of UC. T-P network was generated based on potential targets and UC-related pathways. In the networks, the nodes represented compounds, targets, diseases, and pathways, and the edges displayed the interactions between two nodes. Furthermore, the significance of each node in the networks was assessed via one crucial topological parameter, namely, “degree,” which was defined as the total of edges related with a node [30, 31]. Degree of all nodes was analyzed using plugin NetworkAnalyzer of Cytoscape 3.6.0.


## 2.1. Screening of Potential Bioactive Compounds in Quyushengxin Formula

Traditional Chinese Medicine Systems Pharmacology Database (TCMSP,http://lsp.nwu.edu.cn) is a systems pharmacology platform of Chinese herbal medicines that captures the relationships between drugs, targets, and diseases [20]. Ingredients along with their molecular weight (MW), water partition coefficient (AlogP), number of hydrogen bond donors (Hodn), number of hydrogen acceptors (Hacc), oral bioavailability (OB), Caco-2 permeability (Caco-2), blood-brain barrier (BBB), drug-likeness (DL), fractional negative accessible surface area (FASA) ,and half-life (HL), of all four herbs in Quyushengxin formula were retrieved from TCMSP. Then, absorption, distribution, metabolism, and excretion (ADME) properties, including OB, DL, and HL, were evaluated for screening out bioactive compounds. The potential bioactive compounds in Quyushengxin were predicted and sifted out via an integrated model including PreOB (for prediction of OL), PreDL (for prediction of DL), and PreHL (for prediction of HL) [21, 22]. In detail, OB value was obtained by OBioavail 1.1, and the compounds with OB ≥ 30% were selected out for further analysis [20, 23]. PreDL was utilized to calculate the DL index of compounds, and compounds with DL ≥ 0.18 were included for further research. The DL evaluation approach was constructed via both Tanimoto coefficient and molecular descriptors, and the formula is listed as follows:(1)TX,Y=X⋅YX2+Y2−X⋅Y,where X was the molecular descriptors of herbal ingredients and Y showed the average molecular properties of all molecules in the DrugBank database (http://www.drugbank.ca/).Besides, PreHL was estimated by combining multivariable linear regression model and MLR (mixed logistic regression) algorithm [22], as follows:(2)Yt1/2=13.310±13.31+13.376±13.37×nArCO+7.092±nA7×H7m+0.053±0.007×D/Dr09+19.377±4.052×N−070−7.598±70−7.×C−033−347.423±33−347.×JGI6+32.752±JG2×nRC=N−0.100±nR0×Mor02e,R2=0.65,Q2=0.62,F=27.272,SEE=8.127,Ntraining=126,Ntest=43,where R2 was the correlation coefficient of training set and Q2 was the correlation coefficient of external test sets of the model. SEE was the estimated standard deviation of training set. F was the mean square ratio. Besides, Ntraining indicated the number of chemical compounds in the training set, and Ntest indicated the number of chemical compounds in the test set. It was evidenced that there were eight descriptors satisfying the linear regression as follows: nArCO, H7m, D/Dr09, N-070, C-032, JGI6, nRC=N, and Mor02e. Finally, 4 ≤ HL ≤ 8 was defined as appropriate selection criteria for drug HL evaluation.


## 2.2. Prediction of the Candidate Targets of Bioactive Compounds

Weighted ensemble similarity (WES) algorithm was applied for predicting direct targets of the bioactive compounds via a large scale of drug target relationships [24]. Those targets with likelihood score ≥7 were deemed as direct targets in this study. Thereafter, candidate targets were mapped to Uniprot (http://www.uniprot.org/) for annotation and normalization.


## 2.3. Functional Enrichment Analyses

Gene Ontology- (GO-) biological processes (BPs) and Kyoto Encyclopedia of Genes and Genomes (KEGG,http://www.genome.jp/kegg/) pathways of the candidate targets of bioactive compounds were predicted via the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database [25] with P<0.05 as the criterion for significance.


## 2.4. Prediction of Target-Related Disease

Target-related diseases were predicted by integrating multisource databases, including Comparative Toxicogenomics Database (CTD,http://ctdbase.org/) [26], Therapeutic Target Database (TTD, http://bidd.nus.edu.sg/group/cjttd/) [27], and PharmGKB database (https://www.pharmgkb.org/) [28].


## 2.5. Network Construction

Three kinds of networks in this study were established using Cytoscape 3.6.0 software [29]: compound-target network (C-T network), target-disease network (T-D network), and target-pathway network (T-P network). C-T network was composed of bioactive compounds and their potential targets, which was built to reveal the drug-target interactions. T-D network was built based on the potential targets and their related diseases. The pathway information of targets was selected from the results for KEGG pathway enrichment analysis by excluding those pathways with no relevance to UC based the latest pathological information of UC. T-P network was generated based on potential targets and UC-related pathways. In the networks, the nodes represented compounds, targets, diseases, and pathways, and the edges displayed the interactions between two nodes. Furthermore, the significance of each node in the networks was assessed via one crucial topological parameter, namely, “degree,” which was defined as the total of edges related with a node [30, 31]. Degree of all nodes was analyzed using plugin NetworkAnalyzer of Cytoscape 3.6.0.


