Exploration of Anti-Staphylococcus Aureus Activity and Molecular Mechanism of Wuweixiaoduyin using Network Pharmacology Anti-Staphylococcus Aureus Activity of Wuweixiaoduyin using Network Pharmacology

Background: WWXDY is a traditional Chinese medicine, and it is usually used to treat infections in China. This study aimed to explore the active compounds, therapeutic targets, key pathways, and potential mechanisms of WWXDY in the treatment of S. aureus infection. Materials Methods: Data related to active compounds and therapeutic targets of WWXDY for treating S. aureus were collected from DisGeNET, GeneCards, and DrugBank databases. To explore the roles of the active targets in gene function and signaling pathways, KEGG (Kyoto Gene and Genomics Encyclopedia) pathway enrichment and GO (Gene Ontology) analyses of the 122 target genes in the PPI (protein-protein interaction) network were performed. We further performed NP (network pharmacology) by using a network analyzer to screen 30 key targets. active compounds and therapeutic targets of WWXDY in treating S. aureus infection, as well as its potential mechanisms.


Introduction
S. aureus (Staphylococcus aureus) is a Gram-positive coccus that can cause a variety of infectious diseases, including skin and soft tissue infections, chronic osteomyelitis, and even cause toxic shock syndrome or sepsis [1][2][3]. Basically, 30% of healthy adults' skin and nasal cavity are colonized with S. aureus [4]. Its pathogenicity is determined by bacterial virulence, immune status, etc. Besides, the emergence of MRSA (methicillin-resistant Staphylococcus aureus) is due to increased bacterial resistance, resulting in the treatment failure of several common anti-staphylococcal antibiotics [5]. It may increase the risk of life-threatening infection, and impose a great burden to the society.
S. aureus can express a variety of resistance mechanisms and virulence factors, allowing it to evade host natural defenses. Particularly, S. aureus secretes numerous exotoxins, including polypeptides to damage plasma membrane [6]. The enterotoxin is a protein exotoxin, which can be released by S. aureus and mainly targets the intestines, leading to diarrhea and food poisoning. Besides, α-Hemolysin is the most characterized virulence factor of S. aureus. It can bind to the cell surface and form a β-barrel transmembrane pore, which can abnormally transport ions, such as K + and Ca 2+ [7,8]. Chronic osteomyelitis is a progressive in ammatory process, which is mainly caused by S. aureus, and can result in bone destruction and sequestrum formation [9]. However, the management of chronic osteomyelitis has still remained a main challenge. S. aureus may cause serious damages to multi organs; thus, it is highly essential to explore its corresponding pathogenic mechanism and potential treatments.
In China, TCM (traditional Chinese medicine) has a long history in treating a variety of infectious diseases. Minami et al. demonstrated that Buzhongyiqitang, which is commonly used to improve the immunity of frail elderly people, could effectively treat nasal infection of MRSA in a mouse model [10].
Besides, extract of Angelica dahurica and Rheum o cinale could also inhibit the release of in ammatory cytokines and promote wound healing, which was infected with S. aureus [11]. In TCM, WWXDY (Wuweixiaoduyin) is used to treat patients with symptoms caused by S. aureus infections, such as fever, joint pain, and swelling. Since WWXDY is composed of various medicinal materials, it is characterized by multi-pathway, -target, -component, resulting in unclear material basis and mechanisms of WWDXY in treating S. aureus. It is highly signi cant to e ciently treat infectious diseases caused by S. aureus by exploring WWDXY's active ingredients and targets.
To explore the principles of network theory and systems biology of WWDXY treatment on S. aureus, NP (network pharmacology) was applied to analyze its compound-gene/protein-pathway-disease axis, which is of great importance to describe the connections of biological systems, WWXDY, and S. aureus. In the present study, the compositions and effective components of WWXDY were screened, as well as the predicted component of target proteins. In order to cross-validate the target proteins, PPI (protein-protein interaction) network and drug-compounds-genes-disease network were cross-validated. Finally, we performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment pathway analyses for target proteins. This is the rst study to investigate bioactive ingredients in WWXDY and reveal its potential mechanisms in treating S. aureus infection by using the NP. Based on ADME (absorption, distribution, metabolism, and excretion) parameters, bioactive components were selected. The threshold values for screening of medicinal components were considered as DL (druglikeness) ≥ 0.18 and OB (oral bioavailability) ≥ 30%, which contributed to compounds with higher oral absorption, utilization, and biological properties for further analysis.
2.2 Screening of the targets of active compounds of WWXDY and S. aureus-related genes The SDF structure of the WWXDY components was achieved by using PubChem database. The targets with predicted scores greater than 0 were taken as the target of active compounds of WWXDY. The S. aureus-related genes were screened by DisGeNET, GeneCards, and DrugBank databases through querying the keyword of Staphylococcus aureus. Besides, Venny 2.1 software was used to lter out the co-targets between active compounds of WWXDY and S. aureus.

