The Study of Molecular Mechanisms of Xuanbai Chengqi Decoction(宣白承气汤) in the Treatment of Coronavirus Disease 2019 Based on Network Pharmacology and Molecular Docking Method

LIU Zi-Xiu (刘子修), LIU Qian (刘 茜), HONG Sheng-Wei (洪盛威), YONG Tai-Ping (雍太萍),XU Duo (许 多), ZHOU Yan-Ping (周燕萍), CHEN Yun-Yan (陈云艳), LIU Mei (刘 梅)

1.Department of Pharmacy, Air Force Hospital of Eastern Theater Command, Nanjing 210002, China,

2.Department of Pharmacy, The Affiliated Brain Hospital of Nanjing Medical University, Nanjing 210029 China

3.Nanjing University of Chinese Medicine, Nanjing 210023, China

4.Wannan Medical College, Wuhu 241002, China

ABSTRACT Objective:To explore the molecular mechanism of Xuanbai Chengqi Decoction (宣白承气汤)in the treatment of Coronavirus Disease 2019 (COVID-19) based on network pharmacology, and to verify by molecular docking technology.Methods:The components and targets of Xuanbai Chengqi Decoction (宣白承气汤) were obtained by TCMSP, targets' information was corrected based on the databases such as UniProt and DrugBank, and the software Cytoscape3.7.1 was adopted to construct TCM-Component-Target and Component-Target network.The main targets were mapped to the KEGG pathway and the GO biological process with the help of DAVID to further elucidate the potential relationship between the main targets and Xuanbai Chengqi Decoction (宣白承气汤) therapy for COVID-19.In the end, the Swiss Dock platform was adopted for the molecular docking verification of key components and targets.Results:The Component-Target network consists of 35 components and 106 corresponding targets, the main targets include COX-2, NCOA2,PTGS1, HSP90AB1, PRKACA and PGR, etc.There are 561 GO entries of target mapping (P 0.05), including 155 entries for Biological Processes (BP), 147 entries for Cell Composition (CC), and 259 entries for Molecular Function (MF).There are 38 KEGG mapping pathways (P 0.05), including many aspects of infectious disease, immune system and endocrine system, as well as Calcium signaling pathway, VEGF signaling pathway, PI3K-Akt signaling pathway and other processes.Conclusion:The result of molecular docking shows that the affinity of the key components such as beta-sitosterol and stigmasterol are similar to recommended medications for COVID-19.Its effect in the treatment of middle stage of COVID-19 may be related to the blocking of the binding of COVID-19 virus and ACE2, antivirus, and relieving inflammatory storm.

KEYWORDS Xuanbai Chengqi Decoction (宣白承气汤); Coronavirus Disease 2019 (COVID-19); Network pharmacology; Molecular docking; Angiotensin converting enzyme 2 (ACE2)

lNTRODUCTlON

Since December 2019, Coronavirus Disease 2019 (COVID-19) has erupted, which is caused by a newly discovered β coronavirus[1].The main routes of transmission are via contact and respiratory droplet spread, so COVID-19 is strongly infectious,highly epidemic and generally susceptible.Other transmission routes such as aerosols and fecaloral are not clear[2].The patient's initial symptoms are fever, dry cough, and fatigue.Severe patients can quickly develop into multi-organ dysfunction and even death[3], which brings a huge threat to human health.Many of the currently diagnosed patients suffer from chronic diseases, making it difficult to rely on single-modern medical treatment,while traditional Chinese medicine (TCM) focuses on holistic, dialectical treatment, fully embodies its unique advantages, and has made important contributions to the fight against epidemic.

As early as the beginning of the Ming Dynasty, Wu Youke had discovered the difference in cause, pathogenesis, diagnosis and treatment between warm and cold diseases.In the face of the epidemic, he repeatedly carried out clinical exploration and wrote Wenyi Lun which was the precedent study of infectious disease.In the mid Qing Dynasty, Wu Jutong perfected the system of epidemic febrile diseases which was independent of cold diseases and created the Identification of Warm Diseases(Wen Bing Tiao Bian) which is the iconic book of the warm disease theory.COVID-19 belongs to the warm diseases according to TCM,and the treatment is mainly against the dampness evil and epidemic toxin.Xuanbai Chengqi Decoction(宣白承气汤) is adapted from Identification of Warm Diseases and is composed of raw gypsum (生石膏), Radix et Rhizoma Rhei (rude rhubarb生大黄),Semen Armeniacae Amarum (almond powder 杏仁粉), and Pericarpium Trichosanthis (瓜蒌皮)[4].Some scholars[5]believed that the diagnosed patients had the symptoms of the dampness toxin accumulation and lung qi depression in the middle stage of COVID-19.In the Xuanbai Chengqi prescription, raw gypsum (生石膏) has the effect in clearing heat from the lung, Radix et Rhizoma Rhei (rude rhubarb生大黄) in purging heart and bowels, (almond powder杏仁粉) in ventilating the lung and arresting cough, and Pericarpium Trichosanthis (瓜蒌皮) in moistening the lung for removing phlegm.When combined for use,they have an effect in dispersing and lowering lung qi, clearing lung and eliminating phlegm, capturing fu-viscera[4].

