Study on the mechanism of Wikstroemia indica in the treatment of inflammatory diseases based on network pharmacology

Jiayi Li,Zisong Bai,Tingting Guo,Heng Zhang,Gang Chen,Di Zhou,Ning Li

School of Traditional Chinese Materia Medica;Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug,Shenyang Pharmaceutical University,Shenyang 110016,China

Abstract Wiksroemia indica belongs to the family of Thymelaeaceae.As a material of Traditional Chinese Medicine,it is mainly used to treat inflammation-related diseases.However,due to its complex composition,the scientific basis for its traditional application is still insufficient and the mechanism of action is still unclear.At present,most studies on its anti-inflammatory effect focus on a certain component and its different solvent fractions.There are few reports about the synergistic mechanism of the constituents from W.indica for the treatment of inflammatory diseases.Therefore,the present study aimed to uncover its potential mechanism from a holistic perspective using the network pharmacology approach.Firstly,compounds in W.indica and genes associated with inflammatory diseases were obtained.Then,the active compounds were screened out and their targets were predicted.After that,the key targets for W.indica to treat inflammatory diseases were screened out.Finally,the protein-protein interaction (PPI),Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of key targets were performed to further study the mechanism of W.indica in the treatment of inflammatory diseases.The results showed that the active constituents of W.indica might play an anti-inflammatory role through the key targets CYP19A1 and CYP1B1,which clarified the primary mechanism for the treatment of inflammatory diseases and provided a scientific basis for further study of this herb.

Key words: Wikstroemia indica;inflammation;network pharmacology;mechanism

1 Introduction

Inflammation,a defense response of living tissue with the vascular system to damage factors,is beneficial to the body.In general,inflammatory response plays an important role in the repair of tissue damage [1].But if the body is in a chronic inflammatory state for a long time,it can lead to a series of serious diseases such as cancer,cardiovascular diseases,diabetes,neurodegenerative diseases and so on.Inflammation aggravates the development of these diseases.Therefore,timely treatment of inflammation can reduce the incidence and death rate of these diseases,thereby reducing the huge personal and social healthcare burden.

Currently,adrenal cortical hormones and non-steroid anti-inflammatory-immunity drugs(NSAIDs) are commonly used to treat inflammation in clinic but both of them have severe side effects such as inhibition of pituitary-adrenal function,gastrointestinal adverse reaction,and kidney damage.Long-term clinical and experimental studies have confirmed that Traditional Chinese Medicines(TCM) have good anti-inflammatory effects and less toxic and side effects which may be attributed to the characteristics of low-dose and multiple targets.Network pharmacology studies have found that 11 active compounds in

Astragali Complanati Semen

exerted anti-inflammatory effects by regulating 50 potential targets [2].Therefore,finding antiinflammatory constituents from TCM has become the research trend of new drug development.

Wikstroemia indica

(L.) C.A.Mey is a member of Thymelaeaceae family,mainly distributed in the southeast of China [3].As a traditional Chinese herb,

W.indica

has long been used for the treatment of inflammatory diseases such as bronchitis,pneumonia,and rheumatoid arthritis [4-6].Special attention has been paid to the antiinflammatory effect of

W.indica

[7-11].A wealth of bioactive components such as coumarins,flavonoids,terpenoids,lignans,volatile oils have been isolated from

W.indica

[12].However,the essential constituents of

W.indica

in the treatment of inflammatory diseases have not been determined and the underlying anti-inflammatory mechanism is still unclear.The present study aimed to explore the potential mechanism of

W.indica

in the treatment of inflammatory diseases via network pharmacology approach,a novel research method for the discovery of TCM at the systemic and molecular level [13].The workflow for the network pharmacology study of

W.indica

is shown in Fig.1.

Fig.1 Workflow for the network pharmacology study of W.indica

2 Materials and methods

2.1 Chemical compositions of W.indica

SciFinder database (https://scifinder.cas.org/)was used to search ingredients of

W.indica

.Then structures of the obtained compounds were drawn in ChemDraw 18.0 software and saved to files in simplified molecular input line entry specification(SMILES) format for further analysis.

