Exploring the Mechanisms of Lian Hua Qing Wen Capsule against COVID-19 by Network Pharmacology and Molecular Docking Approach

Background The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or COVID-19) disease has led to a wide-spread global pandemic. There is no specic antiviral drug proven effective for the treatment of patients with COVID-19 at present. Combination of western and traditional Chinese medicine (TCM) is recommended, and Lian Hua Qing Wen (LHQW) capsule is a basic prescription and widely used to treat COVID-19 in China. However, the mechanisms of LHQW capsule treating COVID-19 are not clear. The aim of the study is to explore the mechanisms of LHQW capsule treating COVID-19 based on network pharmacy and molecular docking approach. Methods The active compounds and targets of LHQW capsule were obtained from traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). COVID-19 related target genes were obtained from GeneCards database and OMIM database. Protein–protein interaction (PPI) networks of LHQW capsule targets and COVID-19-related genes were visualized and merged to identify the candidate targets for LHQW capsule treating COVID-19. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were also performed. The hub genes involved in the gene-related pathways were screened and their corresponding compounds were used for in vitro validation of molecular docking predictions. Results A total of 185 active compounds of LHQW capsule were screened out, and 263 targets were predicted. Third hundred and fty-two COVID-19 related target genes were obtained from GeneCards database and OMIM database. GO functional enrichment analysis showed that the biological processes of LHQW capsule treating COVID-19 were closely linked with the regulation of inammation, immunity, cytokines production, vascular permeability, oxidative stress and apoptosis. KEGG enrichment analysis revealed that the pathways of LHQW capsule treating COVID-19 were signicantly enriched in AGE−RAGE signaling pathway in diabetic complications, Kaposi sarcoma−associated herpesvirus infection, TNF, IL−17, and Toll−like receptor (TLR) signaling pathway. The hub targets genes in the gene-related capsule treating COVID-19. The active compounds of LHQW capsule and their targets were rstly identied using the pharmacology analysis platform of the Chinese medicine system (TCMSP). Then, COVID-19 related target genes were obtained from GeneCards database and OMIM database. The mechanisms of LHQW capsule treating COVID-19 were explored by gene ontology (GO) and Kyoto Encyclopedia of gene and genomes (KEGG) pathway enrichment analysis. Molecular docking was used to further verify the mechanisms of herbs intervention in COVID-19. Based on TCM network pharmacology and molecular docking approaches, this study claried the mechanisms of LHQW capsule treating COVID-19 at molecular and cellular levels.


Background
The novel coronavirus 2019 (COVID- 19), also known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the genus β, with envelope, round or elliptic and often pleomorphic form, and 60-140 nm in diameter [1]. The ongoing pandemic of SARS-CoV-2 infections has led to more than 4 900 647 cases and 320 107 deaths globally as of May 20, 2020. This pandemic is still ongoing and mortality is increasing, especially in north America and Europe. Therefore, it is very urgent to nd new preventive and therapeutic agents as soon as possible. However, there are no speci c antiviral drugs or vaccines against COVID-19 at present [2]. According to traditional Chinese medicine (TCM) science, COVID-19 was considered as "plague" and TCM plays important auxiliary roles in the treatment of COVID-19 [3,4].
Lian Hua Qing Wen (LHQW) capsule was prescribed based on aetiology and symptoms of COVID-19 and has been applied widely in China for treating COVID-19, which has shown satisfactory curative effects [5,6]. Moreover, LHQW capsule has been listed in many countries for the prevention and treatment of COVID- 19. LHQW capsule consists of 13 Chinese medicine extracts and has the effects of clearing away the plague and detoxify, promoting the dispersing function of the lung and discharging heat. The herbs in to clarify the effects of compounds and potential mechanisms of compound-target-disease at the molecular level [7,8].
In the present study, we performed bioinformatics investigation and molecular docking approach to elucidate an "ingredient-target-pathway" network of LHQW capsule treating COVID-19. The active compounds of LHQW capsule and their targets were rstly identi ed using the pharmacology analysis platform of the Chinese medicine system (TCMSP). Then, COVID-19 related target genes were obtained from GeneCards database and OMIM database. The mechanisms of LHQW capsule treating COVID-19 were explored by gene ontology (GO) and Kyoto Encyclopedia of gene and genomes (KEGG) pathway enrichment analysis. Molecular docking was used to further verify the mechanisms of herbs intervention in COVID-19. Based on TCM network pharmacology and molecular docking approaches, this study clari ed the mechanisms of LHQW capsule treating COVID-19 at molecular and cellular levels.

Methods
The technical strategy of this research is shown in Fig. 1.

