Yan Zhou,Lei Fan,Du-Hong Xie,Xiao-Ying Han,Bo-Xuan Zhao,Yi Zhang,De-Kun Li,Ai-Chun Ju,Hai-Xia Ji,Ou Qiao,Xin-Yu Zhang,Wen-Zhe Wang,Chang-Xiao Liu,Wen-Yuan Gao*

1School of Pharmaceutical Science and Technology,Tianjin University,Tianjin 300072,China.2School of International Education,Tianjin University,Tianjin 300072,China.3Clinical School of Changsha Health Vocational College,Changsha 410100,China.4Tasly Pride Pharmaceutical Company Limited,Tianjin 300410,China.5Tianjin Pharmaceutical Research Institute,Tianjin 300072,China.

Abstract Background: 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.Yiqi Fumai lyophilized injection (YQFM) is a modern Chinese medicine preparation of the Shengmai decoction.The mechanism of its intervention at the molecular level in the severe stage of COVID-19 remains unclear.Therefore,it is necessary to investigate the mechanism of YQFM in the treatment of patients with severe COVID-19.Methods:The corresponding target genes of the main active ingredients in YQFM and COVID-19 were obtained by using multiple databases and literature retrieval.A protein-protein interaction network was constructed,and enrichment analysis of the target was performed using Cytoscape 3.8.1.Lastly,the docking of all the identified compounds with angiotensin-converting enzyme II was confirmed by applying molecular docking technology.Results:YQFM has anti-inflammatory effects on RAW267.4 macrophages.The main active compounds of YQFM are all effective anti-inflammatory agents,and these active compounds also show beneficial physiological functions,such as anti-oxidation,anti-bacterial,and anticancer activities.Gene Ontology analysis showed enrichment in the following pathways: lipopolysaccharides,interleukins,NF-kappa B,interleukin-2 and others,revealing that YQFM may play a role in the treatment of patients with severe COVID-19 through these pathways.Conclusion:YQFM has multicomponent and multitarget characteristics,and it could reduce lung injury by inhibiting inflammatory reactions,promoting antiviral activities,and regulating immunity,among other functions,to treat patients with severe COVID-19.

Keywords: network pharmacology;novel corona virus;traditional Chinese medicine;natural compounds;inflammation

Background

The coronary pneumonia caused by the novel coronavirus 2019(2019-nCoV) is referred to as coronavirus disease 2019 (COVID-19).The virus is rapidly transmitted and has a relatively long incubation period,which makes the disease especially difficult to counteract in terms of public health programs worldwide.According to the pathological results of the clinical studyPneumonia Diagnosis and Treatment Plan for Novel Coronavirus Infection(Trial Seventh Edition),2019-nCoV can cause degeneration and necrosis of cardiomyocytes,resulting in heart damage.Patients with COVID-19 and heart-related conditions are at high risk.

Yiqi Fumai lyophilized injection (YQFM),which comprises three drugs,namely,Ginseng Radix et RhizomaRubra,Schisandra chinensisandOphiopogon japonicus,originated from the classical prescription Shengmai decoction.Studies have shown that this traditional Chinese medicine compound preparation has significant curative effects in cardiovascular disease and plays an important role in the treatment of new coronary pneumonia.In the present study we used network pharmacology to investigate the mechanism of YQFM in the treatment of heart damage caused by 2019-nCoV.The results provide a basis for elucidating the mechanism of YQFM on severe systemic inflammatory response syndrome ormultiple organ failure caused by 2019-nCoV.

Materials and methods

YQFM ingredients collection and targets acquisition

YQFM consists of 3 Chinese herbal extracts ofGinseng Radix et RhizomaRubra,Schisandra chinensisandOphiopogon japonicus.In our study,specific criteria (oral bioavailability (OB) ≥ 30%,drug similarity (DL) ≥0.18),which indicates that these compounds have high bioavailability [1-2].We obtained the compounds ofGinseng Radix et RhizomaRubra andSchisandra chinensisin YQFM using of traditional Chinese medicine systems pharmacology (TCMSP)database (http://tcmspw.com/tcmsp.php) [3].Because it is impossible to search for a single Chinese medicine (Ophiopogon japonicus) active compounds in TCMSP,so we used the bioinformatics analysis tool formolecular mechanism of traditional Chinese medicine(BATMAN-TCM) database [4] (http://bionet.ncpsb.org/batman-tcm/)to search active compounds.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.

