Network analysis and molecular docking of the mechanism of Shengmai decoction in treating patients with severe novel coronavirus pneumonia

Jia-Ting Su,Xiao-Ling Li,Yu-Ge Huang,Hao-Wen Lin,Hui Luo,Lian-Xiang Luo＊

1The First Clinical College of Guangdong Medical University,Zhanjiang 524000,China;2Animal Experiment Center of Guangdong Medical University,Zhanjiang 524000,China;3Affiliated Hospital of Guangdong Medical University,Zhanjiang 524000,China;4The Marine Biomedical Research Institute,Guangdong Medical University,Zhanjiang 524023,China.

Abstract Background:Studies have shown that,Shengmai injection(Shengmai decoction)which has been included in the diagnosis and treatment of coronavirus disease 2019(COVID-19),is effective in the early treatment of patients with severe COVID-19.However,the mechanism of its intervention in severe stage of COVID-19 at molecular level is still unclear.Therefore,it is necessary to further explore the mechanism of Shengmai decoction in the treatment of patients with severe COVID-19 based on network pharmacology.Methods:The Traditional Chinese Medicine Systems Pharmacology,BATMAN,UniProt databases and the published literatures which contain the reported compounds that have therapeutic effects on COVID-19 were used to screen out the active ingredients and targets of Shengmai decoction,and the“drug-active compound-target”network was constructed.The GeneCards database was used to screen out the targets of COVID-19.The protein-protein interaction network map was constructed by mapping two genes,and the network of active ingredients,targets and disease was constructed using Cytoscape software.Thereafter,Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of the key targets were performed using the Metascape Site Maintenance website and David databases.The Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment of the mapping targets were analyzed.Results:The active component-target network contained 73 main compounds and 457 targets.The first five main active components(hydroquinone,guanosine,ophiopogon C,ophiopogon B,ophiopogon D)were obtained by topological analysis of 33 common targets.Using the Matthews correlation coefficient algorithm,the key targets included albumin,tumor necrosis factor,and cyclic adenosine response element binding.Compounds in Shengmai decoction were found to have good binding effect and strong interaction with caspase-8,caspase-3,apoptosis regulator Bcl-2,tumor necrosis factor,C-C motif chemokine 3,to inhibit the inflammatory response and improve lung injury.Additionally,31 biological processes were obtained by Gene Ontology enrichment,mainly related to lipopolysaccharide response and cytokine-mediated signal pathways,and 71 biological processes were obtained by Kyoto Encyclopedia of Genes and Genomes enrichment,mainly related to pulmonary tuberculosis,hepatitis B,and mitogen-activated protein kinase signal pathways.Conclusion:Shengmai decoction has multicomponent,multitarget and multichannel characteristics,which can provide an important theoretical basis to treat patients with severe COVID-19.

Keywords:Shengmai decoction,Novel coronavirus pneumonia,Network pharmacology,Anti-inflammation,Molecular docking

Background

The outbreak of the novel coronavirus infection caused by the 2019 novel coronavirus has posed a great threat to public health worldwide[1].According to the World Health Organization coronavirus disease 2019(COVID-19)situation report,as of 12:00 on Nov 27,2020,the epidemic of the novel coronavirus has affected 215 countries and regions worldwide.In addition to China,the total number of cumulative confirmed cases of COVID-19 has reached 6.140 million and 0.144 million deaths have been reported.Unfortunately,despite the efforts of health protection systems in each country to control the spread of the epidemic,the global epidemic of COVID-19 is still accelerating and upgrading.Moreover,there is no specific effective drug or vaccine available for the new coronavirus and the therapeutic effects of western treatment,such as antiviral medicine(redcevir,abidol,and ribavirin),immunotherapy and plasma exchange are not promising[2].Therefore,identifying effective drugs or vaccines for COVID-19 is urgent[3].Novel coronavirus pneumonia is an acute respiratory disease caused by COVID-19[4].It is highly infectious and can spread among people via spatter,contact and aerosol.It is widespread at all ages and has a wide circulation[5,6].Upon infection,the infected patients usually present fever,diarrhea,asthenia,cold and others as clinical manifestations.Patients with more severe conditions may have respiratory distress syndrome,septic shock and other symptoms[7,8].

