Network pharmacology combined with molecular docking simulations reveal the mechanism of action of Glycyrrhiza for treating pneumonia

A well‐established mechanism of action for managing pneumonia using Glycyrrhiza is unknown. Using network pharmacology and molecular docking simulations, we investigated the mechanism of action of Glycyrrhiza against pneumonia. To identify the targets of the active components of Glycyrrhiza from the Traditional Chinese Medicine Systems Pharmacology database, oral bioavailability and drug likeness were utilized as indicators. Pneumonia‐associated genes were identified and screened from the databases. Integrated analysis was conducted to elucidate the relationship between the active components of Glycyrrhiza and intersecting genes; a comprehensive Glycyrrhiza active component‐target gene relationship map was constructed. Intersecting genes underwent Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses to examine their biological functions. A protein–protein interaction network map was constructed to identify hub genes. Molecular docking simulations were performed to investigate binding interactions between hub genes and their corresponding active components. Of the 96 overlapping genes, topological analysis revealed 10 hub genes. Glycyrrhiza exerts therapeutic effects through a multi‐target and multipathway approach, suggesting a synergistic treatment for pneumonia. MAPK14 showed a favorable binding affinity with most of the active compounds, indicating that MAPK14 and related compounds in Glycyrrhiza have development potential.

America and Europe, chemically synthesized drugs such as remdesivir are commonly used to treat COVID-19.Although these drugs have shown promising therapeutic effects, their adverse effects cannot be ignored.Research has shown that prolonged or high-dose remdesivir use may lead to cardiotoxicity and sinus bradycardia in SARS-CoV-2 patients. [6,7]] Thus, the development of safe and effective novel antipneumonia drugs is of the utmost importance.
The plague existed in ancient Chinese society before the advent of chemically synthesized drugs.Traditional Chinese medicine (TCM), with its unique theoretical system and focus on observing life, has effectively protected and promoted the survival and growth of the Chinese nation. [11]With societal development, TCM had undergone modernization and development.Modern medical research has identified chemical substances in TCM, leading to significant advancements.For example, Tu Youyou was inspired by ancient TCM books, and successfully isolated artemisinin from Artemisia annua.By modifying its structure, artemisinin has become an effective antimalarial drug, serving as a prime example of TCM modernization. [12]TCM is widely recognized for its high efficiency and few side effects. [13]6] Chinese herbs, many of which are natural medicines, are commonly used within the TCM framework.Among these, Glycyrrhiza, commonly known as licorice-has various medicinal properties, such as spleen toning, Qi benefit, clearing heat, detoxification, relieving cough and phlegm, easing pain, and harmonizing medicines.It is frequently used to treat coughs, bronchitis, and other diseases. [17][19] Modern medical research suggests that Glycyrrhiza contains active components, such as triterpenoid saponins and flavonoids, [20,21] and Glycyrrhiza exhibits promising antiinflammatory, antiviral, antioxidant, and immunomodulatory activity. [22]wever, limited research on the use of Glycyrrhiza for treating pneumonia exists.Therefore, further investigation of the targets and mechanisms of action of Glycyrrhiza in pneumonia treatment is imperative.
The characteristic therapeutic approach in TCM involves modulating multiple targets through multiple pathways using multiple components.Improvements in TCM composition and disease gene databases have enabled the exploration of complex regulatory mechanisms between drugs and diseases using network pharmacology.This field reveals the theoretical mechanism of multicomponent synergistic treatments through the integration of drug-target-gene-disease interaction networks.Network pharmacology facilitates the development of natural drugs with complex components by offering theoretical validation prior to conducting real experiments and reducing trial-and-error costs.This provides robust support for practical experiments and constitutes an essential step for researchers before conducting drug development experiments.This study investigated the mechanism of action of Glycyrrhiza in the treatment of pneumonia.A network pharmacology approach was used to elucidate the targets and signaling pathways of Glycyrrhiza at the molecular level.The aim of this study was to provide a theoretical basis for developing novel antipneumonia drugs.

