Exploring the Mechanism of Wu Ling San plus Flavor for the Treatment of Diabetic Macular Edema Based on Network Pharmacology and Molecular Docking Techniques

Aim: To explore the possible mechanism of action of Wu Ling San Plus for the treatment of diabetic macular edema (DME) through network pharmacology and molecular docking. Methods: DME-related genes were searched in drugbank database, GeneCards database, Online Mendelian Human Genetic (OMIM) database, and TTD database. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was applied to screen for potential chemically active substances and related target proteins in the drug pairs. A “drug-disease target” regulatory network was constructed with Cytoscape (3.7.2) software, and a protein-protein interaction (PPI) network was established by Bisogenet and CytoNCA, followed by GO and KEGG enrichment analysis using the Bioconductor platform and R software. Finally, molecular docking of potential chemically active substances and key targets was performed using MOE software. Results: 63 compounds were screened from Wu Ling San Plus, including 71 targets for the treatment of DME, and the PPI core genes were AKT1, JUN, TP53, IL10, VEGFA, IL6, MMP9, CASP3, CCL2, PTGS2, MAPK8; GO biofunctional analysis contained 2143 enrichment results, mainly involving the hypoxic responses, the KEGG enrichment yielded 122 related signaling pathways consisting of the AGE-RAGE signaling pathway, cellular senescence pathway, TNF signaling pathway, IL-17 signaling pathway, C-type lectin receptor signaling pathway, FoxO signaling pathway, MAPK signaling pathway, T cell receptor signaling pathway, etc. Molecular *Kunmao Ke, Xiaoyun Jiang and Yun Zhang contributed equally to this work. #Corresponding authors. How to cite this paper: Ke, K.M., Jiang, X.Y., Zhang, Y., Zhou, Y.K., Zhao, J., Zhang, J.B., Liu, Y.L. and An, M.X. (2022) Exploring the Mechanism of Wu Ling San plus Flavor for the Treatment of Diabetic Macular Edema Based on Network Pharmacology and Molecular Docking Techniques. Chinese Medicine, 13, 33-50. https://doi.org/10.4236/cm.2022.133004 Received: August 22, 2022 Accepted: September 23, 2022 Published: September 26, 2022 Copyright © 2022 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access RE TR AC TE D


Background
Diabetic macular edema (DME) currently is the primary cause [1]. The prevalent pathogenesis of DME is a disruption in the production and absorption of fluid in the retina, leading to increased accumulation of fluid between the layers of the retina, with exudate accumulating in the macula. The disruption of the

Blood-Retinal Barrier caused by an increase in the Vascular Endothelial Growth
Factor is regarded to be the fundamental pathophysiology of DME (VEGF). Anti-VEGF injections are the most frequent treatment for DME, however, due to variances in staging, severity, and the complexity of the concomitant retinal diseases, some patients have poor outcomes and the oedema is difficult and recurring to remove [2].
"Treatise on Typhoid Fever-Defining the Pulses and Symptoms of Sun Diseases," by Wu Ling San. Only five plants are used in the original formula: Oriental Waterplantain rhizome, Polyporus, Atractylodes, Poria, and Cassia twig, which have the effects of warming yang, transforming qi, promoting water and permeating dampness, and are mainly used to treat the evidence of stagnation of water and dampness in the bladder, thus primary being used in modern times for the evidence of internal stagnation of water and dampness [3]. According to traditional Chinese medicine, the yellowish color of macula is ascribed to the spleen evidence, which prefers dryness and despises dampness, so if water and dampness cease inside the macula for an extended period of time, the middle yang will be obstructed, the spleen will be blocked, the spleen will be unable to transfer fluid, the qi will be weak, the water will flood and produce swelling, and the water and dampness will halt in the macular area, causing macular edema.
Treatment for DME patients with long-term diabetes mellitus should include systemic and fundus identification, as well as the principles of nourishing yin and benefitting qi, as well as dipping dampness and strengthening the spleen [4] [5] [6].
The Wu Ling San formula was supplemented with Rehmannia glutinosa to nourish Yin and promote fluid, Milkvetch root to strengthen the spleen, tonify

R E T R A C T E D
Qi, and promote diuresis to reduce swelling, and fried Coix seed to strengthen the effect of diuresis and dampness to strengthen the spleen in this study. However, the target and mechanism of action remain unknown. Network pharmacology provides a comprehensive explanation of the pharmacological mechanisms underlying drug formulations, enabling time and labor-efficient enhancement of clinical efficacy as well as reduction of toxicity and side effects [7]. Additionally, molecular docking is a theoretical method to study the interaction and recognition between protein receptors and small molecule ligands owing to its capacity to predict the binding mode and affinity strength [8] [9] [10]. Network pharmacology, as a brand new subject based on system biology, bioinformatics, and high throughput histology, is getting more and more motivated [11] [12] [13]. Therefore, this study employed network pharmacology combined with molecular docking to explore the key genes and pathogenesis of Wu Ling San plus flavor for the treatment of DME, with the goal of identifying novel therapeutic targets.

