Explore the mechanism of Yueju pill in treating depressive polycystic ovary syndrome based on internet pharmacology

Jing-Jing Gao,Hai-Quan Liu,Yan-Jin Fu,Qing-Yu Li,Wan-Jun Ming,Lin-Liang Song,Jia-Jia Qin＊

1School of traditional Chinese medicine, Jinan University, Guangzhou, P.R. China; 2Huizhou Hospital of Guangzhou University of Chinese Medicine,Huizhou,P.R.China.

#Jing-Jing Gao and Hai-Quan Liu should be considered joint first authors.

Abstract

Keywords:Depression, Polycystic Ovary Syndrome, Medicine, Chinese Traditional, Molecular Mechanisms of Pharmacological Action

Introduction

Polycystic ovary syndrome (PCOS) is a complex endocrine disease characterized by polycystic ovary,anovulation and hyperandrogenemia, insulin resistance,hyperinsulinemia, pancreatic cell dysfunction, and hyperlipidemia [1]. Depending on the different population studied and the different definition used, its prevalence ranges from 8%to 13%[2,3].Women with PCOS have an increased risk of diabetes, obesity and metabolic syndrome [4, 5]. The prevalence of depression in PCOS woman increases, and the chance of moderate/severe depression symptoms increases as well [6]. A large-scale retrospective study based on Asian women shows that young women with PCOS(rather than middle-aged women) have a higher risk of depression [7]. Depression increases the burden of the disorder for the women with PCOS and may negatively impact efforts at self-care, thus compounding the metabolic consequences [8].At present, the mechanism of the increased prevalence of depression in women with PCOS is still not clear, and there is no specific treatment.

Yueju pill is recorded in the book "Danxi Xin Fa"by Zhu Danxi (a Chinese medical scientist in Yuan Dynasty), and has been widely used to treat six types of Yu (Qi Yu, Blood Yu, Phlegm Yu, Fire Yu,Dampness and Feeding Yu) from ancient times. "Yu, is the coagulation of Qi, without abreaction, which has the similar symptoms with depression." Qi, Blood,Phlegm, Fire, Dampness and Feeding can all get together to cause Yu. The spleen and stomach are the mediation hubs of Qi's ascending and descending,which are mostly susceptible to Qi Yu. Yueju pill can treat six Yu, and Qi Yu is the first to be cured, because Qi movement can lead to the circulation of blood.Once the Qi's movement becomes smooth, Blood Yu,Phlegm Yu, Fire Yu, Dampness and Feeding Yu will disappear quickly. Dr. Zhu used five herbs that individually dealt with the different types of Yu,including Xiang Fu (Cyperus rotundus L.,Cyperaceae.) for Qi Yu, Chuan Xiong (Ligusticum striatum DC., Apiaceae) for Blood Yu, Zhi Zi(Gardenia jasminoides J.Ellis, Rubiaceae) for Fire Yu,Cang Zu (Atractylodes lancea (Thunb.) DC.,Compositae.) for Dampness and Phlegm Yu, and Shen Qu (Massa Medicata Fermentata) for Feeding Yu [9].Xiangfu is commonly used in traditional medicine to treat gynecological or emotional disorders [10]. In the classic prescription Xiang-Fu-Si-Wu Decoction(XFSWD), Xiangfu and Chuanxiong used as a "herbpair" (Yaodui in Chinese) synergistically, so as to have the effects of regulating Qi and blood, activating blood circulation, dissipating blood stasis [11]. Zhi Zi(Gardenia jasminoides J. Ellis, Rubiaceae) is considered to be the main component of the rapid antidepressant effect of Yueju pill. The antidepressant effect of GJ is related to the upregulation of brainderived neurotrophic factor (BDNF) expression in the hippocampus [12]. PCOS is often associated with abdominal obesity, insulin resistance, increased visceral fat, metabolic disorders and cardiovascular risk factors [13]. In which, the traditional Chinese medicine(TCM)syndromes of obese PCOS in TCM is mainly about spleen-deficiency and phlegm-turbid stagnation. With Cang Zu (atractylodes) as the monarch drug in prescription, the Cangfu daotan decoction is widely used in treating the patients with phlegm-dampness stagnation type of PCOS [14].Shenqu, also known as Liushenqu, is a medicine commonly used in TCM formulas to protect the stomach, strengthen the spleen, and stimulate patient's appetite to assist digestion. Current research shows that some digestive enzymes (amylase, protease,glucoamylase), vitamins and other substances play a major role in stimulating appetite and digestion[15].