YQFM does not contain all the ingredients of these three traditional Chinese medicines due to the special property of injections.In the early stage,our research group and other research groups used ultra-fast liquid chromatography coupled with ion trap time-of-flight mass spectrometry and other techniques to identify the composition of YQFM for injection [5-7].The literature reports onGinseng Radix et RhizomaRubra,Schisandra chinensisandOphiopogon japonicuswere reviewed to determine its pharmacologically active ingredients,which have ameliorating effects on inflammation and myocardial damage[8-10].Screen high content,accurate biological activity and important pharmacological activity of compounds [11-16].Then use the Swiss target prediction database(http://www.swisstargetprediction.ch/) to perform target prediction for the collected compounds.

Collection of novel corona virus related targets

In order to construct the network,we next collected target genes related to novel corona virus disease.The potential target of novel corona virus is obtained from the GeneCards database(http://www.genecards.org/).GeneCards is a comprehensive biological information database that provides researchers with a platform and tools to effectively explore human genes,diseases,cells,and proteins[17].Using this database and“novel corona virus”as key words,we obtained the gene names of COVID-19 related targets.Then we used the online Venn diagram tool to identify the overlapping targets of YQFM and COVID-19,so as to obtain the core target of YQFM for COVID-19 treatment.The disease target and the YQFM active ingredient target are taken to intersect and a Venn diagram is made to obtain the potential target of YQFM for the treatment of new coronary pneumonia.

Protein-protein interaction (PPI) network construction

Proteins tend to interact to form macromolecular complexes to perform biological functions.PPI network analysis helps us to study the molecular mechanisms,biological processes and functions of disease treatment from a systematic perspective.Use the online data of the STRING 11.0 analysis platform(https://string-db.org/) to input the overlapping targets of the compound and the disease,set the minimum interaction score to 0.9,hide the individual targets,and obtain the PPI network diagram after analysis.In order to determine the core goal of YQFM for COVID-19 treatment,PPI data was imported into Cytoscape 3.8.1 software,and the closely connected functional modules were selected using the molecular complex detection (MCODE) plug-in.

Enrichment analysis

To further understand the main action process of the common targets,the list of the target protein genes was imported into the Metascape online database for enrichment analysis of Gene Ontology (GO).And the top 15 items in each group of the GO analysis results were visualized in bar chart.

Composition-target molecular docking

Studies have found that the key to the new coronavirus (SARS-CoV-2)infecting human cells is the combination of the coronavirus S protein and human angiotensin-converting enzyme II (ACE2) protein,which allows the virus to invade the human body and cause disease [18].The active ingredients in YQFM are docked with ACE2 protein.At the same time,drugs that may be effective against COVID-19 (remdesivir,lopinavir,ritonavir) are regarded as positive controls.First,we downloaded the pdb format file of the 3D structure of the protein from the RSCB PDB database (https://www.rcsb.org/),and performed hydrogenation,dehydration and generation in Pymol and Auto-Dock software.Grid file used for docking.Secondly,the structure files of active compound and positive control were downloaded from PubChem database (https://pubchem.ncbi.nlm.nih.gov/),and preprocessed with Chem3D and Pymol software.Finally,import the generated protein grid file and the pre-processed ligand into the Auto-Dock module to achieve higher precision docking.Use the docking score of the positive drug as a reference value to compare the docking results of the active compounds.

RAW267.4 cell anti-inflammatory activity experiment

Inflammation is a symptom or complication of many diseases.This new coronary pneumonia is mainly caused by inflammation.Inflammation is a defensive response produced by the body after being stimulated and damaged by various inflammatory factors.It can inactivate and clear these inflammatory factors and create an environment for tissue repair.Viruses and bacteria are the main mediators that induce inflammation [19-20].RAW264.7 macrophages are central cells that initiate the production of inflammatory mediators in the body [21].Regarding the regulation of inflammatory response,the central cell is activated macrophages,and it is also the main source of many inflammatory factors [22].Using Lipopolysaccharides (LPS)-induced RAW264.7 macrophages as a target to simulate the inflammatory response caused by the disease,the anti-inflammatory effect was explored by using YQFM drugs[23].

