Investigation of Potential therapeutic value of Shuanghuanglian Chinese medicine based on network pharmacology for coronavirus pneumonia

Objective: The interaction network between coronavirus pneumonia and Shuanghuanglian was established to explore the potential therapeutic effect of the active ingredients of Shuanghuanglian on coronary virus pneumonia. Methods: Using the TCMSP database, the effective components of Shuanghuanglian were obtained by screening consistent with oral utilization (cid:0) drug similarity and blood-brain barrier permeability thresholds, and the drug target prediction was performed.The SARS treatment target mining was performed through the GeneCards database, and the two data sets of therapeutic target and drug target were analyzed and the intersection was screened, and the wayne map was drawn.The intersection genes were used as potential therapeutic targets. Cytoscape 3.6 software was used to build a drug active ingredient-therapeutic target interaction network and analyze the active ingredient-therapeutic target point network Degree parameters to nd important active ingredients and targets. Using DAVID and String databases to perform GO,KEGG enrichment analysis and protein interaction analysis on the intersection genes to nd out the potential signal pathway of Shuanghuanglian against SARS. Results: 43 effective components against SARS were screened, includingcoptisine (cid:0) Mandenol (cid:0) Ethyl linolenate (cid:0) phytouene (cid:0) wogonin (cid:0) FORSYTHINOL (cid:0) baicalein (cid:0) Moslosooavone (cid:0) Panicolin, etc.A total of 115 intersection genes with coronavirus pneumonia were screened as important treatment targets: based on the protein interaction network, Shuanghuanglian's therapeutic targets for diseases were signicantly enriched in 1763 GO and 133 KEGG signaling pathways; The main action pathways are: responses to steroid hormones and ketones, and development of the reproductive system, responses to lipopolysaccharides, Toxins, responses to bacteria and aging, and effects on epithelial cell proliferation. Conclusion: The active ingredients in Shuanghuanglian Chinese medicine compound can act on the disease-related target of coronavirus pneumonia and have potential therapeutic effects on coronavirus pneumonia. and key parameters of excretion, including drug similarity (DL), oral bioavailability (OB), blood-brain barrier transmission rate (BBB), etc. This study uses the TCMSP analysis platform to identify "Honeysuckle" , "Scutellaria baicalensis"," Forsythia "three Chinese medicine ingredients were searched as Herb name, using oral bioavailability (OB), Drug-likeness (DL) and cell penetration rate assessment (caco-2) as screening indicators, select OB ≥ 30%, DL ≥ 0.18, caco-2 ≥ 0.4as eligible active ingredients. The target corresponding to the active ingredient of Shuanghuanglian is obtained through the TCMSP database and


Introduction
Viral pneumonia is a common respiratory disease, but it is still the leading cause of death in young children and the elderly in developing countries. The second leading cause of death was upper respiratory tract infection in 2013, according to The Global Burden of Diseas Study [1]reports,with an agestandardized mortality of 41.7 (95% CI 37.1-44.1).And the incidence of pneumonia caused by upper respiratory infection is about 1.5-14 per 1,000 people per year [2][3][4] .The incidence of communityacquired pneumonia is U-shaped. It is common in children under 5 years old and adults over 65 years old, and more common in men than in women. In 2008,1.6 million children under 5 years old died of pneumonia [5]. Although Streptococcus pneumoniae is the main pathogen that causes communityacquired pneumonia worldwide [6,7,8] accounting for about 27.3%, but the virus is the most common cause of acute respiratory infections.
Unfortunately, so far no speci c therapies for SARS-CoV, MERS-CoV and other HCoV infections have been found. Symptomatic treatment and supportive treatment are the main means for patients with HCov infection. Although ribavirin and various types of interferon have become the most common treatment attempted by patients with SARS and MERS, The clinical effects are inconsistent when used alone to treat SARS.In vitro studies have shown that the combined use of IFN-β can make these two drugs have better antiviral activity ,but the clinical e cacy is still controversial (16).At present, the 2019-nCov new coronavirus epidemic outbreak in the world From the current reports in various places, the structure, function.clinical symptoms and disease progression of the emerging coronavirus are extremely similar to SARS-CoV.The current treatment measures for 2019-nCov pneumonia are mostly refered to the treatment of SARS, and 2019-nCov and SARS-CoV have high Sequence homology.Therefore, we take SARS-CoV as the main research object, and it is of great signi cance to nd effective and low-toxicity drugs to treat coronavirus pneumonia.
Shuanghuanglian is a traditional Chinese medicine compound, which has the functions of relieving wind and relieving phlegm, clearing heat and detoxifying, and is widely used in China for fever and cold. It is reported that Shuanghuanglian can inhibit the production of proin ammatory cytokines and chemokines and help ght in ammation and immunity Regulating effect in the rat model of chronic Pseudomonas aeruginosa pneumonia. Shuanghuanglian can increase the lung resistance of rats to chronic Pseudomonas aeruginosa. Shuanghuanglian is also an effective drug for upper respiratory tract infection and acute tonsillitis (17)(18)(19)(20). By analyzing the chemical components and the the action genes involved in the compound Chinese herbal medicine Shuanghuanglian and integrate them with viral Pneumonia related genes to build a drug-gene-disease interaction network. Through the analysis of the interaction network, we predict whether Shuanghuanglian can act on coronavirus pneumonia and treat it.
1 Materials And Methods

