WU Hu-Ying,LI Jing,MAO Yi-Zhi,YUAN Zhi-Ying,LI Feng,LI Ling,,HUANG Hui-Yong*

a.Provincial Key Laboratory of TCM Diagnostics,Hunan Engineering Technology Research Center for Medicinal and Functional Food,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China

Keywords

Hunan expert group recommended Chinese medicine prevention No.2 prescription (Pre-No.2)

Coronavirus disease 2019 (COVID-19)

Network pharmacology

Immune system

ABSTRACT

Objective To explore the possible preventive mechanism of Hunan expert group recommended Chinese medicine prescription of No.2 (Pre-No.2) against coronavirus disease 2019(COVID-19) by network pharmacology method.

Methods The target proteins of effective components and active compounds in Pre-No.2 were screened by searching the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP).A component-target-disease interaction network of Pre-No.2 was constructed by Cytoscape 3.7.2,gene ontology (GO) analysis,and Kyoto encyclopedia of genes and genomes (KEGG) analysis of target protein pathway by DAVID.

Results A total of 163 compounds and 278 target protein targets in Pre-No.2 were collected from the TCMSP database.Kaempferol,wogonin,7-methoxy-2-methyl isoflavone,formononetin,isorhamnetin,and licochalcone A were the most frequent targets in the regulatory network.GO enrichment analysis showed that Pre-No.2 regulated response to virus,viral processes,humoral immune responses,defense responses to virus and viral entry into host cells.KEGG enrichment analysis showed that the formula regulated the NF-κB signaling pathway,B cell receptor signaling pathway,viral carcinogenesis,T cell signaling pathway and FcγR-mediated phagocytosis signaling pathway.

Conclusions Pre-No.2 may play a preventive role against COVID-19 through regulation of the Toll-like signaling,T cell signaling,B cell signaling and other signaling pathways.It may regulate the immune system to protect against anti-influenza virus.

1 Introduction

December 2019,an acute pneumonia (COVID-19)caused by the new coronavirus (SARS-CoV-2) has spread worldwide.Data from the World Health Organization revealed that a total of 216 countries or regions were affected by this disease,with 8 708 008 cases and 461 715 deaths confirmed updated to June 21st,2020[1].SARS-CoV-2 can be considered as a new type of human infectiousβ-coronavirus.Although phylogenetic analysis indicated that bats may be the original host of the virus,the intermediate host and the exact transmission route have not been determined[2,3].Epidemiological studies have confirmed that the new viruse can transmitted from person to person through respiratory droplets or close contact,so it's crucial and necessary to adopt prevention practices in public health[4].

Fever,dry cough and fatigue are the main clinical symptoms.Severe patients may develop dyspnea,hypoxemia,and even acute respiratory distress syndrome and septic shock[5].At present,integrative Chinese and western medicine treatment is the main powerful method when specific vaccines are absent.Traditional Chinese medicine (TCM) has accumulated many valuable experiences in the prevention and treatment of epidemic diseases.COVID-19 belongs to the category of “epidemic disease” in the TCM.A series of Chinese medicine prescriptions has been used to defend against COVID-19,effectively controlling the development of the pandemic[6].Prevention is an essential method and a characteristic of Chinese medicine.Therefore,in the early stage of the epidemic,the Hunan Provincial Administration of Traditional Chinese Medicine organized a senior expert group of Chinese medicine with Professor XIONG Ji-Bo as a consultant,and jointly developed the Hunan Province’s New Coronavirus Infected Pneumonia TCM Diagnosis and Treatment Plan (Trial Version 3).The expert group recommended preventive methods for different groups and constitutions in the “Plan”.Pre-No.2 was consisted of Isatidis Radix (Ban Lan Gen,板藍(lán)根),Menthae Haplocalycis Herba (Bo He,薄荷),Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草),Lonicerae Japonicae Flos(Jin Yin Hua,金銀花),Schizonepetae Herba (Jing Jie,荊芥),Forsythiae Fructus (Lian Qiao,連翹),Phragmitis Rhizoma (Lu Gen,蘆根) and Mori Cortex (Sang Bai Pi,桑白皮).The prescription was originated from Yin Qiao Powder,which was mainly used in key populations with strong physique and history of close contact with infected people.

Network pharmacology is a new discipline based on the theory of systems biology used to analyze the network of biological systems and select specific signal nodes for multi-target drug molecular design.TCM is a complex system with multiple compounds,multiple targets and synergies among the compounds.Network pharmacology emphasizes the multi-path regulation of signaling pathways and the multi-compound-target-path regulation[7],which can help improve the efficiency and reduce the costs of new drug development in clinical trials.In this study,we utilized the network pharmacology method to screen the core active compounds and metabolic pathways in Pre-No.2 and to predict its potential preventive mechanisms against COVID-19.