According to the clinical features of novel coronavirus pneumonia and its seasonal,epidemic and infectious characteristics,combined with the local background of Wuhan,novel coronavirus pneumonia belongs to traditional Chinese seasonal diseases considering syndrome differentiation and disease differentiation of traditional Chinese Medicine.Thus,it is a pestilence.Traditional Chinese medicine has been treating epidemic diseases for thousands of years.Through a profound study of its etiology,pathogenesis,syndrome differentiation and treatment[9],we believe that from the theory of traditional Chinese medicine,it is of great significance to explore the ideas and prescriptions for the prevention and treatment of patients with severe COVID-19.Moreover,through many studies domestically and internationally,traditional Chinese medicine has made great achievements in the treatment of COVID-19[10].For example,Lianhua Qingwen(the approved number of China Food and Drug Administration:Z20040063)[11]and other Chinese medicine preparations or proprietary Chinese medicines have alleviated the condition of COVID-19 patients in varying degrees.Therefore,the National Health Commission and State Administration of Traditional Chinese Medicine of China have issued documents on treating coronavirus pneumonia from the perspective of Chinese medicine[12-14].

Shengmaiyin injection(Shengmai decoction)(the approved number of China Food and Drug Administration:Z32021056)is a Chinese herbal formula recommended for patients with severe COVID-19 in the treatment plan.Shengmai decoction is derived from the ancient prescription“Shengmai San”,which was first recorded in theOn the Perplexity of Internal and External Injurieswritten and also finished in Jin Dynasty[15].It comprises three drugs,namely,Hongshen(Red ginseng),Wuweizi(Schisandra chinensis)),and Maidong(Ophiopogon japonicus),which make Shengmai decoction an ancient prescription to tonify Qi(Qi,a concept based on traditional Chinese medicine theory,that is to say,it is an ever-moving fine substance with strong vitality and is widely regarded as the material basis of all things.)and Yin(Yin,a concept based on traditional Chinese medicine theory,that is a very important philosophical concept which opposes and relates to Yang and is an important aspect to maintain one's health.).[16,17].Studies have shown that,Shengmai decoction can play an active role in anti-oxidation,anti-inflammatory,improving immune function and microcirculation by regulating the metabolism of arginine and proline,taurine and taurine[18].In addition,Shengmai decoction has been reported to have a definite effect on stabilizing blood oxygen saturation and improving oxygenation level of patients with severe COVID-19[19].In short,Shengmai decoction can effectively prevent the deterioration of COVID-19 and plays an important role in the clinical treatment of critical diseases[19].

Network pharmacology comprehensively and objectively reveals the interaction among drugs,targets and diseases from the molecular and system levels[20],providing us a new research strategy for translating traditional Chinese medicine from an experience-based medicine to an evidence-based medicine system.To some extent,traditional Chinese medicine can improve the current drug research strategy[21,22].These advantages have allowed network pharmacology to be gradually developed into a breakthrough in the field of traditional Chinese medicine research[23].A case in point is that the mechanism of Lianhua Qingwen for the treatment of novel coronavirus pneumonia can provide theoretical support for clinical medication based on network pharmacology.All the results obtained from the perspective of network pharmacology are expected to lay a certain foundation for the treatment of COVID-19 and drug development[11].Therefore,our study used the methods of network pharmacology and molecular docking to explore the mechanism of Shengmai decoction in the treatment of patients with severe COVID-19,providing theoretical reference for further research and the development of effective drugs.

Methods

Screening of active compounds and acquisition of target proteins

In our study,with the specific criteria(oral bioavailability(OB)≥30% and drug likeness(DL)≥0.18,which indicate that the compounds have high bioavailability and are considered to be drug-like[24,25]),the Traditional Chinese Medicine Systems Pharmacology (TCMSP) [26] database(http://tcmspw.com/tcmsp.php)was used to obtain the chemical compounds ofRed ginsengandSchisandra chinensisin Shengmai decoction.Since the active compounds of the single drug(Ophiopogon japonicus)could not be retrieved in TCMSP,BATMAN-TCM database(http://bionet.ncpsb.org/batman-tcm/)[27]was used to search.The threshold“score cutoff 30”was set because of the principle of drug target similarity,and the mean value in BATMAN-TCM was used as the optimization conditions to screen out the main active compounds.It is worth noting that the paper[25,28-31]containing the reported compounds included in Shengmai decoction were also searched and collected to avoid deviations from the screening of active compounds using only OB and DL as screening conditions.These compounds were also considered to be the active compounds of Shengmai decoction in the treatment of COVID-19.Additionally,the related targets of active compounds in Shengmai decoction were also predicted in TCMSP and BATMAN-TCM,and the names of the target proteins were standardized with the UniProt database(https://www.UniProt.org/).