## 3. Results

### 3.1. Screening of Potential Bioactive Compounds from Four Herbs in Quyushengxin Formula

Quyushengxin formula consists of 4 main herbs:Panax ginseng C.A. Mey. (Araliaceae), Astragalus membranaceus (Fisch) Bunge, Pulsatilla chinensis (Bge.) Regel, and Coptis chinensis Franch. After retrieving from TCMSP, 190, 87, 57, and 48 ingredients were obtained for these four herbs, respectively. Based on the criteria of OB ≥ 30%, DL ≥ 0.18, and 4 ≤ HL ≤ 8, 41 potential bioactive compounds, including quercetin, ursolic acid, kaempferol, β-sitosterol, and rutin, were sifted out (Table 1), which accounted for 10.73% of all 382 ingredients in Quyushengxin.Table 1
Details of 41 bioactive compounds and their biological parameters.
ID
Compounds
Structure
OB (%)
DL
HL
Degree
Herb
mol01
Quercetin
46.43
0.28
14.40
73
Coptis chinensis FranchAstragalus membranaceus (Fisch) Bunge
mol02
Ferulic acid
39.56
0.06
2.38
7
Coptis chinensis Franch
mol03
Palmatine
64.60
0.65
2.25
9
Coptis chinensis Franch
mol04
Jatrorrizine
19.65
0.59
4.21
9
Coptis chinensis Franch
mol05
Berberine
36.86
0.78
6.57
8
Coptis chinensis Franch
mol06
Columbamine
26.94
0.59
5.21
9
Coptis chinensis Franch
mol07
Coptisine
30.67
0.86
9.33
8
Coptis chinensis Franch
mol08
Worenine
45.83
0.87
8.41
6
Coptis chinensis Franch
mol09
Magnoflorine
0.48
0.55
6.22
8
Coptis chinensis Franch
mol10
Berberrubine
35.74
0.73
6.46
8
Coptis chinensis Franch
mol11
Epiberberine
43.09
0.78
6.10
7
Coptis chinensis Franch
mol12
(R)-Canadine
55.37
0.77
6.41
9
Coptis chinensis Franch
mol13
Berlambine
36.68
0.82
7.33
9
Coptis chinensis Franch
mol14
Corchoroside A_qt
104.95
0.78
6.68
2
Coptis chinensis Franch
mol15
Tetrandrine
26.64
0.10
4.77
9
Coptis chinensis Franch
mol16
β-Sitosterol
36.91
0.75
5.36
15
Panax ginseng C.A. Mey. (Araliaceae)Pulsatilla chinensis (Bge.) Regel
mol17
Kaempferol
41.88
0.24
14.74
26
Panax ginseng C.A. Mey. (Araliaceae)
mol18
Stigmasterol
43.83
0.76
5.57
10
Panax ginseng C.A. Mey. (Araliaceae)GPulsatilla chinensis (Bge.) Regel
mol19
β-Elemene
25.63
0.06
6.32
8
Panax ginseng C.A. Mey. (Araliaceae)
mol20
Ginsenoside Ro_qt
17.62
0.76
7.50
1
Panax ginseng C.A. Mey. (Araliaceae)
mol21
Dianthramine
40.45
0.20
5.14
3
Panax ginseng C.A. Mey. (Araliaceae)
mol22
Arachidonate
45.57
0.20
7.56
5
Panax ginseng C.A. Mey. (Araliaceae)
mol23
Ginsenoside La_qt
15.70
0.78
5.20
1
Panax ginseng C.A. Mey. (Araliaceae)
mol24
Ginsenoside rh2
36.32
0.56
11.08
9
Panax ginseng C.A. Mey. (Araliaceae)
mol25
Ginsenoside-Rh3_qt
13.09
0.76
6.22
1
Panax ginseng C.A. Mey. (Araliaceae)
mol26
Ginsenoside-Rh4_qt
31.11
0.78
6.97
1
Panax ginseng C.A. Mey. (Araliaceae)
mol27
Malkangunin
57.71
0.63
4.09
1
Panax ginseng C.A. Mey. (Araliaceae)
mol28
Alexandrin_qt
36.91
0.75
5.53
1
Panax ginseng C.A. Mey. (Araliaceae)
mol29
Ginsenoside rf
17.74
0.24
4.66
5
Panax ginseng C.A. Mey. (Araliaceae)
mol30
Hederagenin
36.91
0.75
5.35
6
Astragalus membranaceus (Fisch) Bunge
mol31
Isorhamnetin
49.60
0.31
14.34
10
Astragalus membranaceus (Fisch) BungePulsatilla chinensis (Bge.) Regel
mol32
7-O-methylisomucronulatol
74.69
0.30
2.98
11
Astragalus membranaceus (Fisch) Bunge
mol33
Rutin
3.20
0.68
6.22
15
Astragalus membranaceus (Fisch) Bunge
mol34
1,7-Dihydroxy-3,9-dimethoxy pterocarpene
39.05
0.48
7.95
5
Astragalus membranaceus (Fisch) Bunge
mol35
Isoferulic acid
50.83
0.06
2.45
7
Astragalus membranaceus (Fisch) Bunge
mol36
Betulinic acid
55.38
0.78
8.87
1
Pulsatilla chinensis (Bge.) Regel
mol37
Oleanolic acid
29.02
0.76
5.56
6
Pulsatilla chinensis (Bge.) Regel
mol38
Sitosteryl acetate
40.39
0.85
6.34
1
Pulsatilla chinensis (Bge.) Regel
mol39
Lanosterol
42.12
0.75
5.84
1
Pulsatilla chinensis (Bge.) Regel
mol40
3-beta,23-Dihydroxy-lup-20(29)-ene-28-O-alpha-L-rhamnopyranosyl-(1-4)-beta-D-glucopyranosyl(1-6)-beta-D-glucopyranoside_qt
37.59
0.79
6.70
1
Pulsatilla chinensis (Bge.) Regel
mol41
Ursolic acid
16.77
0.75
5.28
35
Pulsatilla chinensis (Bge.) Regel