Network pharmacology is a new technology based on receptor theory and biological network technology that explains the role of drugs and their mechanism of action.It has created a new model of complex mesh relationship between multiple components and multiple targets.Research on potential pharmacological mechanisms has opened up new ideas[6].Compared with network pharmacology, molecular docking technology is more mature.Based on three-dimensional structure of the ligand and the spatial structure of the receptor, it can predict and score the affinity and combine mode, and select the best affinity with the target compound[7].Angiotensin-converting enzyme 2 (ACE2) is the receptor of SARS-Cov and recent studies have shown that the binding domain of COVID-19 virus and ACE2 is similar to SARS-Cov[8].It initially revealed that the ACE2 serves as the entry point into the cells (See Figure 1).This study was intended to explore the key active components of Xuanbai Chengqi Decoction (宣白承气汤) for the treatment of COVID-19 with the help of network pharmacology,and to conduct molecular docking between the key components and ACE2, so as to provide a theoretical basis for its clinical application.

Figure 1.3D Structure of Angiotensin-converting Enzyme 2 (ACE2)

METHODS

1.Chemical Components Collection in Xuanbai Chengqi Decoction (宣白承气汤)

With the help of systematic pharmacology analysis platform of Traditional Chinese medicine[9]TCMSP(http://tcmSPW.com/), the four Traditional Chinese medicines in Xuanbai Chengqi Decoction(宣白承气汤)were selected as the retrieval words, and the chemical components were collected and downloaded.

2.Active Components Screening and Targets Collection

In this study, based on ADME (drug absorption,distribution, metabolism, and excretion) model,oral availability (OB) and drug-like property (DL)information of chemical components were obtained using TCMSP database.OB30% and DL0.18 were set as screening conditions, and the targets of active components were derived.Protein target ID and gene name were retrieved through UniProtdatabase[10](https://www.uniprot.org), and target information of Xuanbai Chengqi Decoction(宣白承气汤) was sorted out.

3.Component-Target Network Construction and Analysis

The selected active components of Xuanbai Chengqi Decoction (宣白承气汤) on and their corresponding targets were imported into Cytoscape3.7.1 software to generate the network diagram reflecting the interaction between components and targets, so as to analyze the molecular mechanism of TCM compound treatment.The topology parameters of the component-target network diagram were analyzed using the NetworkAnalyzer.

4.Pathway Analysis of the Targets

The GO (Gene ontology) biological process enrichment analysis and KEGG pathway annotation analysis were carried out on 36 major targets using DAVID[11]online analysis tool, and the threshold valueP0.05 was set.Data visualization was completed using Omicshare cloud platform (https://www.omicshare.com/).

5.Component-target Molecular Docking

3D structure of 4 key active molecules and 5 recommended antiviral drugs in Diagnosis and Treatment Scheme of COVID-19 (Trial Version 5)[1]were obtained from Pubchem, and were stored in sdf format.The sdf format file was transformed into mol2 format by Discovery Studio 4.5.3D structure of ACE2 in PDB format was downloaded from PDB database(https://www.rcsb.org/) and the protein was processed by eliminating water molecules, adding hydrogen, etc through Auto Dock software.The prepared targets and corresponding ligands were uploaded to online molecular docking platform SwissDock with default value for all parameters.The binding energy of protein and ligand was assessed based on the functions of CHARMM22 force field.If the binding energy is less than 0, the ligands and receptor can spontaneously combine.At present, there is no uniform standard for target screening of active molecules.According to literature reports[13], the active components with binding energykcal/mol are selected as the screening basis for the target of Xuanbai Chengqi Decoction (宣白承气汤) to treat COVID-19.

