Pharmacological mechanism of Xiaoyao San in the treatment of polycystic ovary syndrome based on network pharmacology


 Background : Xiaoyao San(XYS) has been widely used in the treatment of polycystic ovary syndrome(PCOS), but its mechanism is not clear. The purpose of this study is to elucidate the mechanism of XYS in the treatment of PCOS from the aspects of active components, targets and pathways. The purpose of the study is to explore the molecular mechanism of XYS in the treatment of PCOS. Methods : TCMSP database, UniProt and Perl were used to screen and collect the active components and targets of XYS. The genes related to PCOS were searched in GeneCards database. Collect the related targets of PCOS and XYS, use STRING database and Cytoscape software to process the data visually and analyze topology, and screen the key components and targets in the network. The key targets were enriched by R Project to predict the mechanism of XYS in the treatment of PCOS. Results : 68 active components and 96 drug targets in XYS were screened out. 3648 PCOS related disease targets were collected. 66 targets of XYS for PCOS treatment were obtained after analysis. 21 key targets of NCOA2, PGR, PTGS1, PPARG and AR were constructed after topology analysis. 63 biological functions and 111 biological pathways were obtained after gene ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) Pathway enrichment analysis. Conclusions : XYS has the characteristics of multi-component, multi-target and multi-path. This study discussed the active components, targets and potential mechanism of XYS in the treatment of PCOS, which provided a new direction for further study of the mechanism of XYS in the treatment of PCOS, and provides more ideas for clinical treatment of PCOS.


Backgroud
PCOS is a common endocrine disorder with reproductive dysfunction and metabolic abnormality. It is characterized by continuous anovulation, hyperandrogenism and polycystic ovarian changes, often accompanied by IR. The global incidence of PCOS is 5% − 10%. TCM in the treatment of PCOS differentiation diagnosis and treatment [6], liver depression is a non obese type of PCOS patients often show the syndrome type [7]. Guangrong Li et al [8] proposed that emotional failure is an important cause of PCOS, and stagnation of liver Qi is an important pathogenesis of PCOS. Modern medicine shows that the pathogenesis of PCOS is very complex, which is mainly related to the mechanism of human hormone secretion and regulation. In clinic, TCM compound prescription is often used to recuperate patients' menstruation. In addition, the study shows that the complications of PCOS, such as cardiovascular and cerebrovascular diseases, diabetes, endometrial cancer and so on, have brought dual physical and mental harm to women.
TCM is a kind of compound Chinese medicine, which can increase the e ciency and reduce the toxicity.
The clinical effect of TCM compound is a comprehensive result of complex biological process in vivo.
The research of effective component group and related drug target group is two important problems faced by the modernization research of TCM compound [10]. In clinic, XYS is widely used in the treatment of PCOS with liver depression, which has a good clinical effect [12][13][14][15], but up to now, the material basis and mechanism of XYS is not clear. Network pharmacology is to study the occurrence and development of diseases from the perspective of biological network, and to understand the interaction between drugs and the body [17]. To explore the relationship between XYS and PCOS, a biological network was constructed by means of network pharmacology and information technology. It is helpful to explain the mechanism of XYS in the treatment of PCOS.
Based on the research methods of network pharmacology and Informatics, the potential active components of XYS were analyzed and screened out under the conditions of OB value and DL value. Through the construction of compound component target disease network, the links and interrelations of nodes in the network were analyzed, and the mechanism of XYS in the treatment of PCOS was clari ed The foundation of the research is established.

The active components of XYS
XYS is composed of six kinds of medicine, such as Liquorice, Radix Bupleuri, Radix Paeoniae Alba, Angelica, Poria cocos and Rhizoma Atractylodis Macrocephalae. The TCM systems pharmacy database and analysis platform (tcmsp) (http://tcmspw.com/tcmsp.php) [19] database was used to nd the chemical components of each single drug in XYS, and then ADME analysis was carried out [20].
According to the condition parameters of oral bioavailability (OB) ≥ 30%, drug like (DL) ≥ 0.18, the active components were screened and collected.

