Network pharmacology and molecular docking study on the treatment of polycystic ovary syndrome with angelica sinensis- radix rehmanniae drug pair

This study aimed to investigate the angelica sinensis - radix rehmanniae (AR) role in polycystic ovary syndrome (PCOS), employing network pharmacology and molecular docking techniques for active ingredient, targets, and pathway prediction. AR active components were obtained through TCMSP platform and literature search. The related targets of AR and PCOS were obtained through the disease and Swiss Target Prediction databases. An “active ingredient-target” network map was constructed using Cytoscape software, and gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis was conducted through Hiplot. Finally, Auto Dock Tools software was used to conduct molecular docking between active ingredients and core targets. The main bioactive ingredients of AR in the treatment of PCOS are acteoside, baicalin, caffeic acid, cistanoside F, geniposide, etc. These ingredients involve 10 core targets, such as SRC, HSP90AA1, STAT3, MAPK1, and JUN. The effect of AR on anti-PCOS mainly involves the AGE-RAGE signaling pathway, Relaxin signaling pathway, TNF signaling pathway, and ErbB signaling pathway. Molecular docking results showed that the main active components and key targets of AR could be stably combined. AR can improve hyperandrogen status, regulate glucose homeostasis, and correct lipid metabolism and other physiological processes through multi-component, multi-target, and multi-pathway. Thus, it could play a significant role in PCOS treatment. The results of our study provide a scientific foundation for basic research and clinical applications of AR for the treatment of PCOS.


Introduction
Polycystic ovary syndrome (PCOS) is the most common gynecological endocrine disorder among women of reproductive age, [1] characterized by irregular menstruation, hyperandrogenism, and polycystic ovaries. [2]PCOS is one of the major causes of infertility in women of reproductive age, [3] accounting for 70% of ovulation-disordered infertility, [4] and its prevalence in women of reproductive age is 6% to 10%. [5]he etiology of polycystic ovary syndrome is complex, mainlycaused by genetic and environmental factors. [6]In recent years, the incidence of PCOS has increased significantly, [7] and it is often accompanied by complications such as diabetes, abnormal uterine bleeding, infertility, and high androgen expression, [8] which has brought a heavy economic burden to patients and society.Therefore, PCOS treatment is a hot topic in current medical research.Currently, the treatment drugs for PCOS mainly include ovulation-stimulating drugs, insulin resistance-improving drugs, and androgen-lowering drugs.Still, these drugs are limited to symptomatic treatment, easy to relapse after drug withdrawal, require repeated treatment, and are prone to ovarian hyperstimulation syndrome, premature ovarian failure, lactic acid poisoning, increased cardiovascular burden, and other side effects. [9]New treatments are urgently needed.Traditional Chinese medicines (TCM) have the advantages of having fewer side effects and acting on multiple targets and pathways.Several clinical studies have demonstrated that TCM has achieved significant clinical effectiveness in restoring the menstrual cycle, regulating sex hormone levels and glucose-lipid metabolism, as well as increasing ovulation rates and conception rates., [10] so the study of TCM used in treating polycystic ovary syndrome has a broad application prospect.
Herbal pairs, the basic constituent units of herbal formulas, have special clinical significance in Chinese-medicine and have gradually become a hotspot of modern research. [11]Compared with a single drug, herbal pairs can act synergistically to promote the treatment of various diseases.Modern research shows that Angelica sinensis has antitumour, liver protection, anti-inflammatory, hypoglycemic, immune regulation, cardiovascular and cerebral vascular system protection, etc, [12] and Radix Rehmanniae has the effect of regulating blood lipids and blood glucose, antiaging, antitumour, antibacterial, and protecting gastric mucosa, etc. [13] The pair "angelica sinensis -radix rehmanniae (AR)" was first recorded in "Taiping Huimin Hejiaobu Fang", which is a commonly used pair in TCM.Angelica sinensis and Radix Rehmanniae are 2 Chinese medicines with the highest frequency of use and the most frequent combinations of medicines in treating PCOS.However, their mechanism of action in PCOS treatment is still poorly understood.
The progress of network pharmacology relies on a more profound grasp of how molecules and proteins interact with each other.There are great opportunities to gain a better understanding of diseases, how they develop TCM syndromes, and their treatment mechanisms.It has been applied to the field of TCM, including drug target discovery, efficacy evaluation, and mechanism research.A variety of applications have been made to TCM, including the discovery of drug targets, evaluation of efficacy, and investigation of mechanisms.In order to better understand the mechanism of action of TCM, network pharmacology is an effective method.In this paper, we explored the active ingredients of AR using network pharmacology methods and predicted their targets and signaling pathways related to PCOS, built a disease-drug-ingredient-target network, and analyzed the targets and signaling pathways that could be involved in the treatment of PCOS by AR.