### 3.2. Establishment of C-T Network

Candidate targets of the 41 bioactive compounds were predicted via WES algorithm. A total of 367 potential targets for these 41 bioactive compounds were obtained. After removing the overlapping targets, 94 candidate proteins were reserved. Then, C-T network was built by Cytoscape 3.6.0 which contains 367 connections between 41 compounds and corresponding 94 candidate targets (Figure1). The degrees of the 41 bioactive compounds in the C-T network were calculated and are displayed in Table 1. The average degree of targets per compound was 4.7, indicating multitarget functions of Quyushengxin formula. Among the 41 bioactive compounds, 8 of them showed a high degree (degree > 10). Quercetin possessed the highest degree of targets (degree = 73), followed by ursolic acid (degree = 35), kaempferol (degree = 26), β-sitosterol (degree = 15), rutin (degree = 15), 7-O-methylisomucronulatol (degree = 11), stigmasterol (degree = 10), and isorhamnetin (degree = 10).Figure 1
Compound-target network. A compound node and a target node are connected.The degree of the candidate targets was also calculated and displayed in Table2. Eight out of the 94 compounds possessed a degree larger than 10, including ESR1 (estrogen receptor 1, degree = 34), PTGS2 (prostaglandin-endoperoxide synthase 2, degree = 27), NOS2 (nitric oxide synthase 2, degree = 25), PTGS1 (degree = 23), PPARG (peroxisome proliferator-activated receptor gamma, degree = 21), NOS3 (degree = 21), ESR2 (degree = 17), and KCNH2 (Potassium Voltage-Gated Channel Subfamily H Member 2, degree = 13).Table 2
Details of 94 UC-related targets of herbs via UniProt.
ID
UniProt
Protein names
Gene names
Degree
Organism
1
P35228
Nitric oxide synthase, inducible
NOS2
25
Homosapiens
2
P23219
Prostaglandin G/H synthase 1
PTGS1
23
Homosapiens
3
P03372
Estrogen receptor
ESR1
34
Homosapiens
4
P37231
Peroxisome proliferator-activated receptor gamma
PPARG
21
Homosapiens
5
P35354
Prostaglandin G/H synthase 2
PTGS2
27
Homosapiens
6
Q92731
Estrogen receptor beta
ESR2
17
Homosapiens
7
P11388
DNA topoisomerase 2-alpha
TOP2A
5
Homosapiens
8
P16389
Potassium voltage-gated channel subfamily H member 2
KCNH2
13
Homosapiens
9
P08709
Coagulation factor VII
F7
6
Homosapiens
10
P29474
Nitric-oxide synthase, endothelial
NOS3
21
Homosapiens
11
P27338
Amine oxidase [flavin-containing] B
MAOB
5
Homosapiens
12
Q04206
Transcription factor p65
RELA
6
Homosapiens
13
P00533
Epidermal growth factor receptor
EGFR
1
Homosapiens
14
P31749
RAC-alpha serine/threonine-protein kinase
AKT1
2
Homosapiens
15
P15692
Vascular endothelial growth factor A
VEGFA
2
Homosapiens
16
P24385
G1/S-specific cyclin-D1
CCND1
3
Homosapiens
17
P10415
Apoptosis regulator Bcl-2
BCL2
5
Homosapiens
18
P01100
Proto-oncogene c-Fos
FOS
3
Homosapiens
19
P38936
Cyclin-dependent kinase inhibitor 1
CDKN1A
4
Homosapiens
20
P55211
Caspase-9
CASP9
4
Homosapiens
21
P00749
Urokinase-type plasminogen activator
PLAU
4
Homosapiens
22
P08253
72 kDa type IV collagenase
MMP2
2
Homosapiens
23
P14780
Matrix metalloproteinase-9
MMP9
2
Homosapiens
24
P22301
Interleukin-10
IL10
1
Homosapiens
25
P01133
Proepidermal growth factor
EGF
1
Homosapiens
26
P06400
Retinoblastoma-associated protein
RB1
2
Homosapiens
27
P01375
Tumor necrosis factor
TNF
6
Homosapiens
28
P05412
Transcription factor AP-1
JUN
4
Homosapiens
29
P05231
Interleukin-6
IL-6
3
Homosapiens
30
P42574
Caspase-3
CASP3
7
Homosapiens
31
P04637
Cellular tumor antigen p53
TP53
4
Homosapiens
32
P11926
Ornithine decarboxylase
ODC1
1
Homosapiens
33
Q14790
Caspase-8
CASP8
3
Homosapiens
34
P00441
Superoxide dismutase [Cu-Zn]
SOD1
2
Homosapiens
35
P17252
Protein kinase C alpha type
PRKCA
2
Homosapiens
36
P03956
Interstitial collagenase
MMP1
3
Homosapiens
37
P42224
Signal transducer and activator of transcription 1-alpha/beta
STAT1
2
Homosapiens
38
P04626
Receptor tyrosine-protein kinase erbB-2
ERBB2
1
Homosapiens
39
P09601
Heme oxygenase 1
HMOX1
3
Homosapiens
40
P05177
Cytochrome P450 1A2
CYP1A2
2
Homosapiens
41
P01106
Myc proto-oncogene protein
MYC
1
Homosapiens
42
P05362
Intercellular adhesion molecule 1
ICAM1
4
Homosapiens
43
P01584
Interleukin-1 beta
IL1B
5
Homosapiens
44
P13500
C-C motif chemokine 2
CCL2
1
Homosapiens
45
P19320
Vascular cell adhesion protein 1
VCAM1
2
Homosapiens
46
P10145
Interleukin-8
CXCL8
2
Homosapiens
47
P05771
Protein kinase C beta type
PRKCB
2
Homosapiens
48
O15392
Baculoviral IAP repeat-containing protein 5
BIRC5
2
Homosapiens
49
P04792
Heat shock protein beta-1
HSPB1
1
Homosapiens
50
P01137
Transforming growth factor beta-1
TGFB1
3
Homosapiens
51
P60568
Interleukin-2
IL2
2
Homosapiens
52
Q16678
Cytochrome P450 1B1
CYP1B1
2
Homosapiens
53
P00750
Tissue-type plasminogen activator
PLAT
1
Homosapiens
54
P01579
Interferon gamma
IFNG
4
Homosapiens
55
P09917
Arachidonate 5-lipoxygenase
ALOX5
3
Homosapiens
56
P60484
Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
PTEN
1
Homosapiens
57
P05164
Myeloperoxidase
MPO
1
Homosapiens
58
Q9UNQ0
ATP-binding cassette subfamily G member 2
ABCG2
1
Homosapiens
59
P09211
Glutathione S-transferase P
GSTP1
3
Homosapiens
60
Q16236
Nuclear factor erythroid 2-related factor 2
NFE2L2
1
Homosapiens
61
P15559
NAD(P)H dehydrogenase [quinone] 1
NQO1
2
Homosapiens
62
P09874
Poly [ADP-ribose] polymerase 1
PARP1
1
Homosapiens
63
P35869
Aryl hydrocarbon receptor
AHR
2
Homosapiens
64
P19875
C-X-C motif chemokine 2
CXCL2
1
Homosapiens
65
O96017
Serine/threonine-protein kinase Chk2
CHEK2
1
Homosapiens
66
Q07869
Peroxisome proliferator-activated receptor alpha
PPARA
1
Homosapiens
67
P02741
C-reactive protein
CRP
1
Homosapiens
68
P02778
C-X-C motif chemokine 10
CXCL10
1
Homosapiens
69
Q9NS23
Ras association domain-containing protein 1
RASSF1
1
Homosapiens
70
P17936
Insulin-like growth factor-binding protein 3
IGFBP3
1
Homosapiens
71
P01344
Insulin-like growth factor II
IGF2
1
Homosapiens
72
P21860
Receptor tyrosine-protein kinase erbB-3
ERBB3
1
Homosapiens
73
P09488
Glutathione S-transferase Mu 1
GSTM1
2
Homosapiens
74
P28223
5-Hydroxytryptamine 2A receptor
HTR2A
4
Homosapiens
75
P84022
Mothers against decapentaplegic homolog 3
SMAD3
1
Homosapiens
76
P08588
Beta-1 adrenergic receptor
ADRB1
3
Homosapiens
77
P29466
Caspase-1
CASP1
2
Homosapiens
78
P18509
Pituitary adenylate cyclase-activating polypeptide
ADCYAP1
1
Homosapiens
79
O95456
Proteasome assembly chaperone 1
PSMG1
1
Homosapiens
80
P05112
Interleukin-4
IL-4
1
Homosapiens
81
P00325
Alcohol dehydrogenase 1B
ADH1B
1
Homosapiens
82
P28702
Retinoic acid receptor RXR-beta
RXRB
1
Homosapiens
83
P04040
Catalase
CAT
1
Homosapiens
84
P01308
Insulin
INS
1
Homosapiens
85
P21731
Thromboxane A2 receptor
TBXA2R
1
Homosapiens
86
P07858
Cathepsin B
CTSB
1
Homosapiens
87
P40763
Signal transducer and activator of transcription 3
STAT3
1
Homosapiens
88
Q00534
Cell division protein kinase 6
CDK6
1
Homosapiens
89
P09038
Heparin-binding growth factor 2
FGF2
1
Homosapiens
90
P15336
Cyclic AMP-dependent transcription factor ATF-2
ATF2
1
Homosapiens
91
P04141
Granulocyte-macrophage colony-stimulating factor
CSF2
1
Homosapiens
92
P17677
Neuromodulin
GAP43
1
Homosapiens
93
P18031
Tyrosine-protein phosphatase nonreceptor type 1
PTPN1
1
Homosapiens
94
P30279
G1/S-specific cyclin-D2
CCND2
1
Homosapiens