The potential targets of XYS
In tcmsp database, the corresponding target points of each avor components of XYS were mined and collected. Using the uniprotkb search function in the protein database (UniProt) (http://www.uniprot.org/), enter the free scatter target and set the species as "human", and collect all the target corrected o cial names (Symbol).

PCOS related targets acquisition
Enter "polycystic ovary syndrome" in the genecards database (https://www.genecards.org/) to search and collect relevant targets of PCOS.

Construction and analysis of "compound-componenttarget-disease" network
The intersection of the collected targets was selected to determine the targets of XYS for the treatment of PCOS. After sorting out the existing data and prediction results, import them into the software of Cytoscape 3.7.2 [21] to get the topology network of "compound composition target disease". Each node in 2.5 Construction and analysis of protein protein interaction (PPI) network The obtained target proteins were imported into the string [22] database (https://string-db.org/), and "Homo sapiens" was selected as the research species. After removing the isolated free nodes, the PPI network was obtained. The key target proteins of XYS in the treatment of PCOS were obtained by analyzing the PPI network.
2.6 Gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis Using R Project [23], Cluster Pro ler [24], Colorspace [25], Dose [26], org.Hs.eg.db [27], Pathview [28] and other packages to program, the names of cross target proteins of XYS and PCOS were transformed into o cial names, and biological function enrichment analysis and biological pathway enrichment analysis were carried out. The threshold which was set P value < 0.05 was used as the enrichment condition.

Collection results of active components in XYS
A total of 928 active components of the six drugs in XYS were collected after screening in tcmsp. Among them, 85 of Radix Paeoniae Alba, 55 from Atractylodes macrocephala, 349 of Radix Bupleuri, 125 from angelica, 250 of Liquorice, and 24 from Poria cocos. Oral bioavailability and chemotaxis are important kinetic parameters for drug screening and evaluation. Set parameters OB ≥ 30%, DL ≥ 0.18 to screen 928 compounds in XYS and get 76 compounds meeting the conditions. After de duplication, 67 active ingredients with research value in XYS are obtained, as shown in Table 1.

Collection results of potential targets of XYS
96 potential targets of XYS were obtained after 67 potential targets of active ingredients were found in TCMSP and repeated targets were removed.

Results of PCOS target gene acquisition
A total of 3648 PCOS related disease targets were obtained by searching "polycystic ovary syndrome" in the GeneCards.

Network construction and analysis results
66 intersection targets were obtained by matching 96 drug targets with 3648 disease targets. After visualization, the results are shown in Fig. 1. 66 potential therapeutic targets of XYS for PCOS were determined. The interaction network of "XYS-active ingredient-target-PCOS" was constructed by using the software of cytocsape 3.7.2, as shown in Fig. 2. There are 93 nodes (25 active component nodes, 66 gene nodes, 1 compound node, 1 disease node) and 373 edges in the network. The larger the degree value of the key active ingredient, the more important it is in the complex network, and the more signi cant it is in the treatment of diseases. After the network analysis, the key chemical components are quercetin kaempferol isorhamnetin beta-sitosterol Stigmasterol sitosterol 3,5,6,7-tetramethoxy-2-(3,4,5trimethoxyphenyl)chromone hederagenin petunidin Areapillin 8β-ethoxy atractylenolide 3βacetoxyatractylone α-spinasterol (+)-catechin and so on. The degree statistics results As shown in Table 2. These chemical components are the key active components of XYS in the treatment of PCOS.

