Network Pharmacology-based Study of Simiao Yongan Decoction for Treatment of Herpes Zoster Infection

Background: Herpes zoster (HZ) is a virus that causes infectious diseases that impact the quality of life of patients. Herein, we applied network pharmacological methods to predict the target of bioactive components in Simiao Yongan Decoction (SYD) that could treat HZ. Methods: We developed a Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMP) and GenneCards databases for screening of bioactive components of SYD, their targets, and HZ related targets. A bioactive component-target network of SYD was constructed using Cytoscape. We also constructed a protein-protein interaction (PPI) network using the Search Tool for the Retrieval of Interacting Genes Database (STRING) to identify potential SYD targets for the treatment of HZ. "ClusterProler" in R-project was used for Gene Ontology (GO) and KEGG pathway enrichment analyses. We screened SYD hub genes based on component-target network topological parameters and conrmed the ndings by molecular docking. We selected 126 bioactive components and 235 targets. Results: By assessing the topological parameters of the degree network, we identied that CDK2, CASP3, JUN, AKT1, and MAPK1 were hub genes related to SYD-based therapy against HZ. The ndings showed that treatment of HZ with SYD mainly involved toll-like receptor, C-type lectin receptor, MAPK, PI3K-Akt, and other signaling pathways. The molecular docking results revealed good binding energy between the SYD bioactive compounds and hub targets. Conclusion: We showed that SYD could effectively treat HZ via multiple targets and pathways. Our results provide theoretical support for treatment of HZ with SYD and a new direction for such treatment using traditional Chinese medicine.


Background
Herpes zoster (HZ) causes a localized infection of the dorsal root ganglia of the spinal/ cranial nerves that spreads like a rash over the corresponding dermatome. It is usually caused speci cally by the varicella-zoster virus [1], which seriously impacts the quality of life of patients. The incidence of HZ is increasing; for example, in the United States, the incidence of HZ infection is 3.2-4.2 per 1 000 personyears [2]. Oral antiviral drugs are the most important basis for treatment of HZ. The oral-based antiviral medications that are approved for HZ treatment include famciclovir, acyclovir, and its derivative, valacyclovir. Meta-analyses have revealed that oral acyclovir substantially decreases HZ-associated symptoms, including intensity, duration, and frequency of zoster-mediated pain. However, this drug does not affect postherpetic neuralgia (PNH) [3], and acyclovir may produce neurological side effects [4]. An ideal agent for treatment of HZ has not been identi ed thus far.
Simiao Yongan Decoction (SYD) is a classic traditional Chinese medicine (TCM,) prescription listed in the "Yan Fang Xin Pian". It includes the Chinese herbs, Jinyinhua, Xuanshen, Gancao, and Danggui. Clinical studies in China have suggested that SYD can treat HZ without causing signi cant side-effects [5]. Others have shown that SYD is effective for treatment of PNH [6]. However, Chinese herbs contain many active ingredients with various pharmacological effects; hence, mechanisms of action should be elucidated for appropriate clari cation.
Network pharmacology involves the construction and analysis of biological networks to study disease pathogenesis [7]. Network pharmacology has been widely utilized to explore the pharmacological mechanisms of Chinese herbs. In the present study, we applied network pharmacology and molecular docking to reveal the core target and main active agents, and possible relationships among them, to provide theoretical support for (PAP) HZ treatment by SYD.

Screening and identi cation of SYD compounds
The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (http://tcmspw.com/tcmsp.php) is a platform based on systems pharmacology that exclusively focuses on Chinese herbal medicine. It shows interactions between drugs, targets, and diseases [8]. We therefore used the TCMSP to identify the main active components of SYD herbs. The screening parameters were OB (oral bioavailability) and DL (drug-like). These parameters are popular approaches for screening of the chemical composition of TCM. The OB is de ned as the relative amount of a drug that enters the bloodstream after extravascular administration, and DL indicates the extent of similarity of a compound with a known drug and the likelihood of the compound to be used as a pharmaceutical agent [9]. Herein, we analyzed compounds with OB ≥ 30% and DL ≥ 0.18.

