The Mechanism of Action of Qihuang Jiangtang Capsule in the Treatment of Type 2 Diabetes Based on Network Pharmacology and Molecular Docking Technology

Objective Our objective was to investigate the potential mechanism of action of Qihuang Jiangtang capsule (QHJTC) in the treatment of type 2 diabetes mellitus (T2DM) through network pharmacology and molecular docking. Methods The active components of materia medica in the formula of QHJTC were searched on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and Encyclopedia of Traditional Chinese Medicine. The targets related to the active components were obtained via PubChem database. The targets related to T2DM were retrieved through the GeneCards database. The targets corresponding to the active components and diabetes mellitus were uploaded to the Venn diagrams website to get the Venn diagram, and the intersecting targets were the potential targets of QHJTC in treating T2DM. The active components and potential targets were imported into Cytoscape 3.7.2 software to construct the active component – potential target network, and the key compounds and targets were screened by the Network Analyzer module in the Tools module. The potential targets were imported into the STRING database to obtain the interaction relationships, so as to analyze and construct the protein – protein interaction (PPI) network by Cytoscape 3.7.2 software. The intersecting targets were introduced into Metascape for gene ontology pathway enrichment analysis. The top 20 signaling pathways obtained by the KEGG pathway enrichment analysis and the related targets and the corresponding targets were analyzed by using Cytoscape 3.7.2 software to construct the “ active component – important target-key pathway network ” for the intervention of T2DM with QHJTC. The molecular docking of active components and core targets was performed with AutoDock software.


Introduction
According to the World Health Organization, diabetes is a chronic metabolic disease characterized by elevated blood sugar levels which can cause serious damage to the heart, blood vessels, eyes, kidneys, and nerves over time. At present, the most common type of diabetes mellitus is type 2 diabetes mellitus (T2DM), which occurs when the body develops resistance to insulin or produces insufficient insulin. In the past 30 years, the prevalence of T2DM has increased significantly, 1 and China has become the country with a large number of diabetes patients. 2,3 At present, the world has reached a goal to inhibit the growth in the number of people with diabetes and obesity by 2025. As the only patented traditional Chinese medicine approved by the China Food and Drug Administration (CFDA) of the People's Republic of China in 2017, Yidaokang Qihuang Jiangtang capsule (QHJTC) can treat diabetes and its complications from the root and has a broad market prospect. Pharmacological and clinical studies have confirmed that QHJTC can repair damaged islet tissue, increase insulin release, and reduce blood glucose. 4 In this study, network pharmacology and molecular docking were used to investigate the potential mechanism of QHJTC in the treatment of T2DM, so as to provide a theoretical basis for further study of the active components of QHJTC.

Screening of Main Active Components and Related Targets of Qihuang Jiangtang Capsule
By searching the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https://old.tcmsp-e.com/tcmsp.php) 5  , and Yinyanghuo (Epimedii Folium) was collected, through literature search. 7,8 The bioactive components of QHJTC with oral bioavailability !30% and herb likeness !0.18 were screened. 9 The related targets of the active components were obtained using PubChem database (https://pubchem. ncbi.nlm.nih.gov).

Type 2 Diabetes Mellitus-Related Targets and Potential Targets of Qihuang Jiangtang Capsule in the Treatment of Type 2 Diabetes Mellitus
The GeneCards database (https://www.genecards.org/) was searched with "type 2 diabetes mellitus" as a keyword to obtain T2DM-related targets. The targets corresponding to the active components of QHJTC and the T2DM targets were imported into the Venn diagrams website (http://bioinformatics.psb.ugent.be/) to draw the Venn diagram. The intersecting targets should be the potential target of QHJTC in the treatment of T2DM.

Construction of Active Component-Potential Target Network Diagram
The active components and potential targets of QHJTC were introduced into Cytoscape 3.7.2 software to construct the active components-potential targets network. The key components and key targets were screened out by topology analysis via the Network Analyzer module in the Tools module.

Construction of Protein-Protein Interaction Network
The potential targets were imported into STRING database, "multiple proteins" was selected, the species was limited to "homo sapines", and the interaction relationship was obtained. The interaction relationship was imported into Cytoscape 3.7.2 software to analyze and construct proteinprotein interaction (PPI) network.