AR active component screening and target prediction
In this study, the compound compositions of Angelica sinensis and Radix Rehmanniae were searched through the TCMSP database (https://tcmsp-e.com/tcmsp.php). [14]Based on pharmacokinetic principles and platform recommendations, the chemical components were obtained as the active ingredients of the 2 Chinese medicines.In the search results, Angelica sinensis and Radix Rehmanniae had oral bioavailability of ≥ 30% and drug-like properties of ≥ 0.18.Also, based on a literature search, ingredients with pharmacodynamic effects were included as active ingredients.A database containing active ingredient information has been integrated with PubChem (https://pubchem.ncbi.nlm.nih.gov/). [15]Download the Smiles information for each component and enter it into the Swiss Target Prediction database (http://www.Swisstargetprediction.ch/). [16]The target proteins corresponding to each component were obtained by setting the attribute as "human" and filtering out targets with "Probability" > 0 as potential targets.

Screening of AR targets for the treatment of PCOS and its network construction
The Genecard database (https://www.genecards.org/) [17]and OMIM database (https://www.ncbi.nlm.nih.gov/omim/) [18]were searched for disease targets related to PCOS using the keywords "polycystic ovary syndrome".Obtaining PCOS-related targets was achieved by combining the results of the 2 databases and de-weighting them.
The drug target and disease target were taken to intersect to obtain the common target, which is the target of AR drug pair for PCOS treatment.In order to construct the drug-active ingredient therapeutic target network, Cytoscape v3.8.0 (www.cytoscape.org/) was utilized. [19]Compounds with the top 10 Degree values were then selected to screen them as the key compounds, considering them to have an essential role in the treatment of PCOS by AR.

Screening for core targets based on protein interaction networks
A String database (https://string-db.org/), [20]was used to construct protein interactions with therapeutic targets, with a threshold value > 0.900 for filtring nonspecific interactions.Cytoscape 3.8.0software was employed for visualizing the protein interaction network.The 10 highest-degree targets are considered core targets for subsequent molecular docking.

Gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis
The Hiplot platform (https://hiplot-academic.com/), [21] was utilized for GO and KEGG pathway enrichment analysis.GO enrichment analysis provided results for Biological Processes, Molecular Functions, and Cellular components.We create a Bubble plot for the top 20 entries based on KEGG enrichment analysis with a P value < .05.

Molecular docking validation
By employing restricted species "homosapiens," with an acceptable resolution of < 2.5, and small molecule compounds as reference criteria, we conducted screening of suitable target protein structures in the RCSB database (http://www.rcsb.org). [22]We used a PubChem database to download SDF files of the 3D structures of the active ingredients.The target protein was dehydrogenated using Auto Dock software. [23]The Box size and position were adjusted to make the ligand and receptor into the optimal conformation, and the results were output in "pdbqt" format.We visualized A molecular docking result with PyMol 2.4.0 software. [24]Our flow chart of the study is shown in Figure 1.

Acquisition of drug-active ingredients and targets
A total of 125 chemical components of Angelica sinensis and 76 chemical components of Radix Rehmanniae were retrieved, and 14 active components of Angelica sinensis and 20 active components of Radix Rehmanniae were obtained after screening, among which 2 active components were common to both Angelica sinensis and Radix Rehmanniae (Table 1).According to the Swiss Target Prediction platform, 421 targets were obtained after removing duplicates, 339 targets were obtained for Angelica sinensis, and 215 for Radix Rehmanniae.

