Potential Molecular Mechanisms of JTW in the Treatment of Diabetes Mellitus and Depression Based on Network Pharmacology

Yueheng Tang Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Hao Su Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Fuer Lu Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Kexin Nie Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Zhi Wang Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Hongzhan Wang Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Wenya Huang Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Hui Dong (  tjhdonghui@163.com ) Huazhong University of Science and Technology https://orcid.org/0000-0003-4771-0846


Introduction
Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia with a rapidly increasing prevalence. According to the International Diabetes Federation (IDF), about 463 million adults worldwide suffered from DM in 2019, and the number is expected to reach 700 million in 2045 [1]. China has the largest number of DM patients in the world, with an incidence of 11.2% [2]. Not only DM itself is seriously harmful to human health, but also the complications will bring a heavy burden to the family and society.
Depression, one of the most prevalent disorders of mental health that limits psychosocial functioning and diminishes quality of life, is a common psychological complication of diabetes [3]. A meta-analysis showed that 14.5% of patients with type 2 diabetes mellitus (T2DM) were complicated with depression [4], and depression is twice as common in people with DM as in the general population [5]. Long-term depression not only affects patients' compliance with treatment, but also causes neuroendocrine dysfunction and increases blood glucose level, and poor blood glucose control will aggravate patients' depression. Therefore, it is urgent to strengthen early identi cation and give corresponding psychological or drug intervention.
At present, the commonly used antidepressants are selective serotonin reuptake inhibitors (SSRIs) and serotonin and noradrenaline reuptake inhibitors (SNRIs). According to reports, psychopharmacological treatment with SSRIs medications has a moderate-to-large effect on depression with lesser effects on glycemic control [6]. Another cohort study showed that antidepressant use is associated with the risk of diabetes onset in a time-and dose-dependent manner, the adjusted hazard ratio is 3.95 for long-term high-dose antidepressant use [7]. Hence in-depth exploration of the pathogenesis and development of drugs with the dual effects of improving of DM and depression is imminent.
Traditional Chinese medicine (TCM) is a holistic medical system for diagnosis, prevention and treatment of diseases which uses experience-based therapies such as acupuncture and herbal medicine [8]. Jiaotai-wan (JTW), originated from the "Han Shi Yi Tong" in the Ming Dynasty, is composed of two herbal medicines: Huanglian (Rhizoma Coptidis) and Rougui (Cinnamon), and it has been used to treat insomnia since ancient times. With the development of science and technology and the veri cation of experiments, the therapeutic effect of JTW on DM has been discovered [9][10][11]. Since insomnia and depression are closely related, in recent years, more and more studies have con rmed that JTW have obvious antidepressant effects in addition to reducing blood glucose level and improving sleep quality.
Experimental research results showed that JTW can signi cantly alleviate the depressive-like behavior of mice, such as shortening the immobility time of the tail suspension test (TST) and forced swimming test (FST) [12,13]. Therefore, JTW may have dual effects in the treatment of DM and depression. However, the mechanism of JTW to treat DM and depression is not yet clear, especially the molecular target mechanisms of its effective components, which needs to be further explored.
Network pharmacology is a new discipline based on the theories of systems biology, bioinformatics and classical pharmacology. The new method for analyzing the targets and mechanisms of drug treatment of diseases from multiple angles provides the possibility to reveal the mechanism of TCM, which is booming in recent years and has been widely used in the eld of TCM [14]. Network pharmacology, as a useful tool, can help us to further understand the role of drugs and how we can improve drug discovery for complex diseases [15]. Thus, this paper used the method of network pharmacological analysis to construct the network of "Drug-Compounds-Targets-Disease (D-C-T-D) " to explore the targets and mechanisms of JTW in the treatment of DM and depression, in order to provide ideas and theoretical basis for the later experimental research and future clinical application. The work ow is shown in Fig. 1 [19,20]. We also chose those two indicators as the screening criteria in our study.
The Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM, http://bionet.ncpsb.org.cn/batman-tcm/) was also used to screen the active compounds and potential targets. The BATMAN-TCM database is the rst online bioinformatics analysis tool specially designed for the research of molecular mechanism of TCM, which is aimed at accelerating the elucidation of TCM's molecular mechanism [21]. According to the parameter information given by the BATMAN-TCM database, the Score cutoff ≥ 30 and P value ≤ 0.05 were considered as indicators when screening the active compounds of JTW.
In addition, we also searched the relevant literature on the compounds of JTW and completed the data collection to avoid omitting some important active compounds and corresponding target information due to the setting of screening criteria.