Construction of the drug-active compound-target network

To explore the pharmacodynamic mechanism of Shengmai decoction,the active compounds and gene list of the corresponding targets were introduced into Cytoscape 3.7.0 software for the drug-active compound-target network construction.The degree value of one node in the network represents the number of the node connected to the others in the network,which can reflect the importance of a network node.The greater the value of the node,the more likely it is to become the key node in the network[32].Thus,the first five important compounds were screened out by the order of the degree value.

Acquisition of the common target genes

The Human Gene Database (GeneCards)(https://www.genecards.org/)is a platform that can provide all known human genes in the genome,proteome,transcription,genetics and function.It can automatically extract relevant information from other multiple databases,a feature that is helpful for researchers to quickly understand the latest knowledge about genes[33,34].Using this database and“novel coronavirus pneumonia”as key words,we obtained the standard gene names of related targets.

The Venny 2.1(https://bioinfogp.cnb.csic.es/tools/venny/)online tool was used to map the active ingredient targets of Shengmai decoction and the targets of COVID-19 and to draw Venn diagrams.

Construction of the compound-common target network and analysis of its protein-protein interaction(PPI)network

The common targets acting on COVID-19 and their corresponding active compounds were imported into Cytoscape 3.7.0 to construct a compound-common target network.Additionally,these common targets were imported into the STRING platform(https://string-db.org/cgi/input.pl), with “Homo sapiens”and a confidence＞0.4 as the screening conditions.Thus,the PPI network was obtained and then the results saved in the TSV format was imported into Cytoscape.Topological analysis was performed using“network analysis”,and the selection of the key targets was performed using the Cytohubba plug-in.

Enrichment analysis of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway

To further understand the main action process of the common targets,the list of the target protein genes was imported into the Metascape Site Maintenance website(https://metascape.org/gp/index.html#/main/step1)for enrichment analysis of GO.Setting“Homo sapiens”as the limitation,a kappa score of 0.3 as the threshold value and aPvalue≤0.05 as the screening condition were the criteria in Metascape Site Maintenance.And the top 10 items in each group of the GO analysis results were visualized in bar chart.To further understand the role of these common targets in the signaling pathway,the common targets were imported into the DAVID database(https://david.ncifcrf.gov/)for enrichment analysis of the KEGG signaling pathway.Thus,the first 20 items were selected owing to the order of thePvalue,and the information on the pathways and corresponding targets were imported into Cytoscape 3.7.0 to build the target-pathway network.Additionally,the targets were imported into KEGG(https://www.genome.jp/kegg/)[35]to obtain the pathway map of target action.

Molecular docking

PubChem database(https://pubchem.ncbi.nlm.nih.gov/)and TCMSP database were used to obtain the SDF format of the compounds in Shengmai decoction.And then ChemOffice was used to construct the 3D structure of the compounds,which were saved as mol2 format and the energy of which was minimized.After that,the PDB format files of 3D structure of caspase-8(CASP8)(PDB ID:2bbr),tumor necrosis factor(TNF)(PDB ID:1d01),caspase-3(CASP3)(PDB ID:1qx3),C-C motif chemokine 3(CCL3)(PDB ID:2x6g)and apoptosis regulator Bcl-2(BCL2)(PDB ID:5jsn)were obtained from the PDB database(https://www.rcsb.org/),and the compounds of Shengmai decoction were connected with the above targets.With pymol2.3 used to remove the water molecules of the targets and Autodock4 used to add hydrogen,Autodock4 was used to save the compound and target in PDBQT format,and then Vina program was used for docking.Finally,pymol2.3 was used to draw the 3D map of the compound with the highest score of each target that has finished docking.

