Network pharmacology based investigation into the bioactive ingredients and molecular mechanisms of QingFeiPaiDu Decoction treating COVID-19


 Objective: To study the QingFeiPaiDu Decoction (QFPDD) in the treatment of Corona Virus Disease 2019 (COVID-19) bioactive ingredient and its potential mechanism. Methods: Combined with the clinical symptoms of COVID-19 patients, a "component-target-disease" network model was constructed based on the network pharmacology method, and potential active components, targets and molecular mechanisms of QFPDD for COVID-19 were screened out through topology parameter analysis.Results: We collected 376 active ingredients of QFPDD from the database, and 18833 potential anti-novel coronaviruses (SARS-CoV-2) targets were analyzed and screened. The principal targets involved PIK3CA, PIK3R1, APP, SRC, MAPK1, MAPK3, AKT1, HSP90AA1, EP300, CDK1, etc. We obtained 574 GO entries by Gene Ontology enrichment analysis and obtained 214 signal pathways with P<0.05 by KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. Among them, the antiviral biological processes of QFPDD included a cellular response to nitrogen compound, protein kinase activity, membrane raft, etc. Pathways involved in the regulation include Pathways in cancer, Endocrine resistance, PI3K-Akt signaling pathway, Proteoglycans in cancer, etc. Molecular docking results showed that the core ingredients of QFPDD have a better affinity to the 2019-nCoV 3CL hydrolytic enzyme (Mpro) and angiotensin-converting enzyme 2 (ACE2). Conclusion: Through network pharmacology research and molecular docking verification, this paper preliminarily explored the potential molecular mechanism and relevant active ingredients of QFPDD playing an anti-SARS-CoV-2 role, providing a reference for the further development and utilization of QFPDD and the development of new specific antiviral drugs.


Screening of main active ingredients and targets of QFPDD
Cytoscape software (version 3.7.2) was used to construct the ingredient-target network diagram [10][11] , and the Cytoscape tool was used to calculate the network topology parameters. The main active ingredients and targets of QFPDD were screened according to the average degree of freedom and the maximum degree of freedom [12] .

Construction and analysis of "QFPDD-COVID-19-Target" network
The main active ingredient targets and disease targets were established in pairs to obtain the potential targets of QFPDD in the treatment of COVID-19. The obtained data were imported into Cytoscape software (version 3.7.2) for visual processing to construct the "QFPDD-COVID-19-Target" network [11,18] . Topological parameters of the constructed network graph were analyzed to explore the interaction of different traditional Chinese medicines in QFPDD in the treatment of COVID-19.

Construction and analysis of protein-protein interaction
The potential targets of QFPDD in the treatment of COVID-19 were imported into the String database (https://string-db.org) [19][20][21] , and the protein-protein interaction (PPI) network of potential targets was obtained, and the interaction targets with confidence score≥0.9 were screened. For further analysis of the interaction between target and screening key targets, the income data import Cytoscape software version (3.7.2) topology parameters analysis, including each node degree, betweenness centrality, closeness centrality, and average shortest path length, with their average as the filter to filter the key targets.

GO enrichment analysis and KEGG pathway analysis
Through Metascape website (http://metascape.org) [22] to get QFPDD potential targets for the treatment of COVID-19 to GO (Gene Ontology) enrichment analysis, at the same time with DAVID website (https://david.ncifcrf.gov/) [23] and KEGG database (Kyoko Encyclopedia of Genes and Genomes) [24][25] signaling pathway enrichment analysis. The potential targets were input, and the species were restricted to be human. The analysis results were screened with P≤0.05 as the index. and Python script were used for molecular docking, and the sequence was sorted according to the optimal binding energy (affinity) of each ligand compound.

Main active ingredients and targets of QFPDD
Although many ingredients of QFPDD are helpful in the treatment of COVID-19, the study of the main ingredients of TCM compounds is an effective way to elucidate the mechanism of its pharmacological action. In this study, network pharmacology was used to explore its core ingredients. We constructed the ingredient-target network ingredients and corresponding targets of 20 Chinese medicines were screened from the ingredient-target perspective, as shown in Table 1 Table

Targets of significant symptoms of COVID-19
To explore the molecular mechanism of the drug's effect, 9,624 targets for fever, cough, and fatigue were collected from the database, details of which are listed in supplement 1(sheet 3). Chinese traditional medicines acting on the same target SHBG. There are four were mutual targets of 13 kinds of Chinese medicines, nine were common targets of 12 Chinese medicines, two were common targets of 11 Chinese medicines, 13 were common targets of 10 Chinese medicines, 14 were common targets of nine Chinese medicines, 14 were common targets of eight kinds of traditional Chinese medicine, 14 were common targets of seven kinds of traditional Chinese medicine, and 14 were common targets of six kinds of traditional Chinese medicine, which fully illustrates the "multi-ingredient-multi-target" of traditional Chinese medicine Point feature.