2.2 Screening of active compounds

SwissADME (http://www.swissadme.ch/) [14]was applied to assess the drug-likeness and gastrointestinal absorption of each compound isolated from

W.indica

so that compounds that were chemically unsuitable for drugs were removed.The candidate compounds satisfied the criteria that at least two of the five drug-likeness rules (Lipinski,Ghose,Veber,Egan,Mugge) were“yes”and the gastrointestinal absorption was“high”.

2.3 Identification of targets associated with inflammatory diseases

GeneCards database (http://www.genecards.org/) [15]and OMIM (Online Mendelian Inheritance in Man) database (http://www.omim.org/) [16]were used to identify the target proteins associated with inflammatory diseases.The targets were searched by using the keywords“hepatitis”,“pneumonia”,“rheumatoid arthritis”,“bronchitis”,“tonsillitis”,“nephritis”,“cellulitis”,“lymphadenitis”,“mumps”and“mastitis”,and the redundancies were discarded.

2.4 Prediction of targets for candidate compounds

The SMILES of active compounds were imported into Swiss Target Prediction web (http://www.swisstargetprediction.ch/) [17].Using this web server,potential targets with official gene symbols of these compounds were identified.Proteins with a probability score ≥ 0.15 were selected as potential targets.

2.5 Construction of protein-protein interaction network

Protein-protein interaction (PPI) network was constructed using STRING 11.0 (http://stringdb.org/) [18],with the species set to“Homo sapiens”and minimum required interaction score set to medium confidence (0.4000).Cytoscape 3.7.2(http://www.cytoscape.org/) was utilized for network topological analysis [19].

2.6 Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)pathways enrichment analyses

GO terms and KEGG pathways enrichment analyses of potential target proteins were performed using DAVID 6.7 (https://david.ncifcrf.gov/),a database for annotation,visualization and integrated discovery [20].

2.7 Construction of“compound-target”and“pathway-target-disease”networks

To facilitate scientific interpretation of complex relationships involved in

W.indica

,“compoundtarget”and“target-pathway-disease”networks were constructed using Cytoscape 3.7.2.In these networks,nodes represent compounds,targets,diseases and pathways,while edges represent the interactions between nodes.Network topological analysis was conducted using the“Network Analyzer”tool of Cytoscape 3.7.2.

3 Results and discussion

3.1 The active compounds of W.indica

About 173 compounds of

W.indica

were retrieved from

literature.Among them,87 compounds satisfied the criteria that at least two of the five drug-likeness rules were“yes”and the gastrointestinal absorption was“high”.

3.2 The targets related to inflammatory diseases

15513 targets related to 10 kinds of inflammatory diseases were obtained from GeneCards database and OMIM database after removing duplicates.

3.3 The targets for active compounds

162 targets for the 87 active compounds were obtained from Swiss Target Prediction web after removing duplicates.In predicting the targets for the compounds,only 39 compounds met the screening criteria with probability score ≥ 0.15 (Table 1).The structures of 39 active compounds are shown in Fig.2.As shown in Fig.2,flavonoids,aliphatic compounds,lignans and aromatic compounds were major structural types in

W.indica

for the treatment of inflammatory diseases.

Table 1 Information of 39 active compounds in W.indica

Continued table 1

Fig.2 Structures of 39 active compounds of W.indica

3.4 The potential targets related to inflammatory diseases for W.indica

In order to obtain the potential targets related to inflammatory diseases for

W.indica

,the targets for 39 active compounds and the targets of inflammatory diseases were imported into Venny 2.1.0 (http://bioinfogp.cnb.csic.es/tools/venny/) at the same time.152 common targets were selected as the targets related to inflammatory diseases for

W.indica

(Fig.3).