RESULTS

1.Active Components Screening

A total of 182 chemical components in Xuanbai Chengqi Decoction (宣白承气汤) were retrieved by TCMSP, including 0 in raw gypsum (生石膏), 60 in Pericarpium Trichosanthis (瓜蒌皮), 46 in rude rhubarbs (生大黄) and 76 in almond powder (杏仁粉).WithOB30% andDL0.18 as the threshold,182 components were screened, and 46 active components of Xuanbai Chengqi Decoction (宣白承气汤) were finally determined, including 0 in raw gypsum (生石膏), 11 in Pericarpium Trichosanthis(瓜蒌皮), 16 in rude rhubarb (生大黄) and 19 in almond powder(杏仁粉).(See Table 1)

2.TCM-Component-Target Network Construction

Topology Network of TCM-component-target Network was constructed with Cytoscape software(See Figure 2).In Figure 2, nodes represented proteins and edges represented the correlation between proteins, involving a total of 3 TCM, 35 chemical components, 106 targets and 388 edges.11 chemical components and 1 TCM were not participated in the network construction.The model showed that there was a complex relationship between the chemical components of Xuanbai Chengqi Decoction (宣白承气汤) and the targets,and this complex network also confirmed the conclusion that Xuanbai Chengqi Decoction (宣白承气汤) exerts its effect through multi-components and multi-targets.

3.Topology Parameter Analysis of Network Model

By the NetworkAnalyzer, the target protein was used as a network node, and the topology parameters of the network model were calculated,such as Betweenness Centrality, Degree and Closeness Centrality, etc.As shown in Figure 3,since the node area to Degree ratio is fixed, the higher Degree and bigger node area indicated that the target is more important in this network.The color of the node represented the classification,yellow, blue, and green represented rude rhubarb(生大黄), almond powder (杏仁粉) and Pericarpium Trichosanthis (瓜蒌皮), respectively, and red represented targets.After a comprehensive consideration of Degree, Betweenness Centrality and Closeness Centrality, top 8 components were selected as key active components.36 major targets were further selected including COX-2, NCOA2,PTGS1, HSP90AB1, PRKACA, PGR, etc., all of which had a Degree4.

Table 1.Basic lnformation on Some Active Components in Xuanbai Chengqi Decoction(宣白承气汤)

4.Biological Function of Target and Target-Pathway Analysis

36 major targets were imported into DAVID database to conduct KEGG pathway and GO enrichment analysis, and the bar charts were drawn via Omicshare platform as shown (Figure 4-5).561 GO entries of target mapping (P0.05)contained 155 entries for biological processes(BP), 147 for Cell composition (CC) and 259 for molecular function (MF), some of which were dramatically significant, such as G proteincoupled amine receptor activity and G proteincoupled acetylcholine receptor activity.38 Mapping KEGG pathways (P0.05) contained calcium, VEGF, PI3K-Akt signaling pathways,infectious disease-related pathways including hepatitis, pertussis, human cytomegalovirus infection, Kaposi sarcoma-associated herpesvirus infection and Epstein-Barr virus infection, immune system related pathways involving Th17 cell differentiation, interleukin-17 (IL-17) signaling pathway, C-type lectin receptor signaling pathway and platelet activation, and nervous system involving cholinergic synapses, serotonin synapses, etc.

Figure 2.TCM-Component-Target Network of Xuanbai Chengqi Decoction (宣白承气汤 ) in Treatment of COVlD-19

Figure 3.Component-Target Network of Xuanbai Chengqi Decoction (宣白承气汤) in Treatment of COVlD-19

5.Molecular Docking Analysi s

It is generally considered that when the conformation of ligand binding to the receptor is stable, the lower the energy, the greater the possibility of action.In this study, the binding energy is-2.0 kcal/mol as the screening standard.The molecular docking results showed that the molecular docking affinity of key active components of Xuanbai Chengqi Decoction (宣白承气汤) and ACE2 was much lower than -2.0 Kcal /mol, and some of them such as β-sitosterol and Stigmasterol were similar to the affinity of recommended COVID-19 drugs.It indicated that the core active components in Xuanbai Chengqi Decoction (宣白承气汤) had well binding activity with ACE2 (See Table 2),

Figure 4.GO Enrichment Analysis of 36 Major Disease Targets

Figure 5.KEGG Pathway Enrichment Analysis of 36 Major Disease Target

Table 2.the Binding Energy of Key ActiveComponents of Xuanbai Chengqi Decoction(宣白承气汤) and Reported Effective Drugs to ACE2

DlSCUSSlON

As of now, no special drugs for COVID-19 have been found, and non-specific interventions have been taken to prevent serious complications.Compared with the long development period of new drugs,safe and reliable TCM shows greater advantages.For patients in the middle stage of COVID-19 with main symptoms of persistent fever or alternation of chills and fever, cough with less or yellow sputum,and abdominal distension or constipation, Xuanbai Chengqi Decoction (宣白承气汤) with addition and subtraction is recommended with the function of clearing lung, relieving asthma, purging heat and defecating according to Diagnosis and Treatment Scheme of COVID-19 (Trial Version 5).