Fig.3 QFPDD-COVID-19-Target Network
(the rhomboid node represents the traditional Chinese medicine, the circular node represents the target, and n represents the number of types of targets distributed in the same circle acting on the same amount of traditional Chinese medicine.)

PPI network
The potential targets of QFPDD in the treatment of COVID-19 were analyzed through the String database, and the PPI network of protein interactions was obtained ( Fig.4).

Fig.4 Protein-protein interaction (PPI) network of potential targets of COVID-19 treated with QFPDD
Through the calculation of the degree of each node, betweenness centrality, closeness centrality, and average shortest path length selected 24 key targets, the target information according to the degree of descending order, are shown in Table 2.  Signaling pathways [26][27][28][29][30][31][32][33][34][35][36][37] have been reported to be associated with pneumonia. The top 20 pathways of all signaling pathways are shown in Fig.4(E). One hundred ninety-six signaling pathways with P<0.01 were screened, and a total of 262 genes were annotated to these pathways. The correlation map of these genes was plotted to obtain the correlation of genes on different pathways, as shown in Fig.6. pathway. Rap1 signaling pathway has been found to play an essential role in the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS), and the regulation of the Rap1 signaling pathway may become a new target for the treatment of ALI/ARDS [38] . The schematic diagram of 24 critical targets involved in the treatment of COVID-19 is shown in Fig.8.   Table 3. To further analyze the interaction between ingredient and protein, 2D and 3D molecular docking diagrams of interaction between the ingredient and 3CLpro and ACE2 were constructed, respectively. The molecular docking diagrams of the ingredient with the best results were selected, as shown in Fig.9. Due to hydrophobic Pi-Alkyl, 3'-Methoxyglabridin parent nucleus mainly binds to amino acid residues CYS145, MET165, and PRO168 of 3CLpro. Meanwhile, hydrogen bonds are formed between phenolic hydroxyl on parent nucleus and amino acid residues GLY143, LEU141, and CYS145. Due to hydrophobic Pi-Alkyl, Alisol,b,23-acetate parent nucleus combines with ACE2 amino acid residues LEU85 and HIS15, and hydroxyl and carbonyl groups on the parent nucleus form hydrogen bonds with amino acid residues GLN101 and ASN103. These interactions increase the binding of molecules to proteins.  The positive nucleic acid negative conversion rate is also significantly improved.

Table2 Topological analysis of QFPDD-COVID-19-Target network
Therefore, it is highly feasible to find and develop anti-SARS-CoV-2 active ingredients from this method.
Network pharmacology is based on the overall and systematic interaction of drugs, targets, and diseases, and uses complex network models to express and analyze the pharmacological properties of the research objects [39] . With the research and development of network pharmacology in the field of traditional Chinese medicine, it provides a new perspective and thinking for the systematic study of the complex ingredients of traditional Chinese medicine [40][41][42] . Therefore, in this study, we constructed and analyzed the target network map of QFPDD and COVID-19, and used these targets for functional enrichment and signal pathway analysis to reveal the potential molecular mechanism of QFPDD in the treatment of COVID-19, and found the main active ingredients and potential target genes of the treatment.

Main active ingredients
The SARS-CoV-2 isolated from Wuhan is the seventh coronavirus to infect humans discovered so far [43][44] . Rao Zihe/Yang Haitao's research group from Shanghai university of science and technology determined the high-resolution crystal three-dimensional structure of the 2019-nCoV 3CLpro. Previous studies have confirmed that SARS-CoV-2, like SARS-CoV, enters human cells through ACE2 and causes infection [45] . Therefore, the ingredient closely related to the hydrolase and  [46] . The cancer-related changes in the PIK3R1 gene will change the regulatory subunit so that it can no longer control the activity of PI3K, thus significantly increasing PI3K signaling. The increase of PI3K signaling seems to promote the uncontrolled cell growth and division characteristic of cancerous tumors [47] . Amyloid precursor protein (APP) is also considered to be one of the molecules involved in the proliferation and invasion of a variety of human malignant tumor cells, and its phosphorylated form (p-APP) is considered to be an effective prognostic factor for patients with Ad and Sq lung cancer [48] . App42-beta activates mononuclear phagocytes in the brain, causing inflammation and promoting Tau aggregation and TPK II mediated phosphorylation.
Laser microdissection neurons extracted from pre-plague APP transgenic rats can produce a variety of effective immune factors at transcription and protein levels [49] .