Establishment of drug-compound-gene-disease network
Based on the data of WWXDY-component-target proteins, interaction network of drug-compound-genedisease was constructed by using Cytoscape 3.8.0 software. This network includes edges and nodes, which respectively represent molecular interactions of compounds, protein targets, signaling pathways, or diseases and molecules.
Network Analyzer was used for topological analysis. The "degree" value indicated the number of correlations between the components and the targets.

Construction of PPI network
The retrieved active ingredient targets of WWXDY are correlated with protein targets of S. aureus by STRING database (https://string-db.org/). The species was set to "Homo sapiens". In addition, the PPI network of the effective components of WWXDY and S. aureus' protein targets was constructed based on the minimum threshold of interaction (0.4). Cystoscap 3.7.2 software was used for PPI network analysis, along with the Network Analyzer for topological analysis. Degree sequencing was performed based on the 4 parameters of degree, betweenness centrality, average path length, and so-called centrality as the reference standards. Genes with scores of top 30 were selected to draw a bar chart by R 3.6.0 programming language.

KEGG pathway enrichment and GO analyses
Additionally, R 3.8.1 programming language was used to carry out the KEGG pathway enrichment and GO functional analyses with a FDR (false discovery rate) <0.2 and P-value <0.05. The GO is a resource that supplies information about gene product function using ontologies to represent biological knowledge. The GO covers three domains: CC (Cellular Component), MF (Molecular Function), and BP (Biological Process).

Effective ingredients of WWXDY and predicted targets
WWXDY consists of Lonicera japonica, Dandelion, Semiaquilegiae Radix, Chrysanthemum, and Viola philippica. In TCMSP database, 92 compounds satis ed the criteria of OB ≥ 30% and DL ≥ 0.18 and were retrieved, including 25 in Lonicera japonica, 49 in Dandelion, 7 in Semiaquilegiae Radix, 12 in Chrysanthemum, and 5 in Viola philippica (Table 1). Moreover, 785 target proteins were found among the 92 active compounds of WWXDY. The results suggested that the main active compounds of WWXDY mainly regulated in ammation-related genes.
To understand the relationship between compounds of WWXDY, therapeutic targets, and S. aureus-related genes, a PPI network was constructed. It was composed of 459 nodes (5 herbs, 74 active compounds of WWXDY, 122 overlapped genes, and 1 S. aureus-related gene) (Fig. 2). The main active compounds for the treatment of S. aureus included secologanic dibutylacetal_qt, desacetylmatricarin, chryseriol, luteolin, quercetin, acacetin, etc.

Identi cation of the hub genes in the constructed PPI network
In order to explore the interaction among 122 overlapped genes, the PPI network was constructed. We found that the network contained 122 nodes and 1,838 edges with interaction score > 0.4 (Fig. 3).
Moreover, 122 nodes represented the overlap among target proteins of active compounds of WWXDYand S. aureus-associated genes, and 1,838 edges represented a relationship among the identi ed genes.
We further performed NP by using a network analyzer to screen 30 key targets. Figure 4 displays the 30 topological properties of WWXDY in the S. aureus-related genes by the PPI network.

KEGG pathway enrichment and GO functional analyses of the target proteins
To con rm the biological responses of S. aureus infection to WWXDY, we performed GO functional analysis of the 122 potential target genes based on MF, CC, and BP. The results showed a total of 1,988 entries. 1,815 entries were sorted in BP analysis, including leukocyte migration, reaction to lipopolysaccharide and reaction to molecule of bacterial origin. In CC analysis, 69 obtained cases were involved in membrane regions membrane microdomains, membrane rafts, etc. Besides, 104 entries were screened after MF analysis. These cases were related to endopeptidase activity, binding receptors to cytokines, serine-type peptidase activity, etc. The top 20 GO terms are listed in Fig. 5.
Additionally, we performed the KEGG pathway enrichment analysis to reveal the biological processes. It was revealed that the genes mentioned above were enriched in 145 pathways with P-value < 0.05 according to the number of the target genes. The top 5 pathways included COVID-19, AGE-RAGE signaling pathway, Pertussis, Chagas disease and CLR (C-type lectin receptor) signaling pathway (Fig. 6).