Evaluation of the disturbance of the network robustness of Shengmai decoction on COVID-19

Presently,many traditional Chinese medicine prescriptions are used in clinical treatment.Through the contrast of Yin and Yang(Yang,a concept based on traditional Chinese medicine theory,that is a very important philosophical concept which opposes and relates to Yin and is an important aspect to maintain one's health.),they can be used as the bases of traditional Chinese medicine clinical medication.The novel coronavirus pneumonia efficacy prediction analysis platform[36](TCMAntiCOVID-19 version 1.0)(http://tcmatcov.bbtcml.com/)was used to evaluate the robustness of the network of Chinese herbs against COVID-19.The positive control in the platform is the prescription that has been proven to affect the new crown,while the negative control is the prescription that has no effect on the new crown from the perspective of traditional Chinese medicine theory.In the platform,based on the COVID-19 severe disease network simulated by SARS,we set the interaction confidence score as 0.5,with Banxia Tianma Baizhu decoction(a traditional Chinese medicine prescription consisting of Banxia(Pinellia ternata(Thunb.)Breit.),Tianma(Rhizoma Gastrodiae),Fuling(Poria Cocos(Schw.)Wolf.),Juhong(Citri ExocarpiumRubrum), Baizhu (Atractylodes Macrocephala Koidz.)and Gancao(Glycyrrhiza uralensis Fisch))as the negative control and Qingfei Paidu decoction(a traditional Chinese medicine prescription consisting of Mahuang(Ephedra sinica Stapf),Zhigancao(Glycyrrhizae.),Xingren(Semen Armeniacae Amarum),Shigao(Gypsum Fibrosum),Guizhi(Ramulus Cinnamomi),Zexie(Rhizoma Alismatis), Zhuling (Polyporus),Atractylodes Macrocephala Koidz.,Poria Cocos(Schw.)Wolf.,Chaihu (RadixBupleuri), Huangqin(Radix Scutellariae),Jiangbanxia(Rhizoma Pinelliae),Shengjiang(Rhizoma Zingiberis Recens),Ziwan(Radix Asteris),Kuandonghua(Flos Farfarae),Shegan(Rhizoma Belamcandae),Shanyao(Radix Paeoniae Rubra),Chenpi(Pericarpium Citri Reticulatae),Huoxiang(Herba Pogostemonis),Zhishi(Fructus Aurantii Immaturus)and Xixin(Herba Asari))as the positive controls to evaluate the stability of the drug intervention disease network in Shengmai decoction.

Results

Collection of active compounds of Shengmai decoction

Forty-nine main active compounds(9Red ginsengand 40Schisandra chinensis)were collected from TCMSP,and the relevant information is shown in Supplementary Table 1.Additionally,the information of 22 main active compounds ofOphiopogon japonicuscollected from BATMAN-TCM is shown in Supplementary Table 2.

Pharmacological studies have defined that the compounds in Shengmai decoction have anti-inflammatory,anti-tumor,antioxidation,antiviral,immunoregulation and other pharmacological effects[37-40].Thus,we can guess that,in the process of treating patients with severe COVID-19,Shengmai decoction can play a related pharmacological role to achieve the effect of treating patients with severe COVID-19.The OB value and DL value of different compounds were different,indicating that the absorption degree of these chemical compounds in human body is different and they may affect the treatment of patients with severe COVID-19 with Shengmai decoction(Supplementary Table 1).The degree value represents the target number of compounds(Supplementary Table 2).The degree values of orchinol,guanosine and others were more prominent,that is,these active compounds have more targets.Thus,we believe that these chemical compounds may play an important role in the treatment of patients with severe COVID-19 with Shengmai decoction.

Analysis of the drug-active compound-target network of Shengmai decoction

The drug-active compound-target network was constructed using Cytoscape 3.7.0 software(Figure 1).The network graph directly reflects the interaction among drugs,compounds and targets.It contains 531 nodes and 292,140 edges:the 531 nodes include three herbal medicines(red V-shaped),71 active compounds(yellow octagon:9 active compounds ofRed ginseng;red circle:40 active compounds ofSchisandra chinensis;green rhombus:22 active compounds ofOphiopogon japonicus)and 457 target genes(pink hexagon).Degree can be used to reflect the number of links between a node and another in the network;this value can be used to track more important nodes.In this network,the higher is the value,the closer is the relationship between the active compounds or targets and other nodes and the more important are the roles they play in the treatment of patients with severe COVID-19 with Shengmai decoction.Thus,the top five active compounds with the highest degree are orchinol(99),guanosine(84),ophiopogon C(70),ophiopogon B(66)and ophiopogon D(66),highlighting the multicomponent and multitarget characteristics of Shengmai decoction in the treatment of severe COVID-19.

Figure 1 Drug-active compound-target gene network.