SRC regulates many important mechanisms in both normal and cancer cells and is
overexpressed in various tumors, including lung cancer, representing a potential target for cancer treatment [50] . MAPK is considered to be an important regulator of the inflammatory response in ALI. MAPK1 can eliminate LPS induced inflammatory injury of A549 cells and has a protective effect on lung injury in ALI mice [51] .
Abnormal expression of MAPK3 is related to invasion, metastasis, and drug resistance of various tumor cells. Overexpression of mir-129 can inhibit the expression of MAPK3, inhibit cell proliferation, induce cell apoptosis, and reduce cisplatin resistance [52] . The AKT1 gene provides a protein specification for the preparation of AKT1 kinase. The protein is found in all cell types in the body and plays a crucial role in many signaling pathways. AKT1 kinase helps regulate cell growth and division, cell survival, and apoptosis. AKT1 gene belongs to a class of genes called oncogenes, which may cause normal cell canceration after mutation [53][54] .
The expression of HSP90AA1 is related to a variety of physiological functions, not only the heat shock response [55] . HSP90AA1 and other genes regulate the pathogenesis of squamous cell lung cancer (SQCLC) caused by chronic obstructive pulmonary disease (COPD). These genes can be used as potential therapeutic targets for the treatment of patients with COPD associated SQCLC [56] . EP300 is a histone acetyltransferase that regulates transcription through chromatin remodeling and plays an important role in cell proliferation and differentiation. EP300 is also a target for viral oncoproteins. Data show that EP300 is mutated in epithelial carcinoma, proving it to be a classic tumor suppressor gene [57] . CDK1 controls the cell cycle, and approximately 75 CDK1 targets have been identified that control key cell cycles such as DNA replication and isolation, transcriptional processes, and cell morphogenesis [58] . JAK2 plays a crucial role in the function of certain types of cytokine receptors [59] , and a small-molecule inhibitor of JAK2 is radiosensitizing in lung cancer models [60] .
EGFR is more widely expressed in lung cancer tissues than in adjacent normal lungs [61] , and pneumonic lung cancer (PLC) has a certain relationship with EGFR gene mutation [62] . F2 plays an important role in protective immunity in fibrin-induced pneumonia and sepsis and enhances platelet-neutrophils interactions [63] . MAPK8 is an important signal molecule in the MAPK signal transduction pathway, and its functions involve various mechanisms such as cell proliferation, cell differentiation, and cell apoptosis [64][65] . RXRA plays a central role in the regulation of many intracellular receptor signaling pathways, and macrophage RXRA plays a key role in the regulation of innate immunity and has become a potential target for sepsis immunotherapy [66] . ESR1 tumor suppressor gene inactivation occurs in many tumor types, including lung cancer [67] . PKCD is an important regulator of human neutrophil pro-inflammatory signaling and plays an important role in regulating neutrophil-endothelial cell interactions and recruitment in the inflamed lung [68] .
Activation of members of the PTK family involved in acute inflammatory responses, such as acute lung injury and sepsis, has been identified as essential for recruitment and activation of monocytes, macrophages, neutrophils, and other immune cells [69] .
RPS6KB1 is a member of the ribosomal S6 kinase family that encodes serine/threonine kinases. The encoded protein responds to mTOR (a mammalian target of rapamycin) signaling to promote protein synthesis, cell growth, and cell proliferation. The activity of this gene is associated with human cancer [70] . MAPK14 may play an important role in the development of ventilator-associated pneumonia (VAP) by altering the immune response and MAPK signaling pathway [71] . NR3C1 is involved in the inflammatory response, cell proliferation, and differentiation of target tissues [72] . PRKCE can be used as a breath regulator to participate in mitochondrial respiration, promote the occurrence of mitochondrial organisms, and reduce organ damage caused by pneumonia [73] . CDK5 plays a crucial role in a variety of cancers by regulating the migration and motility of cancer cells [74] . CDK5 gene is overexpressed in non-small cell lung cancer [75] . PPARA plays an important role in lipid metabolism and inflammatory regulation. Previous studies have demonstrated the immune regulatory effect of PPARA and the immunosuppressive effect of PPARA ligand WY14643 on acute lung transplant rejection in rats [76] . According to the results of enrichment analysis, we found that some important targets can regulate a variety of pathways. Among them, MAPK1, MAPK3, PIK3CA, and PIK3R1 participate in more than 90 signaling pathways, reflecting the synergistic effect of multiple targets.