Remove RAW264.7 macrophages from liquid nitrogen and place them in a constant temperature CO2incubator after rapid recovery.When the cells are fully attached and grow to 80%,they are trypsinized and then subcultured at a ratio of 1:3.When it is fully attached and grows to 80%,it is used for the subsequent CCK-8 method to detect the cell activity of YQFM on RAW264.7 cells and the enzyme-linked immunosorbent assay method to determine the tumor necrosis factor α (TNF-α) and interleukin (IL)-1β in the cell supernatant,inducible nitric oxide synthase (iNOS),granulocyte macrophage colony stimulating factor (GM-CSF),IL-10 content.An enzyme immunoassay kit (Shanghai FANKEL Industrial Co.,Ltd.,Shanghai,China) was used to measure the content changes of TNF-α IL-1β,GM-CSF,IL-10 and iNOS in the serum according to the manufacturing scheme.In the CCK-8 experiment,after 3 passages,after the cells are stable,6 × 103cells are pipetted into a 96-well plate with 100 μL cell suspension per well to ensure that the cells in each well are even and the number is basically the same.Incubate in a constant temperature CO2incubator for 24 hours,then treat the cells with different concentrations of YQFM (0.625,1.25,2.5,5,10 mg/mL),incubate for 24 hours,add 10 μL of CCK-8 place it in the incubator and wait for 2 h,and measure the absorbance A at 450 nm with a microplate reader (Fnpmite zoopro TW,TECAN,Switzerland).When determining the factors in the cell supernatant,add the corresponding concentration of YQFM.After 24 hours of culture,add 100 μL of 10 μg/mL LPS to each group except the normal group.Continue to incubate in the box for 24 h,and then transfer the supernatant in the petri dish to the EP tube for later use.The contents of TNF-α,IL-1β,iNOS,GM-CSF and IL-10 were determined.

Results

Collection of active compounds of YQFM

Twelve main active compounds (four types of ginseng and eight types ofSchisandra) were isolated from TCMSP.In addition,information on the main active substances of 21 kinds ofOphiopogon japonicuscollected from BATMAN-TCM is shown in Table 1.Pharmacological studies have determined that the compounds in YQFM have anti-inflammatory,antitumor,anti-oxidant,antiviral,immunomodulatory,and other pharmacological effects [24-27].Therefore,we can speculate that these effects are also evident in the treatment of severe COVID-19.Based on our search for relevant literature,we screened 11 compounds with high content,accurate biological activity reports,and important pharmacological activities.The 11 screened compounds were subjected to Swiss target prediction.The target information for each target compound is shown in Figure 1 and Supplementary Information 1.

Collection of targets related to novel coronavirus

In total,791 targets related to COVID-19 (Supplementary Information 2) were mapped to 334 targets of YQFM,as shown in the Venn diagram in Figure 2,in which the blue circle represents the targets of YQFM and the yellow circle the targets of COVID-19;the 53 common targets in the intersection are the potential targets of YQFM in the treatment of COVID-19.

PPI network analysis and screening of key targets

Based on the potential targets of YQFM in the treatment of COVID-19,we used online data from the STRING 11.0 analysis platform to input53 target points,set the minimum interaction score to 0.9,hide the individual target points,and obtain the PPI network diagram after analysis.The PPI data were imported into Cytoscape software,and the MCODE plug-in was used to filter the two most closely connected functional modules,as shown in Figure 3.These two MCODE can be considered as key targets for YQFM in the treatment of patients with severe novel coronavirus pneumonia.These targets include TNF,synthetase (NOS2),vascular endothelial growth factor A (VEGFA),mitogen-activated protein kinase 14 (MAPK14),mitogen-activated protein kinase 6 (MAPK6),Janus kinase(JAK),and signal transducers and activators of transcription (STAT).

Table 1 Basic information of active compounds in YQFM

Figure 1 Target information of the target compound A) Ginsenoside Rg1,B)Ginsenoside Rb1,C) Schisandrol B,D) Schisandrin B

Figure 2 Disease target and YQFM active ingredient target. In total,791 targets related to COVID-19 were mapped to 334 action targets of YQFM.The blue circle represents the targets of YQFM,the yellow circle represents the targets of COVID-19,and the 53 common targets in the intersection part are the potential targets of YQFM in the treatment of COVID-19.YQFM,Yiqi Fumai lyophilized injection;COVID-19,coronavirus disease 2019.

