Identification of Potential Molecular Targets and Active Ingredients of Mingmu Dihuang Pill for the Treatment of Diabetic Retinopathy Based on Network Pharmacology

Objective Mingmu Dihuang Pill (MMDHP) is a traditional Chinese formula that has shown remarkable improvements of dry eyes, tearing, and blurry vision; however, the mechanisms underlying MMDHP treatment for diabetic retinopathy have not been fully understood. This study is aimed at identifying the molecular targets and active ingredients of MMDHP for the treatment of diabetic retinopathy based on network pharmacology. Methods All active ingredients of MMDHP were retrieved from TCMSP and BATMAN-TCM databases, and the targets of active ingredients of MMDHP were predicted on the SwissTargetPrediction website. Diabetic retinopathy-related target sets were retrieved from GeneCards and OMIM databases, and the intersecting targets between targets of active ingredients of MMDHP and potential therapeutic targets of diabetic retinopathy were collected to generate the traditional Chinese medicine-ingredient-target-diabetic retinopathy network and to create the protein-protein interaction network. In addition, GO terms and KEGG pathway enrichment analyses were performed to identify the potential pathways, and molecular docking was employed to verify the binding of active ingredients of MMDHP to key targets of diabetic retinopathy. Results Network pharmacology predicted 183 active ingredients and 904 targets from MMDHP, and 203 targets were intersected with the therapeutic targets of diabetic retinopathy. The top 10 hub targets included PIK3RA, TP53, SRC, JUN, HRAS, AKT1, VEGFA, EGFR, ESR1, and PI3KCA. GO terms and KEGG pathway enrichment analyses identified AGE-RAGE, PI3K-AKT, and Rap1 signaling pathways as major pathways involved in MMDHP treatment for diabetic retinopathy. Molecular docking confirmed a good binding affinity of active ingredients of MMDHP, including luteolin, acacetin, naringenin, and alisol B, with AKT1, SRC, and VEGFA as the three key targets of diabetic retinopathy. Conclusion MMDHP may be effective for the treatment of diabetic retinopathy through active ingredients luteolin, acacetin, naringenin, and alisol B via AKT1, SRC, and VEGFA in AGE-RAGE, PI3K-AKT, and Rap1 signaling pathways.


Introduction
Diabetes mellitus, a group of metabolic diseases characterized by chronic hyperglycemia, is a major health problem worldwide, which greatly threatens human health and global economy [1]. The global number of people living with diabetes and global health expenditures due to diabetes were esti-mated to be 536.6 million USD and 966 billion USD in 2021, and these figures were projected to be 783.2 million USD and 1,054 billion USD in 2045 [2]. As one of the leading causes of mortality and disability worldwide [3], diabetes was the 9th leading cause of mortality worldwide and approximately 1.5 million people died of this chronic metabolic disorder in 2019 [4], while the death was predicted to increase to 1.59 million using an autoregressive integrated moving average (ARIMA) model in 2025 [5].
Diabetic retinopathy, one of the most common and most severe complications of diabetes, is a chronic, progressive microvascular disorder that may lead to vision-threatening damages to the retina and even blindness [6]. There are two types of diabetic retinopathy, including nonproliferative diabetic retinopathy (NPDR) at the early stage, which is characterized by increased vascular permeability, retinal hemorrhage, and pericyte exfoliation from retinal vessels; and proliferative diabetic retinopathy (PDR) at the late stage, which is characterized by pathologic neovascularization, retinal scarring, and detachment and vitreous hemorrhage [6]. A recent meta-analysis showed a 22.27% (95% confidential interval (CI): 19.73% to 25.03%) global prevalence rate of diabetic retinopathy among individuals living with diabetes mellitus and 103.12 million adults with diabetic retinopathy in 2020 and 160.5 million adults were predicted to have with diabetic retinopathy in 2045 [7]. In China, the prevalence of diabetic retinopathy was estimated to be 1.14% among general populations and 18.45% among individuals with diabetes mellitus [8]. These data imply the urgent need of interventions targeting diabetic retinopathy.
Mingmu Dihuang pill, a traditional Chinese formula that consists of multiple medicinal plants in optimal prescriptions, including Chrysanthemum morifolium, Lycium barbarum, Moutan cortex, Dioscoreae rhizoma, Poria cocos, Alisma orientalis, Cornus officinalis, Paeonia lactiflora, Tribulus terrestris , and Concha haliotidis [9], has shown remarkable improvements of dry eyes, tearing, and blurry vision [10]. Previous studies have demonstrated that Mingmu Dihuang pills are effective to reduce the area of ecchymosis and number of retinal microangioma and to improve the vision sight among patients with diabetic retinopathy [10][11][12][13][14]. However, the targets and active ingredients of Mingmu Dihuang pill for the treatment of diabetic retinopathy have not been fully understood until now [15].
In the present study, the active ingredients and potential molecular targets of Mingmu Dihuang pill for the treatment of diabetic retinopathy were screened using network pharmacology, and the key targets and signaling pathways involved in Mingmu Dihuang pill treatment for diabetic retinopathy were identified and verified using molecular docking [16][17][18][19][20] (Figure 1). Our findings may provide insights into the development of novel agents for the treatment for diabetic retinopathy.

