Identification of the Active Constituents and Significant Pathways of Cangfu Daotan Decoction for the Treatment of PCOS Based on Network Pharmacology

Background Polycystic ovary syndrome (PCOS) is the most common female endocrine disease. Cangfu Daotan Decoction (CDD) can effectively relieve the clinical symptoms of PCOS patients. Methods To explore the active ingredients and related pathways of CDD for treating PCOS, a network pharmacology-based analysis was carried out. The active ingredients of CDD and their potential targets were obtained from the TCM system pharmacology analysis platform. The obtained PCOS-related genes from OMIM and GeneCards were imported to establish protein-protein interaction networks in STRING. Finally, GO analysis and significant pathway analysis were conducted with the RStudio (Bioconductor) database. Results A total of 111 active compounds were obtained from 1433 ingredients present in the CDD, related to 118 protein targets. In addition, 736 genes were found to be closely related to PCOS, of which 44 overlapped with CDD and were thus considered therapeutically relevant. Pathway enrichment analysis identified the AGE-RAGE signalling pathway in diabetic complications, endocrine resistance, the IL-17 signalling pathway, the prolactin signalling pathway, and the HIF-1 signalling pathway. Moreover, PI3K-Akt, insulin resistance, Toll-like receptor, MAPK, and AGE-RAGE were related to PCOS and treatment. Conclusions CDD can effectively improve the symptoms of PCOS, and our network pharmacological analysis lays the foundation for future clinical research.


Background
Polycystic ovary syndrome (PCOS) is the most common endocrine disease in women and is characterized by abnormal adrenal and ovarian androgen secretion, ovulatory dysfunction, menstrual irregularity, acne [1], and polycystic ovarian morphological features, and in a significant proportion of patients, insulin resistance, with a prevalence between 5% and 15% [2,3]. Although for a long period, studies on PCOS have focused on reproductive disorders [4], recent evidence suggests that PCOS is a heterogeneous disorder associated with a large number of severe metabolic implications as well as cardiovascular disease for affected women [5], which brings a heavy burden to the patient's family and to society. However, there is a generally poor understanding of its aetiology [6]. Genome-wide and molecular mechanism studies have identified certain candidate gene targets, although their role in the development of PCOS is still largely unknown; thus, early diagnosis, effective treatment, and the elucidation of underlying mechanisms are necessary [7,8].
In the past few decades, oral contraceptive pills (OCPs) have been widely used in PCOS patients to regulate their menstrual cycles and reduce hyperandrogenism [4]. Clomiphene citrate and letrozole have both been used to induce ovulation in PCOS patients with fertility problems [9][10][11]. However, reports have been coming in regarding the side effects of OCPs in increasing the risk of venous thromboembolism (VTE) [12,13]. Moreover, some experts have called attention to the unclear long-term risk-benefit ratio [11,14,15]. Due to the limitations of current treatments, traditional Chinese medicine (TCM) treatment for PCOS has become an important alternative therapy.
ere is no specific Chinese medical term referring to "PCOS" in the ancient books of Chinese medicine. According to the clinical characteristics of PCOS, it is attributed to the category of irregular menstruation, infertility, obesity, etc. For thousands of years, TCM has been used to treat menstrual disorders, infertility, and obesity. ere are a number of herbal formulas in treating the above diseases. A study showed that herbal medicine administration significantly relieved some of the symptoms of PCOS [16]. Specifically, the CDD consists of nine herbs: Atractylodes lancea ( unb.) Dc (Cangzhu), Cyperi Rhizoma (Xiangfu), Arisaematis Rhizoma (Dannanxing), Arum ternatum unb (Banxia), Zingiber officinale Roscoe (Shengjiang), Citrus reticulata (Chenpi), Aurantii Fructus (Zhike), Poria cocos (Schw.) Wolf (Fuling), and Licorice (Gancao) and is one of the most common prescriptions for phlegm and dampnesstype PCOS patients [17]. Another study showed that the CDD can significantly improve the pregnancy rate of infertile patients with PCOS, which might be associated with reduced insulin resistance, improved endometrial blood flow, and finally improved endometrial receptivity [18]. Although the CDD has been used clinically for gynaecological diseases for a long time, its mechanism of action is unclear because of its complex composition. erefore, it is necessary to clarify the biological basis and molecular nature of the TCM decoction. Network pharmacology has recently been developed as a new strategy and technique for elucidating complex pharmacological problems for new drug discovery [19]. In recent years, the TCM Systems Pharmacology (TCMSP) database and analysis platform has emerged as an ideal information convergence of the absorption, distribution, metabolism, and excretion (ADME) properties, drug-likeness, drug targets, associated diseases, and interaction networks of traditional medicines [20].
TCM decoctions have complex ingredients and multiple targets, and network pharmacology can predict novel compound targets and the potential pathways of action based on existing TCM decoctions and has helped clarify the mechanism of several TCM decoctions so far [21][22][23].
In this study, we used the network pharmacology approach to explore the potential mechanism of action of CDD in treating PCOS. We first filtered the TCMSP database for active compounds of CDD and identified the targets, followed by mining for disease-related genes and network analysis of those genes ( Figure 1).