| MATERIALS AND METHODS
The flowchart of the study is shown in Figure 1.The MeSH keyword search (https://www.ncbi.nlm.nih.gov/mesh) for this study encompassed "Glycyrrhiza" and "pneumonia."

| Collection and screening target proteins for the active components of Glycyrrhiza
The Traditional Chinese Medicine Systems Pharmacology (TCMSP) (https://old.tcmsp-e.com/tcmsp.php)database is extensively used in network pharmacology research on TCM. [23]This database encompasses comprehensive information on TCM, including active components and their associated targets, along with essential data such as the oral bioavailability (OB) and drug likeness (DL) of these components.By employing "Glycyrrhiza" as the keyword, active components of Glycyrrhiza were obtained from the TCMSP database using screening conditions of OB ≥ 30% and DL ≥ 0.18. [24]bsequently, the active components of Glycyrrhiza were crossreferenced to identify their respective targets.These targets were converted into gene symbols using the UniProt (https://www.uniprot.org/)database. [25]

| Construction of effective components of Glycyrrhiza and pneumonia target gene network
The active components of Glycyrrhiza were screened without deduplication using pneumonia target genes as references.The results were then imported into the Cytoscape software (version 3.8.0,Java 11.0.7,AdoptOpenJDK) to construct a network.

| Protein-protein interaction (PPI) network construction and hub genes screening
The PPI network was created using the STRING (https://cn.string-db.org/) database with Homo sapiens as the organism. [31]Set the minimum interaction threshold to "high confidence" (>0.900).Stray nodes were excluded from the analysis.CytoNCA software (version 2.1.6)was used to calculate six parameters: degree centralities (DC), betweenness centralities (BC), closeness centralities, eigenvector centralities (EC), network centralities (NC), and local average connectivity-based method centralities (LAC).Nodes were identified as keys in the network if their values for all six parameters exceeded the median value.The screening process was repeated twice to identify hub genes in the PPI network.

| Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses
GO function enrichment and KEGG pathway analyses were performed on the crossover genes using the "org.Hs.eg.db" software package.GO functional enrichment analysis involved three aspects: biological processes (BP), cellular components (CC), and molecular functions (MF).Significance was determined based on p-and q-values less than 0.05.

| Molecular docking stimulations
The three-dimensional structures of the hub genes were obtained from the RCSB Protein Data Bank (RCSB PDB) (https://www.rcsb.org/) database.Using pyMol software (version 2.5.4), the structures were dehydrated, and ligands were removed.Two-dimensional structural data for the active components associated with the hub genes were obtained from the TCMSP database.The chem3D software (version 20.0.0.41) was used to optimize these two-dimensional F I G U R E 1 Investigation process for investigating the antipneumonia mechanism of Glycyrrhiza by combining network pharmacology and molecular docking simulation.
structures into three-dimensional structures with minimal energy.Molecular docking simulations were conducted using AutoDockTools software (version 1.5.6), and the results were visualized using PyMol software (version 2.5.4).

| Potential targets of effective components of Glycyrrhiza
According to the TCMSP database, Glycyrrhiza contained 92 active components and 2506 targets.After matching and screening the active components with the targets, we included a total of 1769 targets in subsequent analyses.Upon converting the full names of these targets into gene symbols, 1497 potential targets of the active components of Glycyrrhiza were identified (Table S1).

| Overlapping targets of effective components of Glycyrrhiza and pneumonia
We searched the GeneCards, OMIM, PharmGkb, TTD, and Drug-Bank databases using the keyword "pneumonia," and the search results were concatenated to obtain a total of 1537 targets, as shown in Table S2 (Figure 2).The targets of the active components of Glycyrrhiza were de-emphasized, and the results obtained were then used to intersect the pneumonia target genes, resulting in 96 overlapping targets (Figure 3).Table S3 shows information on the 96 targets.