Prediction of Targets of Active Ingredients of Mingmu
Dihuang Pill. To predict the targets of active ingredients of Mingmu Dihuang pill, the sdf files regarding the active ingredients of Mingmu Dihuang pill were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) or the mol2 files were downloaded and converted into smiles files using the software Open Babel. Then, the sdf or smiles files were loaded into the SwissTargetPrediction web server (http://swisstargetprediction.ch/). The targets of active ingredients acquired from the SwissTargetPrediction web server with a probability of 0 and higher were selected as potential targets.

Prediction of Targets for Diabetic Retinopathy.
Diabetic retinopathy-related genes were retrieved from the Gene-Cards (https://www.genecards.org) and Online Mendelian Inheritance in Man (OMIM) databases (http://www.omim .org) using the term "diabetic retinopathy".

Generation of the Traditional Chinese
Medicine-Ingredient-Target-Diabetic Retinopathy Network. Diabetic retinopathy-related genes and all targets of active ingredients of Mingmu Dihuang pill were input into the WeiShengxin database (http://bioinformatics.cn), in order to screen the intersection between the diabetic retinopathy-related gene set and the drug target set, and the Venn diagram was plotted. The names of intersecting targets and names and ingredients of Mingmu Dihuang pill were loaded into the network visualization platform Cytoscape version 3.2.1, in order to generate the traditional Chinese medicine-ingredient-target-diabetic retinopathy network.

Creation of a Protein-Protein Interaction (PPI) Network.
To create a PPI network, the intersecting targets of Mingmu Dihuang pill and diabetic retinopathy were input into the STRING 11.5 platform (https://string-db.org/cgi/), and the protein type was defined as "Homo sapiens" using the multiple protein tool and was saved as a TSV-format file. The PPI network was then visualized using the software Cytoscape version 3.2.1.

Gene Ontology (GO) Terms and Kyoto Encyclopedia of
Genes and Genomes (KEGG) Pathway Enrichment Analyses. GO terms and KEGG pathway enrichment analyses were performed to investigate the biological processes and metabolic pathways of all intersecting targets between Mingmu Dihuang pill and diabetic retinopathy, using the online Metascape platform (http://metascape.org/gp/index.html).

