Xianwei Jiang,kangmei Qiu,Zhanping Ma

1 Shaanxi University of Chinese Medicine,Shaanxi 712046,China.

2 Shaanxi traditional Chinese medicine Hospital,Shaanxi 710003,China.

Abstract Objective: To explore the mechanism of Lianqiao-Heye (Fructus Forsythiae and Folium Nelumbinis, FF-FN)drug pair in treating acute pharyngitis (AP) by network pharmacology.Methods: The chemical components and potential therapeutic targets of FF-FN drug pair were obtained from TCMSP firstly,and databases GeneCards,DrugBank,OMIM and PharmGKB were utilized to get the genes related to AP and the intersection of the results was obtained.Then Cytoscape software was used to construct drug-component-target network diagram to screen out the key compounds.Protein interaction network (PPI)was established using String database,and the core targets were screened by CytoNCA topology analysis.R language software was used for GO biological function analysis and KEGG pathway analysis.Finally,the docking verification of key compounds and core target molecules was carried out by AutoDock software.Results: A total of 38 active compounds and 917 potential therapeutic targets were obtained from FF-FN drug pair,while a total of 1534 targets were screened out for AP,among which,the number of intersection targets was 117.7 core targets were screened out from PPI core network,with JUN,TP53,CXCL8 and RELA included.A total of 2487 biological processes were involved in GO enrichment analysis,and 157 related pathways were screened out by KEGG.Based on results of molecular docking verification,the key compounds such as quercetin,luteolin and wogonin in FF-FN drug pair were proved capable of binding to the core targets and a good affinity was shown.Conclusion: FF-FN pair can intervene AP through multiple targets and multiple pathways,including PI3KAkts signaling pathway,AGE-RAGE signaling pathway and IL-17 signaling pathway.The combination of quercetin,luteolin,wogonin,kaempferol and β-sitosterol with JUN,RELA,MAPK1,TNF and MYC can possibly be one of the mechanisms regarding to the therapeutic effect.

Keywords: Network pharmacology,Molecular docking,Fructus Forsythiae, Folium Nelumbinis,Acute pharyngitis

Background

Acute Pharyngitis (AP) is a common clinical disease,which refers to the acute inflammation of the pharyngeal mucosa and submucosa.Clinically,it is usually manifested as pharyngeal pain,burning sensation,or even systemic inflammatory reactions such as fever [1].Its pathogenesis is mainly the inflammatory reaction caused by virus,bacteria,or physical and chemical factors to stimulate the throat [2].Antibiotics,antivirus,or hormones are widely used in clinical treatment.However,the use of antibiotics unspecific may cause adverse effects like abdominal distension,even drug resistance due to long-term use,which is a troubling clinical problem with no doubt [3].In recent years,studies have found that Chinese herbal compounds have the characteristics of definite efficacy and simple non-corrupt in the treatment of this disease[4],therefore,Chinese herbal medicine has become a new trend in the intervention of acute pharyngitis.According to traditional Chinese medicine theory,acute pharyngitis belongs to the category of "acute laryngitis" and "wind-heat laryngitis".It is mostly caused by wind-heat evil gas attacking the lungs and stomach; therefore,the principle of clearing wind,clearing heat,and relieving pain for pharynx become the basis of treatment for acute pharyngitis [5,6].

The combination of Lianqiao-Heye (Fructus ForsythiaeandFolium Nelumbinis,FF-FN) was designed based on clinical practice by the author's tutor Ma Zhanping,a famous traditional Chinese medicine doctor in Shaanxi Province,China.Owing to his deep insight into the concepts of "Epidemic Febrile Diseases" and "dissipate excessive stagnation of fire",which was used to treat the syndrome of exogenous wind and heat of upper coke in clinical practice,he substitutedFolium Nelumbinisfor mint to promote the Qingyang.It turned out to have a very good effect.FFFNare commonly used clinical drugs,among whichFolium Nelumbinishas the effect of clearing heat and dampness and promoting the Qingyang.Fructus Forsythiaehas the effect of evacuating wind and heat,clearing heat and detoxification,and is mostly used to clear away heat of upper coke [7].In modern pharmacological experiments,it has been found to have good antibacterial,antiviral,and anti-inflammatory effects [8-10].For example,Yan Qingzi et al.[11]showed thatFolium Nelumbiniscan achieve antiinflammatory effect by reducing the levels of TNF-α,IL-6 and NO in the LPS-induced mouse macrophage RAW26 4.7 cell model.Duan Linjian et al.[12] showed that in vitro cell experiment,Fructus Forsythiae,forsythia alcohol extract and forsythia water decoction all have inhibitory effects on the expression of influenza A virus nucleoprotein.

