Using a System Pharmacology Method to Search for the Potential Targets and Pathways of Yinqiaosan against COVID-19

The first reported case of coronavirus disease 2019 (COVID-19) occurred in Wuhan, Hubei, China. Thereafter, it spread through China and worldwide in only a few months, reaching a pandemic level. It can cause severe respiratory illnesses such as pneumonia and lung failure. Since the onset of the disease, the rapid response and intervention of traditional Chinese medicine (TCM) have played a significant role in the effective control of the epidemic. Yinqiaosan (YQS) was used to treat COVID-19 pneumonia, with good curative effects. However, a systematic overview of its active compounds and the therapeutic mechanisms underlying its action has yet to be performed. The purpose of the current study is to explore the compounds and mechanism of YQS in treating COVID-19 pneumonia using system pharmacology. A system pharmacology method involving drug-likeness assessment, oral bioavailability forecasting, virtual docking, and network analysis was applied to estimate the active compounds, hub targets, and key pathways of YQS in the treatment of COVID-19 pneumonia. With this method, 117 active compounds were successfully identified in YQS, and 77 potential targets were obtained from the targets of 95 compounds and COVID-19 pneumonia. The results show that YQS may act in treating COVID-19 pneumonia and its complications (atherosclerosis and nephropathy) through Kaposi sarcoma-related herpesvirus infection and the AGE-RAGE signaling pathway in diabetic complications and pathways in cancer. We distinguished the hub molecular targets within pathways such as TNF, GAPDH, MAPK3, MAPK1, EGFR, CASP3, MAPK8, mTOR, IL-2, and MAPK14. Five of the more highly active compounds (acacetin, kaempferol, luteolin, naringenin, and quercetin) have anti-inflammatory and antioxidative properties. In summary, by introducing a systematic network pharmacology method, our research perfectly forecasts the active compounds, potential targets, and key pathways of YQS applied to COVID-19 and helps to comprehensively clarify its mechanism of action.


Introduction
Coronavirus infectious disease 2019 (COVID-19) is caused by 2019-nCoV virus and it seriously endangers human health. On January 12, 2020, the World Health Organization (WHO) confirmed and named its pathogen 2019-nCoV [1], which belongs to the novel coronavirus pneumonia of genus β. On January 31, 2020, it was designated a Public Health Emergency of International Concern (PHEIC). According to the latest statistics of the World Health Organization, as of October 28, 2021, there were approximately 244385444 confirmed cases and more than 4961489 deaths worldwide, including over 7053806 confirmed cases and over 108334 deaths in the United States. e main clinical features of these cases are a high body temperature, cough without mucous, breathing difficulties (dyspnea), headache, and pneumonia. Disease attack may lead to progressive respiratory failure due to alveolar damage (which has been observed by transverse chest computerized tomography images) and cytokine storm syndrome and it could also develop a severe form with sometimes fatal outcomes [2,3]. e cause of the disease was identified by clinicians as virus-induced pneumonia in line with the clinical signs and other standards, including increased body temperature, decreased lymphocytes and white blood cells (although the levels of the latter seemed normal sometimes), new pulmonary infiltrates on chest radiography, and no evident improvement after treatment with antibiotics for three days [4].
In traditional Chinese medicine (TCM), COVID-19 falls under the category of "pestilences," which occur because of direct or indirect contact with epidemic pathogens. Since the onset of the disease, the rapid response and intervention of TCM have played a vital role in effectively controlling the epidemic, and the classic prescription has become one of the highlights in preventing and controlling the epidemic [5,6]. Yinqiaosan (YQS) is a traditional prescription for treating the influenza virus from the "Wen Bing Tiao Bian." YQS is a herbal formula comprised of 10  Tang et al. [7], based on the TCMATCOV platform, analyzed the potential effects of commonly used traditional formulations for treating COVID-19 and discovered that YQS is a frequently used therapy for pneumonia. For the past few years, systems pharmacology [8,9] means have been widely introduced in the prescription and mechanism of TCM. System pharmacology is an emerging systematic methodology and is guided by the "multicomponent multitarget network" theory, which conforms to the integrity and systematicity of TCM prescriptions. In addition, it combines oral bioavailability screening and multiple drug target prediction, providing an overall method for exploring the targets and potential mechanisms of TCM, and it can be used as an alternative strategy to analyze the therapeutic effects of the active ingredients [10,11]. Wang et al. [12] discussed the targets and signaling pathways of Maxing Shigan decoction for treating pneumonia through systems pharmacology and found that the possible targets were IL-6, TNF, MAPK8, etc. Xia et al. found that Akt1 was a potential target of Lianhua Qingwen to treat and prevent COVID-19 using network pharmacology and molecular docking analyses [13].
Aiming to explore the possibility and mechanism of YQS in treating COVID-19, this paper uses systems pharmacology techniques and approaches to forecast the possible targets and signaling pathways of YQS and provides data for subsequent experimental research.
rough the TCMSP database and the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) combined with the Swiss Target Prediction database, the targets of the active ingredients were obtained. UniProt KB (http://www. UniProt.org/), which prevents, for example, overannotation of semblable proteins such as paralogs and putative products of pseudogenes, was utilized to normalize gene names and organisms. rough the UniProt database (https://www.UniProt.org/), the species was set as "Homo sapiens (Human)" to standardize the obtained drug targets.
e UniProt database was applied to standardize the obtained disease targets in Homo sapiens.