Molecular Docking Verification.
According to the degree values of active ingredients in the traditional Chinese medicine-ingredient-target-diabetic retinopathy network, the degree value of the node in the PPI network and GO terms and KEGG pathway enrichment analysis results, four major active ingredients (alisol B, luteolin, naringenin, and 2 BioMed Research International acacetin) and three key targets AKT1 (PDB ID: 4EJN), SRC (PDB ID: 4U5J), and VEGFA (PDB ID: 3BDY) were selected for molecular docking. The AKT1 protein was fixed at the grid center set to 32, 42, and 13 for x, y, and z and at the grid box size set at 64, 64, and 64 Å for x, y, and z, respectively, and the SRC protein was fixed at the grid center set to −3.1, 51, and 25 for x, y, and z and at the grid box size set at 90, 77, and 104 Å for x, y, and z, respectively, while the VEGFA protein was fixed at the grid center set to −39, −57, and −3 for x, y, and z and at the grid box size set at 65, 138, and −365 Å for x, y, and z, respectively. The grid box size was set using the software AutoDock Vina version 1.5.6, and the docking results were visualized using the PyMOL software [21]. A negative binding energy indicates a high likelihood of the binding between the ligand and receptor [15,22,23].    3 BioMed Research International officinalis, 2 active ingredients of A. sinensis, 13 active ingredients of P. lactiflora, 12 active ingredients of T. terrestris, and 4 active ingredients of C. haliotidis. Following removal of the active ingredients with unidentified targets, the active ingredients of Mingmu Dihuang pill were finally predicted (Supplementary Table 1). A total of 13 common ingredients were identified, including C. morifolium and T. terrestris (A); C. morifolium, M. cortex, P. lactiflora, and T. terrestris (B); C. morifolium, L. barbarum, and M. cortex (C1); C. morifolium, L. barbarum, R. rehmanniae preparata, C. officinalis, A. sinensis, and P. lactiflora (C2); C. morifolium and C. officinalis

Common Targets between Diabetic Retinopathy Treatment
and Mingmu Dihuang Pill Actions. A total of 1,021 diabetic retinopathy-associated target genes were retrieved in the Gene-Cards database, and 59 target genes were retrieved in the OMIM database. Following target gene merging and removal of repeated targets, a total of 1,079 diabetic retinopathyassociated target genes were identified. Then, these targets were intersected with 904 potential targets of Mingmu Dihuang pill, and 203 potential targets were finally yielded ( Figure 2). The traditional Chinese medicine components of Mingmu Dihuang pill and their corresponding active ingredients and potential targets of diabetic retinopathy were input into the Cytoscape software, so as to generate a traditional Chinese medicineingredient-target-diabetic retinopathy network ( Figure 3). Network topology analysis showed that the seven chemicals with the highest node degree included luteolin, alisol B, acacetin, naringenin, chryseriol, isorhamnetin, and kaempferol, with degrees of freedom of 36,36,35,35,34,34, and 34, respectively.

Topology
Analysis of the PPI Network. The 203 predicted potential targets were input into the STRING platform to generate a PPI network, which contained 203 nodes and 1,169 lines. In this study, the targets with a confidence score of higher than the median were defined as the core targets of Mingmu Dihuang pill, and following screening of the median three times, the targets with a confidence score of > 0.979, 5 were identified as core targets of Mingmu Dihuang pill. The screened targets were visualized using the Cytoscape software and subjected to topology analysis, and a PPI network, which contained 111 nodes and 171 lines, was generated ( Figure 4). The top 10 hub proteins included PIK3RA, TP53, SRC, JUN, HRAS, AKT1, VEGFA, EGFR, ESR1, and PI3KCA.

Molecular Docking of Potential Active Compounds
with Key Targets of Diabetic Retinopathy. AKT1, SRC, and VEGFA were found to have the binding energy of < −5 kcal/mol with all key targets of diabetic retinopathy (Table 4), suggesting the good binding energy towards the key targets of diabetic retinopathy. Next, molecular docking results with the good binding energy were visualized ( Figure 7). Luteolin was found to bind to Ser205, Asp292, Gln79, and Asn54 of AKT1, which increased the structural stability and had good receptor-ligand binding energy, and acacetin bound to Asn54 and Gln79 amino acid residues of AKT1, while alisol B was embedded in the SRC protein cavity to form a stable structure. In addition, acacetin interacted with Tyr340, Met341, Leu273, and Thr338 of the SRC protein, which had a high binding affinity and stable binding mode, and alisol B bound to Ser168 and Asp167 of the VEGFA protein, while acacetin interacted with Gln107, Gly41, Gln38, and Gly42 of the VEGFA protein.