By using network pharmacology and molecular docking mechanism,this study explored the mechanism of action ofFF-FNin the treatment of acute pharyngitis,aiming to provide new evidence from traditional Chinese medicine for the intervention of acute pharyngitis,and to promote certain clinical drug research and development.

Methods

Acquisition of main compounds and mining of potential targets for FF-FN

The TCMSP (https://tcmspw.com/tcmsp.php) platform was used to search all chemical components ofFF-FNof Traditional Chinese Medicine,oral bioavailability(OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were selected as screening indexes to obtain eligible active ingredients in the pharmacokinetic characteristics of TCM.Meantime,potential Targets of the drug were obtained in the Related Targets column of the platform,while Uniprot database was used to convert all target points into GeneSymbol IDs.

Acquisition of targets related to acute pharyngitis

Relevant targets of AP were found in databases,DrugBank (https://go.drugbank.com/),GeneCards(https://www.genecards.org/),PharmGKB(https://www.pharmgkb.org/) and OMIM(https://omim.org/),with "acute pharyngitis" as keyword.

Construction of drug-component-target network

The obtained drug targets and disease targets were screened.After the elimination of the duplication,R software was used to get the intersection and draw the Venn diagram.Then,the intersection genes and major drug compounds were imported into Cytoscape3.8.0 software to construct drug-component-target network diagram.In the network diagram,Node 1 represents the drug,Node 2 represents the target,and Edge represents the relationship between different nodes,which is classified according to the size of the network degree.The greater the degree value,the more important the role it plays in the network.

Construction of PPI network and the acquisition of core network

The obtained intersection genes were uploaded to the STRING network platform（https://string-db.org/） to construct the PPI network.The PPI network interaction model diagram was constructed with the lowest interaction threshold set as (＞ 0.9),and the free targets removed.The obtained network was imported into Cytoscape software,and the CytoNCA plug-in was used for topology analysis to obtain the relevant core genes.

GO biological enrichment analysis and KEGG enrichment analysis

The GO biological function and KEGG pathway enrichment analysis of the intersection genes were carried out by R language software.With P value ＜ 0.05 and the correctedPvalue (qvalue) ＜ 0.05 set as the screening conditions,the analysis was conducted,the results were output,and the corresponding histogram was drawn.The vertical axis in the figure is the name of biological process or pathway,the horizontal axis represents the number of genes enriched in it,and the color represents the significance of enrichment.The darker the color is,the stronger the significance of gene enrichment in the biological process or pathway.

Molecular docking verification

Firstly,small molecule 2D structures of the main compounds inFF-FNdrug pair were obtained from PubChem database,and imported into ChemM Office software.They were converted into 3D structures and optimized.The core protein receptor structure was obtained by using RCSB PDB platform,while the water molecules and small molecule ligands were removed by using PyMol software.Then the obtained structure was imported into AutoDockTools software for hydrogenation and other pretreatments.Finally,the binding activity of the structure was verified by the conduction of molecular docking using VINA software.

Results

Compounds and potential targets of FF-FN were obtained

According to the OB and DL,23 active components ofFructus Forsythiaeand 15 active components ofFolium Nelumbiniswere obtained by screening and mining from the TCMSP platform.At the same time,917 potential targets ofFF-FNwere obtained from this platform as well,and 99 active targets were obtained after Uniprot database annotation and elimination of repeated targets.

Obtaining of intersection genes and Venn map plotting

After the search in the corresponding database,the disease-related targets were obtained,including 23 DrugBank,1593 GeneCards,16 PharmGKB and 130 OMIM.After integrating the target information obtained from the four databases,with duplicates removed,,1534 disease-related genes were obtained.Then,R software was used to process the corresponding drug targets and disease targets.As a result,117 intersection genes were obtained,and the corresponding Venn diagram was drawn (Figure 1).

Drug-component-target network

The obtained intersection genes and the co mpounds in theFF-FNdrug pair were used to construct a regulatory network (Figure 2) and ranked by degree.FF-FN,in which the circle represents the drug (red representsFructus Forsythiae,yellow representsFolium Nelumbinis),and the square represents the intersection genes.According to the ranking,the top five compounds were quercetin (MOL000098),luteolin(MOL000006),kaempferol (MOL000422),wogonin(MOL000173) and β-sitosterol (MOL000358),as shown in Table 1.