2 Materials and Methods

2.1 Collection of active compounds and targets of Pre-No.2

The TCM Database@Taiwan[8]and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database[9]were searched for “Isatidis Radix” “Menthae Haplocalycis Herba” “Glycyrrhizae Radix Et Rhizoma” “Lonicerae Japonicae Flos” “Schizonepetae Herba” “Forsythiae Fructus” “Phragmitis Rhizoma” and “Mori Cortex” to collect the compounds of Pre-No.2 and then to investigate the pharmacokinetic parameters and targets of each active compound in TCMSP.The pharmacokinetic parameters included oral bioavailability (OB),Caco-2 permeability,and drug-like(DL),according to the standards provided by the TCMSP database,and Tanimoto and OBioavail,which are embedded in the database,were used to obtain predicted and calculated DL and OB.OB > 30%,Caco-2 > ? 0.4,and DL > 0.18 were used as standards to screen orally absorbable active compounds with pharmacological activity.The protein name of the collected target was entered into UniProtKB(http://www.uniprot.org/).The restricted species is“Homo sapiens”,thus correcting the protein name to the official symbol.

2.2 Collection of COVID-19-related targets and data on protein-protein interactions (PPI)

The Online Mendelian Inheritance in Man (OMIM)database[10]and Genecards[11]were used to collect SARS-CoV-2-related targets in infected humans.The search results of the two databases were summarized and deduplicated.The String database (http://stringdb.org/)[12]is usually used to search for interactions between known proteins and predicted proteins,as well as to predict direct and indirect interactions from prokaryotic cells to various other eukaryotes.In this study,we used the String database to collect data on PPI of Pre-No.2 and the related targets of COVID-19 in infected cells,with confidence > 0.4.

2.3 Network construction and analysis

Cytoscape 3.7.2[13]was used as a tool to visualize the PPI network.Four main networks were constructed:(1) an active compound-action target network of Pre-No.2; (2) an active target of Pre-No.2-COVID-19-related target protein interaction network; (3) an active compound-biological process/cell component/molecular function-target network; and (4) an active compound-signal pathway-target network.In the network,nodes represent active compounds,targets,or pathways and nodes are connected by edges.Degree represents the number of connections between the nodes and the degree of betweenness represents the shortest path through the nodes.

2.4 Pathway enrichment analysis

Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG)pathway annotation analysis can reveal the importance of different biological processes,cell components,molecular functions and signaling pathways in a PPI network.In this study,COVID-19-related targets and the targets of Pre-No.2 were introduced into the DAVID platform version 6.8(https://david-d.ncifcrf.gov)[14]for GO enrichment and KEGG enrichment analysis,and the results in the related network were displayed in tables and bubble graphs.

3 Results

3.1 Active compound-action target network of Pre-No.2

A total of 163 active compounds in Pre-No.2 and their targets were collected.The details are shown in Table 1.The active compounds and targets were introduced into Cytoscape 3.7.2 to build a network of active compound-targets of Pre-No.2.The network consists of 163 active component nodes,278 active target nodes and 2 567 edges.In this network,the size of a node is positively related to its degree.For example,CALM1 has a larger node and can be controlled by active compounds such as Kaempferol,Wogonin,7-Methoxy-2-methyl isoflavone,Formononetin,Isorhamnetin,Licochalcone A,Medicarpin,4'-Methoxyglabridin,1-Methoxyphaseollidin,Vestitol and Licoagrocarpin,etc.,while the 1-Monolinolein node is small and can only regulate PTGS1 (Figure 1).

3.2 Pre-No.2 target-COVID-19-related target PPI network

Pre-No.2 targets (i.e.,drug target),COVID-19-related targets (i.e.,viral target),and PPI information wereimported into Cytoscape 3.7.2 to construct a Pre-No.2 target-COVID-19-related target PPI network.The network consisted of 298 nodes (267 drug target nodes,23 virus target nodes,and 8 drug-virus target nodes) and 6 440 edges.Among them,31 COVID-19-related targets can be divided into:(1) targets directly regulated by relapse (i.e.,drug-virus co-acting targets):CASP3 (131 edges),FOS (119 edges),IL-10(98 edges),CCL2 (97 edges),BCL2L1 (84 edges),IFNG (72 edges),CXCL10 (66 edges) and DPP4 (31 edges); (2) indirectly regulated targets:CREB1 (108 edges),CCL5 (77 edges),CREBBP (65 edges),IRF3(46 edges),IFNB1 (43 edges),ATF2 (31 edges),LCN2(31 edges),IFNA1 ( 27 sides),BAG3 (20 sides),MX1(20 sides),CASP6 (19 sides),ACE2 (18 sides),CD209(18 sides),HLA-A (17 sides),HLA-DRB1 (14 sides),ICAM3 (14 sides),MBL2 (14 sides),HLA-B (11 sides),HELLS (10 sides),SH2D3A (6 sides),FCER2 (5 sides),CLEC12A (3 sides) and CLEC4M (3 sides) (Figure 2).