Chemical Components of Each Herb in CDD.
To screen the active ingredients of CDD, we used the TCMSP database (http://tcmspw.com/tcmsp.php) [20]. TCMSP is a unique systems pharmacology platform specifically designed for Chinese medicine that contains information on their ADME characteristics and captures the relationships between disease, targets, ingredients, and drugs [24].

Pharmacokinetic Prediction of CDD.
CDD active ingredients were filtered mainly on the basis of oral bioavailability (OB), Caco-2 permeability (Caco-2), and drug-likeness (DL). OB, Caco-2, and DL are the three most key indicators of pharmacology. Specifically, the ingredients contained in the CDD meeting the criteria of OB ≥ 30%, Caco-2 ≥ 0.4, and DL ≥ 0.18 were chosen as candidate ingredients for further analysis.
OB refers to the percentage and rate of the release and absorption of active ingredients into the systemic blood circulation and is an important pharmacokinetic index of oral drugs [25]. It is also an important index to objectively evaluate the intrinsic quality of oral drugs [26], which is particularly important in the drug administration of most oral Chinese herbal formulas [27]. DL is defined as a complex balance of structural features and various molecular properties, which determine whether the particular molecule is similar to the known drugs [26]. ese parameters, such as hydrogen bonding characteristics and hydrophobicity, mainly influence the behaviour of molecules in living organisms, which ultimately affects their transport properties, affinity for proteins, metabolic stability, and many other properties. Caco-2 permeability is widely used as a standard permeability screening assay for oral drug absorption related to drug permeability, which can predict the intestinal absorption of the ingredients and the fraction of the oral dose absorbed in humans [28].
In this study, OB ≥ 30%, DL ≥ 0.18, and Caco-2 ≥ 0.4 were regarded as a threshold for filtering possible candidate drugs.

Potential Targets of the Chemical Components of CDD.
We chose the TCMSP database as the main source of component-target data and obtained the target protein names of each herb in CDD. Only the proteins that had interactions with the bioactive components in CDD we had already obtained were selected. en, we converted the target protein names of the bioactive components of CDD into gene names with the species limited into "Homo sapiens" with the UniProt Knowledgebase (UniProtKB, http:// www.uniprot.org).

Known
erapeutic Targets for PCOS. We collected PCOS targets from two sources. One is the GeneCards database (https://www.genecards.org/), which is a searchable, integrative database that predicts human genes. e knowledgebase automatically integrates gene-centric data from ∼150 web sources, including genomic, transcriptomic, proteomic, genetic, clinical, and functional information [29]. Another resource was the gene map in the Online Mendelian Inheritance in Man (OMIM) database [30] (https://omim. org/, updated on November 15, 2019), which is a comprehensive, authoritative, and timely knowledgebase of human genes and genetic disorders in the human genome. We searched the OMIM database with the keyword "PCOS."

Protein-Protein Interactions.
To illustrate the possible interaction between PCOS-related targets and the potential

Target Gene Prediction of CDD.
A total of 1433 potential targets from the 111 ingredients were retrieved from the TCMSP database ( Figure 2). After eliminating the overlapping proteins, 118 related proteins were obtained and converted into gene names with the species limited into "Homo sapiens" based on the UniProtKB.