PPI network construction and core gene prediction

Figure 1.Venn plot of drug composition and disease target

Figure 2.Regulatory network of TCM compounds

The intersection genes were introduced into the string network platform to construct PPI network (Figure 3),in which the nodes represent the targets,and the lines represent the protein interaction relationships.The stronger the relationship is,the thicker the line is.At the same time,different colors in the network represent different evidences.With acquired PPI network,the DC,BC,CC,EC，NC and LAC values of the network nodes were further calculated by using CytoNCA,and the median of all the above values were also calculated.Targets,less than the median,were removed.As a result,a total of 38 important targets were screened out,including 7 core targets (Figure 4): JUN,TP53,CXCL8,RELA,MAPK1,TNF and MYC,which play an important role in the network.

Go enrichment analysis and KEGG pathway enrichment analysis

From GO enrichment analysis (Figure 5),a total of 2,487 enrichment results were obtained,among which 2,220 biological processes (BP) mainly involve in reactions to reactive oxygen species,antibiotics,cell responses to oxidative stress,bacteriogenic molecules,and lipopolysaccharides.While 111 cell components(CC) mainly participate in cyclin-dependent protein kinase holoenzyme complex,serine/threonine protein kinase complex,intrinsic components of synaptic membrane,etc.There were 176 molecular functions(MF) chiefly regarding to signal receptor activator activity,ubiquitin protein ligase binding,cytokine receptor binding,etc.

According to KEGG enrichment analysis results(Figure 6),approximate 156 results received are mainly related to fluid shear stress and atherosclerosis,IL - 17 signaling pathways,TNF signal pathway,human papillomavirus (HPV) infection,Hepatitis b,the AGE- RAGE signaling pathways,PI3K-Akt signaling pathways,human cytomegalovirus infection,Kaposi’s sarcoma associated herpesvirus infections.It is suggested thatFF-FNmay play an intervening role in acute pharyngitis through these pathways.

Docking verification of active compounds with core target molecules

The top five compounds in the regulatory network of important compounds,quercetin,luteolin,kaempferol,wogonin and β-sitosterol were molecularly docking with the top five core genes,JUN,RELA,MAPK1,TNF and MYC,respectively,and the results were shown in Table 2.According to literature reports,generally,it indicates a good binding effect that the binding energy score ≤ 5.0 kcal·mol-1[13].The results showed that the binding energies of quercetin,luteolin,kaempferol,wogonin and β-sitosterol with the core genes JUN,RELA,MAPK1,TNF and MYC are all less than -5.0 kcal·mol-1,suggesting that they have a good binding ability (Figure 7).

Figure 3.PPI protein interaction network

Table 1.chemical composition of the top five degree values

Figure 4.Core genes of PPI network

Figure 5.Biological enrichment analysis of GO

Table 2.Binding energy between active compound and core target

Figure 6.Enrichment analysis of KEGG signaling pathway

Figure 7.Molecular docking diagram of compounds and targets (A,Quercetin and Jun; B,Quercetin and TNF; C,Luteolin and Jun; D,kaempferol and Jun; E,Wogonin and MAPK1; F β-sitosterol and MAPK1)

Discussion and Conclusion

FF-FNdrug pair is commonly used in clinical treatment in our department with good efficacy,however,further systematic studies are necessary for verification.This paper used network pharmacology and molecular docking technology to screen its active components and targets,in the hope of providing support and help for further research.In this study,five important components were obtained,including quercetin,luteolin,kaempferol,wogonin and betasitosterol.Modern research has found a significant association of these ingredients with acute pharyngitis.For example,quercetin has anti-tumor,antioxidant,anti-inflammatory,antiviral effects,among which antiinflammatory effect is related to quercetin pharmacological activities most [14,15].Quercetin can be reduced by increasing Treg cells differentiation,differentiation of Th17 cell and balance restoring of Th17/Treg,leading to the improvement of inflammation expression [16].Quercetin can also interact with the HA2 subunit on the cell membrane to block the fusion of the virus and thus achieve the inhibitory effect on the virus [17].The antiinflammatory effect of Luteolin is mainly achieved through the regulation of inflammatory mediators.Luteolin can also play an antioxidant role through signaling pathways such as MAPK [18].Regarding kaempferol,in addition to inhibiting the expression of inflammatory factors through MAPK and JAK/STAT pathways,it also has a good inhibitory effect on Staphylococcus aureus,Pseudomonas aeruginosa and other pathogenic bacteria [19,20].Wogonin and βsitosterol have obvious anti-inflammatory and antibacterial effects as well [21,22].The results above indicated thatFF-FNcould play a role in treating acute pharyngitis through multi-component and multi-path synergistic action.