Table 1 Information of 158 active compounds in Pre-No.2

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

3.3 GO enrichment analysis

The results of GO enrichment include biological processes (BP),cellular components (CC),and molecular functions (MF).After enrichment analysis,a total of 28 BP,9 CC and 11 MF were obtained,all of which were related to virus interventions (Figure 3).For example,response to viruses,viral processes,B cell proliferation,defense against viruses,viral entry into host cells,modulation of immune responses,T cell receptor signaling pathways,and neutrophil chemotaxis.The anti-virus-related cell components were extracellular space,plasma membrane,cytosol,extracellular region and membrane raft.The antiviral-related MF included protein homodimerization activity,nitric oxide synthase regulator activity,RNA polymerase II transcription factor activity,activated sequence-specific DNA binding,protein kinase binding,and viral receptor activity(Figure 4).These biological processes and the targets they contain may be the key to the preventive effect of Pre-No.2.In addition,in this network,Lonicerae Japonicae Flos (Jin Yin Hua,金銀花) can regulate 196 targets; Forsythiae Fructus (Lian Qiao,連翹) can regulate 137 targets; Menthae Haplocalycis Herba(Bo He,薄荷) can regulate 105 targets; Schizonepetae Herba (Jing Jie,荊芥) can regulate 179 targets;Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草) can regulate 219 targets; Isatidis Radix (Ban Lan Gen,板藍(lán)根) can regulate 71 targets; Phragmitis Rhizoma(Lu Gen,蘆根) can regulate 28 targets; and Mori Cortex (Sang Bai Pi,桑白皮) can regulate 174 targets.

3.4 KEGG pathway enrichment analysis

After KEGG pathway enrichment analysis,23 signaling pathways related to virus interventions were obtained (Figure 5).For example,NF-κB signaling,B cell receptor signaling,viral carcinogenesis,mTOR signaling and FcγR-mediated phagocytosis pathways (Figure 6).These signaling pathways and the targets may be closely related to the mechanism of Pre-No.2 against COVID-19.Moreover,in this network,Lonicerae Japonicae Flos (Jin Yin Hua,金銀花),Forsythiae Fructus (Lian Qiao,連翹),Menthae Haplocalycis Herba (Bo He,薄荷),Schizonepetae Herba (Jing Jie,荊芥),Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草),Isatidis Radix(Ban Lan Gen,板藍(lán)根),Phragmitis Rhizoma (Lu Gen,蘆根) and Mori Cortex (Sang Bai Pi,桑白皮) can regulate 101,79,65,89,108,39,12 and 90 targets,respectively.

4 Discussion

Pre-No.2 is a prescription revised from Yin Qiao Powder and has the effect of clearing heat and detoxification.In the present study,Lonicerae Japonicae Flos (Jin Yin Hua,金銀花),Forsythiae Fructus (Lian Qiao,連翹),Menthae Haplocalycis Herba (Bo He,薄荷),Schizonepetae Herba (Jing Jie,荊芥),Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草),Isatidis Radix (Ban Lan Gen,板藍(lán)根),Mori Cortex (Sang Bai Pi,桑白皮) and Phragmitis Rhizoma(Lu Gen,蘆根) were shown to affect 101,79,65,89,108,39,12 and 90 targets,respectively.Current research reports revealed that Lonicerae Japonicae Flos (Jin Yin Hua,金銀花) acts on various viruses,such as H1N1 influenza virus,antiviral myocarditis,anti-herpes virus and anti-adenovirus[15].Pharmacological experiments of Forsythiae Fructus (Lian Qiao,連翹) proved that it has antiviral,antitumor,antidepressant,anti-inflammatory,antioxidant and immune function-enhancing effects[16].Pharmacological studies on Menthae Haplocalycis Herba (Bo He,薄荷) have shown that it can enhance the body’s resistance to influenza viruses by adjusting the body’s immune status[17].The pharmacological studies of the active compounds of Schizonepetae Herba (Jing Jie,荊芥) showed that it has anti-inflammatory,immunity-enhancing,and anti-influenza virus pneumonia effects[18].Various triterpenoids in licorice have been shown to have a significant and broad-spectrum antiviral activity[19].The extract of Isatidis Radix (Ban Lan Gen,板藍(lán)根)and its active compounds have antiviral,antiendotoxin,antibacterial,anti-inflammatory and immune function-enhancing effects[20].Study showed that Mori Cortex (Sang Bai Pi,桑白皮) has antihypertensive,diuretic and diarrhea,antibacterial and anti-inflammatory,antitussive,anti-edema,sedative and anticonvulsant effects[21].