Enrichment Analysis
The gene ontology (GO) functional enrichment analysis and Kyoto encyclopedia of gene and genome (KEGG) pathway enrichment analysis of potential targets were carried out through Metascape (https://metascape.org/).
T2DM. There were 32 key components and 49 key targets identified by the active component-potential target network topology analysis. There were 471 terms obtained from GO functional enrichment analysis, among which 248 related to biological processes, 125 related to molecular functions, and 98 related to cellular components. There were 299 signaling pathways obtained from KEGG pathway enrichment analysis. The active components of QHJTC were found spontaneously binding to the core targets.

Construction of Active Components-Important Target-Key Pathway Network
The top 20 signal pathways obtained by KEGG pathway enrichment analysis and the corresponding active components of targets and related targets were imported into Cytoscape 3.7.2 software to construct the "active components-important targets-key pathways" network of QHJTC in treating T2DM.

Molecular Docking
The structure of the key components was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and saved in SDF format. The SDF format file was transformed into Ã mol2 format file by Chem 3D software and imported into Autodock Tools 1.5.6 for hydrogenation, charging, and other processing. The protein conformations of important targets were screened from RCSB PDB database (https://www.rcsb.org/). There were three screening conditions: the biological source of protein structure was human; the protein structure was obtained by X-crystal diffraction; and the crystal resolution of protein was <3Å. The screened proteins were treated by removing water and small molecules, and hydrogenation, charging, and combining nonpolar hydrogen were carried out in Autodock Tools 1.5.6 software. Molecular docking was carried out with Autodock Vina 1.5.6. The receptor ligand was sorted and screened according to the binding affinity. 10,11 When the binding energy was <0 kcal·mol À1 , it was considered that the active component could bind and interact with the target protein spontaneously. The lower the energy was, the more stable the molecular conformation was. The binding energy 5.0 kcal·mol À1 was considered to have a good binding effect. Visualization was conducted by using Pymol 2.3.2 software.

Type 2 Diabetes Mellitus Targets and Potential Targets of Qihuang Jiangtang Capsule in the Treatment of Type 2 Diabetes Mellitus
A total of 1,362 T2DM-related targets were found through GeneCards database. The targets corresponding to the active components of QHJTC and the T2DM targets were imported into the Venn diagrams platform to get the Venn diagram. There were 155 intersecting targets, which were the potential targets of QHJTC in treating T2DM (►Fig. 1).

Protein-Protein Interaction Network
The interaction relationship was obtained by importing the 155 potential targets into STRING database, and the PPI network was analyzed and constructed by Cytoscape 3.7.2 software. The network consisted of 155 nodes and 3,266 lines. The node represented the target protein, and the line represented the interaction between proteins. The degree value represented the number of lines connected to one node, and the more the lines, the greater the correlation. It can be used to evaluate the importance of each node in the network. The larger the node is and the darker the color is in ►Fig. 3, the greater the value. The average degree value of nodes in the network was 42.14, of which 63 nodes were  greater than the average value, including AKT serine/threonine kinase 1 (AKT1), interleukin-6 (IL-6), VEGFA, TNF, TP53, caspase 3 (CASP3), mitogen-activated protein kinase 1 (MAPK1), PTGS2, matrix metalloproteinase-9, MAPK8, etc.