| Construction of "active components-targets" correlation network
The network diagram in Figure 4 illustrates the constructed "active components-targets" network, consisting of 180 nodes and 516 edges.
Pink rectangles represent the active components of Glycyrrhiza, whereas blue rectangles represent pneumonia target genes.Each edge connects the active component of Glycyrrhiza to a target gene in pneumonia.Active components are connected to one or more targets, implicitly suggesting that Glycyrrhiza provides a multicomponent and multi-target synergistic regulation of disease states.Detailed data corresponding to the figure were listed in Table S4.

| Target protein interaction network analysis and hub genes screening
The common targets related to pneumonia and Glycyrrhiza treatment were imported into the STRING database to obtain their interaction relationships (Figure 5).1).These 10 nodes were identified as hub genes (Figure 6).

| GO and KEGG pathway enrichment analyses
To systematically study the complex regulatory mechanisms of Glycyrrhiza in pneumonia, we performed GO functional enrichment and KEGG pathway analyses using the R software (version 4.2.2).GO functional enrichment analysis was structured around three areas: BP, CC, and MF.In total, 2441 GO enrichment results were obtained, comprising 2255 results from BP, 61 from CC, and 125 from MF. Figure 7a

| Molecular docking simulation analysis
One of the 10 hub genes, TNF, has two extensively studied types: TNF-α and TNF-β, both of which are associated with inflammation.
This study included both types in the molecular docking simulations.Thus, 11 protein receptors and 64 active components were involved in the molecular docking simulation.All docking results were arranged in ascending order of free energy to obtain Table 2.In general, when the binding energy is less than À5 kcal/mol, the two entities have good binding activity; when the binding energy is less than À7 kcal/ mol, the docking activity becomes very strong. [32]In our results, all receptor-ligand binding energies were less than À5 kcal/mol, and some binding energies were less than À7 kcal/mol, indicating that the 64 active compounds had satisfactory binding affinities for the corresponding protein receptors.The 66 results listed in Table 2 were F I G U R E 6 Process of topological screening for the PPI network.
further refined.As shown in Figure 8, each protein with the lowest free energy required to dock with the corresponding compound was selected for visualization.The docking free energies of RELA with MOL000422 or MOL000497 were both À7.0 kcal/mol; however, the degree of MOL000422 was higher (Table S5).Therefore, the RELA-MOL000422 group was chosen for the model demonstration.