Enrichment analysis of target

The Metascape online database was used to annotate the 53 key targets selected for GO biological function and to visually analyze the first 15 items.In Figure 4 and 5,the ordinate represents the enriched item and the abscissa represents the number of genes enriched in this item.The main enrichment items are signaling by interleukins,cell response to organic nitrogen compounds,response to lipopolysaccharide,transmembrane receptor protein tyrosine kinase signaling pathway,cytokine-mediated signaling pathway,and response to inorganic substances.

Results of molecular docking

In Table 2,we present the docking scores of the three positive drugs and their major active compounds.The pattern diagrams of the combination of ACE2 and YQFM compounds with the highest ranking can be seen in Figure 6.

Figure 3 A)PPI network diagram;B) The most compact functional module1;C) The most compact functional module2.These two MCODE can be considered key targets for YQFM in the treatment of patients with severe novel coronavirus pneumonia.YQFM,Yiqi Fumai lyophilized injection;MCODE,molecular complex detection;PPI,protein-protein interaction.

Figure 4 Summary of enrichment analysis in DisGeNET.The ordinate represents the enriched item,and the abscissa represents the number of genes enriched in this item.

Figure 5 Key targets are annotated with GO biological function.The main enrichment items are signaling by interleukins,cell response to organic nitrogen compounds,response to lipopolysaccharide,transmembrane receptor protein tyrosine kinase signaling pathway,cytokine-mediated signaling pathway,response to inorganic substances,etc.GO,Gene Ontology.

Figure 6 Pattern diagrams of the combination of important target and YQFM compounds with the highest ranking.YQFM,Yiqi Fumai lyophilized injection.

Because the A and B forms of schisandrin are similar,so are the A and B forms of schisandrol,the Rg1 and Rg3 forms of ginsenoside,and the A and B forms ofOphiopogon japonicus,we chose only one form in each of these pairs for the molecular docking tests.Schisandrin B,schisandrol B,and opogonanone A have three compounds whose absolute value of docking binding force is smaller than that of the positive drugs and does not reach the ideal value.The absolute value of the binding capacity of the four compounds,ginsenoside Rb1,ginsenoside Rg1,ginsenoside Re,and ophiopogonin D,was significantly higher than that of the positive control.In molecular docking,a binding energy of less than 0 indicates that ligand molecules can spontaneously bind to the receptormolecules,and the smaller the binding energy,the more stable the binding conformation.Therefore,we can conclude from the molecular docking results that most of the active compounds in YQFM had good binding activity with ACE2 and therefore play corresponding physiological roles.

Activity of YQFM on RAW264.7 cells

The CCK-8 method was used to observe changes in cell proliferation after the cells were treated with different concentrations of YQFM.The results in Figure 7 show that,compared with the normal group,YQFM (at any of the doses) had no significant effect on the proliferation of RAW264.7 macrophages (P＞0.05),indicating that the drug does not affect cell proliferation within this concentration range and there is no cell toxicity.

Enzyme-linked immunosorbent assay was used to observe changes in inflammatory factors in the cells.As shown in Figure 8,compared with the control group,the expression levels of the above-mentioned inflammatory factors in RAW264.7 macrophages induced by LPS in the model group were significantly increased(＊P＜0.05,＊＊P＜0.01,＊＊＊P＜0.001);compared with the model group,the YQFM treatment significantly down-regulated the expression of the inflammatory factors in the cell supernatant(TNF-α,iNOS,IL-1β,IL-6,GM-CSF)and up-regulated the expression of the above-mentioned anti-inflammatory factors (IL-10) in the cell supernatant (#P＜0.05,##P＜0.01,###P＜0.001).

Discussion

SARS-CoV-2 uses ACE2 and the transmembrane serine protease 2(TMPRSS2) receptor on macrophages to attach to cells and then transmits its genetic material (single-stranded RNA) into the cytoplasmic compartment of the infected cell.The RNA immediately begins to transcribe and amplify the early genes and then uses the host cell machinery [28] for its own genome amplification and packaging.Viral infection by alveolar and dendritic cells causes many cell deaths[29].The release of damage-associated molecular patterns (DAMPs)containing ATP,DNA,RNA,and apoptosis-related dot-like protein(ASC) oligomers further triggers the pro-inflammatory response of neighboring cells,including endothelial cells,macrophages,and epithelial cells [30].

The ACE2 target was selected formolecular docking with the core compounds of YQFM.The results showed that the binding energies of these targets with the core compounds of YQFM were small,indicating that the compounds in YQFM have good binding and strong interactions with the important target and suggesting that the target genes could play an important role in the pathogenesis of COVID-19.