Screening of Candidate Targets.
e acting targets of the active ingredients of YQS and the disease targets of COVID-19 were processed through Venny2.1 software (http:// bioinfogp.cnb.csic.es/tools/venny/index.html) to acquire their common targets, which were taken as alternative targets of YQS for treating COVID-19, and its Venny map was also acquired.

Protein-Protein Interaction (PPI).
e retrieved YQS active ingredient targets were linked to COVID-19 targets by the STRING (https://string-db.org/, ver.11.0) database [18]. e requirement was restrained to "Homo sapiens." In this paper, a high confidence score with a correlation degree ≥0.400 [19] as the cutoff value was set to obtain the network.

GO and KEGG Pathway Enrichment Analysis. Gene
Ontology (GO) enrichment analysis was conducted using the OmicShare tools (http://www.omicshare.com/tools). e KOBAS 3.0 database was applied to analyze the KEGG pathway enrichment of the candidate targets, and the RStudio 3.6.3 and ggplot2 packages were applied to illustrate the results.
2.6. Networks Construction and Analysis. Cytoscape 3.5.1, an open-source software platform to visualize complex networks, was employed to illustrate the networks. With this software, the network building was handled as follows: (1) compound-COVID19 PPI network, (2) compound-targets network, and (3) YQS (Yinqiaosan)-compound-targetspathway network. In addition, four topological features of the hub network, such as "Degree" and "Betweenness Centrality," were determined to choose YQS alternative targets with topological significance [20,21]. To determine the relationship between network clusters and to identify high-connectivity hub genes, "Cytohubba" (a plug-in of Cytoscape) was introduced to check the node element. e top 10 nodes were ranked by EPC [22].

Molecular Docking.
For the sake of validation of the drug-targeted correlations, the molecular docking simulation further proceeded on every drug docking with their targets. Each drug molecule, such as luteolin, naringenin, acacetin, kaempferol, and quercetin, was downloaded from the TCMSP database and converted to a PDBQT file by Chimera (version 1.10.2). e structures of the proteins in each alternative target were downloaded from the RCBS Protein Data Bank (http:// www.rcsb.org/pdb), and every protein file was opened with ADT (version 1.5.6), a free GUI for AutoDock. In every file, the water molecules were canceled, the polar hydrogen atoms were increased, and then they were written to a PDBQT file. e intersection of possible alternative targets and drug targets of PCOS was chosen for in-depth investigations. To validate the interaction and observe the docking sites between drugs and targets, molecular docking was performed by an open-source program called Autodock vina 1.1.2. e docking results were observed with ADT. e foundation for developing medications for the clinical therapy of COVID-19 is shown in Figure 1

Active Components and Target Proteins of Yinqiaosan.
By a systematic search of the public databases, 141 components were retrieved in JYH (17), LQ (11), JG (7), BH (10), DDC (2), DZY (4), NBZ (4), JG (9), LG (1), and GC (76), and the specific information of these components can be found in Table 1. After deleting the duplicate values, 117 active ingredients were acquired. Refer to the results in Figure S1. rough observation, it was discovered that some special compounds in this network interacted with multiple targets and took part in the regulation of multiple targets, such as luteolin, naringenin, acacetin, kaempferol, and quercetin.
A total of 810 targets were obtained from public databases, but not all information on all 810 targets is shown. e YQS compounds may jointly affect all of these targets, producing pharmacological impacts on COVID-19.