| DISCUSSION
Pneumonia is a respiratory disease that manifests as inflammation and infection of the alveoli and bronchi. [33]The extensive use of antibiotics and other traditional methods to treat pneumonia can lead to pathogen resistance, thereby complicating its treatment.Compared to the use of individual compounds prone to causing drug resistance to pathogens, TCM offers a multicomponent, multipathway, synergistic regulatory network with clear benefits.TCM treatment is less likely to result in drug resistance and has a higher efficacy rate, with minimal side effects. [34,35]Recent scientific research has revealed significant pharmacological effects of Glycyrrhiza, such as the inhibition of inflammatory responses.However, the specific mechanisms underlying the treatment of pneumonia remain unknown.This study utilized Chinese medicine composition and disease gene databases to perform target acquisition and screening of effective components of Glycyrrhiza and pneumonia-related targets.The intersections between these parts were examined as potential targets of Glycyrrhiza in the treatment of pneumonia.In addition, we obtained relationship maps of the Glycyrrhiza components and their respective targets.We also mapped and analyzed the PPI network of potential therapeutic targets and screened hub genes.The potential targets were subjected to GO and KEGG enrichment analyses before drawing bar graphs.Hub genes and corresponding active components of Glycyrrhiza were simulated using molecular docking.We used the results of the PPI network analysis to select hub genes, including MAPK3, MAPK1, TNF, IL-6, MAPK14, RELA, TP53, STAT1, IL-1β, and STAT3.The KEGG results indicated the hub genes existed in multiple pathways, suggesting that the therapeutic effect of Glycyrrhiza on pneumonia is achieved through the synergistic action of multiple components and targets participating in multiple pathways.
The KEGG enrichment pathway analysis revealed a significant association between the lipid and atherosclerosis pathway and pneumonia.In macrophages, cholesterol in the form of cholesterol crystals (CCs) or oxidized low-density lipoprotein (ox-LDL) can activate NLR family pyrin domain-containing 3 (NLRP3), which induces an inflammatory state and creates favorable conditions for lung infection. [36]In in vitro studies, the fusion protein of the virus interacts with cholesterol and facilitated the entry of SARS-CoV-2 into host cells. [36]evated cholesterol can also impair the intrinsic defense mechanisms of the lungs, exacerbating the condition of pneumonia patients.
Therefore, lipid-lowering therapy is considered necessary. [36]In contrast to hyperlipidemia, which can increase susceptibility to pneumonia, hypolipidemia, the other extreme, can also impair the lungs' early innate defense mechanisms against both Gram-positive and Gram-negative pathogens, thus affecting pneumonia susceptibility. [37]udies have shown that lower baseline high-density lipoprotein cholesterol (HDL-C) and higher triglyceride levels are associated with an increased risk of long-term pneumonia hospitalization. [38]Various indications suggest that abnormal serum lipid levels can contribute to an inflammatory environment and weaken the body's innate lung immunity.Therefore, it is essential to maintain normal serum lipid levels to protect lung defenses.Furthermore, lipids are recognized as a significant regulator of the immune response.Lipids represented by HDL can enhance the body's ability to combat pathogens by inducing an oxidative state.This may also be a promising approach to treating pneumonia. [38]Glycyrrhiza regulatory genes are closely correlated with the lipid and atherosclerosis pathway.Glycyrrhiza may have a therapeutic role in treating pneumonia by inducing and stabilizing the body's lipid levels back to the normal range.However, further research is needed to determine its specific role.