Through analysis of PPI network after JUN,TP53,CXCL8,RELA,MAPK1,TNF,MYC,seven core target genes,transcription factor,AP-1 (JUN) is found mainly involved in cell proliferation,differentiation,and apoptosis process.It is also involved in physical inflammatory response by multipath.Meanwhile,the initial T cells can be adjusted to the effect of T cell differentiation to affect the immune response and inflammatory process.At the same time,some studies have shown that the overexpression of AP-1 can increase glucocorticoid resistance,causing further increasing of the expression of inflammation [23-24].Transcription factor p65 (RelA) is an important member of the NF-κB family.Activated NF-κB can promote the expression of related inflammatory cytokines,and activation of NF-κB is closely related to transcription factor p65 [25].Modern studies have shown that MAPK signaling pathway is the upstream signal transduction component of the expression of various inflammatory factors,which can mediate cellular immune regulation,inflammatory response,cell proliferation and apoptosis,and is also involved in the signal transduction of various stress responses [26].TNF,as a classic inflammatory factor,plays an important role in inflammatory expression and immune response.These results suggested thatFF-FNmight play a therapeutic role in treating acute pharyngitis from the targets above.

GO biological enrichment analysis showed that the treatment mechanisms are related to response to the reactive oxygen species,response to the antibiotic,cell's response to oxidative stress,the bacteria source molecular reaction,and response to lipopolysaccharide.Existing studies have shown that these biological processes are closely related to the occurrence of acute pharyngitis development,such as reactive oxygen species (ROS),an excessive accumulation of situation can cause oxidative stress and cellular damage [27].It was also reported that lipopolysaccharide (LPS) can significantly promote apoptosis,inflammatory response,and oxidative stress injury [28].Oxidative stress,bacterial infection and other biological processes can eventually induce the expression of inflammatory factors in vivo,further promoting the onset of acute pharyngitis.

KEGG enrichment analysis showed that the main pathways involved in the therapeutic effect of FF-FN drug were IL-17 signaling pathway,TNF signaling pathway,human papillomavirus infection,hepatitis B,AGE-RAGE signaling pathway,PI3K-Akt signaling pathway,human cytomegalovirus infection,etc.AGERAGE signaling pathway can induce the expression of a large number of adhesion molecules and proinflammatory cytokines by activating NF-κB,including IL-6,IL-8,TNF-α,etc.,thereby causing cell and tissue damage.Meanwhile,activation of NF-κB can also enhance the expression of RAGE,continuously aggravating the injury [29].AGE-RAGE can also activate MAPK,P38 and other signaling pathways through enhanced oxidative stress to promote NF-κB phosphorylation,promote the release and expression of the above factors,and induce acute pharyngitis [30].Studies have shown that IL-18 can stimulate related cells to secrete IL-1 and TNF-α through the p13K-Akt signaling pathway to form an inflammatory cascade [31].IL-17 and TNF are important inflammatory pathways.It has been reported that IL-17 is mainly secreted by Th17 cells and is involved in various inflammatory responses and immune inflammatory aggregation processes in the body.Secreted IL-17 can stimulate the corresponding cells to secrete a large number of various proinflammatory cytokines,while the secretion of these pro-inflammatory cells can in turn promote IL-17 secretion [32].Animal experiments have shown that the expression of TNF-α can be inhibited,making it possible to activate NF-κB,reduce the secretion of proinflammatory factors,and improve the symptoms of rat model of pharyngitis [33].Modern studies [34,35] have shown that the treatment of acute pharyngitis is mainly by regulating TNF,NF-κB,MAPK and other signaling pathways to evoke anti-inflammatory and antioxidative stress response.Therefore,it can be speculated thatFF-FNcan play a role in reducing oxidative stress response and regulating the expression of inflammatory factors,explaining its AP treatment mechanisms.Meanwhile,the above analysis showed that all the pathways mentioned previously were involved in mediating the inflammatory response in vivo,and there were many common targets in the pathways,which could conduct signaling pathway dialogue,reflecting the effect ofFF-FNon the coordinated multi-pathways treatment of acute pharyngitis.

Molecular docking results showed that quercetin,luteolin,kaempferol,wogonin,β-sitosterol have good binding force with JUN,RELA,MAPK1,TNF,and MYC,which can further verify the research results and provided support for thisFF-FNstudy.

To sum up,in this research,by using the method of network pharmacology,the main chemical composition,the main targets and pathways related toFF-FNwere analyzed and the reflects on its composition,multiple targets,multiple pathways coordinate treatment function characteristics were predicted.The results obtained laid the foundation for subsequent systematic research,providing the direction for future research.However,this conclusion has only been verified by molecular docking technology and further verification and research are necessary in the future.