Acquisition of Active Components and Target Genes of Shuanghuanglian
In this study, the traditional Chinese medicine system pharmacology analysis platform (http://tcmspw.com/tcmsp.php) was used to search for "Hydronic honeysuckle", "Scutellaria baicalensis". "Forsythia" three avors of traditional Chinese medicine ingredients as Herb name, using oral bioavailability (OB ). Drug-like properties (DL) and cell penetration rate assessment (caco-2) are used as screening indicators, and OB ≥ 30%, DL ≥ 0.18, and caco-2 ≥ 0.4 are selected as the active ingredients that meet the conditions. The activity of Shuanghuanglian was obtained through the TCMSP database. The target corresponding to the component is entered into the uniprot database (https://www.uniprot.org/) to obtain the standard gene name of the drug target.
1.2 Screening of target genes for severe acute respiratory syndrome (Severe Acute Respiratory Syndromes, SARS) Search the Gene Cards (https://www.genecards.org) database for "Severe Acute Respiratory Syndromes" as a keyword to search for target genes related to severe acute respiratory syndrome.

Core target screening
Use Venny2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) online tool to draw a Wayne diagram of Shuanghuanglian related active ingredient target and Sars target to obtain its intersection target.
Investigate the targeting effect of the active ingredients of Shuanghuanglian on Sars target.

Component-target network construction and topology analysis
The compound-target interaction network and the Shuanghuanglian pharmacological component-target interaction network were constructed by Cytoscape 3.6 software. Traditional Chinese medicine components or targets are represented as nodes in the network, and their relationship is represented as edges.Using Net-workAnalyzer computational node degree (Degree) parameters in Cytoscape3.6 software to evaluate the importance of medicinal ingredients and targets.

Construction of Shuanghuanglian target protein interaction (PPI) network
Enter the obtained intersection target genes into the String database (http://string-db.org), set the species to "Homo sapiens", select the data with a con dence level higher than 0.7, and obtain the protein interaction information corresponding to the intersection genes, and use the online tool to draw the protein mutual aid network diagram.

Enrichment analysis and pathway analysis
The obtained intersection genes were fed into the DAVID database (https://david.ncifcrf.gov/) for gene ontology (GO) biological process enrichment analysis and KEGG pathway enrichment analysis (p < 0.05). GO and KEGG are displayed in the form of bubble charts through R-studio software.

6-methoxy avanone rivularin
According to the chemical composition information of Shuanghuanglian, its target targets were predicted, and 122 standard gene names corresponding to molecular targets were obtained, including The GeneCards (https://www.genecards.org) database was used to search for target genes related to severe acute respiratory syndrome using "Severe Acute Respiratory Syndromes" as keywords, and 7820 gene targets were obtained.

Core target screening
Using Venny2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) online tool to draw a Venn diagram of Shuanghuanglian related active ingredient target and Sars target to obtain the intersection target 115 (Figure 1

Component-target network construction and topology analysis
Compound-target interaction network and Shuanghuanglian medicinal ingredient-target interaction network were constructed by Cytoscape 3.6 software (see Figure 2). Traditional Chinese medicine ingredients or targets are represented as nodes in the network, a total of 159 between them. The connections are represented as edges, a total of 599.

Construction of Shuanghuanglian target protein interaction (PPI) network
Enter the obtained intersection target genes into the String database (http://string-db.org), set the species to "Homo sapiens", select the data with a con dence level higher than 0.7, and obtain the protein interaction information corresponding to the intersection genes and 5 cluster interaction networks(  Table 1. We noticed that some biological processes may be related to steroid hormones, ketones, lipids. polysaccharides, toxic substances, bacterial origin molecules and reproductive systems, aging and epithelial cell proliferation were related; Shuanghuanglian's resistance to Sars may be related to the membrane cell composition such as cell membranes and pre-synaptic membranes, which may be related to DNA transcription ,nuclearreceptor. steroid hormone receptors, G-protein-coupled amine receptors, or neurotransmitter receptor activity. Analysis of target KEGG pathway in acute respiratory distress syndrome DAVID database was used to perform KEGG signal pathway enrichment analysis on core targets in the acute respiratory distress syndrome network, and visualized after sorting by P value and Gene ratio( Figure  4B). In the analysis of KEGG signal pathway, the P value and GeneRatio of the top 20 signal pathways are shown in Table 2. The related parameters in the drawn bubble chart are shown in