### 3.3. GO-BP Analysis

To further validate whether biological processes enriched by candidate targets as mentioned above were correlated with UC, GO-BP enrichment analysis was performed via DAVID. The top 20 significant GO-BP terms are shown in Figure2. Most of them were strongly associated with inflammatory- and immune-related BPs such as “positive regulation of interleukin-6 biosynthetic process,” “regulation of inflammatory response,” “immune response,” and “positive regulation of T-cell proliferation.” In short, the 41 bioactive compounds in Quyushengxin formula may act on 94 candidate targets with inflammatory- and immune-related effects to affect UC pathogenesis.Figure 2
Gene Ontology biological process analysis. They-axis shows significantly enriched “Biological Processes” categories, and the x-axis shows the enrichment scores of these terms.


### 3.4. Establishment of T-D Network

Target-related diseases were predicted by mapping them to integrating multisource databases, including CTD, TTD, and PharmGKB. A T-D network consisting of 90 targets and 4 kinds of diseases was built (Figure3). The four diseases were digestive system disease (degree = 60), pathology (degree = 49), cancer (degree = 23), and signs and symptoms (degree = 14).Figure 3
Target-disease network. Red square represents disease and green circle represents target.


### 3.5. T-P Network Evaluation

KEGG pathway enrichment analysis was performed for the 94 targets, and T-P network was built. Results displayed that 79 targets could be further mapped to 78 pathways, including “mTOR signaling pathway,” “T-cell receptor signaling pathway,” “JAK-STAT signaling pathway,” and “FOXO signaling pathway” (Figure4). The average degree of targets was 6.85, and the average degree of pathway was 2.8. In addition, 71 candidate targets could be mapped to several pathways (≥5), suggesting that these targets might mediate the cross-talk and interactions between different pathways. Besides, those pathways (70/78) mapped by multiple targets (≥8) might be the main factors for UC development and progression. These pathways were further divided into five function modules, including inflammatory regulation, immune regulation, metabolic regulation, bacterial infection or mycosis and other function.Figure 4
Target-pathway network. Red square represents pathway and purple circle represents targets.


### 3.6. Establishment of Compound-Target-Function Module Network

By combing the networks above, a compound-target-function module network was built, which included 140 nodes (5 function modules, 41 compounds and 95 targets) and 653 edges (Figure5).Figure 5
Compound-target-function module network. Green circle represents compound, blue square represents target, and red hexagon represents function module.


### 3.7. Details of 4 UC-Related Pathways from T-P Network Analysis

To further unveil the multi-targets mechanisms of Quyushengxin formula in the treatment of UC, an integrated “UC-related pathway” was established according to the key pathways from the T-P network analysis. UC-related pathways as shown in Figure6 were composed of four pathways, including “T cell receptor signaling pathway” (hsa04660), “FOXO signaling pathway” (hsa04068), “JAK-STAT signaling pathway” (hsa04630) and “mTOR signaling pathway” (hsa04150). Those targets of the integrated “UC-related pathways” displayed the functional relationship with the UC-related proteins. UC-related pathways can be divided into three modules: immunology module, metabolism module and cell apoptosis-related module. Immunology module consisted of “T cell receptor signaling pathway” (hsa04660), and metabolism module consisted of “FOXO signaling pathway” (hsa04068). Cell apoptosis-related module was comprised of “JAK-STAT signaling pathway” (hsa04630) and “mTOR signaling pathway” (hsa04150). Taken together, Quyushengxin formula may well regulate immunology progress, metabolism progress and cell apoptosis progress to suppress UC progression.Figure 6
Distribution of targets of Quyushengxin formula in the “UC-related pathway.” Arrow shows activation effect; T-shaped arrow shows inhibition effect, and dotted arrow represents indirect activation effect or inhibition effect.