Construction of PPI network and results of key target screening
In order to further analyze the mechanism of XYS in the treatment of PCOS, 66 intersection targets were analyzed. 66 target genes were imported into the string database to retrieve protein interaction information. The con dence level was set to 0.4 and isolated target proteins were eliminated. The obtained protein interaction information is visualized and network topology analysis is carried out, and the PPI network related to the effect of XYS on PCOS is constructed as shown in Fig. 3. The nodes represent the targets, while the edges represent the interaction between the targets. After processing the protein interaction information, the results are as shown in Figs. 4 and 24 core targets are selected as shown in Table 3 Caspase-9(CASP9), Retinoblastoma-associated protein(RB1), Estrogen receptor beta(ESR2), Intercellular adhesion molecule 1(ICAM1), Nuclear factor erythroid 2-related factor 2(NFE2L2), Insulin-like growth factor II(IGF2), NAD(P)H dehydrogenase [quinone] 1(NQO1). It is speculated that these targets may be the key targets of XYS in the treatment of PCOS.  Fig. 5, and the speci c information is shown in   Fig. 6, and the speci c information is shown in   4. Discussion PCOS is one of the most common gynecological endocrine disorders. In western medicine, insulin sensitizers, compound cyproterone acetate, laparoscopic ovarian drilling or follicular puncture are often used to treat PCOS. However, due to the side effects of hormone drugs, patients are prone to have adverse symptoms. In recent years, TCM has been widely used in the treatment of PCOS, with signi cant therapeutic effect and high patient satisfaction. TCM can effectively regulate the menstrual cycle of women, improve the related symptoms of patients with PCOS, and restore normal ovulation [33].
As one of the famous prescriptions of TCM, XYS has the remarkable effect of soothing the liver and relieving the depression, nourishing the blood and strengthening the spleen. It is widely used in the treatment of PCOS with liver depression as the main treatment, and has a signi cant effect [39]. In the treatment of polycystic ovary model rats with modi ed or Modi ed XYS, it was found that Modi ed XYS could up regulate the expression of FSHR and LHR protein in granulosa cells of polycystic ovary rats, and reduce the levels of serum testosterone (T), anti Mullerian hormone (AMH) and egg yolk The expression intensity of AMH protein in the nest. Therefore, AMH can be regulated to reduce the androgen level in rats and improve PCOS [40].
TCM plays an important role in disease prevention and treatment through multi-component and multitarget, which makes the research on the material basis of the e cacy of TCM complex extremely di cult.
Network pharmacology can systematically predict and reveal the action and mechanism of different drug molecules. In this study, the mechanism of action of TCM compound was comprehensively analyzed and explained from the molecular network level, and then integrated with disease analysis to achieve the overall analysis of medical theory [45].
In this study, TCM holistic view, principle of syndrome differentiation and treatment, new mode of network target network pharmacology were effectively combined. Using TCMSP, GeneCards, R Project and Cytoscape, the active ingredients and targets were screened and collected, and "XYS-Active ingredients-Target-PCOS" Physical network was constructed. In order to fully elucidate the potential mechanism of XYS in the treatment of PCOS, the key nodes in the network were analyzed and the related biological functions and pathways were studied. The results of KEGG analysis showed that many biological pathways were involved in many targets regulated by XYS. After analyzing the existing research work, it is found that the collected biological pathways are closely related to the potential therapeutic mechanism and pathogenesis of PCOS. The main biological pathways regulated by the collected cross-linked targets (the cross-linked targets are lled with red in the pathway map, logFC = 1) are: AGE-RAGE signaling pathway in diabetic complications ( Fig. 7), Fluid shear stress and atherosclerosis (Fig. 8), Colorectal cancer, Proteoglycans in cancer, Bladder cancer, Breast cancer, Hepatitis B, Endocrine resistance, Hepatocellular carcinoma, Estrogen signaling pathway (Fig. 9), EGFR tyrosine kinase inhibitor resistance, Thyroid hormone signaling pathway, Prolactin signaling pathway, p53 signaling pathway, Platinum drug resistance, Apoptosis, MicroRNAs in cancer, PI3-Akt signaling pathway, NF-kappa B signaling pathway, MAPK signaling pathway, Wnt signaling pathway, etc. Among them, the biological pathways that have been con rmed include: ages receptor (RAGE) is a member of immunoglobulin superfamily, rage on cell membrane can activate several signal pathways after binding with corresponding ligands, and it is expressed in many kinds of cells, which is an  In this study, the mechanism of XYS in the treatment of PCOS was preliminarily elucidated, but it is di cult to determine whether the data is comprehensive because the components and target information of XYS are all from the existing database. In this study, we did not take into account the deeper chemical reactions of various chemical components of the drug, and ignored the in uence of XYS type, dose, and the complexity and difference of human body on drug metabolism and response.

Conclusions
Based on the network pharmacology method and information tool platform, this study analyzes the       Estrogen signaling pathway