Functional enrichment and pathway analysis
The enrichment analysis of GO function includes three parts: biological process, molecular function, and cell ingredient [77] . Biological processes include cellular response to nitrogen compound, positive regulation of transferase activity, etc., among which cellular response to nitrogen compound has the largest enrichment proportion in the biological process. Studies have shown that inhibition of nitric oxide can lead to an increase in the release of pro-inflammatory cytokines, leading to worsening of lung injury. Nitric oxide may have a protective effect on acute lung injury [78] . Molecular functions are mainly concentrated in protein kinase activity, protein tyrosine kinase activity, and kinase binding, among which protein kinase activity has the largest enrichment proportion in molecular functions. Experimental results showed that the mitochondrial antioxidant pathway mediated by protein kinase D1 plays an important role in the early stage of bleomycin-induced pulmonary fibrosis in rats [79] . Similarly, the expression of protein kinase CK2 is related to the prognosis of patients with lung cancer, suggesting that protein kinase CK2 may be a potential target for the treatment of lung cancer, and the inhibition of CK2 kinase activity may be an effective method for the treatment of lung cancer [80] . The cell ingredients are mainly concentrated in the membrane, the receptor complex, and the dendrite, among which the membrane raft has the largest concentration in the cell ingredients. Experiments have shown that lipid raft protein stomatin with low oxygen, and Dex upregulation can stabilize the cytoskeleton connected by the membrane and increase the barrier function of lung epithelial cells, thus having a protective effect on lung tissue cells [81] . According to the enrichment analysis results, we found that the target mainly improved the symptoms of COVID-19 patients through cellular response to nitrogen compound, protein kinase activity, and membrane raft. Therefore, it is speculated that the treatment of COVID-19 by QFPDD may be most closely related to the above pathways.
The KEGG database is a comprehensive database for the systematic analysis of the metabolic pathways of gene products and compounds in cells and the functions of these gene products for functional annotation of the genome or transcriptome of a times that of the general population [82] . Combined with the results of this study, the reasons were analyzed. On the one hand, the immune function of cancer patients was low, during which they were more likely to be infected with the virus. On the other hand, it may be related to key target genes. Most of the 24 potential targets predicted in this study are closely related to cancer [83] . A large amount of experimental evidence indicates that the carcinogenic signal transduction pathway is the basis of the Endocrine resistance, including PI3K-Akt-mTOR，MAPK-ERK，Src，CDK4-CDK6， ER itself and so on. A combination of targeted ER and such pathways is probably the most effective way to combat resistance to antiestrogens, and clinical trials testing such strategies have shown promising results. The PI3K-Akt signaling pathway is involved in the regulation of cellular inflammatory response, stimulating the expression of endothelial nitric oxide synthase (eNOS) and increasing the production of nitric oxide (NO), to increase vascular permeability, promote the infiltration of inflammatory cells, stimulate the activation of inflammatory cells, and secrete a large number of inflammatory factors through the activity of NO [84] . Studies have shown that PI3K inhibitor has A significant inhibitory effect on NO production and inflammatory response in the lung tissues of viral pneumonia mice induced by Influenza A virus (IAV) infection [85] . In this fight against the epidemic, in addition to the remarkable curative effect of Chinese medicine on COVID-19, some western drugs against the virus (HIV or Ebola) have also been found to have a certain curative effect. Through KEGG analysis, we found that these 24 genes are also involved in the transduction of the signaling pathway of Human immunodeficiency virus one infection, which may also be the reason why drugs such as remdesivir, lopinavir/ritonavir, which can fight HIV infection, have some effect on COVID-19.

Conclusion
In this study, 39 active ingredients of QFPDD in the treatment of COVID-19 were found through network pharmacology, and 24 potential target genes were identified for the treatment, which was verified by enrichment and pathway analysis to verify the reliability of these ingredients and targets used to evaluate the treatment of COVID-19. Besides, the molecular mechanism of QFPDD in the treatment of COVID-19 was illustrated. According to the results of enrichment analysis, we revealed the synergistic characteristics of multiple targets and found that the targets