## 3.1. Screening of Potential Bioactive Compounds from Four Herbs in Quyushengxin Formula

Quyushengxin formula consists of 4 main herbs:Panax ginseng C.A. Mey. (Araliaceae), Astragalus membranaceus (Fisch) Bunge, Pulsatilla chinensis (Bge.) Regel, and Coptis chinensis Franch. After retrieving from TCMSP, 190, 87, 57, and 48 ingredients were obtained for these four herbs, respectively. Based on the criteria of OB ≥ 30%, DL ≥ 0.18, and 4 ≤ HL ≤ 8, 41 potential bioactive compounds, including quercetin, ursolic acid, kaempferol, β-sitosterol, and rutin, were sifted out (Table 1), which accounted for 10.73% of all 382 ingredients in Quyushengxin.Table 1
Details of 41 bioactive compounds and their biological parameters.
ID
Compounds
Structure
OB (%)
DL
HL
Degree
Herb
mol01
Quercetin
46.43
0.28
14.40
73
Coptis chinensis FranchAstragalus membranaceus (Fisch) Bunge
mol02
Ferulic acid
39.56
0.06
2.38
7
Coptis chinensis Franch
mol03
Palmatine
64.60
0.65
2.25
9
Coptis chinensis Franch
mol04
Jatrorrizine
19.65
0.59
4.21
9
Coptis chinensis Franch
mol05
Berberine
36.86
0.78
6.57
8
Coptis chinensis Franch
mol06
Columbamine
26.94
0.59
5.21
9
Coptis chinensis Franch
mol07
Coptisine
30.67
0.86
9.33
8
Coptis chinensis Franch
mol08
Worenine
45.83
0.87
8.41
6
Coptis chinensis Franch
mol09
Magnoflorine
0.48
0.55
6.22
8
Coptis chinensis Franch
mol10
Berberrubine
35.74
0.73
6.46
8
Coptis chinensis Franch
mol11
Epiberberine
43.09
0.78
6.10
7
Coptis chinensis Franch
mol12
(R)-Canadine
55.37
0.77
6.41
9
Coptis chinensis Franch
mol13
Berlambine
36.68
0.82
7.33
9
Coptis chinensis Franch
mol14
Corchoroside A_qt
104.95
0.78
6.68
2
Coptis chinensis Franch
mol15
Tetrandrine
26.64
0.10
4.77
9
Coptis chinensis Franch
mol16
β-Sitosterol
36.91
0.75
5.36
15
Panax ginseng C.A. Mey. (Araliaceae)Pulsatilla chinensis (Bge.) Regel
mol17
Kaempferol
41.88
0.24
14.74
26
Panax ginseng C.A. Mey. (Araliaceae)
mol18
Stigmasterol
43.83
0.76
5.57
10
Panax ginseng C.A. Mey. (Araliaceae)GPulsatilla chinensis (Bge.) Regel
mol19
β-Elemene
25.63
0.06
6.32
8
Panax ginseng C.A. Mey. (Araliaceae)
mol20
Ginsenoside Ro_qt
17.62
0.76
7.50
1
Panax ginseng C.A. Mey. (Araliaceae)
mol21
Dianthramine
40.45
0.20
5.14
3
Panax ginseng C.A. Mey. (Araliaceae)
mol22
Arachidonate
45.57
0.20
7.56
5
Panax ginseng C.A. Mey. (Araliaceae)
mol23
Ginsenoside La_qt
15.70
0.78
5.20
1
Panax ginseng C.A. Mey. (Araliaceae)
mol24
Ginsenoside rh2
36.32
0.56
11.08
9
Panax ginseng C.A. Mey. (Araliaceae)
mol25
Ginsenoside-Rh3_qt
13.09
0.76
6.22
1
Panax ginseng C.A. Mey. (Araliaceae)
mol26
Ginsenoside-Rh4_qt
31.11
0.78
6.97
1
Panax ginseng C.A. Mey. (Araliaceae)
mol27
Malkangunin
57.71
0.63
4.09
1
Panax ginseng C.A. Mey. (Araliaceae)
mol28
Alexandrin_qt
36.91
0.75
5.53
1
Panax ginseng C.A. Mey. (Araliaceae)
mol29
Ginsenoside rf
17.74
0.24
4.66
5
Panax ginseng C.A. Mey. (Araliaceae)
mol30
Hederagenin
36.91
0.75
5.35
6
Astragalus membranaceus (Fisch) Bunge
mol31
Isorhamnetin
49.60
0.31
14.34
10
Astragalus membranaceus (Fisch) BungePulsatilla chinensis (Bge.) Regel
mol32
7-O-methylisomucronulatol
74.69
0.30
2.98
11
Astragalus membranaceus (Fisch) Bunge
mol33
Rutin
3.20
0.68
6.22
15
Astragalus membranaceus (Fisch) Bunge
mol34
1,7-Dihydroxy-3,9-dimethoxy pterocarpene
39.05
0.48
7.95
5
Astragalus membranaceus (Fisch) Bunge
mol35
Isoferulic acid
50.83
0.06
2.45
7
Astragalus membranaceus (Fisch) Bunge
mol36
Betulinic acid
55.38
0.78
8.87
1
Pulsatilla chinensis (Bge.) Regel
mol37
Oleanolic acid
29.02
0.76
5.56
6
Pulsatilla chinensis (Bge.) Regel
mol38
Sitosteryl acetate
40.39
0.85
6.34
1
Pulsatilla chinensis (Bge.) Regel
mol39
Lanosterol
42.12
0.75
5.84
1
Pulsatilla chinensis (Bge.) Regel
mol40
3-beta,23-Dihydroxy-lup-20(29)-ene-28-O-alpha-L-rhamnopyranosyl-(1-4)-beta-D-glucopyranosyl(1-6)-beta-D-glucopyranoside_qt
37.59
0.79
6.70
1
Pulsatilla chinensis (Bge.) Regel
mol41
Ursolic acid
16.77
0.75
5.28
35
Pulsatilla chinensis (Bge.) Regel