Discussion
In the past decade or so, several previously undiscovered and con rmed viruses have appeared around the world, which cause respiratory diseases that are highly contagious and highly lethal. Especially new coronaviruses such as: SARS-CoV, H1N1-CoV, H5N1-CoV, MERS-CoV can cause tens of thousands of people worldwide infect, hundreds of people die, and because of its infectiousness, whenever pneumonia caused by this virus breaks out, in order to stop the infection sources and routes of transmission inevitably take various isolation measures, which often lead to people's panic and the abnormal functioning of society, causing inestimable losses. The histopathological changes of viral pneumonia vary, and may be related to viral infection and complications. Usually, interstitial pneumonia with lymphocytic in ltration can be seen in viral pneumonia [21]. And fatal cases of Sars and H5N1 were found to be characterized by alveolar damage, peeling of lung cells, edema and the formation of transparent membranes [ 22,23]. Syndrome are similar to those involving fever, cough, dyspnea and pneumonia. Infection may rapidly develop into acute respiratory distress syndrome, multiple organ failure, and death. Compared with SARS, MERS progresses to respiratory failure and causes is faster and causes acute kidney damage (27). What is more frightening is for new coronaviruses like SARS-CoV. MERS-CoV have so far lacked effective clinical antiviral drugs.These patients mainly received supportive treatment.Although during the SARS-MERS outbreak, ribavirin and in ammatory corticosteroids or IFNα with immunoglobulin.Thymosin in combination therapy (28)(29)(30)(31)(32)(33)(34), but these treatments have not been tested in clinical trials, their e cacy is di cult to assess and retrospective analysis has not draw an effective treatment combination. In addition, according to previous reports, it is unknown whether ribavirin has a greater bene t to the disease than it does to the body when it is used alone. However,IFN was proved to be effective against MERS-CoV (35)(36)(37) in vitro, and demonstrated in the MERS rhesus (38) model, the combined use of IFNα and ribovirus can alleviate disease, but it also brings such things as: fatigue, depression, anemia and other side effects.These greatly limit the use of IFNα as a rst-line drug (39)(40)(41). Therefore, it is particularly important to nd drugs that have inhibitory or therapeutic effects on such viral pneumonia and have fewer side effects. Since Professor Tu Youyou won the Nobel Prize in Physiology or Medicine for discovering the therapeutic effect of artemisinin on malaria in 2015, the effect of Chinese medicine on the disease has quickly attracted the attention of the medical community (42,43). In addition to artemisinin many traditional Chinese medicines have been shown to play an important role in the disease, for example: curcumin in turmeric has been shown to inhibit ovarian cancer, skin cancer, colorectal cancer, head and neck squamous cell carcinoma, and gastric cancer (44)(45)(46)(47)(48). Ginsenosides extracted from Fax Chen, Panax ginseng, and cinnamon (Cinnamomum cassia Presl) can inhibit cell proliferation, migration, angiogenesis, and anti-drug resistance (49-57) Plays anti-cancer effects in colorectal cancer, breast cancer, liver cancer and lung cancer (32)(33)(34)(35). Emodin can be isolated from rhubarb palm, Polygonum cuspidatum, Polygonum multi orum and Cassia seed, which has anti-in ammatory, anti-oxidant, prevent hepatic fat accumulation and DNA damage (58)(59)(60)(61)(62)(63)(64) has been shown to slow nasopharyngeal cancer, gallbladder cancer, lung cancer, liver cancer, colorectal cancer, oral cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, gastric and pancreatic cancer (65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77).
Shuanghuanglian is a traditional Chinese medicine compound consisting of honeysuckle, scutellaria baicalensis and forsythia, which has been widely used in China for a long time. Shuanghuanglian injection can inhibit the in ammation associated with viral encephalitis, and it has been reported that Shuanghuanglian can play anti-in ammatory and antioxidant roles in alveolar macrophages of rats. Shuanghuanglian injection can prevent H5N1 virus infection by inhibiting viral replication and reducing lung injury and can effectively exert anti-HAdV virus effects by inhibiting virus penetration (78)(79). We analyze the effective compounds in Shuanghuanglian traditional Chinese medicine compound, explore the proteins and corresponding genes that they act on, and compare the genes that act with SARS-related genes, and nd that these two types of genes have 151 common gene action targets. Then we used Shuanghuanglian active ingredients and disease target genes to build a drug-disease-gene interaction network map. We use the intersection target genes as potential target genes for Shuanghuanglian treatment of coronavirus pneumonia.These target genes were enriched by gene function and action pathways,and the results were found: the main pathways of action are the response to steroid hormones and ketones, the development of the reproductive system, the response to lipopolysaccharides. Toxins, the response to bacteria and aging, and the effect on epithelial cell proliferation. Coronavirus often causes viral pneumonia, destroys normal alveolar tissue, proliferates broblasts and aggregates a large number of extracellular matrices, resulting in pulmonary brosis, resulting in severe loss of lung function,Corticosteroids, anti-in ammatory and supportive treatments are currently for diseases caused by the kind of coronavirus like SARS-CoV, but it is well known that large doses and prolonged use of corticosteroids can cause obesity, immune system disorders, corticosteroid signs, electrolyte disorders, gastrointestinal ulcers, and severe necrosis of the femoral head. According to our research, the active    Shuanghuanglian active ingredient-drug gene target-disease gene target interaction network 3A: protein interaction network for drug gene targets acting on diseases 3B-F: 5 cluster interaction networks