Collection of targets of DM and depression
The targets of DM and depression were also obtained by searching the databases, namely the GeneCards (https://www.genecards.org/,version 5.1), the Online Mendelian Inheritance in Man (OMIM, https://omim.org/), the DrugBank (https://go.drugbank.com/, version 5.1.8) and the Therapeutic Target Database (TTD, http://db.idrblab.net/ttd/). The GeneCards is a database that integrates gene-centric data from about 150 web sources, including genomic, transcriptomic, proteomic, genetic, clinical and functional information [22]. The OMIM database focuses on the relationship between phenotype and genotype and contains information on all known mendelian disorders and over 15,000 genes [23]. The DrugBank is a web-enabled database containing comprehensive molecular information about drugs, their mechanisms, their interactions and their targets, which combines detailed drug data with comprehensive drug target information [24]. The TTD database provides information about the known and explored therapeutic protein and nucleic acid targets, the targeted disease, pathway information and the corresponding drugs directed at each of these targets [25]. With the key words including "depression", "depressive", "depressive disorder", "depressive illness", "diabetes" and "diabetes mellitus", targets related to DM and depression were founded in the above databases.

Construction of the protein-protein interaction (PPI) network
The intersection targets of JTW, DM and depression were uploaded to the STRING database (https://www.string-db.org/, version 11.0) to construct the PPI network of protein-protein interaction and the key targets were screened and analyzed subsequently. The STRING is a database which aims to achieve a comprehensive and objective global network, including direct (physical) as well as indirect (functional) interactions. We set the scoring condition to > 0.90, and the selected target proteins were limited to "Homo sapiens". In the PPI network, the edges represent protein-protein associations, and the more lines, the greater the correlation [28].

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses
The database for annotation, visualization and integrated discovery (DAVID) (https://david.ncifcrf.gov/, version 6.8) was used to carry out GO enrichment analysis and KEGG enrichment analysis on the intersection targets. The DAVID database is a web-based online bioinformatics resource that can be used to explain the functions of large lists of genes/proteins [29]. The GO enrichment analysis includes three different biological aspects: biological process (BP), molecular function (MF) and cellular component (CC) [30]. KEGG is a knowledge base for systematic analysis of gene functions [31].

Collection of therapeutic targets of DM and depression
The key words such as "depression", "depressive", "depressive disorder" and "depressive illness" were used to search for the therapeutic targets of depression in the GeneCards, OMIM, DrugBank, and TTD databases. After simplifying the results of each database and screening and removing duplicate targets, a total of 1842 targets of depression were obtained. Similarly, 1407 targets of DM were obtained by using "diabetes" and "diabetes mellitus" as the key words to search in the databases.

Screening of the intersection targets and constructing the D-C-T-D network
Through taking the intersection of 484 drug targets, 1842 depression targets and 1407 DM targets, we obtained a total of 117 targets ( Fig. 2A), which correspond to 17 active compounds (Table 1).
Subsequently, we input the information of the intersection targets into Cytoscape software for analysis in order to explore the possible mechanism of the therapeutic effect of JTW. Figure 2B shows the D-C-T-D network constructed by the Cytoscape, which fully reveals the multi-component and multi-target mechanism of JTW in the treatment of DM and depression. In addition, the analyzer tool that comes with the software was used to analyze the active compounds. The results showed that the active compounds with the highest number of targets included quercetin, styrene, cinnamic acid, ethyl-cinnamate, (R)-Canadine, palmatine, berberine, etc., indicating that the above compounds may be the main components of JTW in treating DM and depression.

Construction and analysis of the PPI Network
117 intersection targets were uploaded to the STRING database to obtain the PPI network. There are 117 nodes and 1907 edges in the PPI network (Fig. 3A), which suggests that there may be a complex relationship between these targets, and JTW may regulate these targets in a variety of ways. Since the more lines in the PPI network, the greater the correlation, we analyzed each node and screened out the top 10 targets (Fig. 3B). Therefore, we speculate that the active compounds of JTW may play a role in the treatment of DM and depression through targets such as insulin (INS), protein kinase B1 (AKT1), interleukin-6 (IL-6), vascular endothelial growth factor A (VEGF-A), tumor necrosis factor (TNF) and so on.