Screening for DME-Related
Genes DME-related targets were collected in the drugbank database, NCBI Gene database, GeneCards database, OMIM database, and TTD database, respectively, using the Diabetic Macular Edema keyword, and Merge was performed after removing duplicates.

Active Ingredient Screening and Target Prediction
TCMSP was used to screen the compounds for the activity of Polyporus, Atractylodes, Poria, Cassia twig, Rehmannia glutinosa, Milkvetch root, and Coix seeds using bioavailability (OB) and drug-like properties (DL). The activity screening was performed to select those that satisfied both OB ≥ 30% and DL ≥ 0.18 [14] as potential active ingredients. The TCMSP was then adopted to identify the active component targets in each herbal remedy.

Regulatory Network
The interaction targets, the prospective targets of action of Wu Ling San plus flavor on potential active ingredients associated with DME, and the disease differential genes acquired from the screening were compared and analyzed using Perl language. The compound names, target gene names, main biological functions, and signaling pathways were then imported into Cytoscape 3.7.2 software to map the regulatory network of Wu Ling San plus flavor.

Protein-Protein Interaction Network Construction
The identities of interacting target genes of drugs and diseases were imported into Cytoscape 3.7.2 software, and the Bisogenet package was installed to construct protein interaction (PPI) networks [15], followed by the CytoNCA package for network topology analysis.

GO Biofunctional Enrichment Analysis
The ID-transformed target genes acquired after network merging were then submitted to GO enrichment analysis using the Bioconductor platform and R software to assess the protein groups included in each GO annotation with a p-value and indicate the relevance of the protein biological functions. Biological process (BP), molecular function (MF), and cellular composition (CC) were selected. Bubble maps were plotted in 3 modules.

KEGG Metabolic Pathway Enrichment Analysis
The Bioconductor platform and R software were applied to perform KEGG metabolic pathway enrichment analysis on the target genes of drug-disease interaction to determine key metabolic pathways of action of Wu Ling San plus flavor for the treatment of DME.

Molecular Docking Validation
The chemical structures of the active ingredients of Wu Ling San plus flavor in mol2 format were obtained by searching in the TCMSP database, the protein crystal structures were obtained from the RCSB PDB database

Acquisition of DME-Related Targets
DME-related targets were collected from five databases, namely drugbank database, NCBI Gene database, GeneCards database, OMIM database, and TTD database with the Diabetic Macular Edema keyword, respectively, and 35, 33, 11, 905, and 17 DME-related targets were combined after removing duplicates to obtain a total of 957 DME-related targets. Milkvetch Root with 20, Rehmannia glutinosa with 2, and Coix seeds with 9, as shown in Table 1. The target proteins of the selected compounds were obtained from the TCMSP database, and their corresponding gene names were searched in the UniProt database, the DME-related genes got from the joint screening of related databases, and 61 genes were acquired from the intersection of the two, as shown in Figure 1.

Therapeutic DME Regulatory Network Construction
A drug compound-acting target disease network with 35 nodes was constructed using Cytoscape 3.7.2 software (Figure 2), in which purple, yellow, green, cyan, blue, red, and pink nodes represent the main active compounds of DME, C. multiflora, Poria, Cassia, Dioscorea, Astragalus, and Coix seeds, respectively, and blue nodes represent potential targets, with each edge indicating compound and target The interaction relationship between compounds and targets. It can be seen that MOL000098 (quercetin) and MOL000422 (kaempferol) play a key role in the whole network and may be the core compounds of the Angelica-Chuanxiong drug pair for the treatment of DME.

PPI Network Analysis
The 71 intersecting target genes obtained from drug-disease mapping were imported into the String database for PPI network analysis before the topological analysis was performed by CytoNCA in Cytoscape 3.7.2 (Figure 3

GO Enrichment Analysis
To further elucidate the possible roles of candidate targets, GO enrichment analysis was performed using the Bioconductor platform and R software, and

KEGG Enrichment Analysis
KEGG enrichment analysis was performed on 71 putative action targets using the Bio conductor platform and R language, yielding a total of 122 entries. The top 20 entries with small P-values are shown in Figure 5, which are presented in the form of bar graph plots. The length and color of the bar graph show significance; the greater the length, the more genes are represented, suggesting that the entry is more important. It can be seen that the relevant mechanisms mainly involve the AGE-RAGE signaling pathway, cellular senescence pathway, TNF signaling pathway, IL-17 signaling pathway, C-type lectin receptor signaling pathway, FoxO signaling pathway, MAPK signaling pathway, T cell receptor signaling pathway, etc.