With the rapid development of bioinformatics,systems biology and multi-pharmacology, networkbased drug discovery is considered to be a promising and more cost-effective method of drug development[16-18]. The research of "Traditional Chinese Medicine Network Pharmacology" aims to reveal the mystery of TCM prescription from the systematic and molecular level, and promote the research of TCM from the current "single target, single drug" model to the "network target, multi-component drug" model[19]. The "prediction and discovery" of active ingredient or synergistic ingredient in TCM prescription is considered to be the main goal of TCM network pharmacology [20]. Based on network pharmacology tools, combined with multiple types of data sets, including multi-level high-throughput omics data, biomedical literature and human interactive group maps and databases, it is possible to further construct a biomolecular network for diseases or TCM syndromes[21].The development of a powerful multilayer computing network with integrated data may help us to study the complex biological pathways,or to better understand the complex biological mechanisms affected by drug therapy [22]. There are many reports on PCOS and depression, but there are few studies on PCOS with depression. Owing to their vague relationship and unknown molecular mechanism, we entitled the disease as "depressive PCOS" (PCOSDepression) for the moment. Therefore, this study will screen the active ingredients of Yueju pill through network pharmacology tools, construct the protein to protein interaction network, and analyze its biological processes and mediated signaling pathways. Endeavor to provide references for further research on the role of Yueju pill in the treatment of depressive PCOS.

Material and Methods

Chemical composition and related targets for Yueju pill

The Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP, http://tcmspw. com/tcmsp. php) [23] was utilized to search the main active ingredients of Xiangfu, Chuanxiong, Cangzhu, Zhizi, Shenqu in sequence, and constructed chemical composition database of Yueju pill. The active ingredients of Yueju pill were screened by adopting the absorption,distribution, metabolism, and excretion (ADME)criteria in pharmacokinetics. Oral Bioavailability(OB)≥30%,drug-likeness(DL)≥0.18 were selected as the criteria for screening active ingredients of TCM[24]. OB refers to the speed and degree of drug absorption into the human circulation, while DL refers to the similarity of the compound to known drugs [25,26].They are significant indicators for selecting active ingredients of drugs. The TCMSP platform would be continually used to predict the targets of chemical compositions from Yueju pill. All the chemical components obtained their corresponding targets except for "Shenqu". The Bioconductor package and Uniprot database were applied to correct the target proteins obtained above, converted them into the unified gene names, and then eliminated the repetition and coaction data. In final, the database of chemical composition and targets of action (C-T) from Yueju pill were obtained.

Targets for PCOS and depression

Different gene targets associated with the keywords of"Polycystic ovary and polycystic ovarian syndrome,PCOS", "depression, depressive and depressed" were collected respectively from the GeneCards database(http://www. genecards. org/) and OMIM Database(http://www.ncbi.nlm.nih.gov/omim). Genecards is a comprehensive database of human genes, it provides a concise genome, proteome, transcription, genetic and function of all known and predicted human genes[27].OMIM is the primary repository of comprehensive,curated information on genes and genetic phenotypes and the relationships between them [28]. The obtained disease targets from the two datebases were imported into UniProt [29] (http://www.uniprot.org/) to correct their gene names. After we obtained the official gene symbols, we utilized the Venn online tool to gain the intersected disease genes.