Screening Results of COVID-19-Related Disease Targets.
e GeneCards database, as an online catalog of human genes and genetic illness, can enable effective navigation of gene-disease linkages [23]. e OMIM database deals with over 15,500 gene entries and concentrates on elaborating gene-phenotype correlations [24]. rough the two databases, we obtained 435 genes in total (not all shown).

Screening
Results of Candidate Targets. Altogether, 77 overlapping genes were acquired by searching the overlaps of the aforementioned compound targets with the 435 COVID-19 gene targets. ese genes included PIK3CD, PLA2G5, and ECE1 (Table S1, Figure 2(a)).
By importing compound-disease cotarget information into STRING, a compound-COVID-19 target PPI network was obtained. Taking a score ≥0.4 as the limiting condition [19], the interaction relationship of 77 targets obtained from the String network database was screened. en, a network diagram of 77 targets protein interactions was obtained. Combined with Cytoscape 3.5.1 software, we obtained an interaction network graph of alternative targets of YQS for treating COVID-19, including 77 nodes and 719 edges. e node colors are illustrated from red to yellow in descending order of degree values (Figure 2(b)). Among the targets of YQS for treating COVID-19, the 5 targets with the highest degree were TNF, GAPDH, MAPK1, MAPK3, and EGFR.

Results of the Construction of the Compound-Target
Network.
e data of the active ingredients of YQS as well as its targets for treating COVID-19 were input into Cytoscape 3.5.1 software to obtain the network chart of active ingredient-acting targets. e active ingredients of YQS were represented by their Mol IDs. ere were 173 nodes and 831 edges forming the compound-target network. e diamonds represent 95 active ingredients, and the ellipse indicates 77 targets of active ingredients ( Figure 3).

Enrichment Analysis Results of Biological Processes and
Pathways. To carry out an in-depth recognition of the biological functions of the above 77 potential targets in biological networks, a GO enrichment analysis was implemented with the OmicShare tools, a free online platform for data analysis (http://www.omicshare.com/ tools) [25]. As shown in Figure 4, the results fell into three strata: biological processes (BP), cellular components (CC), and molecular functions (MF). e limiting condition was P < 0.01, where the smaller the P value is, the closer the relationship with YQS will be in treating pneumonia. e results show that the BP analysis mainly includes the response to organic substances, the cellular response to organic substances, and the cellular response to chemical stimulus (Figure 4(a)). e CC analysis mainly included membrane microdomains, membrane rafts, and membrane regions (Figure 4(b)). e MF analysis was mainly related to enzyme binding, phosphotransferase activities, alcohol groups as acceptors, and kinase activities (Figure 4(c)).    Journal of Healthcare Engineering 5 Utilizing the online platform KOBAS 3.0, 190 important KEGG pathways (P < 0.01) were found on the basis of differentially expressed coding transcripts. e most important pathways included Kaposi sarcoma-related herpesvirus infection, AGE-RAGE signaling pathways in diabetic complications, and pathways in cancer. e result was constructed by KOBAS 3.0, and the top 20 enrichment pathways were visualized by RStudio ( Figure 5).
is indicated that YQS might be a candidate for treating COVID-19 through these pathways.
e top ten channels in the enrichment analysis of the KEGG pathway and the compound and compound targets were imported into Cytoscape 3.5.1 software to build a "YQS-compounds-targets-pathway" network ( Figure 6). e figure includes 194 nodes and 1185 edges. e red diamonds denote YQS, yellow hexagons represent the traditional Chinese herbs in YQS, green rectangles represent the potential active ingredients, blue ellipses represent YQS candidate treatment targets for COVID-19, and purple triangles represent the possible signaling pathways for the active ingredients to function. It can be seen from the figure that YQS includes 10 herbs and 95 active ingredients. It mainly acts on TNF, GAPDH, MAPK3, MAPK1, EGFR, CASP3, MAPK8, MTOR, IL-2, and MAPK14 and uses Kaposi sarcoma-related herpesvirus infection, AGE-RAGE signaling pathways in diabetic complications, pathways in cancer, apoptosis, hepatitis B, human cytomegalovirus signaling pathways such as infection, measles, and hepatitis C to treat pneumonia.