PPI Network Analysis.
Among the above 736 PCOSrelated targets, CDD shared 44 common targets with PCOS ( Figure 3). e 44 putative therapeutic targets were imported into the STRING database to establish the putative therapeutic target PPI network. e "string_interaction.tsv" file was then imported into Cytoscape 3.7.2 to perform network analysis. e network had 44 nodes, which interacted with 361 edges. e average node degree is 16.4, and the average local clustering coefficient is 0.698. From yellow to green, the degrees of freedom increase, and the thicker edges suggest stronger interactions ( Figure 4). Our results indicated that the top mutual target proteins have various beneficial functions for treating PCOS at the molecular level.
In our network, the degree of the greenness of the nodes is high, and the numbers of edges of the nodes are 37 for AKT1; 32 for IL-6; 29 for VEGFA; 28 for CCND1, STAT3, MAPK1, and MAPK8; 27 for ESR1; 26 for FN1; 25 for MMP9, CXCL8, and MAPK14; 23 for AR; and 22 for PPARG and HIF1A. ese results demonstrate that these targets are closely related to others in the network and consequently may play key roles in PCOS.
AKTs have been confirmed to play an important role in granulosa-lutein cell (GC) proliferation, and there is reciprocal feedback between AKT and androgen. is study showed that the high expression of AKT1 and AKT2 may cause GC dysfunction in PCOS patients through a possible relation with androgen [32]. Elevated serum levels of IL-6 in PCOS patients reflect low-grade chronic inflammation, which has been attributed to insulin resistance in PCOS [33]. In a PCOS rat model, increased expression of IL-6 and IL-11 was found to be associated with the AKT/STAT3 pathway. Increased levels of IL-6 and IL-11 stimulated adipocytes from adipose tissue of the PCOS rat model, which may activate AKT/STAT3 signalling and promote cell proliferation. Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of many diseases. PCOS was also considered to be associated with high expression levels of VEGF. It was found that VEGF was a pivotal mediator of other factors to control ovary angiogenesis in women who underwent assisted reproductive technology (ART) procedures [34]. e MAPK signalling pathway is one of the most important signal transduction pathways in organisms and mediates physiological and pathological processes, such as the growth, development, and differentiation of organisms. Studies have shown that the MAPK signalling pathway plays an important role in the proliferation of ovarian granulosa cells [35,36].
Research confirmed that PPARG rs709154 and ESR1 rs1999805 are significantly associated with PCOS risk in a Chinese population [37]. MMP9 may also be involved in the pathogenesis of PCOS [38]. HIF1A can induce glycolysis gene expression and promote anaerobic metabolism [39]. HIF-1a signalling was confirmed to be inhibited in a rat model with PCOS by increasing PHD activity [40].

Putative erapeutic Compound-Putative erapeutic Target Network Construction.
e Merge function of Cytoscape 3.7.2 software was used to combine the Drug-Mol-Gene target network and the PCOS-Drug-Gene interaction network. e results showed that 111 compounds all together play a role in treating PCOS in the composite ( Figure 5). e targets of 111 putative therapeutic targets in the treatment of PCOS include AKT1, IL-6, VEGFA, CCND1, STAT3, MAPK1, MAPK8, ESR1, FN1, MMP9, CXCL8, MAPK14, AR, PPARG, and HIF1A.