Protein-Protein interaction(PPI)network

STRING11.0 [30] (http://string-db. org/cgi/input. pl)was adopted to construct the drug-disease targets network diagram of Yueju pill. String is a database for searching known and predicted protein interactions.The database can be applied to 2031 species,including 9.6 million proteins and 13.8 million protein interactions.The parameters were set to be:the species was "Homo sapiens" and "medium confidence ＞0.4".Then "hide disconnected nodes in the network" was ticked for update. The document of protein to protein interaction (PPI) network was saved in TSV format and the picture was saved in "PNG" format. In the PPI network, each circle represents a key target gene, and the dot in the center shows the protein structure of the target gene. The connection between two target genes represents a meaning, such as protein homology, gene co-expression, gene co-evolution, adjacency between genes,etc.

Cytoscape network diagram

Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Cytoscape 3.7.2 (http://cytoscape.org/) [31], the java based open source software was adopted to construct the Yueju pill and PCOS-Depression components-potential target network. Firstly, we used Perl script to generate the files for the preparation of building the network:network. txt, genelist. txt, node. txt. Secondly, we uploaded the three files to Cytoscape3.7.2 one after another. Thirdly, we adjusted the shapes, colors and character's size accordingly. Finally, we obtained the"active ingredient-potential target network"diagram.

GO and KEGG enrichment analysis

The Gene Ontology resource (http://geneontology.org)is the most comprehensive and widely used knowledgebase concerning the functions of genes and gene products [32]. While KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database resource for systematic analysis of gene functions, linking genomic

information with higher order functional information[33].We prepared the intersection target genes"merge.txt"of Yueju pill and PCOS-Depression were prepared.Then we utilized the Bioconductor packageto carry out the enrichment analysis of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG).The P-value was calculated and P<0.05 suggested the enrichment degree had statistically significant.

Results

The chemical composition and targets of Yueju pill

There were 37 drug compounds for Yueju pill(Table 1). Among them, 18 from Xiangfu, 7 from Chuanxiong,9 from Cangzhu,5 from Zhizi and 0 from Shenqu. A further search of the drug's action targets revealed that there were 495 for Xiangfu, 61 for Chuanxiong, 65 for Cangzhu, and 141 for Zhizi.Uniprot performed gene correction, and the venn online tool extracted the common genes.After deleting the duplicate values, 111 Yueju pill targets were abtained.

The related targets of PCOS-Depression

After retrieving by the key words of "Polycystic ovary and polycystic ovarian syndrome,PCOS","depression,depressive and depressed" respectively, 4,910 targets were acquired for PCOS and 11, 449 targets were acquired for depression. Removed the duplicate data and false positive genes, and then taken the intersection, a total of 2787 disease-related targets were acquired(Figure 1).

The common targets of Yueju pill and PCOSDepression

R × 64 3.6.1 venny.R package was adopted to obtain the intersecting target genes of Yueju pill and PCOSDepression, which extracted 83 common target genes.Then the merge. txt document was established(Figure 2).

PPI network of drug and disease

The network contains number of nodes: 83, number of edges: 719, expected number of edges: 234, PPI enrichment p-value: ＜1.0e-16 (Figure 3). According to the number of nodes, Bioconductor package was applied to get the top 30 targets of interactions(Figure 4). Barplot diagram showed that IL6(49 edges), VEGFA (45 edges), EGF (42 edges), JUN(42 edges), MAPK (141 edges), PTGS2 (41 edges),ESR1 (40 edges), MAPK8 (40 edges), EGFR(39 edges),IL1B(38 edges)ranked high as the leading 10 core targets.

Cytoscape network of drug and disease

In the network, 83 common target genes and 37 active components were introduced into Cytoscape 3.7.2 to construct a visualized Yueju pill active ingredients-PCOS-Depression target network(Figure 5).There were 105 nodes and 982 edges showed in Figure 5.The red color represented 83 common target genes of drug and disease; the green color represented 37 active component of a chemical composition; the blue color represented disease and the orange color represented drug. According to network analysis, each effective active ingredient acted on at least one target gene, and each gene was regulated by at least two active ingredients, which might illustrate the action characteristics of Yueju pill's multiple active ingredients and targets well. Through the analysis of MCODE, we obtained 4 clusters (Table 2), then the resulted genes were analyzed with Metascape [34].Since each cluster was analyzed and no data was returned separately, the genes of the four clusters were combined for analysis.Finally,we found that the genes were mainly enriched in the Nuclear Receptor transcription pathway and regulation of blood pressure function (Figure 6). In which, AR, ESR1, MAPK14,GSK3B, NR3C1 proteins were most densely connected(Figure 7).