Molecular Docking
The ligands in the proteins were isolated using Pymol (version 2.3.4), and the 11 proteins in Figure 3(c) were docked to quercetin and kaempferol compounds using AutoDock Vina, with binding energies (affinity) −7.0 kcal•mol −1 indicating good binding activity [16] and the lower the binding energy, the better the docking is indicated. As shown in Table 2, a total of 2 out of 11 proteins have binding energy < −7.0 kcal•mol −1 to the corresponding compounds, indicating that these proteins have a strong affinity for the compounds.

Discussion
Network pharmacology, which is based on the theory of systems biology, uses various methods such as histology and network visualization to create system network models to examine the intervention and impact of drugs on diseases from a holistic perspective and reveal the complex relationships between drugs, diseases, targets, and pathways, which are important for understanding the mechanism of action, the material basis of pharmacological effects, and the development of new drugs [17] [18] [19] [20] [21].
In the present study, TCM active component-target network analysis revealed that quercetin, kaempferol, and other active ingredients can act on multiple targets in the network. This data implies that these components are significant for Wu Ling San Plus's therapeutic impact on DME and should be investigated further. Quercetin has the most potential targets. According to previous reports, quercetin, one of the most prominent members of the natural flavonoid family, has been reported to be beneficial in age-related ophthalmopathy and retinal ischemia-reperfusion injury [22] [23], whose protective effect on retinopathy was mainly attributed to its anti-oxidative, anti-inflammatory, anti-apoptotic, anti-angiogenic and neuroprotective effects in retinal cells [24] [25] [26].
The main proteins of the PPI network, AKT1, JUN, VEGFA, MMP9, and CASP3, were evaluated in this study, revealing that the active components of Wu Ling San Plus had strong binding activity to them and can be exploited as prospective targets for the treatment of DME. Anti-VEGF therapy is currently a popular therapeutic option for DME. CASP3, a crucial executor downstream of the apoptotic pathway, performs a last executive function in the JUN apoptotic pathway [27] [28] [29]. MMPs are a family of Zn 2+ -dependent endopeptidases that degrade all ECM components except polysaccharides, promote cell proliferation, neovascularization, and tissue remodeling, and MMP-9 is one of the most important members of the MMPs family, playing an essential role in the formation of DR.
The GO enrichment study produced intriguing findings and illustrated the complexity of DME pathogenesis. First, the target genes were found to be enriched in response to hypoxic stress, which may be directly influenced by sugar metabolism. Clinical and experimental studies have demonstrated that hyperglycemia is the primary factor leading to the pathogenesis of diabetic complications [30]. Oxidative stress can damage cell membrane integrity [31], nducing apoptosis, microvascular damage, and barrier damage and ultimately leading to DR development. 10 most important GO (BP) entries are mainly related to hypoxic response, oxygen level reduction response, apoptotic signaling pathway regulation, metal ion response, regulation of DNA-binding transcription factor activity, regulation of reactive oxygen metabolic processes, cellular oxidative stress response, and reproductive system development. Related studies have shown that the occurrence and development of DME are associated with cellular dysfunction and injury, chronic inflammatory infiltration, and oxidative stress [32]  (a crucial downstream growth factor in angiogenesis) and monocyte chemoattractant protein-1 (MCP-1), both in vivo and in vitro [37]. Another common and well-known pathway, the TNF signaling pathway, is closely related to inflammation, which is a crucial process in DME progression [38]. Gao's study revealed that hypoxia inducible factor subtype 1α in diabetic retina is likely to play a role in dysfunction and vulnerability related to DR progression via TNF-α [39].

Conclusion
The treatment of DME with Wu Ling San Plus shows the multi-component and multi-target features of traditional Chinese medicine, which may achieve the management of DME's developing process through many routes, offering certain insights and a foundation for further research.

Authors' Contributions
Yanli Liu and Meixia An had full access to all study data and take responsibility for its integrity and the accuracy of the analysis. Kunmao Ke, Xiaoyun Jiang and Yun Zhang were responsible for the study concept and design. Yekai Zhou, Jian Zhao and Junbiao Zhang responsible for data acquisition, extraction and drafting the paper, which was revised by Yanli Liu and Meixia An. Yanli Liu and Meixia An supervised the study. All authors read and approved the final manuscript.