Fig.3 Venn diagram of targets for W.indica to treat inflammatory diseases

3.5 Compound-target network analysis

This network includes 191 nodes (39 compounds,152 targets) and 570 edges (Fig.4).The hexagons,circles and the edges represent the targets,active compounds and interactions between compounds and targets,respectively.Targets in the outer circles show more interactions with the compounds than those in the inner circles.The complexity of the network indicated that

W.indica

exerted its anti-inflammatory effect by regulating multiple targets with multiple compounds.The 39 active compounds are listed in Table 2 according to their degree,betweenness and closeness in the network.As shown in Table 2,kaempferol was the constituent that displayed the most target interactions (degree=95),followed by quercetin(degree=94),luteolin (degree=91),apigenin(degree=60) and thevetiaflavone (degree=32).It indicated that flavonoids might play a more important role in the anti-inflammatory effect of

W.indica

.Meanwhile,aliphatic compounds,lignans,coumarins and other compounds also exerted antiinflammatory effects.The top ten targets in the network are CA7,CA2,CA1,CYP19A1,CA9,ESR2,CA4,CSNK2A1,ESR1 and CYP1B1 with the corresponding degree of 13,12,12,11,11,10,10,8,8,and 8.Of the 152 targets,26 had greater degrees than the average degree of neighbors (5.969)in the network (Table 3),indicating they might be the potential target proteins for

W.indica

to treat inflammatory diseases.

Fig.4 Compound-target network of W.indica.This network consists of 39 compounds and 152 targets.The hexagons represent the targets,and the circles represent the active compounds.The edges represent the interaction between them and the node size is proportional to the degree

Table 2 Topological information of 39 compounds in the compound-target network

Continued table 2

Table 3 Topological information of 26 targets in the compound-target network

Continued table 3

3.6 PPI network analysis

In order to further understand the action mechanism of

W.indica

,a protein-protein interaction(PPI) network of 26 potential targets was constructed in the STRING 11.0 database.The network contains 26 nodes and 35 edges.The average values of betweenness,closeness,and degree are 0.093484,0.3772 and 3.333,respectively.As shown in Fig.5,except for CA1,CA2,CA3,CA7 and CA5A,the remaining 21 proteins had different degrees of interaction at the medium confidence level (0.400).The information of nodes is presented in Table 4.Since CA1,CA2 and CA3,three of the top ten targets in the compound-target network,don’t have interactions and PPI means the interaction between these targets,we would give priority to the targets with interaction in the PPI network.

Fig.5 PPI network of the key targets for W.indica

Table 4 Information of nodes in the PPI network

3.7 Enrichment analysis

In order to further understand the molecular mechanism of

W.indica

in the treatment of inflammatory diseases,GO enrichment analysis and KEGG pathway enrichment analysis of key targets were performed.

3.7.1 GO enrichment analysis

A total of 101 GO terms were obtained(

P

＜0.05),of which 50 were related to biological processes,22 to cell composition and 29 to molecular function.The top ten GO entries of biological processes,cell composition and molecular function are shown in Fig.6.

In the top ten entries of biological processes,one-carbon metabolic process,oxidation reduction,response to steroid hormone stimulus,protein amino acid autophosphorylation,response to drug,response to organic substance,negative regulation of apoptosis,negative regulation of programmed cell death is related to inflammation.Cell composition includes neuron projection,plasma membrane,basolateral plasma membrane,cell projection,plasma membrane part and so on.In the top ten entries of molecular function,carbonate dehydratase activity,transition metal ion binding,electron carrier activity,iron ion binding,oxygen binding,estrogen receptor activity and xenobiotic-transporting adenosine triphosphatase (ATPase) activity are related to inflammation.

3.7.2 KEGG pathway enrichment analysis

The results of KEGG pathway enrichment analysis revealed 3 pathways (

P

＜0.05).The three pathways are presented in Fig.6.The nitrogen metabolism pathway (hsa00910) is related to energy metabolism and is the most significant pathway.The ABC transporter pathway (hsa02010) is associated with membrane transport.The steroid hormone biosynthesis pathway (hsa00140) is involved in lipid metabolism.To better understand the major relationship between pathways,targets and inflammatory diseases,the pathway-target-disease network with 25 nodes (3 pathways,13 targets and 9 inflammatory diseases) and 85 edges was constructed (Fig.7).It indicated that

W.indica

mainly acted on these 13 targets through the three pathways to achieve a good therapeutic effect in the treatment of inflammatory diseases.The degree value of these diseases in the network showed that hepatitis,pneumonia,rheumatoid arthritis and tonsillitis might be the four major inflammatory diseases treated with

W.indica

.