Construction of the compound-common target-disease network of Shengmai decoction and the screening of the key genes

In total,259 targets related to COVID-19 were mapped to 457 action targets of Shengmai decoction,as shown in the Venn diagram(Figure 2A).The pink circle represents the targets of Shengmai decoction,the green circle represents the targets of COVID-19,and the 33 common targets in the intersection part are the potential targets of Shengmai decoction in the treatment of COVID-19.The information on these 33 targets is shown in Supplementary Table 3.

Additionally,with the PPI network which can provide rigorous evaluation and integration of protein-protein interactions(including direct(physical)and indirect(functional)correlations)[41],we constructed a compound-common target-disease network(Figure 2B)comprising the disease COVID-19(light blue rhombus),33 common targets(orange and red V-shaped,yellow round),and 54 active compounds(green oval).The connection between the nodes indicates the interaction between the two.We performed topological analysis of the network,which revealed 88 nodes and 7,656 edges,with a clustering coefficient of 0.835 and each node comprising a mean of 20.625 adjacent targets.These data showed that the nodes were closely related.The Matthews correlation coefficient algorithm can rank the nodes according to the given concept of importance so that some important information can be found.Therefore,we used this algorithm to predict 10 core targets.In Figure 2B,the color of the 33 targets changes from yellow to red.The closer it is to red,the greater is the value of the algorithm.These 10 targets can be considered key targets for Shengmai decoction in the treatment of patients with severe novel coronavirus pneumonia.The 10 targets are albumin(ALB),TNF,cyclic adenosine response element binding protein(CREB1),CASP3,transforming growth factor β1(TGFB1),recombinant human basic fibroblast growth factor(FGF2),prostaglandin G/h synthetase 2(PTGS2),nitric oxide synthetase(NOS2),catalase(CAT)and peroxisome proliferator activated receptor γ(PPARG).Details are shown in Supplementary Table 4.

Figure 3 Bar chart of GO enrichment analysis of common targets.BP,biological processes;MF,molecular functions;CC,cell components.

Figure 4 Regulation of Shengmai decoction in some signaling pathways.(A)Regulation of Shengmai decoction in themitogen-activated protein kinase signaling pathway.(B)Regulation of Shengmai decoction in tuberculosis pathway.(C)Regulation of Shengmai decoction in apoptosis pathway.(D)Regulation of Shengmai decoction in TNF signaling pathway.(E)Regulation of Shengmai decoction in Hepatitis B pathway.(F)Regulation of Shengmai decoction in cancer-related pathways.

GO enrichment analysis of common targets of Shengmai decoction in the treatment of COVID-19

In this study,352 items were collected by GO enrichment analysis,including 264 biological processes(BP),57 molecular functions(MF)and 31 cell components(CC).As can be seen from Figure 3,the vertical coordinate representsPvalue,the horizontal coordinate represents GO terms.The main enrichment items of common targets in BP include lipopolysaccharide-mediated signaling pathway,response to drug,and inflammatory response.The main enrichment items of the common targets in CC include extracellular space,cytosol,extracellular region,and external side of plasma membrane.And the main enrichment items of common targets in MF are cytokine activity,identical protein binding,and protein binding.

KEGG enrichment analysis of common targets of Shengmai decoction in the treatment of COVID-19

Seventy signaling pathways(P＜0.05)were obtained after KEGG enrichment analysis of common targets of Shengmai decoction in the treatment of COVID-19,whose relevant information is shown in Supplementary Table 5.The two pathways with the largest number of enriched targets are pathways in cancer and tuberculosis pathway.The former suggests that,when the human body is infected with COVID-19,it may have a significant impact on the treatment of diseases such as cancer[42],while Shengmai decoction may play a role in the treatment of these diseases.The latter shows that Shengmai decoction can play a role in the treatment of related lung diseases,suggesting that Shengmai decoction has an effect on patients with severe COVID-19 in theory.Furthermore,according to relevant studies[43],the mitogen-activated protein kinase(MAPK)signaling pathway in the KEGG analysis results is related to immune inflammation in the lung.Therefore,we focused much on the expression of targets in this pathway(Figure 4A).As can be seen,the targets of COVID-19 can activate the expression of TNF and other inflammatory factors in various ways,while Shengmai decoction can directly or indirectly affect these signaling pathways and other biological processes by blocking the expression of some factors.Thus,Shengmai decoction may play a role in the treatment of patients with severe COVID-19 through this mechanism.