GO functional enrichment analysis(Top 20)

The enrichment analysis of biological functions showed that 106 GO items have been identified in the biological process, mainly related to steroid hormone receptor activity, nuclear receptor activity, transcription factor activity, direct ligand regulated sequencespecific DNA binding, steroid binding, neurotransmitter receptor activity and so on.

KEGG pathway enrichment analysis (KEGG top 20)

Through the analysis of Bioconductor package, a total of 129 signal pathways were identified finally.The top 20 pathways enriched in the number of genes were screened out. The degree of enrichment in the path image was expressed by the shade of color, the darker the color was, the better the degree of enrichment.The signaling pathways mainly included Fluid shear stress and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling pathway,Endocrine resistance,and etc.

Table 1 The active ingredients of Yueju pill

Discussion

Table 2 Clusters of Yueju pill active ingredients and PCOS-Depression targets

Through the retrieval of Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP), we found there were 37 compounds in the prescription of Yueju pill, such as Luteolin,quercetin,isorhamnetin, beta-sitosterol, sitosterol, kaempferol,Stigmasterol, Chryseriol and etc. Most of the compounds are natural flavonoids which often act its effects as the antioxidant, anti-Inflammatory and neuroprotective agents. These compounds have a comprehensive biological efficacy. For example,Luteolin luteolin, 3', 4', 5, 7-tetrahydroxyflavone,whose various biological functions including antiinflammatory, anti-allergy, and anti-cancer [35].Quercetin is also a member of the flavonoid family and has significant heart-related benefits,such as inhibition of low density lipoprotein (LDL) oxidation, nonendothelial vasodilation,reduction of adhesion molecules and other inflammatory markers, protects nitric oxide and endothelial function under oxidative stress, and prevents neuronal oxidation and inflammation damage and anti-aggregation effect of platelets [36].Isorhamnetin (Iso) has the various biological effects of antioxidant, anti-inflammatory, anti-cancer, cardiovascular protection and anti-tuberculosis [37-40]. The pharmacological properties of kaempferol exhibit antioxidants, anti-inflammatory [41], and anti-cancer activities [42]. Ingestion of foods containing kaempferol phenol is associated with reduced the incidence and mortality of myocardial infarction, and lower the severity of cerebrovascular disease [43, 44].Previously, an in vitro study showed that kaempferol could improve hyperglycemia by improving insulinstimulated fat cells to take up glucose. β-sitosterol is a plant-derived steroid, and the related study revealed that the antidepressant-like effect of β-sitosterol may be mediated by elevated levels of 5-HT and NE in the central nervous system [45]. These abundant pharmacological manifestations demonstrated great potential in the treatment of depressive PCOS.Through the analysis of PPI network and Count.R package, we found the leading hub proteins including IL6, VEGFA, EGF, JUN, MAPK, PTGS2, ESR1,MAPK8, and EGFR. Among them, interleukin-6 (IL-6) is a glycosylated polypeptide chain, which was first discovered as a B-cell differentiation factor (BSF-2), it can induce B cells to mature into antibody-producing cells [46].As an important pro-inflammatory cytokine,IL-6 has been shown to affect ovarian function,ovulation, fertilization and implantation in women with PCOS [47-50]. VEGF plays a crucial role in ovarian follicle formation and normal reproductive function [51]. A study evaluated VEGFA variants in 382 Bahraini Arab women with PCOS and 393 age and race-matched control women. The correlation between VEGF levels and changes in PCOS confirmed the role of VEGF secretion changes in the pathogenesis of PCOS caused by genetic variation of the VEGFA gene[52]. Studies have shown that EGF can stimulate VEGFA secretion through the EGF receptor (EGFR)and stimulate cell proliferation by activating the VEGF receptor VEGFR-2 [53]. Meanwhile, in the Cytoscape network, we applied the MCODE function to produce 4 clusters, and the produced genes were analyzed by Metascape.As a result,AR, ESR1, MAPK14, GSK3B and NR3C1 proteins are most densely connected. The Nuclear Receptor transcription pathway and regulation of blood pressure function were highest enriched.Thus, the gene ESR1 and MAPK should be attached great importance. The human ESR1 gene locates on chromosome 6q25.1.The wild-type ESR1 gene is 140 kb in length and consists of eight exons separated by seven introns. Upregulation of ESR1 in the uterus may be the basis for metabolic syndrome dysfunction during the transition period from pre-pubertal to puberty and later. The signal cascade driven by ESR1 will increase PI3K, thus shaping the PCOS glucotoxic and lipotoxicity environment [54, 55]. MAPK plays a strategic role in signal processing, mediating stress response and guiding the transformation and development of cell fate [56, 57]. Many evidences confirm that differently expressed MAPK8 or MAPK8 phosphorylation is related to apoptosis [58]. MAPK14 can be activated by various environmental stresses and pro-inflammatory cytokines, and plays an important role in DNA damage and repair [59].The activation of MAPK14 signaling cascade is pleiotropic and affects a variety of biological processes. For example, due to impaired expression of certain proinflammatory cytokines, MAPK14-deficient mice in macrophages were protected by LPS-induced sepsis. However, these mice showed enhanced macrophage apoptosis and advanced atherosclerosis in the ApoE-/-experimental atherosclerosis model[60,61].