According to a study [31] on the pathogenicity of COVID-19 based on the analysis of network pharmacology and the report [32] 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 decrease significantly but showed an over-activated state,consistent with the “inflammatory storm”.A major cause of severe COVID-19 symptoms is the excess release of inflammatory factors,which induces systemic inflammatory response syndrome.The pathogenesis of systemic inflammatory response syndrome is the“cytokine storm” [33]: changes occur in immune cells and inflammatory factors,such as leukocytes,which secrete many cytokines in tissues and organs,leading to acute respiratory distresssyndrome and/ormultiple organ dysfunction syndrome,ultimately leading to death [34].Given the anti-inflammatory activity of YQFM,it could be useful in the treatment of patients with severe COVID-19.In RAW264.7 cells,YQFM significantly downregulated the expression of inflammatory factors in the cell supernatant (TNF-α,iNOS,IL-1β,IL-6,and GM-CSF).

Table 2 Results of molecular docking

Figure 7 Toxicity of YQFM to RAW267.4 macrophages.YQFM,Yiqi Fumai lyophilized injection.

Figure 8 Effect of YQFM on A) IL-1β,B) TNF-α,C) GM-CSF,D)iNOS,E) IL-10 release of RAW267.4 macrophages. ＊P ＜0.05,＊＊P＜0.01,＊＊＊P ＜0.001 vs conrol,#P ＜0.05,##P ＜0.01,###P ＜0.001 vs model.YQFM,Yiqi Fumai lyophilized injection;TNF-α,tumor necrosis factor α;IL,interleukin;iNOS,inducible nitric oxide synthase;GM-CSF,granulocyte macrophage colony stimulating factor.

According to related literature,certain compounds of YQFM have anti-inflammatory effects.Ophiopogonin D has many pharmacological effects,such as anti-inflammatory and anti-tumor effects,and improves humoral and cellular immunity [35].Ophiopogonin D can achieve an ideal immune-enhancing effect by inducing stronger antibody responses and Th1/Th17-based CD4 T-cell immune responses in an methicillin-resistantStaphylococcus aureussepsis model.CD4 T cells are important immune cells in the human body that are involved in viral infection and inflammation [36].Thus,YQFM may treat patients with severe COVID-19 by enhancing immunity and inhibiting the inflammatory response.Ginsenosides have antiviral,anti-inflammatory,anticancer,antitumor,and other effects.Its immunomodulatory properties are determined by the expression of several cytokines (IL-1,IL-4,IL-10,IL-12,and TNF-α)[37],among which TNF-α is the target of YQFM used to treat COVID-19.Ginsenosides 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 YQFM are all effective anti-inflammatory agents,and these active compounds also show beneficial physiological functions,such as anti-oxidation,anti-bacterial,and anticancer activities.

GO analysis showed enrichment in the following pathways:lipopolysaccharides,interleukins,NF-kappa B,IL-2 and others,revealing that YQFM may play a role in the treatment of patients with severe COVID-19 through these pathways.

In summary,we have described that YQFM has multicomponent and multitarget characteristics,and we predicted that it could reduce lung injury by inhibiting inflammatory reactions,promoting antiviral activities,and regulating immunity,among other functions,to treat patients with severe COVID-19.It has been suggested that YQFM may affect IL-1β,iNOS,GM-CSF,TNF-α,and IL-2 targets,thereby inhibiting excessive inflammation in COVID-19 cases and reducing the risk of cytokine storm.The effect of YQFM involves a variety of biological processes,mainly signal transduction pathways,such as the TNF signal transduction pathway,toll-like receptor signal transduction pathway,and PI3K-Akt signal transduction pathway.We have shown that the therapeutic effect of YQFM on COVID-19 is manifested through multiple components,targets,and channels.However,the limitations in this study should be taken into consideration.Owing to the limitation of experimental conditions,when studying the mechanism of action of YQFM,we had no way to use SARS-CoV-2 to induce macrophage activation.Therefore,we used LPS to induce macrophage activation,which cannot completely mimic the macrophage activation caused by SARS-CoV-2.However,given the fact that LPS is a commonly used immune cell activator,we can conclude from our experimental results that treatment with YQFM inhibits the activation of macrophages and has a strong anti-inflammatory effect.