In this study,Pre-No.2 was shown to contain 158 chemical compounds,according to the screening of OB,Caco-2 permeability,and DL through network pharmacology strategy and network topology analysis,thereby providing the substance basis of the action of Pre-No.2.In addition,GO annotation showed that Pre-No.2 mainly regulated immune BP,such as the proliferation and chemotaxis of immune cells(mononuclear macrophages,T cells and B cells) and immune factors (IFN-γand IFN-α); inflammatory response processes,such as inflammatory factors and inflammatory signaling pathways mediated by them;chemotactic inflammatory cells (white blood cells and macrophages); and other BP.The enrichment of KEGG signaling pathways showed that the relevant action pathways related to the immune system mainly included Toll-like receptor (TLR) signaling,T cell receptor signaling,B cell receptor signaling,and Jak-STAT signaling pathways.Recent studies have shown that innate immunity is an important body immunity,and the pattern recognition receptor family (PRR) is necessary for the natural immune response to recognize microbial pathogens; the multiple conservative points of pathogen recognition are called pathogen-related molecular patterns(PAMP)[22].TLRs,the first members of the PRR family widely present in the immune system,can specifically identify pathogenic microorganisms and transmit extracellular antigen recognition information into cells[23].The TLR signaling pathway includes myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways[24].TLRs can recognize various PAMPs and recruit specific linker molecules to bind TLR functional domains,such as MyD88 and TRIF,and then through a series of signal transduction,the cells will eventually produce inflammatory factors,type I interferon,chemokines and antimicrobial peptides[25].Intact pathogen microorganisms usually contain a large amount of PAMP,which can activate various PRRs,TLRs and PRRs to coordinate with each other to activate the host’s immune responses to resist infection[26].Therefore,TLRs are essential in the natural immune response against the invasion of pathogenic microorganisms (bacteria,viruses and fungi).In addition,T cell receptors (TCRs) can activate many signal transduction cascades and ultimately determine cell fate by regulating cytokine products,cell survival,proliferation and differentiation[27].The early event of TCR activation is the phosphorylation of lymphocyte protein tyrosine kinase (Lck) on the immunoreceptor tyrosine-dependent activation motifs on the cytoplasmic side of the TCR/CD3 complex[28].

Owing to the advancement of research on COVID-19,it has been elucidated that the key to the defense against and treatment of new coronavirus infections is the non-specific and specific immunity as well as the subsequent inflammation waterfall factors of host infection[29,30].In terms of the innate immune system’s defense against COVID-19 infections,the main mechanism involves PRRs,TLRs,RIG-I-like receptors (RLRs),NOD-like receptors (NLRs),C-type lectin-like receptors,interferon and defensin[31].Furthermore,T cell immune responses and humoral immune responses in the adaptive immune response are equally important,especially CD4+T cells and CD8+T cells,which play an important antiviral role by balancing the risk of fighting pathogens and developing autoimmunity or suppressing inflammatory responses[32].In addition,the exhaustion of CD4+T cells is related to the reduction of lung lymphocyte recruitment and the production of neutralizing antibodies and cytokines,which in turn leads to strong immune-mediated clearance of interstitial pneumonia and coronavirus from the lung[33].Laboratory results revealed that the total number of peripheral white blood cells in COVID-19 patients during the early stage was normal or decreased,whereas lymphocyte count decreased; in contrast,in severe cases,D-dimers increase and peripheral blood lymphocytes progressively decrease[5].Moreover,results of pathological examination of deceased COVID-19 patients showed serious damage to the lungs caused by inflammatory factor storm and collapsed immune system[34].Cytokine storm refers to rapid and massive production of cytokines,(such as TNF-α,IL-1,IL-6,IL-12,IFN-α,IFN-β,IFN-γ,MCP-1 and IL-8),caused by infection of microorganisms in the body fluids,and it is an important cause of acute respiratory distress syndrome and multiple organ failure[35].Current research reports showed that the main elevated inflammatory factors in patients with COVID-19 are IL-2,IL-1B,IL-1RA,IL-7,IL-8,IL-9,IL-10,TNF-αand VEGF[5,36].In this study,we found that Pre-No.2 regulated innate and adaptive immune responses,such as NOD-like receptor signaling,TLR signaling,T cell receptor signaling,and B cell receptor signaling pathways which targeted IL-10,IL-6,TNF and VEGFA.

In summary,Pre-No.2 may play a preventive role against COVID-19 through regulation of the Toll-like signaling,T cell signaling,B cell signaling and other signaling pathways.It may regulate the immune system to protect against anti-influenza virus according to the network pharmacology.

Acknowledgements

We thank for the funding support from the Scientific Research Fund of Hunan Administration of TCM(No.KYGG06 and No.KYGG07).

Competing Interests

The authors declare no conflict of interest.