RAGE, a common mediator of pulmonary diseases, has been extensively studied. [39]RAGE is typically classified into two types: membrane-bound RAGE (mRAGE) and soluble RAGE (sRAGE). [40]AGE activates inflammation transcription factors, including NF-κB, indirectly, by binding to mRAGE.sRAGE acts as a decoy receptor, blocking ligand binding to mRAGE and preventing inflammatory responses. [39,41,42]According to a study by Li et al, [43] 18βglycyrrhetinic acid (18β-GA) enhances sRAGE secretion.Moreover, the AGE-RAGE signaling pathway stimulates the expression or secretion of vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β1), monocyte chemotactic protein (MCP-1), and other cytokines, aggravating the inflammatory response and causing cellular and tissue damage. [44,45]Additionally, the AGE-RAGE signaling pathway activates NF-κB, which belongs to a family of transcription factors known to have crucial functions in regulating both innate and adaptive immune processes. [46]When activated, NF-κB triggers the release of multiple pro-inflammatory cytokines.Therefore, it is a key target for inhibiting inflammation. [47,48]Licochalcone A suppresses the activation of NF-κB by inhibiting p65 phosphorylation, leading to its inhibitory effects on cytokines. [49]Furthermore, a study on acute pneumonia conducted by Ko et al [50] used immunofluorescence analysis to demonstrate that Gancaonin N inhibits the nuclear translocation of NF-κB p65 in lipopolysaccharide (LPS)-induced cells.This finding implies that Gancaonin N possesses anti-inflammatory properties via inhibition of the NF-κB signaling pathway.We believe that Glycyrrhiza has the potential to treat pneumonia through the mechanisms mentioned above.
Furthermore, according to the KEGG pathway enrichment analysis, the IL-17 and TNF-α signaling pathways are also important for pneumonia treatment.IL-17 stimulates the secretion of proinflammatory molecules and recruits neutrophils and monocytes to participate in the inflammatory response.Thus, excessive IL-17 expression activates and enhances the inflammatory response, which disrupts the lung's physiological environment. [51]Studies have documented the overexpression of IL-17 in the immune response of children diagnosed with Mycoplasma pneumoniae pneumonia (MPP).
Anti-IL-17 strategies have shown significant potential for the management of refractory Mycoplasma pneumoniae pneumonia (RMPP). [52]Li et al. [53] used an LPS-induced murine model to demonstrate a positive correlation between the severity of acute lung injury (ALI) and the upregulation of IL-17, and IL-17 inhibition effectively reduced pulmonary inflammation.Patients with severe pneumonia have higher IL-17 levels in their peripheral blood than healthy individuals, and IL-17 levels increased with disease progression. [54]Evidence suggests that IL-17 is crucial in the occurrence and progression of pneumonia.
TNF is a central inflammatory cytokine that initiates the body's inflammatory response by inducing the expression of inflammatory genes.It also indirectly promotes inflammation by stimulating immune responses and inducing cell death pathways. [55]The excessive activation of M1 macrophages resulting in an inflammatory cytokine storm is a significant pathogenic factor in SARS-CoV-2 pneumonia.Lung macrophages are the main sources of TNF-α.TNF-α is significantly associated with various inflammatory-mediated lung diseases.Alleviating pulmonary inflammation can be achieved by inhibiting or antagonizing TNF-α. [56]Licochalcone A has immunomodulatory effects on the production of IL-17 and TNF-α in experimental autoimmune encephalomyelitis (EAE) mice cells induced by myelin oligodendrocyte glycoprotein peptide (MOG35-55).It is capable of inhibiting their production and reducing disease severity. [57]According to the pathway To further investigate the material basis of the antipneumonia effects of Glycyrrhiza, we conducted molecular docking simulations of the hub genes and their corresponding active components.According to our docking results, the free energy of the binding of the active components to targets related to pneumonia was less than À5.0 kcal/ mol.Moreover, certain free energies were lower than À7.0 kcal/mol.This implies that the selected active components in Glycyrrhiza are capable of spontaneously binding to relevant targets, which may provide theoretical support for treating pneumonia using Glycyrrhiza.
Interestingly, among the 10 hub genes, the 47 components associated with MAPK14 were the most numerous.When sorting the molecular docking free energies from largest to smallest, almost all MAPK14-associated docking results were ranked at the top.This observation suggests that various active compounds in Glycyrrhiza MAPK pathway inhibitors are potential anti-inflammatory drug candidates for the aforementioned reasons. [58]The active constituents of Glycyrrhiza show good binding activity with proteins such as MAPK14, indicating their potential as targets in the mechanism of action of using Glycyrrhiza for pneumonia treatment.