Afterwards, the enrichment analysis of biological functions showed that 106 GO items have been identified, mainly related to steroid hormone receptor activity, nuclear receptor activity, transcription factor activity, direct ligand regulated sequence-specific DNA binding, steroid binding, neurotransmitter receptor activity and so on. Steroid hormones can regulate many important developmental and physiological functions in different organs. Women with PCOS exhibit a hypothalamic-pituitary-ovarian axis disorder, which changes the steroid pathway.Women with PCOS display a dysregulation of the Hypothalamic-Pituitary-Ovarian axis, which alters the steroid pathway [62]. Nuclear receptors are ligand-activated transcription factors whose different biological functions are classically regulated by small cholesterol molecules. Phosphatidylcholine,phosphatidylinositol, and sphingomyelin belong to a small part of many phospholipid-like molecules.Studies have shown that they can regulate nuclear receptors very specifically in mouse models, cell lines,and in vitro [63].Transcription factors, or proteins that bind DNA to regulate transcription, are frequently aberrant in disease [64]. A study on the role of neurotransmitters in PCOS suggests that the increase in GnRH and LH pulsatility under PCOS may be due to the stimulatory nature of the hypothalamic-pituitary center GnRH and the cumulative effect of inhibitory neurotransmitter changes. Disorders of neurotransmitter mass spectrum in patients with PCOS may also be the cause of low self-esteem, anxiety,frequent mood swings and depression, these characteristics are closely related to the symptoms of woman with PCOS [65]. From the analysis above, we know that the enriched hubs genes and the biological functions play a crucial part in the treatment of depressive PCOS.