Target prediction
The targets of effective components of SYD collected using the TCMSP were veri ed using the Uniprot protein sequence resource (http://www.Uniprot.org). We excluded the bioactive compounds that lacked potential target information. Herpes zoster was used as a keyword to collect disease targets in the GenneCards database (https://www.genecards.org/) [10]. Finally, we matched these targets of SYD and HZ, and selected 31 overlapping targets of SYD for treatment of HZ by illustrating a Venn diagram using R-project version 3.6.3 with the VennDiagram package.

Protein-protein interaction (PPI) network construction and Hub gene analysis
Data on the possible related targets of SYD for treatment of HZ were used as input for STRING (http://stringdb.org) [11] for PPI analysis, with the selected species, Homo sapiens, and a network map of PPI was constructed. We then downloaded PPI data from STRING for further investigation. We also imported data from STRING into the Cytoscape software V3.72 [12] to analyze topological attributes.
Degrees (DC) calculated using CytoNCA indicate numbers of connections between nodes. Finally, we considered genes with the top three DC values as Hub genes.

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The GO (Gene Ontology) project provides crucial information regarding gene functions [13]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database provides information that facilitates understanding of the functions and roles of biological systems, namely cells, organisms, and ecosystems. It contains large-scale molecular datasets obtained via genome sequencing or highly automated technologies [14]. Here, we used the clusterPro ler, DOSE, org.Hs.eg.db, and enrichplot packages for GO and KEGG analysis. We set PvalueCutoff = 0.05 and qvalueCutoff = 0.05 in R-project. A Bubble Chart was plotted using the ggplot2 package.

Molecular docking
We performed molecular docking using the open-source software, AutoDock Vina [15]. We selected compounds and targets with the top three degree values in the compounds-targets network for dock stimulation. All compound structures were downloaded from TCMSP and the 3D structures of the targets were retrieved from PBD (http://www.rcsb.org/).

Identi cation of HZ-related targets in SYD
After removing duplicate targets, we screened 2,056 targets related to the bioactive compounds of SYD from TCMSP. Among these 55, 1,543, 402, and 56 targets were related to Danggui, Gancao, Jinyinhua, and Xuanshen, respectively. We also screened 328 targets corresponding to HZ. Finally, 235 targets were identi ed that interacted with 126 bioactive compounds of SYD, and 31 targets that were associated with HZ ( Fig. 1).

Compound-target network
The compound-target network established by Cytoscape had 98 nodes and 122 edges (Fig. 2). The degree value indicates links between the targets and bioactive compounds. Table 2 shows the following degree values of potential targets and bioactive compounds: MOL000098 (quercetin, degree = 21), MOL000006 (luteolin, degree = 13), and MOL000422 (Kaempferol, degree = 11) Target name Degree Compound Degree

Protein-protein interaction
We obtained 31 nodes and 318 interactions from the PPI network analysis (medium con dence ≥ 0.4; Fig. 3). The average node degree of the PPI network was 20.5 and the local clustering coe cient was 0.848. Based on these ndings, the three nodes with the highest degree were considered as Hub genes, and included CDK2, CASP3, and JUN. These target proteins might be signi cant in the SYD-based treatment of HZ.

GO and KEGG pathway enrichment analyses
We applied R-project for GO_BP enrichment analysis. The bubble chart shown in Fig. 4A shows the top 20 ranked entries. The PPI network targets were mostly involved in response to lipopolysaccharides, molecules of bacterial origin, and reactive oxygen species, as well as leukocyte cell-cell adhesion, modulation of DNA-binding transcription factor activity, T cell activation, and other molecular functions.
We also applied R-project for KEGG pathway enrichment analysis. The bubble chart shown in Fig. 4B shows the top 20 ranked entries. The ndings indicated targets that were mostly associated with the Tolllike receptor signaling pathway, C-type lectin receptor signaling pathway, Endocrine resistance, Osteoclast differentiation, and the FoxO, and MAPK signaling pathways. The Toll-like receptor signaling pathway was more signi cant, and thus we mapped the pathway (Fig. 5). Both GO functional and KEGG pathway enrichment analyses suggested that multiple targets of SYD could act on multiple biological processes for treatment of HZ.