Enrichment Analysis
The 155 intersecting targets were imported into Metascape database for GO functional enrichment analysis and KEGG pathway enrichment analysis. A total of 471 items were obtained by GO analysis, including 248 involved in biological processes (BP), mainly related to response to inorganic substances, trauma, lipopolysaccharide, organic circulation complex cell response, apoptosis pathway, extracellular stimulation response, oxygen level response, positive regulation of cell migration, active oxygen metabolism process, steroid hormone response, regulation of cell stress response, and negative regulation of cell proliferation;125 involved in molecular functions (MF), including protein domain specific binding, protein kinase binding, DNA-transcription factor binding, oxidoreductase activity, serine hydrolase activity, kinase activity, growth factor binding, adrenergic receptor activity, antioxidant activity, and TNF receptor superfamily binding; 98 involved in cell components, mainly related to membrane raft, cystic cavity, extracellular matrix, receptor complex, perinuclear region, cell membrane, dendrites, adhesion plaque, and so on. There were 299 signal pathways obtained by KEGG pathway enrichment analysis, mainly related to cancer pathway, advanced glycosylation end products (AGEs)-receptor of AGEs (RAGE) signal transduction pathway, IL-17 signal pathway, p53 signal pathway, insulin resistance and nuclear factor-kappa B (NF-κB) signal pathway, VEGF signal pathway, thyroid hormone signal pathway, estrogen signal pathway, sphingolipid signal pathway, and so on. Bar and bubble charts of the top 20 items were drawn (►Figs. 4 and 5).   PRSS1, ADRB2, AR, SCN5A, ESR1, NR3C2, GSK3B, CASP9, and so on. There were 18 signal pathways with the degree value greater than the average value, which were proteoglycan in cancer, cancer pathway, IL-17 signal pathway, sphingolipid signal pathway, VEGF signal pathway, AGE-RAGE signal transduction pathway in diabetic complications, platinum resistance, estrogen signal pathway, malaria, measles, p53 signal pathway, thyroid hormone signal pathway, gap junction, insulin resistance, serotonin-containing synapses, transcriptional disorders in cancer, longevity regulation pathway, and NF-κ B signal pathway (►Fig. 6).