## 3.2. Establishment of C-T Network

Candidate targets of the 41 bioactive compounds were predicted via WES algorithm. A total of 367 potential targets for these 41 bioactive compounds were obtained. After removing the overlapping targets, 94 candidate proteins were reserved. Then, C-T network was built by Cytoscape 3.6.0 which contains 367 connections between 41 compounds and corresponding 94 candidate targets (Figure1). The degrees of the 41 bioactive compounds in the C-T network were calculated and are displayed in Table 1. The average degree of targets per compound was 4.7, indicating multitarget functions of Quyushengxin formula. Among the 41 bioactive compounds, 8 of them showed a high degree (degree > 10). Quercetin possessed the highest degree of targets (degree = 73), followed by ursolic acid (degree = 35), kaempferol (degree = 26), β-sitosterol (degree = 15), rutin (degree = 15), 7-O-methylisomucronulatol (degree = 11), stigmasterol (degree = 10), and isorhamnetin (degree = 10).Figure 1
Compound-target network. A compound node and a target node are connected.The degree of the candidate targets was also calculated and displayed in Table2. Eight out of the 94 compounds possessed a degree larger than 10, including ESR1 (estrogen receptor 1, degree = 34), PTGS2 (prostaglandin-endoperoxide synthase 2, degree = 27), NOS2 (nitric oxide synthase 2, degree = 25), PTGS1 (degree = 23), PPARG (peroxisome proliferator-activated receptor gamma, degree = 21), NOS3 (degree = 21), ESR2 (degree = 17), and KCNH2 (Potassium Voltage-Gated Channel Subfamily H Member 2, degree = 13).Table 2
Details of 94 UC-related targets of herbs via UniProt.
ID
UniProt
Protein names
Gene names
Degree
Organism
1
P35228
Nitric oxide synthase, inducible
NOS2
25
Homosapiens
2
P23219
Prostaglandin G/H synthase 1
PTGS1
23
Homosapiens
3
P03372
Estrogen receptor
ESR1
34
Homosapiens
4
P37231
Peroxisome proliferator-activated receptor gamma
PPARG
21
Homosapiens
5
P35354
Prostaglandin G/H synthase 2
PTGS2
27
Homosapiens
6
Q92731
Estrogen receptor beta
ESR2
17
Homosapiens
7
P11388
DNA topoisomerase 2-alpha
TOP2A
5
Homosapiens
8
P16389
Potassium voltage-gated channel subfamily H member 2
KCNH2
13
Homosapiens
9
P08709
Coagulation factor VII
F7
6
Homosapiens
10
P29474
Nitric-oxide synthase, endothelial
NOS3
21
Homosapiens
11
P27338
Amine oxidase [flavin-containing] B
MAOB
5
Homosapiens
12
Q04206
Transcription factor p65
RELA
6
Homosapiens
13
P00533
Epidermal growth factor receptor
EGFR
1
Homosapiens
14
P31749
RAC-alpha serine/threonine-protein kinase
AKT1
2
Homosapiens
15
P15692
Vascular endothelial growth factor A
VEGFA
2
Homosapiens
16
P24385
G1/S-specific cyclin-D1
CCND1
3
Homosapiens
17
P10415
Apoptosis regulator Bcl-2
BCL2
5
Homosapiens
18
P01100
Proto-oncogene c-Fos
FOS
3
Homosapiens
19
P38936
Cyclin-dependent kinase inhibitor 1
CDKN1A
4
Homosapiens
20
P55211
Caspase-9
CASP9
4
Homosapiens
21
P00749
Urokinase-type plasminogen activator
PLAU
4
Homosapiens
22
P08253
72 kDa type IV collagenase
MMP2
2
Homosapiens
23
P14780
Matrix metalloproteinase-9
MMP9
2
Homosapiens
24
P22301
Interleukin-10
IL10
1
Homosapiens
25
P01133
Proepidermal growth factor
EGF
1
Homosapiens
26
P06400
Retinoblastoma-associated protein
RB1
2
Homosapiens
27
P01375
Tumor necrosis factor
TNF
6
Homosapiens
28
P05412
Transcription factor AP-1
JUN
4
Homosapiens
29
P05231
Interleukin-6
IL-6
3
Homosapiens
30
P42574
Caspase-3
CASP3
7
Homosapiens
31
P04637
Cellular tumor antigen p53
TP53
4
Homosapiens
32
P11926
Ornithine decarboxylase
ODC1
1
Homosapiens
33
Q14790
Caspase-8
CASP8
3
Homosapiens
34
P00441
Superoxide dismutase [Cu-Zn]
SOD1
2
Homosapiens
35
P17252
Protein kinase C alpha type
PRKCA
2
Homosapiens
36
P03956
Interstitial collagenase
MMP1
3
Homosapiens
37
P42224
Signal transducer and activator of transcription 1-alpha/beta
STAT1
2
Homosapiens
38
P04626
Receptor tyrosine-protein kinase erbB-2
ERBB2
1
Homosapiens
39
P09601
Heme oxygenase 1
HMOX1
3
Homosapiens
40
P05177
Cytochrome P450 1A2
CYP1A2
2
Homosapiens
41
P01106
Myc proto-oncogene protein
MYC
1
Homosapiens
42
P05362
Intercellular adhesion molecule 1
ICAM1
4
Homosapiens
43
P01584
Interleukin-1 beta
IL1B
5
Homosapiens
44
P13500
C-C motif chemokine 2
CCL2
1
Homosapiens
45
P19320
Vascular cell adhesion protein 1
VCAM1
2
Homosapiens
46
P10145
Interleukin-8
CXCL8
2
Homosapiens
47
P05771
Protein kinase C beta type
PRKCB
2
Homosapiens
48
O15392
Baculoviral IAP repeat-containing protein 5
BIRC5
2
Homosapiens
49
P04792
Heat shock protein beta-1
HSPB1
1
Homosapiens
50
P01137
Transforming growth factor beta-1
TGFB1
3
Homosapiens
51
P60568
Interleukin-2
IL2
2
Homosapiens
52
Q16678
Cytochrome P450 1B1
CYP1B1
2
Homosapiens
53
P00750
Tissue-type plasminogen activator
PLAT
1
Homosapiens
54
P01579
Interferon gamma
IFNG
4
Homosapiens
55
P09917
Arachidonate 5-lipoxygenase
ALOX5
3
Homosapiens
56
P60484
Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
PTEN
1
Homosapiens
57
P05164
Myeloperoxidase
MPO
1
Homosapiens
58
Q9UNQ0
ATP-binding cassette subfamily G member 2
ABCG2
1
Homosapiens
59
P09211
Glutathione S-transferase P
GSTP1
3
Homosapiens
60
Q16236
Nuclear factor erythroid 2-related factor 2
NFE2L2
1
Homosapiens
61
P15559
NAD(P)H dehydrogenase [quinone] 1
NQO1
2
Homosapiens
62
P09874
Poly [ADP-ribose] polymerase 1
PARP1
1
Homosapiens
63
P35869
Aryl hydrocarbon receptor
AHR
2
Homosapiens
64
P19875
C-X-C motif chemokine 2
CXCL2
1
Homosapiens
65
O96017
Serine/threonine-protein kinase Chk2
CHEK2
1
Homosapiens
66
Q07869
Peroxisome proliferator-activated receptor alpha
PPARA
1
Homosapiens
67
P02741
C-reactive protein
CRP
1
Homosapiens
68
P02778
C-X-C motif chemokine 10
CXCL10
1
Homosapiens
69
Q9NS23
Ras association domain-containing protein 1
RASSF1
1
Homosapiens
70
P17936
Insulin-like growth factor-binding protein 3
IGFBP3
1
Homosapiens
71
P01344
Insulin-like growth factor II
IGF2
1
Homosapiens
72
P21860
Receptor tyrosine-protein kinase erbB-3
ERBB3
1
Homosapiens
73
P09488
Glutathione S-transferase Mu 1
GSTM1
2
Homosapiens
74
P28223
5-Hydroxytryptamine 2A receptor
HTR2A
4
Homosapiens
75
P84022
Mothers against decapentaplegic homolog 3
SMAD3
1
Homosapiens
76
P08588
Beta-1 adrenergic receptor
ADRB1
3
Homosapiens
77
P29466
Caspase-1
CASP1
2
Homosapiens
78
P18509
Pituitary adenylate cyclase-activating polypeptide
ADCYAP1
1
Homosapiens
79
O95456
Proteasome assembly chaperone 1
PSMG1
1
Homosapiens
80
P05112
Interleukin-4
IL-4
1
Homosapiens
81
P00325
Alcohol dehydrogenase 1B
ADH1B
1
Homosapiens
82
P28702
Retinoic acid receptor RXR-beta
RXRB
1
Homosapiens
83
P04040
Catalase
CAT
1
Homosapiens
84
P01308
Insulin
INS
1
Homosapiens
85
P21731
Thromboxane A2 receptor
TBXA2R
1
Homosapiens
86
P07858
Cathepsin B
CTSB
1
Homosapiens
87
P40763
Signal transducer and activator of transcription 3
STAT3
1
Homosapiens
88
Q00534
Cell division protein kinase 6
CDK6
1
Homosapiens
89
P09038
Heparin-binding growth factor 2
FGF2
1
Homosapiens
90
P15336
Cyclic AMP-dependent transcription factor ATF-2
ATF2
1
Homosapiens
91
P04141
Granulocyte-macrophage colony-stimulating factor
CSF2
1
Homosapiens
92
P17677
Neuromodulin
GAP43
1
Homosapiens
93
P18031
Tyrosine-protein phosphatase nonreceptor type 1
PTPN1
1
Homosapiens
94
P30279
G1/S-specific cyclin-D2
CCND2
1
Homosapiens