Screening the bioactive ingredients
The candidate ingredients of LHQW capsule were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform [9] (TCMSP, http://tcmspw.com/tcmsp.php) and screened with the criteria of oral bioavailability (OB) ≥ 30% and drug-likeness ≥ 0.18 [10]. In total, 230 eligible compounds were obtained, 23 in FF, 23 in LJF, 22 in EH, 19 in ACV, 39 in IR, 28 in FBR, 7 in HH, 11 in PCB, 16 in RRER, 10 in MH, 91 in LI. Eventually, a total of 244 candidate compounds were obtained after removing the duplications.
Four thousand targets were collected, 450 in FF, 403 in LJF, 406 in EH, 183 in ACV, 254 in IR, 104 in FBR, 204 in HH, 219 in PCB, 88 in RRER, 165 in MH, 1524 in LI. After deleting duplicates, there were 263 targets in total. The 244 candidate compounds were imported into the DrugBank database (https://www.drugbank.ca/) [11] and 185 compounds were obtained after removing 59 compounds which did not link to any targets.

PPI Network Construction
The compound-target networks were established to explore the complex interactions between the compounds of QFPDD and the targets of COVID-19 through using STRING and Cytoscape 3.7.2 (https://cytoscape.org/) [14]. In the network, nodes separately represented compounds or targets, and edges showed the interactions of the inter-node. The conditions of topological analysis included Betweenness Centrality (BC), Closeness Centrality (CC), Neighborhood Connectivity (NC), Number of Directed Edges (NDE), (Topological Coe cient (TC).

Bioinformatic Analysis
Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov, v6.8) [15] was used to perform GO analysis including biological process, cellular component, and molecular functions. Functional categories were enriched within genes (FDR < 0.05) and the top 20 GO functional categories were selected. DAVID database was also used to carry out KEGG pathway analysis [15]. Pathways that had signi cant changes of FDR < 0.05 were identi ed for further analysis and the top 20 pathways were selected. The gene-pathway network was constructed to screen the key target genes of QFPDD treating COVID-19.

Molecular docking
PubChem and PDB were used to nd the chemical and conformational information of the relevant proteins and small-molecule compounds [16,17]. The AutoTools software was used to remove the redundant protein chains, ligands and water molecules with hydrogenation before running docking experiments [18]. The AutoGrid software was used to calculate the energy lattice points with the grid box coordinates of 15 × 15 × 15 [18]. AutoDock Vina was used to simulate the docking condition between proteins and small molecules [18]. The Schrodinger software was used to analyse the preferential conformation and map the simulation.

Results
Compound-Target Network of LHQW capsule

PPI Networks Analysis of LHQW capsule and COVID-19
To reveal the mechanisms of LHQW capsule against COVID-19, 50 shared target genes were added to STRING and Cytoscape to construct a PPI network for exploring the interaction relationships with each other (Fig. 3). Network Analyzer in cytoscape was used to analyse the degree of each target for further identifying the more important targets in the network. The results and parameters from the PPI topological analysis are listed in Table 2. MAPK3, MAPK8, CASP3, MAPK1, IL6 and RELA were the top 6 genes based on degree.   Gene-pathway network analysis As genes cannot directly exhibit their biological and pharmacological activities independently, a genepathway network was established to further elucidate the molecular mechanisms of LHQW capsule treating COVID. According to the degree of the pathways, the study selected the top 30 pathways and 42 genes to perform topological analysis with BC. Figure 5 identi