It is noteworthy that,charts of KEGG analysis of the common target proteins associated with COVID-19 are shown in Figure 5.On the one hand,the results of the KEGG analysis of the 33 targets is shown in the form of the bubble graph(Figure 5A).As is shown,the vertical coordinate is the pathway name,the abscissa is the enrichment degree,the size of bubble represents the number of targets in the path,and the color of bubbles represents the size ofPvalue.These KEGG pathways mainly involve tuberculosis,apoptosis,TNF signaling pathway,Hepatitis B,pathways in cancer and so on.The pathway processes of these pathways are also shown in order in Figure 4(A-F).In Figure 4,because the pink squares are the common targets,the pink squares and yellow squares belong to COVID-19 target genes,and the pink squares and orange squares belong to target genes of Shengmai decoction.Other related information can also be seen from the legend in the figure.On another hand,the chord diagram(Figure 5B)is constructed with the first 20 KEGG paths and the corresponding targets,which shows the correlation between the two.The light gray squares represent the associated targets,the remaining colored squares represent the main pathways obtained from KEGG enrichment analysis,the connection between squares represents the interaction between the main path and target,and the width of a square represents the degree of connection to other squares.

Results of molecular docking

The results have shown that,the binding energy of Shengmai decoction is lower than-30.21 kJ/mol,so the whole docking result of Shengmai decoction is satisfactory.In molecular docking,that the binding energy less than 0 indicates that ligand molecules can spontaneously bind to the receptor molecules,and the smaller the binding energy,the more stable the binding conformation.Therefore,it can be concluded that the core active compounds in Shengmai decoction have good binding activity with the receptor molecules,so as to play corresponding physiological roles.We used Figure 6(A-E)to respectively show the pattern diagrams of the combination of CASP8,CASP3,BCL2,TNF,CCL3 and Shengmai decoction compounds with the highest ranking.Among them,CASP8,TNF and CCL3 were all connected with 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxa n-2-yl)oxychromen-4-one:the binding energy of CASP8 is-41.25 kJ/mol,and three hydrogen bonds are formed(Figure 6A);two hydrogen bonds are formed after TNF docking,and the binding energy is-39.66 kJ/mol(Figure 6B);three hydrogen bonds are formed after CCL3 docking,and the binding energy is-40.66 kJ/mol(Figure 6C).CASP3 and BCL2 were both combined with(-)-Gomisin L1:the binding of CASP3 formed six hydrogen bonds with binding energy of-47.35 kJ/mol(Figure 6D),and binding energy of BCL2 is-38.66 kJ/mol(Figure 6E).Therefore,compounds in Shengmai decoction has good binding effect and strong interaction with these 5 important targets,suggesting that these 5 target genes included in targets of Shengmai decoction could play an important role in COVID-19 pathogenesis.

Figure 5 Charts of Kyoto Encyclopedia of Genes and Genomes analysis of the common target proteins associated with coronavirus disease 2019.(A)Bubble graph of the first 20 KEGG paths.(B)Chord diagram of the first 20 KEGG paths and the corresponding targets.

Figure 6 Pattern diagrams of the combination of 5 important targets and Shengmai decoction compounds with the highest ranking.(A)Pattern diagram of caspase-8 and 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl)oxychromen-4-one.(B)Pattern diagram of tumor necrosis factor and 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl)oxychromen-4-one.(C)Pattern diagram of C-C motif chemokine 3 and 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-y l)oxychromen-4-one.(D)Pattern diagram of caspase-3 and(-)-Gomisin L1.(E)Pattern diagram of apoptosis regulator Bcl-2 and(-)-Gomisin L1.

Analysis of the network robustness disturbance of Shengmai decoction in COVID-19

The result of Yin and Yang analysis is shown in Supplementary Table 6.The smaller the average shortest path is,the greater the average connectivity,connectivity centrality and compactness centrality are,and the more robust the network is.Based on the above four values,the total disturbance rate evaluates the disturbance effect of drugs on the network from the overall level.The higher the value is,the higher the damage degree of drugs to the network stability is,and the greater the influence of drugs is.Therefore,from the table,the total disturbance score of Shengmai decoction to COVID-19 network robustness is greater than that of Banxia Tianma Baizhu decoction but inferior to Qingfei Paidu decoction.This finding may explain why Shengmai decoction is currently recommended only for patients with severe COVID-19,and Qingfei Paidu decoction is almost suitable for different degrees of patients[12-14].