## 3.3. GO-BP Analysis

To further validate whether biological processes enriched by candidate targets as mentioned above were correlated with UC, GO-BP enrichment analysis was performed via DAVID. The top 20 significant GO-BP terms are shown in Figure2. Most of them were strongly associated with inflammatory- and immune-related BPs such as “positive regulation of interleukin-6 biosynthetic process,” “regulation of inflammatory response,” “immune response,” and “positive regulation of T-cell proliferation.” In short, the 41 bioactive compounds in Quyushengxin formula may act on 94 candidate targets with inflammatory- and immune-related effects to affect UC pathogenesis.Figure 2
Gene Ontology biological process analysis. They-axis shows significantly enriched “Biological Processes” categories, and the x-axis shows the enrichment scores of these terms.


## 3.4. Establishment of T-D Network

Target-related diseases were predicted by mapping them to integrating multisource databases, including CTD, TTD, and PharmGKB. A T-D network consisting of 90 targets and 4 kinds of diseases was built (Figure3). The four diseases were digestive system disease (degree = 60), pathology (degree = 49), cancer (degree = 23), and signs and symptoms (degree = 14).Figure 3
Target-disease network. Red square represents disease and green circle represents target.


## 3.5. T-P Network Evaluation

KEGG pathway enrichment analysis was performed for the 94 targets, and T-P network was built. Results displayed that 79 targets could be further mapped to 78 pathways, including “mTOR signaling pathway,” “T-cell receptor signaling pathway,” “JAK-STAT signaling pathway,” and “FOXO signaling pathway” (Figure4). The average degree of targets was 6.85, and the average degree of pathway was 2.8. In addition, 71 candidate targets could be mapped to several pathways (≥5), suggesting that these targets might mediate the cross-talk and interactions between different pathways. Besides, those pathways (70/78) mapped by multiple targets (≥8) might be the main factors for UC development and progression. These pathways were further divided into five function modules, including inflammatory regulation, immune regulation, metabolic regulation, bacterial infection or mycosis and other function.Figure 4
Target-pathway network. Red square represents pathway and purple circle represents targets.


## 3.6. Establishment of Compound-Target-Function Module Network

By combing the networks above, a compound-target-function module network was built, which included 140 nodes (5 function modules, 41 compounds and 95 targets) and 653 edges (Figure5).Figure 5
Compound-target-function module network. Green circle represents compound, blue square represents target, and red hexagon represents function module.


## 3.7. Details of 4 UC-Related Pathways from T-P Network Analysis

To further unveil the multi-targets mechanisms of Quyushengxin formula in the treatment of UC, an integrated “UC-related pathway” was established according to the key pathways from the T-P network analysis. UC-related pathways as shown in Figure6 were composed of four pathways, including “T cell receptor signaling pathway” (hsa04660), “FOXO signaling pathway” (hsa04068), “JAK-STAT signaling pathway” (hsa04630) and “mTOR signaling pathway” (hsa04150). Those targets of the integrated “UC-related pathways” displayed the functional relationship with the UC-related proteins. UC-related pathways can be divided into three modules: immunology module, metabolism module and cell apoptosis-related module. Immunology module consisted of “T cell receptor signaling pathway” (hsa04660), and metabolism module consisted of “FOXO signaling pathway” (hsa04068). Cell apoptosis-related module was comprised of “JAK-STAT signaling pathway” (hsa04630) and “mTOR signaling pathway” (hsa04150). Taken together, Quyushengxin formula may well regulate immunology progress, metabolism progress and cell apoptosis progress to suppress UC progression.Figure 6
Distribution of targets of Quyushengxin formula in the “UC-related pathway.” Arrow shows activation effect; T-shaped arrow shows inhibition effect, and dotted arrow represents indirect activation effect or inhibition effect.