GO enrichment analysis
The DAVID database was used to perform GO enrichment analysis of 117 intersection targets to explore the relationship between these targets and diseases, including three aspects of BP, MF and CC. The top ten results were output after ranking according to the P value from small to large (Fig. 4). It can be seen that the occurrence of DM and depression involves many biological processes, and JTW can achieve the purpose of treatment by regulating multiple biological processes, among which the most closely related

KEGG enrichment analysis
We also conducted KEGG enrichment analysis of the 117 intersection targets through the DAVID database, and the results showed that these targets involved 124 pathways. We selected the top 20 according to the P value from small to large for further analysis (Fig. 5). It can be seen that JTW mainly regulates Chagas disease (American trypanosomiasis) (hsa05142), HIF-1 signaling pathway (hsa04066), pathways in cancer (hsa05200), Hepatitis B (hsa05161) and so on to treat DM and depression. In addition, there are also many targets enriched in pathways such as TNF signaling pathway (hsa04668), PI3K-Akt signaling pathway (hsa04151) and MAPK signaling pathway (hsa04010), indicating that they may also play an important role in the treatment.
Afterwards, in order to explore the relationship between the top 20 pathways, drugs and intersection targets, we integrated the collected information and conducted a network by using the Cytoscape (Fig. 6). It is obvious from the network that each pathway can correspond to multiple targets, and each target can also connect to multiple pathways. Different pathways can be connected to each other through the intersection targets, which fully re ects the multi-component, multi-target, and multi-pathway mechanism of JTW in treating DM and depression.