Fig.6 GO enrichment analysis and KEGG pathway enrichment analysis of the key targets.Gene ratio was calculated from the associated genes/total genes.The size of the bubbles represents the gene counts of this entry,and the colors from warm to cold represent the -log10(P-value) from small to large.Each bubble chart is sorted by -log10(P-value)

Fig.7 Pathway-target-disease network for W.indica.The hexagons represent the targets,the circles indicate the inflammatory diseases and the triangles represent the pathways.The edges represent the interaction between them and the node size is proportional to the degree

4 Conclusion

Multiple components and multiple targets are the intrinsic properties of TCMs.Network pharmacology can not only explore the relationship between drugs and diseases,but also find drug targets,guiding the discovery and development of innovative drugs from a holistic perspective.In terms of methodology,network pharmacology has the characteristics of integrity and systematicness,which is in line with the properties of TCMs [21].In the present study,we applied the network pharmacology method to clarify the material basis and the action mechanism of

W.indica

in the treatment of inflammatory diseases.According to the literature research and database retrieval,we found that 39 active compounds in

W.indica

interacted with 152 targets related to inflammatory diseases.Compound-target network of

W.indica

indicated that flavonoids might play a more important role in anti-inflammatory effect of

W.indica

.Kaempferol,quercetin,luteolin and apigenin might be the main active ingredients in the treatment of inflammatory diseases

of

W.indica

,which is consistent with the reports that luteolin,kaempferol,apigenin,quercetin have strong antiinflammatory effects

in vitro

[22-24].Targets CYP19A1 and CYP1B1 in these three pathways,with higher degrees both in compoundtarget network and PPI network,might play important roles in the anti-inflammatory effect of

W.indica

.CYP19A1 has been declared to encode aromatase and inflammatory and neuro-angiogenesis factors associated with pain and progression of endometriosis [25].CYP1B1 is a cytochrome P450 monooxygenase involved in the metabolism of various endogenous substrates,including fatty acids,steroid hormones and vitamins.It is reported that both CYP1B1 deficiency and high-fat-diet (HFD)induced macrophage infiltration,but CYP1B1 deficiency could inhibit the proinflammatory gene expression induced by HFD in epididymal fat pad of adult mice [26].Therefore,compared with classical clinical drugs,

W.indica

does have certain advantages in the treatment of inflammatory diseases.NSAID drugs exert an anti-inflammatory effect by inhibiting cyclooxygenase (COX),but non selective NSAID inhibits COX-1 and COX-2 at the same time,which easily leads to gastrointestinal injury,inhibition of platelet function and other side effects.Many PLA2 inhibitors have been reported to have anti-inflammatory activity

in vitro

,but most of them only have local anti-inflammatory activities due to their hydrophobicity.Since

W.indica

might exert anti-inflammatory activity through the key targets CYP1B1 and CYP19A1,these side effects or limitations might be avoided.The result of GO enrichment analysis showed that the potential targets for active components in

W.indica

are mostly distributed in the neuron projection and plasma membrane.

W.indica

also plays an important role in the treatment of inflammatory diseases through biological processes such as one-carbon metabolic process,oxidation reduction,and molecular functions such as carbonate dehydratase activity,transition metal ion binding.KEGG pathway analysis showed that the nitrogen metabolism pathway is the most significant pathway,but there is still no clear report that the nitrogen metabolism pathway is related to inflammation.Therefore,

W.indica

might regulate the inflammatory response by altering the ABC transporter pathway and the steroid hormone biosynthesis pathway,which are related to inflammation.ABCA1 and ABCG1 are two ABC transporters.The studies have shown that cholesterol accumulation in the plasma membrane of Abcg1Abca1and Abcg1macrophages leads to an increased inflammatory response after exposure to lipopolysaccharide [27].Steroid hormones can regulate the expression of pro-inflammatory and antiinflammatory cytokines

in vivo

[28].Since the seven targets for

W.indica

on the nitrogen metabolism pathway are all related to inflammation,we can explore the relationship between this pathway and inflammation in the future.In summary,we predicted the active compounds and potential targets of

W.indica

related to inflammatory diseases using network pharmacology method for the first time,providing a new insight into its action mechanism in the treatment of inflammatory diseases and strong support for further study of this herb.