Molecular docking
We selected compounds and targets with the top three degrees in the compound-target network to dock stimulation (Table 3). Lower energy of binding of a ligand-receptor indicates better binding activity between them. Figure 6 shows partial molecular docking processes.

Discussion
The virus Herpes zoster (HZ) causes infections like chicken pox and shingles, and it is classi ed under "Snake sore" and "girdling re cinnabar" in traditional Chinese medicine (TCM). Simiao Yongan Decoction is a famous TCM prescription. Several clinical trials have shown that SYD is effective in patients with HZ infection and PNH 5 6 . Therefore, we applied TCM network pharmacological approaches to elucidate these mechanisms.
The present ndings showed that the major bioactive compounds of SYD were quercetin, luteolin, and kaempferol. Quercetin is a bio avonoid with potent antioxidant and anti-in ammatory activities that is found in various vegetables and fruits [16,17]. Quercetin can relieve in ammatory-induced pain in animal models [18] and it exerts neuroprotective effects [19]. Luteolin protects the nervous system [20] and exerts anti-in ammatory [21], and antioxidant [22] effects. Kaempferol is a avonoid with many health bene ts, particularly against in ammatory diseases [23]. Kaempferol attenuates in ammatory pathways by modulating NF-κB [24]. The PPI network analysis showed that CDK2, CASP3, JUN, AKT1, and MAPK1 had the highest degree values; CDK2 participates in cell cycle regulation (RefSeq, Aug 2020); CASP3 participates in apoptosis, in ammation, and necrosis-related signaling pathways (RefSeq, Aug 2017); AKT1 plays a vital role in the regulation of cell survival, angiogenesis, tumor formation, and insulin signaling; JUN functions in growth and differentiation [25], and MAPK1 plays essential roles in neuropathic pain and in ammatory reactions [26,27]. All these suggest that the bioactive compounds of SYD and the targets of these compounds play crucial roles in the treatment of HZ and PNH.
Toll-like receptor, C-type lectin receptor, MAPK, PI3K-Akt signaling pathway, and other KEGG signaling pathways were enriched. Toll-like receptors perceive conserved microbial structures, such as bacterial lipopolysaccharide or viral double-stranded RNA. Upon perception, they induce various signaling pathways related to immune responses against microbial infections [28]. C-type lectin receptors are expressed mainly on myeloid cells and are involved in antifungal immunity. The MAPK signaling pathway is vital in the mediation of multiple cellular processes, which include proliferation, stress response, differentiation, motility, survival, growth, and death [29]. The PI3K-Akt signaling pathway plays a vital role in mediating survival signals in different types of neuronal cells. The PI3K-Akt signaling pathway may suppress cell death by regulating cytoplasmic cell death machinery, as well as the expression of genes that facilitate cell death and survival [30]. Therefore, we postulate that SYD clears HZ through the Toll-like receptor signaling pathway, ampli es immunity through the C-type lectin receptor signaling pathway, and regulates cell apoptosis via the MAPK and PI3K-Akt signaling pathways. The molecular docking results showed good binding energy between SYD bioactive compounds and hub targets, suggesting that our ndings demonstrated high reference value.

Conclusion
We found that SYD was effective against HZ via multiple targets and pathways. Our results provide theoretical support for the treatment of HZ and a new direction for such treatment by TCM. However, experimental validation is warranted before SYD can be realized as a viable pharmaceutical treatment for HZ infection.

Declarations
Ethics approval and consent to participate Not applicable

Consent for publication
Not applicable Availability of data and materials Please contact author for data requests.

Funding
This study did not receive any funding.
Guanyan Chen and Zhenhai Wu provided Chinese medicines. Yixian Li is a consultant dermatologist. Hongtao Liu and Qiuqin Tang analyzed the results and prepared the manuscript. All authors read and approved the nal version of the manuscript.