Discussion
COVID-19 has brought a tremendous threat to public health, especially the elderly over 60 years old being more likely to die after infection [1]. The clinical symptoms of COVID-19 have been reportedly ranged from mild to severe, ultimately leading to death. The mild symptoms included fever, cough, shortness of breath and pneumonia after viral infection, while the severe cases would rapidly developed to acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation dysfunction and multiple organ failure that eventually brought about mortality [19,20]. There has been no effective antiviral drug to treat COVID-19 until now, even though signi cant efforts are used to develop therapeutic interventions against coronavirus infection [2]. Fortunately, the application of TCM in the auxiliary treatment of COVID-19 has achieve satisfactory therapeutic effects in China [21,22]. LHQW capsule has been recommended for the treatment of patients with mild symptoms in 7th edition of COVID-19 treatment guidelines and widely used in the clinical treatment of COVID-19 in China [23]. LHQW capsule has been demonstrated to have anti-infection or antiviral effects in vitro or in vivo [24][25][26]. In the present study, network pharmacological and molecular docking approaches were used to explore the mechanisms of LHQW capsule treating COVID-19.
According to the GO analysis, the LHQW capsule treating COVID-19 is widely involved in regulating in ammation, virus infection, endothelial barrier and cytokine storm via mediating these processes such as response to molecule of bacterial origin, response to oxidative stress, membrane raft, membrane microdomain, cytokine receptor binding and cytokine activity, which were signi cantly enriched in the GO analysis. Molecule of bacterial origin is involved in Toll-Like Receptor Signaling, which represents a largely evolutionarily conserved pathogen recognition machinery responsible for recognition of bacterial, fungal, protozoan, and viral pathogen associated microbial patterns and initiation of in ammatory response [27]. Oxidative stress is an imbalance between oxidants and antioxidants when the organism exposures to adverse stimuli, playing important roles in virus infection [28]. Membrane raft and membrane microdomain are not only closely associated with inter-endothelial junctions and adhesion of endothelium, but also involved in immune cell adhesion and migration across endothelium [29]. Cytokine storm, emerged from prolonged cytokine/chemokine responses during COVID-19, causes ARDS or multiple-organ dysfunction, ultimately leading to physiological deterioration and death [30]. Quercetin, a typical avonoid, is one of the major compounds of FF, LIF, EH, HH, PCB, and LI in LHQW capsule and has been reported to have the effects of anti-in ammation, antiviral, strengthening the endothelial barrier integrity and inhibiting the expression of cytokines [31][32][33][34]. Kaempferol is avonoid found in FF, LIF, EH, FBR, HH, and LI of LHQW capsule, and can protect against in ammation, virus infection, endothelial barrier dysfunction, and cytokines release [35][36][37][38]. Another important compounds, such as luteolin, naringenin, and beta-sitosterol, in LHQW capsule also have these protective effects [39][40][41][42] [43]. IL-17 signaling can not only activate downstream pathways that include NF-κB, MAPKs and C/EBPs to induce the expression of anti-microbial peptides, cytokines and chemokines, but also is critical for protection against a variety of fungal and bacterial infections [44,45]. TLRs are a well-known family of pattern recognition receptors that play important roles in host immune system and in ammation [46]. TLR signaling activation can result in the production of cytokines, chemokines and interferons and transcription factor NF-κB [47,48], thus inducing in ammatory and immune response. Previous studies have demonstrated that quercetin not only exerted the effect of anti-in ammation by regulating IL-17 signaling [49,50], but also effectively promoted the immunoregulatory effect by activating the TLR-3 pathway and inhibiting downstream cytokines production [51,52]. Similarly to quercetin, another important compound also have these effects. As a result, LHQW capsule treating COVID-19 may directly interfere with IL-17 and TLR signaling, and further regulate the downstream signaling pathways, such as the NF-κB signaling pathway, leading to the inhibition of cytokines production, including TNF-α, IL-1β, IL-8.
Based on gene-pathway network analysis, MAPK3, MAPK1, RELA, IL-6, and CASP8 are the more important genes in LHQW capsule treating COVID-19. Clinical and laboratory data of COVID-19 showed evidence of in ammatory and immune injury, con rming that cytokine storms could be a crucial factor linked to the severity and mortality of COVID-19. MAPK3 and MAPK1, belong to serine/threonine protein kinase family, and the activation of MAPK promotes virus infection and in ammatory response [53][54][55]. REL/NF-κB family of transcription factors plays a central role in initiation and resolution of in ammatory responses [56,57]. IL-6, promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, in ammatory and immune reactions [58,59]. Caspase-8 is the initiator caspase of extrinsic apoptosis and required for the activation of NF-κB and secretion of cytokines in response to activated antigen receptors [60]. Quercetin, kaempferol, luteolin and naringenin, all can inhibit the activation of MAPK and RELA, decrease the expression of IL-6 and CASP8 [61][62][63][64][65]. Moreover, the most critical targets had good binding activities to main compounds, which indicated that pharmacodynamic mechanisms of LHQW capsule had su cient material basis through preliminary analysis.

Conclusion
This study investigated the effective active ingredients and molecular mechanisms of LHQW capsule in the treatment of COVID-19 from the perspective of network pharmacology and molecular docking. The therapeutic effects potentially involved in inhibiting in ammatory response, cytokine storm and virus infection, and regulating immune reactions, apoptosis and endothelial barrier.  Visualization of the PPI of the target genes using STRING and Cytoscape. Size of the spot represents number of genes and color represents FDR value.

Figure 5
Gene-Pathway Network of LHQW capsule against COVID-19. The topological analysis of 30 pathways and 42 genes was carried out with betweenness centrality (BC). The green prismatic represent target genes and the orange hexagon represent pathways. Big size represents the larger BC.