## 4. Discussion

TCM has the advantages of high treatment efficacy and low treatment cost and side effect in the treatment of several diseases, including UC in China for several thousands of years [32–34]. After preliminary screening based on ADME properties, 41 potential bioactive compounds of Quyushengxin were screened out. Thereafter, 94 candidate targets of these 41 bioactive compounds were predicted for further analysis. Functional enrichment analyses suggested that these targets were closely related with inflammatory- and immune-related biological processes. Besides, a C-T network, a T-D network, a T-P network, and a compound-target-function module network were built. These networks indicated that the therapeutic effects of Quyushengxin on UC may be achieved through the synergistic and additive effects on multiple molecules and multiple pathways with immune and inflammatory effects to treat UC.Previous reports showed that the TCMSP-based method was reliable for screening out bioactive compounds of TCM for treatment of thrombosis [35], gastric precancerous lesions [36], cardiocerebrovascular disease [37], and rheumatoid arthritis [38]. In this study, 41 bioactive compounds of Quyushengxin formula were selected out by using TCMSP database in combination with ADME properties. Most of the 41 compounds have been reported to have anti-inflammatory and immune-regulatory effects. For example, quercetin (mol01, OB = 46.43%, DL = 0.28, HL = 14.40) could inhibit lipopolysaccharide- (LPS-) induced interleukin- (IL-) 6 production [39], TNF-α production, and IL-8 production [40, 41] to exert anti-inflammatory effect. Besides, ursolic acid (mol17, OB = 16.77%, DL = 0.75, HL = 5.28) was reported to have human neutrophil elastase inhibitory effect both in vitro and in vivo [42]. Kaempferol (mol17, OB = 41.88%, DL = 0.24, HL = 14.74) was reported to significantly reduce the overproduction of TNF-α, IL-1β, IL-6, intercellular adhesion molecule- (ICAM-) 1, and vascular cell adhesion molecule- (VCAM-) 1 induced by LPS [43]. In addition, β-sitosterol (mol16, OB = 36.91%, DL = 0.75, HL = 5.36) and rutin (mol33, OB = 3.2%, DL = 0.68, HL = 6.22) were shared with significant anti-inflammatory activity [44, 45]. Above all, TCMSP-based systems pharmacology sifted out 41 potential bioactive compounds in Quyushengxin formula for treatment of UC.Eight of the 94 targets have degree larger than 10 in the C-T network, including ESR1, PTGS2, NOS2, PTGS1, PPARG, NOS3, ESR2, and KCNH2. ESR1 was targeted by 34 compounds, which contributed to T-cell-mediated autoimmune inflammation by promoting T-cell activation and proliferation [46]. Besides, PTGS2 with the second highest degree played a critical role in the pathogenesis of gut inflammation [47, 48]. Moreover, PPARG was demonstrated to be able to downregulate proinflammatory cytokines production, such as IL-4, -5, and -6. In addition, PPARG could also enable to interfere with profibrotic molecules, such as platelet-derived growth factor (PDGF), IL-1, and transforming growth factor beta (TGF-β) [49]. These results suggested that Quyushengxin formula could probably treat UC by regulating anti-inflammatory action and the immune system.In this study, 94 targets were utilized to perform T-P network analysis, and the results showed that 79 targets could be further mapped to 78 pathways. Meanwhile, numerous pathways mapped by multiple targets might be the main factors for UC progression. Four pathways including “T-cell receptor signaling pathway,” “FOXO signaling pathway,” “JAK-STAT signaling pathway,” and “mTOR signaling pathway” were closely associated with immune and inflammatory effects. T-cell receptors play significant role in function of T cells and formation of the immunological synapse, and they connected T cells and the antigen-presenting cells [50]. T-cell receptor pathway was reported to be important in regulation of UC [51, 52]. FOXO pathway plays a key role in regulating the expression of genes related to cell function such as apoptosis, cell cycle, oxidative stress, and differentiation [53–55]. FOXO3a was shown to control the inflammatory response and help maintain the homeostasis of the intestinal mucosa, which may also be a protective factor in the gut, and maintain a balance between the mucosal immune hemostasis against intravascular bacteria and inflammatory cytokines [56]. Besides, JAK-STAT pathway is the fulcrum for many important cellular processes, including cell survival, differentiation, proliferation, and regulation of immune function [57]. The mTOR pathway plays an important role in regulation of cell metabolism, proliferation, and autophagy. It is reported that mTOR signaling pathway was activated in bacteria-induced colitis in mice [58]. Inhibitors of mTOR signaling pathway are effective as anti-inflammatory drugs in treating colitis [59–61]. Therefore, Quyushengxin might suppress UC progression through targeting these anti-inflammation, autophagy, and immunoregulation pathways.Nevertheless, limitations in this study could never be neglected. First, results in this study were mainly based on known chemical components in Quyushengxin, related targets, and pathways in UC. With the development of science and technology, new components in Quyushengxin, as well as new targets and pathways in UC will be further discovered, which will supply us with more theoretical evidences for further elucidation of underlying mechanisms of UC pathology. Second, the interaction relationships of the nodes in the networks, such as the action type, e.g., activation, inhibition, binding, and catalysis, and the action effect, e.g., positive, negative, and unspecified, are not investigated due to lack of these data. Third, due to the complex interaction between TCM and the human body, many of its acting mechanisms still needed to be further elucidated via pharmacokinetic test and other experiments.


## 5. Conclusion

In short, network pharmacology analysis of Quyushengxin showed that 41 bioactive components of Quyushengxin may act on 94 immune and inflammation-related targets to suppress UC progression in a synergistic and additive manner, which may provide us with a new starting point for a more detailed knowledge of mechanisms of UC pathogenesis.



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*Source: 7870424-2019-12-28.xml*