Discussion
S. aureus is one of the common pathogenic bacteria that causes numerous infectious diseases, such as mastitis, chronic osteomyelitis, pneumonia, etc. [12][13][14]. The results of previous research have shown that the treatment of S. aureus is accompanied by several drawbacks. Although the antibiotic treatment may be signi cant for bacterial infections to a certain extent, the abuse of antibiotics not only leads to the emergence of drug-resistant strains-MRSA, but also causes antibiotic residues, seriously threatening food safety [15]. Thus, there is a main challenge to nd out an appropriate substitute for antibiotic treatment of S. aureus infection.
With the increased clinical application of TCM and its extracts in prevention and treatment of S. aureus infection, its therapeutic effects have been extensively con rmed. Lan et al. demonstrated that avonoids from Artemisia rupestris L., a traditional Chinese herb, had antibacterial effects against S. aureus [16].The antibacterial activity of Sophora avescens extract, Angelica sinensis extract, S. avescens extract and herb pair A. sinensis was investigated, and it was found that S. avescens extract showed a strong antibacterial effect on Chalmers, Escherichia coli, Shigella castellani, and Staphylococcus aureus [17]. In TCM, Scutellaria baicalensis is widely used to treat infections with a symptom of fever. As an active compound of Scutellaria baicalensis, baicalin can effectively reduce the virulence of S. aureus, and cooperate with penicillin G in anti-infection treatment with an improved e cacy of 75% [18]. WWXDY is a kind of TCM, which is often used to treat bacterial infection. It is composed of Lonicera japonica, Dandelion, Semiaquilegiae Radix, Chrysanthemum, and Viola philippica. In our previous study, we attempted to assess the clinical e cacy of the combination of TCM and Western medicine on the traumatic chronic tibial osteomyelitis (CO). We found that combination of the TCM and Western medicine could mitigate the local lesion of traumatic CO and ameliorate the general status [19]. However, the active compounds of WWXDY and its potential molecular mechanisms for the treatment of S. aureus have still remained elusive.
The promising applications of PPI networks to disease datasets are mainly 4 areas: (i) identi cation of disease-related subnetworks, (ii) network-based disease classi cation, (iii) the identi cation of genes and proteins associated with diseases, and (iv) the study of network properties and their relation to disease status. By constructing PPI network, it was found that IL-6, TNF-α, VEGFA, AKT1, CXCL8, MAPK3, TLR4, IL-1β, MMP9 and EGFR were the top 10 hub genes, and they were mainly enriched in in ammation-related signaling pathways. It is signi cant to deepen the understanding of the functional relationship between proteins. In macrophages and epithelial cells, S. aureus could enhance the expression levels of proin ammatory cytokines, including IL-1β, IL-6, and TNF-α [26,27], which were highly correlated with therapeutic targets of WWXDY. Wu et al. [28] demonstrated that Chryseriol, which is a main active compound of WWXDY, could signi cantly inhibit IL-6, TNF-α, and IL-1β in in ammatory diseases.
To explore the roles of the active targets in gene function and signaling pathways, KEGG pathway enrichment and GO functional analyses of the 122 target genes in the PPI network were performed. The effects of WWXDY in the treatment of S. aureus infection are mainly attributed to the regulation of endopeptidase activity, binding receptors to cytokines, and serine-type peptidase activity. Besides,KEGG pathway enrichment analyses indicated that identi ed genes were mainly enriched in COVID-19, CKR signaling pathway, AGE-RAGE signaling pathway, Pertussis, and Chagas disease. The advantages of TCM in the treatment of COVID-19 include effective relief of symptoms, improvement of cure rate, reduction of mortality, and promotion of rehabilitation [29]. COVID-19 patients have a high probability of co-infection with S. aureus [30]. Our data suggest that WWXDY may provide a new therapeutic option for the treatment of COVID-19. The AGE-RAGE signaling pathway was rstly recognized to be involved in S. aureus infection. The complications of atherosclerosis and nephropathy in type 2 diabetes (T2DM) might be treated with TCM mainly through the AGE-RAGE signaling pathway [31]. We found that WWXDY could treat S. aureus infection, which was correlated with the regulation of AGE-RAGE signaling pathway.

Conclusions
In summary, our ndings revealed the effective ingredients of WWXDY and their targets in the treatment of S. aureus infection. Secologanic dibutylacetal_qt, desacetylmatricarin, chryseriol, and other main active compounds may play therapeutic roles in regulating their targets, including IL-6, TNF, VEGFA, AKT1, CXCL8, etc. However, further studies should be performed to clarify the speci c therapeutic mechanisms of the active compounds of WWXDY. The results of the current research may be signi cant for effective drug selection and accurate treatment of S. aureus infection.     GO functional analysis of the target proteins, including BP, CC, and MF terms.