This study is based on the research methods of network pharmacology, and explores the molecular mechanism for the treatment of COVID-19 by Xuanbai Chengqi Decoction (宣白承气汤).After analyzing the component-target network, 4 key active components in rude rhubarb (生大黄), almond powder (杏仁粉) and Pericarpium Trichosanthis (瓜蒌皮) were screened out, and they were compared with recommended antiviral drugs by molecular docking.It is worth noting that although raw gypsum (生石膏) didn't participate in the network construction, its effect in clearing heat, discharging fire, relieving restlessness and eliminating thirst cannot be ignored.Modern research shows that the main components of raw gypsum (生石膏) are CaSO4·2H2O and trace amounts of iron and magnesium which have antipyretic effects,enhancing phagocytic ability and shortening coagulation time[14]to relieve high fever in middle stage of COVID-19.

The latest research shows that ACE2 is a key receptor for both COVID-19 virus and SARS-CoV[15],and the binding of S-protein expressed by COVID-19 virus and ACE2 cause virus invasion and the disease[16].Blocking the binding is a prerequisite for preventing virus infections[15], and ACE2 receptors will become a key target for the development of antiviral drugs[17].The molecular docking results show that the four key active components can be effectively combined with ACE2, particularly betasitosterol and Stigmasterol, which have higher absolute value of binding energy than that of the recommended drugs Chloroquine diphosphate and Arbidol.In order to develop the promising ACE2 blockers in treatment of COVID-19, and whether the effect of Xuanbai Chengqi Decoction(宣白承气汤)against COVID-19 is depended on combination with ACE2 is needed to be verified.

KEGG enrichment analysis has shown that 36 main targets are enriched in 6 infectious diseases,4 of which are caused by virus (Kaposi's sarcomaassociated herpesvirus, human cytomegalovirus,Hepatitis C virus and Epstein-Barr virus), and involve multiple targets such as COX-2, PRKACA,CALM1, RXRA.In the Component-target network,COX-2 has the highest value of Degree and can bind 20 components at the same time.Research has shown that COX-2, as an important medium for inflammation after virus infection, participates in the replication process of various viruses such as hepatitis C virus[18-19]and human cytomegalovirus[20].It has a great significance in discovering for antivirus compounds with suppression of virus-induced COX-2 expression and Xuanbai Chengqi Decoction(宣白承气汤) may have an antivirus effect in treatment of COVID-19.In addition, the main targets are also concentrated on the signaling pathway of biological processes such as immune system, endocrine system, circulatory system,cardiovascular system, signal transduction, etc.

Inflammatory Storm is an important node for the conversion of mild to severe diseases for COVID-19 patients.After lung infection by COVID-19 virus through the ACE2 receptors,activated immunocyte releases a large number of cell factors, which lead to inflammatory storm by positive feedback loop mechanism.Multiple immune cells and exudates accumulate in lung, and then trigger respiratory distress.The enrichment analysis results show that the IL-17 signaling pathway, Th17 cell differentiation, platelet activation, and C-type lectin receptor signaling pathway play the important roles in the immune regulation of Xuanbai Chengqi Decoction (宣白承气汤).Th17 cell mediates inflammatory response and mainly secretes IL-17 which stimulates production of chemical inducers by vascular endothelial cells to help immune cells escape from blood to tissue.In recent years, many researches have shown that activated platelets,as inflammatory cells, can synthesize and release inflammatory mediator.It is an intermediate process in the formation and development of multiple diseases.Therefore, platelets can also be used as a target for the treatment of inflammation[21].C-type lectin receptors, as pattern recognition receptors in the inherent immune system, play a key role in the identification and regulation of respiratory pathogens[22].Xuanbai Chengqi Decoction (宣白承气汤) may affect Th17 cell and platelet activation,inhibit release of cell factors and activation of related receptors, so as to achieve the purpose of relieving over-inflammatory response.

CONCLUSlON

The result of molecular docking showed that the affinity of the key components such as betasitosterol and stigmasterol are similar to recommended medications for COVID-19.Xuanbai Chengqi Decoction (宣白承气汤) is indeed effective in the treatment of middle stage of COVID-19 and the effect may be related to the blocking of the binding of COVID-19 virus and ACE2, antivirus, and relieving inflammatory storm, etc., which provide a reference for study of molecular mechanism.

Financial support and sponsorship:

Wannan Medical College Youth Fund, No.WK201804

Conflicts of interest

There are no conflicts of interest.