Discussion

Previous studies[44,45]have demonstrated that the pathogenesis of novel coronavirus pneumonia is highly complex,and no clinically effective treatment is available to date.By contrast,traditional Chinese medicine can achieve a considerable curative effect for both suspected cases in the medical observation period and individuals who have been diagnosed with serious or critical basic diseases.Therefore,in the national diagnosis and treatment program,Shengmaiyin injection(Shengmai decoction)and other traditional Chinese medicine are recommended for different treatment stages.However,the mechanism of Shengmai decoction in the treatment of severe COVID-19 patients is unclear.Thus,in this work,we used the methods and technologies of network pharmacology and molecular docking to analyze and explore the mechanism of Shengmai decoction in the treatment of patients with severe COVID-19.

According to the study[46]on the pathogenicity of COVID-19 based on the analysis of network pharmacology and the report[47]on the pathological anatomy of the first patient who died from COVID-19 in China,the number of lymphocytes in the peripheral blood of severe patients decreased significantly but showed an over activated state,consistent with the“inflammatory storm”.Therefore,an important reason for the occurrence of patients with severe COVID-19 is the over release of inflammatory factors,which induce systemic inflammatory response syndrome(SIRS).The pathogenesis of SIRS is the“cytokine storm”[48]:changes occur in immune cells and inflammatory factors,such as leukocytes,which secrete a large number of cytokines in tissues and organs,leading to acute respiratory distress syndrome and/or multiple organ dysfunction syndrome,and then leads to death[49].Therefore,Shengmai decoction may play an anti-inflammatory role in the treatment of patients with severe COVID-19.

According to related literature,we found that the certain compounds of Shengmai decoction have anti-inflammatory effects.Ophiopogonin D has many pharmacological effects,such as anti-inflammatory and anti-tumor effects,improving humoral immunity and cellular immunity[50].Ophiopogonin D can achieve the ideal immune-enhancing effect by inducing stronger antibody responses and Th1/Th17-biased CD4 T-cell immune responses through the MRSA sepsis model.CD4 T cells are very important immune cells in the human body that are related to virus infection and inflammation[51].Thus,Shengmai decoction may treat patients with severe COVID-19 by enhancing the body’s immunity and inhibiting the inflammatory response.Guanosine and its derivatives have anti-tumor and anti-proliferation effects[52].Moreover,some studies have shown that the new guanosine derivatives have a great prospect in the treatment of Middle East Respiratory Syndrome coronavirus[53].Both Middle East Respiratory Syndrome coronavirus and novel coronavirus pneumonias are caused by coronaviruses,indicating that guanosine and its derivatives may have great potential in the treatment of severe novel coronavirus pneumonia. N-trans-feruloyltyramine has demonstrated enhanced antioxidant,antibacterial,anti-inflammatory, anti-melanin production,anti-cancer and anti-inflammatory effects[54,55].Ginsenoside RF can alleviate neuropathic pain and related depression,restore the balance between pro-inflammatory and anti-inflammatory cytokines[56],and has anti-injury and anti-inflammatory effects[57].Diosgenin has antiviral,anti-inflammatory,hypoglycemic,anticancer,antitumor and other effects.Its immunomodulatory properties are determined by detecting the expression of several cytokines(interleukin-1, interleukin-4, interleukin-10,interleukin-12,and TNF-α)[58],among which,TNF-α is the target of Shengmai decoction used to treat COVID-19.Therefore,diosgenin may play an anti-inflammatory role by regulating TNF-α to alleviate the condition of patients with severe COVID-19.In summary,the main active compounds of Shengmai decoction are all good anti-inflammatory agents,and these active compounds also show good physiological functions such as antioxidation,anti-bacterial,anti-melanin production and anti-cancer activities.According to a previous study[59],patients with severe COVID-19 often have other symptoms,such as nervous system imbalance;therefore,Shengmai decoction can play a holistic and dialectical role in the treatment of patients with severe COVID-19.