Discussion
T2DM belongs to the category of "dispersion-thirst" in TCM 12 and is the syndrome of root vacuity and tip repletion due to the lack of congenital constitution, emotional imbalance, improper diet, and so on. The pathogenesis of dispersion thirst has been understood in traditional Chinese medicine, which holds that yin deficiency and dryness heat are its basic pathogenesis. The more deficient yin is, the more exuberant dryness heat is, and the more exuberant dryness heat is, the more deficient yin is, so the two are cause and effect to each other throughout the whole process of the disease.  shown that Renshen Baihu decoction can reduce blood sugar, blood lipids, and promote the improvement of quality of life. 13 Xiaoke formula can continuously inhibit the activity of adenosine 5'-monophosphate (AMP)-activated protein kinase by Sirtuin 1 (SIRT1) to decrease the activity of oxidase and oxidative stress, and then reduce blood glucose and promote microcirculation. 14 Yu Nyu decoction combined with Shashen Maidong decoction has a definite effect on diabetic patients of fire excess from yin deficiency, which can reduce the levels of serum Vaspin and Omentin-1 and improve blood glucose metabolism. 15 Liuwei Dihuang Pill is a classic prescription for nourishing yin and tonifying the kidney. It plays a role in the prevention and treatment of diabetes, such as antioxidant injury, reducing blood glucose, improving insulin resistance, and alleviating diabetic complications and has a significant protective effect on the vascular system of diabetes patients. Its mechanism may be related to increasing the level of serum adiponectin or upregulating the expression of adiponectin receptors (Adi-poR1 and AdipoR2). 16 In this study, a total of 237 active components and 281 related targets were obtained from QHJTC. Through Gene-Cards database, 1,362 T2DM targets were found, including 24 from Gouqizi (Lycii Fructus), 18   Xuanshen (Scrophulariae Radix), 1 from Shanzhuyu (Corni Fructus), 1 from Nyu Zhenzi (Ligustri Lucidi Fructus), 1 from Maidong (Ophiopogonis Radix). Quercetin belonging to the active component of Yinyanghuo (Epimedii Folium) had the greatest degree value, so it is speculated that the key herb of QHJTC in treating T2DM may be Yinyanghuo (Epimedii Folium). The related chemical constituents of Shigao (gypsum fibrosum), Kuguagan (dried bitter gourd) and Ji'neijin (Galli Gigerii Endothelium Corneum) were not found in TCMSP and ETCM database, and the related chemical components were not found in the literature, but in the clinical application of QHJTC, Shigao (Gypsum Fibrosum) as minister herbs, Kuguagan (dried bitter gourd) as assistant herb, and Ji'neijin (galli Gigerii Endothelium Corneum) as guide herb were essential herbs to assist Canjian (silkworm cocoon), Huangqi (Astragali Radix), Shanyao (Dioscoreae Rhizoma), and Xiyangshen (Panacis Quinquefolii Radix) to replenish qi and nourish yin. Network topology analysis showed that PTGS2, PTGS1, PRKACA, ADRB2, SCN5A, PRSS1, and so on were the core targets. PTGS2, also known as cyclooxygenase 2, plays a major role in the occurrence and development of T2DM. PTGS2 produces prostaglandins, which negatively regulate glucose-stimulated insulin secretion and act as mediators of inflammatory response. 17 Genetic correlation studies have shown that ADRBs gene variation is associated with T2DM. 18 Quercetin, kaempferol, β-sitosterol, luteolin, phytosterol, anhydroicaritin, diosgenin, isorhamnetin, formononetin, and baicalein are the main active components. Quercetin is a kind of flavonol compound, which is widely distributed in the plant world. It has a variety of biological activities and extensive pharmacological effects, such as antioxidation, anti-inflammation, antivirus, antitumor, hypoglycemic, lipid-lowering, immune regulation, and so on, which is of very important clinical significance in the treatment of bacterial infection, viral infection, tumor, diabetes, hyperlipidemia, and immune system diseases. 19 Modern  pharmacological studies have shown that quercetin can activate fibroblast growth factor 21 (FGF21)/ MAPK signal pathway to effectively reduce peripheral insulin resistance and blood glucose in T2DM rats. 20 Based on network pharmacology and molecular docking technology, it is shown that quercetin may act on NOS3, CYP1B1, NOS2, and other core targets to regulate toll-like receptor signal pathway, MAPK signal pathway, insulin signal pathway, and so on. 21 A total of 471 items were obtained by GO functional enrichment analysis of 155 intersecting targets. The BP are mainly involved in the responses to inorganic substances, trauma, lipopolysaccharide, cellular responses to nitrogen compounds, apoptosis pathway, active oxygen metabolism, and so on. MF are mainly about protein domain-specific binding, protein kinase binding, DNA-transcription factor binding, cytokine receptor binding, oxidoreductase activity, serine hydrolase activity, nuclear receptor activity, protease binding, phosphatase binding, and so on. Cell components are mainly membrane raft, capsule cavity, extracellular matrix, endoplasmic reticulum cavity, protein kinase complex, cell membrane, dendrite, adhesion spot, lipid vacuole, organelle membrane cavity and so on. A total of 299 signal pathways were obtained by KEGG pathway enrichment analysis, mainly related to cancer pathway, AGE-RAGE signal transduction pathway in diabetic complications, IL-17 signal pathway, p53 signal pathway, insulin resistance, and so on. The pathogenesis of T2DM is related to insulin resistance (IR) and β-cell dysfunction. IR refers to the decrease of the biological effect of insulin, which leads to the decrease of glucose uptake and metabolism, including the decrease of insulin sensitivity and responsiveness. It is the initial factor of T2DM, which runs through the whole disease course, and its mechanism is complex. Traditional Chinese medicine can act on multiple targets related to the pathogenesis of IR, so as to slow down and prevent IR. 22 QHJTC can effectively reduce the levels of blood glucose and blood lipids in patients with mild T2DM by reducing IR and improving islet function. 23 It can also improve hemorheological indexes and has a significant effect on the prevention and treatment of diabetic complications. 24 Diabetic model rats were used to explore the hypoglycemic effect of QHJTC and its effect on islet function and pancreatic tissue changes. The results showed that QHJTC could significantly reduce blood glucose in alloxan-induced hyperglycemic rats, significantly reduced the glucose tolerance curve of diabetic rats, and significantly improve the results of glucose tolerance in diabetic rats. It could also promote insulin secretion in rats with high glucose, improve the islet function of diabetic rats, and repair pancreatic tissue damage caused by alloxan. 25 AGE-RAGE signal pathway is an important link in the occurrence and development of diabetic nephropathy. 26 Traditional Chinese medicine has the advantages of overall regulation, multichannel and multitarget in the treatment of diabetic nephropathy, which can improve the progression of diabetic nephropathy by blocking AGEs-RAGE signal pathway, but the mechanism and target are not clear. Quercetin, kaempferol, βsitosterol, luteolin, and baicalein docked with AKT1, BAX, BCL2, CASP3, PTGS2, CCND1, IL6, and MTOR all have docking binding energies <0 kcal·mol À1 , indicating that the key components of QHJTC could spontaneously bind to the core target.

Conclusion
QHJTC can treat T2DM through multicomponents, multitargets, and multipathways, which provides references and a theoretical basis for further revealing the pharmacological mechanism of QHJTC.