Last but not least, the enrichment analysis of KEGG was carried out, and the top ten enriched signaling pathways included Fluid shear stress and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling pathway,Endocrine resistance, Chagas disease (American trypanosomiasis), Relaxin signaling pathway, Th17 cell differentiation, Estrogen signaling pathway, TNF signaling pathway, Prostate cancer. The P-value of Fluid shear stress and atherosclerosis pathway was the smallest. Endothelial cells regulate intracellular signals in response to changes in local shear stress,resulting in changes in gene expression, cell morphology, and structural reconstruction [66]. The endothelium forms different adaptive phenotypes and co-exists with other risk factors, such as high cholesterol, hypertension,obesity, diabetes and smoking, to respond differently,thereby promoting site-specific susceptibility to the occurrence and development of atherosclerosis [67].The second enriched signaling pathway is AGE-RAGE signaling pathway in diabetic complications.Advanced glycation end products(AGEs)are the final products of a chemical process called the Maillard reaction[68, 69],which usually form slowly under physiological conditions. The accumulation of AGEs is related to various pathologies, such as T2DM, methionine,cardiovascular disease, aging (including ovarian aging), inflammation, neurodegenerative diseases,obesity, and even PCOS [70, 71]. Evidence from human, animal, and in vitro studies suggests that advanced glycation end products (AGEs) may contribute to the pathogenesis of PCOS and its metabolic and reproductive consequences. AGEs can induce the production of oxidative stress and proinflammatory cytokines by activating key intracellular signaling pathways, thereby promoting the adverse health effects of PCOS [72]. RAGE is an immunoglobulin superfamily molecule, and its multiple ligands have been proven to accumulate in diabetic tissues.RAGE has been found to be a receptor for AGEs, such as carboxymethyllysine (CML) [73].AGEs mainly activate the signaling mechanism through RAGE, which leads to cell stress, cell dysfunction, damage to target organs, and complications [74]. The results of studies on the interaction of RAGE with non-AGE ligands, such as S100/ calcium granule protein and high mobility group protein 1 (HMGB1) [75], emphasize that RAGE is not only associated with diabetes complications, but also related with the cause of type 1 and 2 diabetes.Women with PCOS exhibit functional changes in downstream components of the insulin signaling cascade, such as GLUT-4, which is necessary for glucose absorption into cells [76-78]. AGEs and RAGE affect insulin signaling and glucose metabolism in insulin-sensitive cells and tissues (including ovarian cells). RAGE overexpression-induced changes in adipocyte morphology are associated with decreased GLUT4 gene expression and decreased insulin signal[79]. In addition, there is a significant correlation between the blood pressure level of women with PCOS and the IL-17 pro-inflammatory cytokine secreted by Th17 cells. The IL-17 serum level was directly associated with the increase in blood pressure.Increasing the level of these factors can enhance patients' related complications. Therefore, controlling inflammation and preventing autoimmune processes through treatment and interrupting the cascade of inflammation can affect the disease and its complications [80]. PCOS is also associated with a variety of clinical complications,which mainly involve the endocrine system (including increased incidence of diabetes) and the cardiovascular system (including hyperlipidemia)[81,82].

Yueju pill was used in the treatment of six "Yu",especially in Qi Yu traditionally, but after the analysis above, we concluded that Yueju pill has relatively few targets and pathways related to depression, which was beyond our expectation. We speculated that Yueju pill,as a representative prescription for relieving depression, mainly relieves depressive symptoms through the improvement of systemic symptoms, not just through stimulation of depression-related targets and pathways to achieve antidepressant effects.Research has indicated that there is an intrinsic link between depression, inflammation and gut microbiota[83]. This is consistent with the view that depression itself is not a purely mental disorder. On the contrary,it has a deep connection with the physical balance of the body. Therefore, a holistic approach using herbal mixtures such as Yueju pill that involves both the brain and the body may be more promising to improve the treatment outcome of major depressive disorder(MDD) [9]. Furthermore, the more targets and pathways we found were closely correlated to the main features and related complications of PCOS, which provided strong support for the usage of Yueju pill to treat depressive PCOS.

Conclusion

In summary, network pharmacological data mining results and analysis show that many related targets and pathways are strongly support Yueju pill in the treatment of PCOS. Meanwhile, the treatment of depression may be through the relief of systemic symptoms to achieve the purpose of anti-depression.Therefore, it is feasible to use Yueju pill for the treatment of depressive PCOS. This study will provide fresh insights for expanding the clinical application of Yueju pill.