For one thing,according to the above,most compounds of Shengmai decoction have anti-inflammatory effect;for another,according to the related paper,CASP8[60],CASP3[61],CCL3[62,63],BCL2[64,65],TNF[66-69]are common targets related to inflammatory process,and therefore,these 5 targets were selected for molecular docking with the core compounds of Shengmai decoction.The results showed that the binding energy of these targets with the core compounds of Shengmai decoction were small,indicating that the compounds in Shengmai decoction have good binding and strong interaction with the five important targets and suggesting that these five target genes could play an important role in the pathogenesis of COVID-19.Caspase-8(CASP8)plays an important role in infectious inflammation[70].It can produce inflammatory factors such as interleukin-1β and interleukin-18 by Fas activation.Under the stimulation of LPS,it can also regulate the activation of the NLRP3 inflammatory corpuscle.Additionally,it can activate almost all downstream apoptosis cascade reactions and induce the cell to undergo apoptosis[60].Therefore, Shengmai decoction can inhibit inflammation and reduce lung injury by regulating CASP8 and other genes to treat patients with severe COVID-19 effectively.CASP3 is the main effector in the apoptosis cascade and is the most critical target to carry out the physiological process of apoptosis[60].Additionally,CASP3 plays an important role in the process of inflammation[61].CCL3 is a member of the CC subfamily of chemokines,which demonstrate strong chemotaxis to neutrophils,monocytes,mast cells,dendritic cells,NK cells,and T and B lymphocytes,among others[62].While chemokines and their receptors play an important role in the pathogenesis of acute lung injury and acute respiratory distress syndrome,increased CCL3 expression aggravates the pulmonary inflammatory response[63].The BCL2 family can determine the apoptosis of platelets through the balance between pro-apoptotic factors and pro-survival factors[64],while the accumulation and activation of platelets can maintain the balance of the lung defense and integrity.Otherwise,the reduction in the number of platelets will lead to the risk of serious infection[65].Therefore,the analysis suggests that Shengmai decoction can be used to treat patients with severe COVID-19 in various ways.Some studies have shown that coronavirus can inhibit the apoptosis of infected cells through its E protein;thus,the virus can complete physiological processes such as virus assembly in the host cell[71].Therefore,Shengmai decoction can induce the apoptosis of infected cells by regulating CASP3 and other apoptosis-related genes to prevent the release and transmission of the virus.Shengmai decoction can also regulate the expression of CASP8,BCL2,CCL3 and other genes to inhibit the inflammatory response,improve lung injury,and alleviate the symptoms of patients with severe COVID-19.

Additionally,TNF is not only directly related to tumors but also has the function of anti-infection,inhibiting virus replication and killing virus-infected cells[72].It is noteworthy that the TNF target is closely related to the lipopolysaccharide pathway in the result of GO enrichment analysis.According to the relevant studies, two mechanisms of the lipopolysaccharide pathway exist.In one,LPS upregulates the expression of interleukin-1β,TNF and other factors after stimulating pulmonary vascular endothelial cells,and it can work together with TNF-α to increase vascular endothelial permeability,leading to the aggregation of inflammatory factors in the pulmonary interstitium and causing and aggravating lung injury[66].TNF-α and other factors belong to the targets of Shengmai decoction in the treatment of COVID-19.Therefore,for this mechanism,the treatment of Shengmai decoction in patients with severe COVID-19 may achieve effective treatment by inhibiting the expression of TNF-α and other proinflammatory factors,reducing the damage to the lung and its tissues.However,some studies have shown that LPS can induce lung tissue to produce a large number of reactive oxygen species[67],which can induce macrophages and bronchial epithelial cells to release inflammatory factors,and induce the inflammatory response in lung tissue[68].Therefore,inhibiting this biological process may be one way that Shengmai decoction works in the treatment of patients with severe COVID-19.

KEGG enrichment analysis results were mainly involved in the following pathways:pathways in cancer,tuberculosis,hepatitis B,the MAPK signaling pathway and others,revealing that Shengmai decoction may play a role in the treatment of patients with severe COVID-19 through these pathways.The MAPK signaling pathway plays an important role in the regulation of cell proliferation,differentiation,the inflammatory response and other processes.According to previous studies[73],the MAPK signaling pathway may play an anti-inflammatory role by inhibiting the expression of TNF-α and other proinflammatory factors.

Conclusion

In summary,in this study,we described that Shengmai decoction has multicomponent and multitarget characteristics,and we predicted that it can reduce lung injury by inhibiting inflammatory reactions,promoting antiviral activities,and regulating immunity,among other functions,to treat patients with severe COVID-19.However,few pharmacological studies have investigated Shengmai decoction;thus,the results of this study reflect the overall regulatory effect of Shengmai decoction on the body at a certain level and can provide certain theoretical support for the treatment of patients with severe COVID-19.Due to the limitations of network pharmacology,it is necessary to verify the main active compounds,key targets and pathways in the later stage to provide theoretical and experimental bases for the development of drugs.