| CONCLUSIONS
In conclusion, we used network pharmacology techniques combined with molecular docking simulations to conduct a preliminary and systematic investigation of the antipneumonia mechanism of Glycyrrhiza.
The results show that multiple components, targets, and pathways of Glycyrrhiza act synergistically to achieve a therapeutic effect against pneumonia.These findings offer valuable directions and references for subsequent research aimed at developing new antipneumonia drugs.

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I G U R E 4 Active component-target network of Glycyrrhiza.F I G U R E 5 The role of the Glycyrrhiza PPI network in the treatment of pneumonia.activator of transcription 1 (STAT1), interleukin 1-beta (IL-1β), and signal transducer and activator of transcription 3 (STAT3) (Table displays the top 10 results for each category of BP, CC, and MF.BP was primarily involved in responding to lipopolysaccharides, bacterial molecules, and oxidative stress.CC was primarily linked to membrane rafts, membrane microdomains, and the vesicle lumen.MF was primarily associated with binding cytokine receptors, cytokine activity, and activation of signaling receptors.An algorithm in the R programming language was used to obtain 173 KEGG pathways, and the top 30 results were visualized as bar graphs.The related target pathways were mainly enriched for lipids and atherosclerosis (Has05417), advanced glycation end product-receptor for advanced glycation end product (AGE-RAGE) signaling pathway in diabetic complications (Hsa04933), fluid shear stress and atherosclerosis (Hsa05418), IL-17 signaling pathway (Hsa04657), and TNF signaling pathway (Hsa04668).Other enriched pathways included toxoplasmosis (Hsa05145), Chagas disease (Hsa05142), Kaposi sarcomaassociated herpesvirus infection (Hsa05167), prostate cancer (Hsa05215), and toll-like receptor signaling pathway (Hsa04620) (Figure 7b).

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I G U R E 7 (a) Common targets of GO enrichment for the treatment of pneumonia by active components of Glycyrrhiza.(b) Enrichment of potential targets for treating pneumonia from the main active components of the Glycyrrhiza KEGG pathways.

F I G U R E 8
The molecular docking models.Panels show the molecular docking model between (a) MAPK14 and MOL004805.(b) TNF-α and MOL000098.(c) MAPK1 and MOL000098.(d) STAT3 and MOL000497.(e) RELA and MOL000422.(f) MAPK3 and MOL004328.(g) STAT1 and MOL000098.(h) TP53 and MOL000098.(i) IL-1β and MOL000098.(j) IL-6 and MOL000098.enrichmentresults obtained in this study, Glycyrrhiza may exert therapeutic effects on pneumonia by inhibiting IL-17 and TNF-α.In addition, as suggested by the KEGG enrichment analysis, pathways involving fluid shear stress and atherosclerosis, toxoplasmosis, Chagas disease, Kaposi sarcoma-associated herpesvirus infection, prostate cancer, and the toll-like receptor signaling pathway may also contribute to the onset and progression of pneumonia, potentially providing therapeutic options.This study revealed that Glycyrrhiza can synergistically utilize these pathways to form an integrated network that may provide new insights into the comprehensive and multifaceted treatment of pneumonia.This highlights the potential of Glycyrrhiza as a research focus for pneumonia treatment.
exert their antipneumonia effects by regulating MAPK14.Therefore, MAPK14 plays a crucial role in the antipneumonia activity of Glycyrrhiza.Furthermore, studies have demonstrated the role of MAPK in regulating various essential inflammatory mediators within the body.MAPK inhibitors, such as SB 203580, SB 220025, and SB 239063, have been shown to downregulate inflammatory cytokine levels and inhibit inflammatory responses in models of rheumatoid arthritis, systemic inflammation, chronic gastrointestinal disorders, encephalitis, and stroke.The p38 MAPK pathway can be activated by cytokines such as IL-17 and TNF-α.Specifically inhibiting MAPK-related signaling pathways may be an effective approach for treating pneumonia.
The emergence of network pharmacology techniques has provided new methods and a focus on drug development.Integrating technologies such as systems biology, computer science, and big data enables the expedient and intuitive prediction and simulation of a compound's efficacy and mechanisms, surpassing traditional laboratory methods.This integration has the potential to enhance the efficiency of drug research and development.However, the limitations of network pharmacology techniques must be acknowledged.Network pharmacology techniques rely on relevant drug and disease databases.Consequently, research outcomes are limited by factors such as the data quality and comprehensiveness.This study presents an initial, innovative examination of the antipneumonia mechanism of Glycyrrhiza.However, further confirmatory experiments are required.This study establishes a theoretical foundation for future experimental validation and offers valuable references and guidance for subsequent research.Given the rapid advancement in scientific technology and ongoing expansion of research, our understanding of drugs and diseases will inevitably become more thorough, further broadening the prospects and momentum of network pharmacology techniques.
Characteristics of the 10 hub genes.
T A B L E 2 The docking results.