Discussion
Both DM and depression are serious chronic diseases, which can lead to a serious decline in the quality of life, increase functional disability and costs of care than many other chronic diseases [35]. The bidirectional link between DM and depression has been con rmed. An epidemiological study has shown that the prevalence rate of depression is more than three times higher in people with type 1 diabetes mellitus (T1DM) and nearly twice as high in people with T2DM than those without [36]. Another research also indicated that the presence of diabetes doubles the odds of comorbid depression [37]. The results of a meta-analysis carried out by Mezuk et al. showed that compared with people without diabetes, people with T2DM had a 15% increased risk of depression, while those with depression had a 60% increased risk of developing T2DM [38]. However, the current treatments for these two diseases are relatively single and there is a lack of the comprehensive treatment needed to improve clinical outcomes [39].
JTW, one of the most classical Chinese prescription, has been used to treat insomnia for hundreds of years. In recent years, more and more experiments have con rmed that JTW can not only improve the quality of sleep, but also has the dual effects of hypoglycemic and antidepressant [40][41][42]. In the traditional theories of TCM, different diseases may have similar etiology, pathogenesis, symptoms, and disease location during the occurrence and development, so that different diseases can be cured with the same prescription, which fully re ects the advantages of TCM in syndrome differentiation, holistic treatment and comprehensive treatment [43]. Our study is to explore the targets and mechanisms of the dual effects of JTW through the novel analysis method called network pharmacology.
The results of analysis using Cytoscape software showed that the active compounds with the highest number of targets included quercetin, styrene, cinnamic acid, ethyl-cinnamate, (R)-Canadine, palmatine, berberine and so on. First of all, quercetin, a naturally occurring avonoid, is one of the active compounds of Huanglian. A number of research results have con rmed that quercetin has multiple pharmacological effects such as antioxidant, anti-tumor, anti-in ammatory, antimicrobial, regulating immune function, and protecting cardiovascular function, etc. [44][45][46][47] [49]. Not only that, quercetin has also been found to alleviate LPS-induced depression-like behaviors in rats [50], and can dose-dependently decrease the immobility time of diabetic mice in FST and this effect is comparable to that of uoxetine, a traditional antidepressant [51,52].
Berberine, a natural isoquinoline alkaloid, is also an active compound of Huanglian. Berberine has a wide range of pharmacological effects, such as antimicrobial, anti-in ammatory, anti-tumor, antioxidant, neuroprotective, etc. [53,54], and can be used to treat many gastrointestinal disorders [55,56]. The results of a clinical trial showed that berberine can signi cantly reduce FBG and hemoglobin A1c (HbA1c) in patients with diabetes, and its hypoglycemic effect was similar to that of metformin [57]. In addition, other active compounds of JTW are also considered to have the effect of improving DM and depression. For example, palmatine can protect cells against reactive oxygen species and endoplasmic stress, thus exhibiting antidiabetic properties [70]. The results of behavioral tests such as sucrose preference test (SPT), FST and open-eld test (OFT) in rats also con rmed the antidepressant effect of it [71].
In ammatory mediators have always been considered to be important factors in promoting the development of insulin resistance (IR), which will lead to the occurrence of T2DM. The results of our PPI network analysis that IL-6 and TNF are the top ve targets also con rmed this view, indicating that they may play an important role in the treatment of DM and depression. It was found that the levels of cytokines such as TNF-α and IL-6 were highest in non-treated diabetic rats, and decreased signi cantly following quercetin or glibenclamide treatments [48]. The systematic review conducted by Esser et al. directly showed that immune system activation and chronic low-grade in ammation are involved in the pathogenesis of IR and diabetes [72]. In ammation is also thought to have a bidirectional relationship with depression [73]. A meta-analysis demonstrated that there was a signi cant correlation between depression and C-reactive protein (CRP) and IL-6 in children and adolescents [74]. IL-6 knockout mice exhibit resistance to stress-induced depression-like behavior and showed reduced despair in FST and TST [75]. Vascular endothelial growth factor (VEGF) is a key driver of neovascularization and vascular permeability [76,77]. Prakash et al. revealed that diabetes-induced cerebral neovascularization is accompanied by increased expression of VEGF-A and activation of VEGF receptor-2 (VEGFR-2) [78]. A case-control study showed that altered VEGF secretion, caused by genetic variation in VEGF-A gene, is involved in T2DM pathogenesis [79]. VEGF is also related to other diabetic complications such as diabetic retinopathy (DR) and diabetic foot ulcer ( . It can be seen that VEGF is closely related to DM and depression. The correlation between INS and DM has long been recognized worldwide, and AKT1, as a key factor in the PI3K-Akt signaling pathway, has been con rmed by many researches on its relationship with DM and depression, which will be discussed in detail below. The results of KEGG enrichment analysis showed that the active compounds of JTW may play a therapeutic effect on DM and depression by regulating multiple pathways. The intersection targets are mainly enriched in HIF-1 signaling pathway, pathways in cancer, TNF signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway, etc. Many studies have shown that HIF-1 signaling pathway is associated with DM and depression. Hypoxia-inducible factor 1-alpha (HIF-1α) is important for maintaining the function and survival of pancreatic β cells [89] and the expression of hypoxiainducible factor 1-beta (HIF-1β) mRNA in patients with T2DM is decreased [90]. Glucose-induced inhibition of HIF-1α protein stability may also accelerate the deterioration of β cell function and speed progression to diabetes [91]. Myocyte HIF-1α is necessary for normal muscle glucose uptake and insulin sensitivity [92]. Furthermore, HIF-1 is also associated with diabetic complications such as DR and diabetes kidney disease (DKD) [82,93,94]. In conclusion, HIFs play a role in the pathogenesis of β cell dysfunction and diabetes [92]. In terms of depression, Li et al. established a depression model using chronic unpredictable mild stress (CUMS) and found that FG-4592 can reverse depressive behaviors by activating HIF-1 signaling pathway [95]. The study conducted by Shibata et al. indicated that the altered expression of HIF-1 and its target genes mRNA in peripheral blood cells are associated with mood disorders, especially with MDD [96]. Kang et al. also proposed that interventions including the intermittent hypoxia conditioning and hyperbaric oxygen therapy to elevate the level of HIF-1 in the brain might be considered as new additional treatments for depression [97].
PI3K-Akt signaling pathway is the main downstream molecular pathway of insulin, which plays a crucial role in regulating glucose and lipid metabolism. PI3K-Akt signaling pathway block and abnormal function of downstream target proteins can cause IR [98]. Lots of studies have con rmed that the IR of diabetic mice can be improved by regulating the PI3K-Akt signaling pathway [99][100][101]. AKT1 has been considered as a key mediator of insulin-stimulated glucose uptake, suppression of apoptosis, stimulation of glycolysis and the activation of glycogen and protein synthesis [102]. The activation of PI3K-Akt pathway can protect pancreatic β cells from the in uence of different apoptotic stimuli [103]. What's more, Cao et al. found that the expression of PI3K in depressed rats were attenuated signi cantly [104]. inhibiting the occurrence and development of IR and diabetes [113]. Gelidium elegans extract can ameliorate T2DM via regulation of MAPK and PI3K-Akt signaling pathways [114].

Conclusion
In summary, this study based on network pharmacological analysis revealed the active compounds and corresponding targets and pathways of JTW, a classic TCM, in the treatment of DM and depression. The multi-component, multi-target, and multi-pathway mechanism of JTW provides a reference for in-depth study of the pharmacological effects. However, the pathogenesis of DM and depression is more complicated, and the targets and pathways are interrelated and regulate each other, in addition, due to the limitations of some databases, we cannot collect all the active compounds and targets of JTW. In the future, more in-depth and comprehensive study is still needed, and in vivo and in vitro experiments are necessary for exploring and verifying more extensive pharmacological effects and mechanisms of JTW.

Availability of data and materials
All data are available in the manuscript and they are showed in gures and tables.

Competing interests
The authors declare no con ict of interest.