Pathway Analysis.
To clarify the biological actions of these targets, we performed KEGG analysis and pathway enrichment analysis with the RStudio (Bioconductor) database. We listed the genes of major PCOS putative therapeutic targets and imported them into RStudio to generate relevant pathways that might have an important influence on the biological process of CDD in treating PCOS. Pathways with an adjusted p value < 0.05 were considered significant.
A total of 123 signalling pathways were significantly enriched through pathway enrichment analysis. In the histogram and bubble diagram (Figure 9), the colour, size, and length of the nodes and bars were determined according to the numbers and p values of the related genes. e colour  from blue to red reflects the adjusted p values from large to small, and the node size and bar length indicate the number of related genes. e histogram and bubble chart were separately arranged according to the p value and the number of related genes. According to the p value, the AGE-RAGE signalling pathway in diabetic complications, Kaposi sarcoma-associated herpesvirus infection, proteoglycans in cancer, endocrine resistance, hepatitis B, the IL-17 signalling pathway, prostate cancer, the prolactin signalling pathway, human cytomegalovirus infection, pancreatic cancer, the HIF-1 signalling pathway, and EGFR tyrosine kinase inhibitor resistance were within the top 12 terms. According to the number of related genes, Kaposi sarcoma-associated herpesvirus infection, proteoglycans in cancer, the PI3K-Akt signalling pathway, the AGE-RAGE signalling pathway in diabetic complications, human cytomegalovirus infection, hepatitis B, endocrine resistance, the IL-17 signalling pathway, prostate cancer, the HIF-1 signalling pathway, Epstein-Barr virus infection, and the prolactin signalling pathway were in the top 12 terms. e overlapping terms of the above lists included the AGE-RAGE signalling pathway in diabetic complications, endocrine resistance, the IL-17 signalling pathway, the prolactin signalling pathway, and the HIF-1 signalling pathway.
Prolactin (PRL) is involved in a variety of biological functions, including osmotic regulation, reproduction, lactation, endocrinology and metabolism, growth and development, and immunomodulation. It is a polypeptide hormone secreted by the pituitary gland that regulates the female reproductive and endocrine system by activating pathways, including the MAPK and PI3K pathways [46]. Insulin resistance is a common type of endocrine resistance and is related to PCOS by deregulating the IRS-PI3K-Akt  Evidence-Based Complementary and Alternative Medicine signalling axis that integrates aberrant inflammatory responses [47]. A previous study also revealed that hypoxia-inducible factor-1a (HIF-1a) mediated endothelin-2 (ET-2) signalling and plays an important role in ovulation in rats and indicated that HIF-1a signalling is inhibited in a PCOS rat model by increasing PHD activity [40].

Discussion
TCM plays a crucial role in the pharmaceutical industry, alternative medicine, and many other fields [48][49][50]. However, due to the complex chemical composition and unclear pharmacological mechanism of action of TCMs, it faces great obstacles in pharmacological research, quality control and supervision, modernization, and internationalization [51].
TCM gives priority to the concept of holism in diagnosis and pays more attention to the overall changes in function. e treatment is based on syndrome differentiation, emphasizing the recovery of overall function, which is similar to the network concept of network pharmacology [52].
Traditional research methods on TCM mainly rely on pharmacochemical and pharmacological experiments, which have many problems, such as low data flux, low precision, long cycles, and high costs. e construction and development of a network pharmacology analysis platform, TCM database, and target prediction technology has brought new ideas and strategies for the study on the basis and mechanism of the pharmacodynamic substances of TCM to clarify characteristics such as the good curative effects, high safety profile, and multicomponents and multitargets of TCM [53]. ese developments also lead to a new direction for the modernization and internationalization of TCM [54]. e combination of bioinformatics, computer technology, and experiments in the study of the pharmacodynamic components and potential targets of TCM broke through the deficiency of traditional studies is more rapid, flexible, and accurate and has been widely used in research on the network pharmacology of TCM [55].
TCM has a long history in treating menstrual diseases and infertility related to PCOS and has achieved good effects   [56]. In this study, several network pharmacology-based methods were used to predict potential targets. is approach provides new clues to exploring ethnopharmacology and herbal or even TCM formulas. rough the analysis of the putative therapeutic target network and biological functions in this study, the potential pharmacological and molecular mechanisms of CDD in treating PCOS were preliminary revealed.
In this study, the preliminary analysis based on network pharmacology provided a basis for subsequent studies on the pharmacodynamic ingredients and mechanisms of CDD. However, due to the limitations of the screening conditions and the TCM database, this study also has some shortcomings. Although a large number of targets and pathways were obtained through network pharmacology screening, these results must be verified by subsequent pharmacological experiments. In this study, the active components of CDD and their molecular mechanisms in PCOS were predicted overall, and a variety of potential therapeutic targets were explored. us, this study lays a good theoretical foundation for the next step of experimental verification and provides directions for further research on the molecular mechanism of PCOS.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request. Disclosure e funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
XWT designed the study, analyzed the data, and wrote the manuscript; WLH supervised the study and revised the  Evidence-Based Complementary and Alternative Medicine 13 manuscript; TMY and WJH obtained and analyzed the data. All authors have read and agreed to publish this manuscript.