Molecular
Docking. Molecular docking was further utilized in our research to estimate the interaction between components and targets to reduce the complexity and increase the precision of the constituent target network [26].   Figure 2: Overlapping targets (a) and the protein-protein network (b). Node colors change gradually from red to yellow, and node sizes are in proportion to their degree. e cytoHubba control panel is a useful tool to retrieve subnetworks from the whole large PPI set [27]. Hub gene recognition was processed by the cytoHubba plug-in in Cytoscape software. All active ingredients in the top 5 online pharmacology degrees of freedom were taken as the research object. To determine the valid connecting effects between YQS-main active ingredients and their predicted targets, molecular docking was assessed using the YQS-hub genes connecting the energy. Molecular docking analyzed 10 hub genes (CASP3, EGFR, GAPDH, IL-2, MAPK1, MAPK14, MAPK3, MAPK8, mTOR, and TNF) and 5 compounds (acacetin, kaempferol, luteolin, naringenin, and quercetin).
Zhou et al. [3] ascertained that 2019-nCoV makes use of the same cell entry receptor, angiotensin transforming enzyme II (ACE2), as SARS-CoV. 3CL hydrolase is the core protease of single-stranded RNA virus precursor polyproteolysis and it plays a vital role when replicating the single-stranded RNA virus [28]. erefore, in this study, the 5 compounds were also molecularly docked with SARS-CoV-2 3CL hydrolase (Mpro) and ACE2 to provide references for treating COVID-19 by TCM. e lower the binding energy, the more stable the conformation of the ligand-receptor binding [29]. When the binding energy is less than −5.0 kcal/mol, this suggests that the compound can bind to the target. When the binding energy is less than -7.0 kcal/mol, this suggests that the compound has a good ability to bind to the target. We selected the conformation with the lowest binding energy to analyze the docking binding mode and used Discovery Studio for mapping.
Molecular docking results showed that all five active ingredients could be combined with 10 hub genes (Table S2), Mpro or ACE2 (Table 2, Figure 7). erefore, the 5 compounds could efficiently act on the 10 targets, especially TNF, MAPK14, MAPK3, and MAPK8. Kaempferol (Figures 7(a) and 7(b)) and Mpro have a better binding capacity, which is consistent with the docking results of the molecules discovered by Ling [30]. Acacetin (Figures 7(c) and 7(d)), luteolin (Figures 7(e) and 7(f )), and ACE2 have a better combining ability.