Discussion
Mingmu Dihuang pill, a traditional Chinese formula based on Liuwei Dihuang pill with addition of C. morifolium, L. barbarum, Catsia tora, P. lactiflora, A. sinensis, and T. terrestris in optimal prescriptions, has been used for the clinical treatment of eye dryness, photophobia, blurry eyes, and tears in wind [9,10]. In rats with diabetic retinopathy, Mingmu Dihuang pill was found to protect the injured retina and improve retinal lesions through promoting the production of autophagosome in retinal tissues, improving retinal microcirculation, and promoting blood flow [11]. In addition, Mingmu Dihuang pill was reported to increase the antioxidative damages in the retina and effectively reduce the damages of oxidative stress to retinal cells in diabetic rats, thereby protecting retinal injury [24]. However, the active ingredients and targets of actions of Mingmu Dihuang pills have not been fully understood.
In this study, we identified 183 active ingredients, 904 targets of Mingmu Dihuang pill, and 203 targets that were intersected with the therapeutic targets of diabetic retinopathy using network pharmacology and molecular docking. PPI network analysis identified 111 hub targets, and the top 10 hub targets included PIK3RA, TP53, SRC, JUN, HRAS, AKT1, VEGFA, EGFR, ESR1, and PI3KCA. In addition, GO terms and KEGG pathway enrichment analyses revealed that Mingmu Dihuang pill may be effective for treatment of diabetic retinopathy through biological processes of positive regulation of cell migration, response to inorganic substance, transmembrane receptor protein tyrosine kinase signaling pathway via the AGE-RAGE signaling pathway in diabetic complications, PI3K-AKT signaling pathway, Rap1 signaling pathway, HIF-1 signaling pathway, VEGF signaling pathway, Erb B signaling pathway, and Foxo signaling pathway. It is therefore hypothesized that Mingmu Dihuang pill is active to mediate cell growth, participate in glucose metabolism, suppress cell apoptosis, and inhibit angiogenesis. Our data indicate that compound Chinese medicines function through multi-ingredient, multitarget, and multipathway actions. Molecular docking confirmed a good binding affinity of active ingredients of Mingmu Dihuang pill, including luteolin, acacetin, naringenin, and alisol B, with AKT1, SRC, and VEGFA, three hub targets of diabetic retinopathy. Of the four critical active ingredients The hub target AKT1 is highly expressed in insulinsensitive tissues and triggers the downstream glucose transport protein to increase glucose uptake, thereby mediating glucose metabolism [25,26]. The hub target SRC, a nonreceptor tyrosine kinase, plays a critical role in cell adhesion, cell cycle, and cell migration [27]. SRC was identified as a key gene associated with the risk of type 1 diabetes mellitus using a network biology approach [28], and inhibition of SRC activation resulted in a reduction in endogenous reactive oxygen species (ROS) production and an increase in ATP production in obese diabetic mouse models with hyperlipidemia [29]. In addition, selective SRC family kinase inhibition may serve as a novel attractive therapeutic intervention for retinal vascular pathology [30]. The hub target VEGFA is widely expressed in arterial, venous, and lymphatic microvessels and endothelial cells of large vessels [31], and VEGF inhibitors or anti-VEGR agents have been proven to be highly active against diabetic retinopathy [32,33]. Our findings further demonstrate the feasibility and reliability of network pharmacology to investigate the mechanisms of drug actions.
Our findings showed that the three hub targets AKT1, VEGFA, and SRC were all significantly enriched in the Rap1 signaling pathway, and AKT1 and VEGFA were both significantly enriched in the AGE-RAGE signaling pathway in diabetic complications and PI3K-AKT signaling pathway. It is therefore hypothesized that Mingmu Dihuang pill may