Discussion
At present, COVID-19 has expanded to over 200 countries and areas, and the lives and health of people around the world are under serious threat. Special drugs against COVID-19 are still under development. TCM adopts the principle of dialectical treatment with the characteristics of multiple targets and multiple pathways, and it plays a vital role in antiepidemic treatment [31][32][33]. e "COVID-19 Diagnosis and Treatment Program (Trial Ver. 3)" issued by the National Health Commission of the People's Republic of China pointed out that COVID-19 is categorized as a "pestile" in TCM, which happens under direct or indirect contact with epidemic pathogens. e main features are "damp, heat, poisonous, stasis." COVID-19 is divided into four types of TCM syndromes: cold-dampness stagnating in the lung, damp-heat accumulating in the lung, damp toxin stagnating in the lung, and internal blocking causing external collapse.
e clinical symptoms of "damp-heat accumulating in the lung" are low body temperature or no fever, mild aversion to cold, fatigue, a heavy sensation in the head and body, dry coughs with scanty phlegm, sore throat, and a dry mouth with no wish to drink water. Alternatively, chest tightness, epigastric fullness, absence of sweating or  Journal of Healthcare Engineering inhibited sweating, vomiting, a poor appetite, and loose stools or sticky stools with a feeling of incomplete bowel movement may also be present. e tongue is pale red with a white, thick, and greasy or thin, yellow coating. e pulse is soft or slippery and rapid. e recommended prescriptions are Maxing shigantang and YQS. YQS was described by Wu in "Wen Bing Tiao Bian." He is a famous infectious febrile disease specialist. Part of the composition of the Lianhua Qingwen capsule comes from YQS, which is particularly effective in treating pneumonia. YQS is comprised of ten Chinese herbs, JYH, LQ, JG, BH, DDC, DZY, NBZ, JJ, LG, and GC, and mainly treats the manifestations caused by diseases such as damp-heat accumulation in the lung. Bohe and Niubangzi have flavor and cool properties and can help sore throats. Jingjie and Dandouchi can help the main medicine drive away damp heat. Dan Zhuye and Lugen can clear the heat, and Jiegeng and Gancao can relieve cough. YQS is commonly used in the clinic against influenza A virus infection [34][35][36].
is study uses network pharmacology to forecast the main active components and potential molecular mechanism of YQS against COVID-19.
rough the TCMSP database, 141 active ingredients of each single medicine in Yinqiao powder were obtained, of which the five most powerful components were acacetin, kaempferol, luteolin, naringenin, and quercetin. Studies have shown that luteolin may suppress the infection of respiratory epithelial cells by Escherichia coli through immunomodulation [37]. Wu et al. established a mouse model of Staphylococcus aureus pneumonia and found that naringenin could significantly alleviate the disease [38]. Acacia has antibacterial, antioxidant, anti-inflammatory, antiviral, antipyretic, and analgesic effects [39]. It can protect against atherosclerosis by antagonizing the LysoPC-induced apoptosis of vascular smooth muscle cells and stimulating the antioxidant regulatory factor Nrf2 [40]. Kaempferol has an extensive scope of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, etc. [41], and its derivatives are Top 20 of GO-BP terms Enrichment response to organic substance cellular response to organic substance cellular response to chemical stimulus response to chemical response to external stimulus response to oxygen-containing compound response to stress apoptotic process positive regulation of response to stimulus . Rich factors are proportional to the degree of enrichment. e node size shows the number of genes, and the larger the node is, the more the genes enriched in the term are. e color depth of the node demonstrates the significance level, and the redder the node is, the higher the significance will be to the term. used as antiviral drugs against coronavirus 3a channel protein [42]. Quercetin and its derivatives also have strong anti-inflammatory and analgesic pharmacological activities [43]. Wang et al. [12] found that active compounds such as quercetin, kaempferol, naringenin, and luteolin also played a pivotal role when treating COVID-19 using Maxing Shigan decoction. Kong Figure 5: Analysis of the 20 most enriched KEGG pathways of candidate targets of Yinqiaosan (YQS) for COVID-19. e node size denotes the number of target genes that belong to the pathway. e X-axis denotes the enrichment factor (rich factor), that is, the number of genes in the pathway in the target gene/the number of genes contained in the pathway in the background gene set. e Y-axis denotes the pathway. e color depth of the node demonstrates the significance level, and the redder the node is, the higher the significance will be to the pathway.  Figure 6: Yinqiaosan (YQS)-compound-target-pathway network. e red bubbles denote YQS (Yinqiaosan), the yellow bubbles denote Chinese herbs, the pink bubbles denote compounds, and the violet bubbles denote pathways.  MAPK8, mTOR, IL-2, and MAPK14 are important targets for YQS in COVID-19 treatment. COVID-19 is similar to other viral pneumonia conditions. Some scholars have found that cytokine storms (CSs) may affect the progression of COVID-19 illness [45]. e cytokines involved in CS mainly include six categories: tumor necrosis factor (TNF), interleukin (IL), interferon (IFN), colony stimulating factor (CSF), and growth factor (GF). e cytokine storm induced by SARS-CoV-2 includes the cytokine IL-2, and the TNF-αand H7N9-induced cytokine storms include cytokines, including IL-2 [46]. Studies have shown that, in throat swabs and sputum specimens of 4 cases of COVID-19, the housekeeping gene GAPDH of human cells showed a typical amplification signal curve [47]. Staphylococcus aureus pneumonia and virulence factor A can stimulate the AMPK signaling pathway and inhibit the mTOR pathway to induce autophagy in alveolar epithelial cells, an important mechanism during early infection of S. pneumoniae [48]. Liu et al. [49] predicted MAPK1, MAPK3, and MAPK14 through network pharmacology as important targets for Dayuanyin to treat COVID-19. Huang et al. [4] predicted that MAPK1 and MAPK14 were important targets for Huanglian Jiedu decoction to treat COVID-19. Other studies have shown that EGFR may be a key factor in the inflammation caused by Klebsiella pneumoniae infection [50]. Furthermore, molecular docking results showed that the 5 compounds could efficiently act on the 10 targets, especially TNF, MAPK14, MAPK3, and MAPK8. Kaempferol and Mpro have a better binding capacity. Acacetin, luteolin, and ACE2 have a better combining ability. Whether they are docked with molecules of hub genes or docked with the proven targets of pneumonia, this shows the feasibility of using these five compounds to treat pneumonia. Reportedly, AGEs modulate COVID-19 pathogenesis and related comorbidities through their interactions with RAGE, among other molecules [51]. Interestingly, Yalcin Kehribar et al. [52] found that the RAGE pathway plays an important role in the aggravation of COVID-19. ese reports provide strong evidence for our data.
In the next step, further in vitro and in vivo experimental validation will be carried out to advance our research. e follow-up plan of our research group is to carry out more indepth experimental research on YQS and to fully explore the mechanism of its efficacy.

Data Availability
All data included in this study are available upon request by contact with the corresponding author.