Screening of AR for the treatment of PCOS targets and its network construction
The Genecard and OMIM databases were searched for disease targets related to PCOS using the keyword "polycystic ovary syndrome".After de-emphasis, 6675 disease targets were obtained; the intersection of component targets and disease targets was taken to get 222 potential targets of AR for PCOS treatment (Fig. 2A).Cytoscape 3.8.0software was used to construct a network diagram of "traditional Chinese medicine -active ingredients -therapeutic targets -diseases".The network contains 253 nodes and 907 edges, with yellow representing the active ingredients and blue representing the targets (Fig. 2B).The protein-protein interaction results of these targets were obtained through the STRING database, and the highest confidence results with a screening threshold higher than 0.9 were screened (Fig. 2C).The network topology analysis of this protein interaction information was performed through the Network Analyzer  function of Cytoscape 3.8.0software, which contained 222 nodes and 610 edges, and colors from light to dark represent Degree values from small to large (Fig. 2D).The targets with the top 10 degree values were screened out as the core targets of AR for the treatment of PCOS, as shown in Table 2.The compounds corresponding to them are acteoside, Baicalin, Caffeic acid, Cistanoside F, geniposide, Isoacteoside, the Kaempferol-3-O-glucuronide, Rehmannioside A, Rehmapicroside, Reineckiagenin, Coniferyl ferulate, ferulic acid, levistolide A, ligustilide sedanolide, Senkyunolide A, Senkyunolide B, can be used as core compounds.

Enrichment analysis results
With P value < .05 as the screening condition, a total of 4525 entries were obtained for GO enrichment, and the top-ranked results were visualized in Figure 3.The results of the enrichment analysis showed that these targets could affect biological processes through the regulation of hormone levels, response to xenobiotic stimulus, steroid metabolic process, regulation of body fluid levels, response to lipopolysaccharide, etc; and affect biological processes through membrane raft, membrane microdomain, vesicle lumen, cytoplasmic vesicle lumen, etc; affects cellular composition through protein serine/threonine/tyrosine kinase activity protein serine/ threonine kinase activity, oxidoreductase activity, heme binding.
A total of 278 KEGG enrichment analysis results were screened with P value < .05,highlighting higher-ranking pathways such as 20 AGE-RAGE signaling pathways, Relaxin signaling pathways, TNF signaling pathways, ErbB signaling pathways, and others''.The angelica-ripened dihuang pair may play a role in these pathways for treating PCOS, and the results are shown in Figure 4A.

Molecular docking results
Molecular docking was performed to analyze how the core compounds interact with the core targets.The docking results will then be combined and displayed through heatmaps.PyMol Table 2 The core targets of AR for the treatment of PCOS.

Discussion
PCOS is a polygenic, multifactorial, systemic, inflammatory autoimmune disease.Its pathogenesis is not fully clear. [25][28] It is found that most PCOS patients show increased androgen content, high blood sugar, and lipid metabolism disorders. [29]TCM plays a role in treating PCOS through the advantages of multi-targets and multi-pathways.
Clinical studies have shown that the pair of AR and the formula containing AR can significantly improve hyperandrogenism, regulate glucose homeostasis, and correct lipid metabolism abnormality. [30,31]It is therefore necessary to clarify the molecular basis and biological basis of AR against PCOS.This study could provide new directions for further research on the molecular mechanism of PCOS using network pharmacology, a new method for elucidating complex pharmacological problems.
According to the PPI study based on the STRING database and network topology parameters, we further screened 10 core targets: SRC, HSP90AA1, STAT3, MAPK1, JUN, HRAS, EP300, CREBBP, PIK3CA, and EGFR.C-src is a proto-oncogene that regulates cell proliferation, differentiation, and apoptosis.It promotes the activation of primordial follicles through the PI3K, PKC, and MAPK signaling pathways. [32]HSP90AA1, a member of the heat shock family, encodes inducible molecular chaperones that aid in the folding of specific target proteins.HSP90 is mainly found in ovarian granulosa cells, with significantly higher levels in dominant follicles.It plays a role in promoting follicle maturation regulation. [33]STAT3 is an intracellular signaling transcription factor that plays a crucial role in the immune response, cell cycle regulation, and cell survival.Up-regulation of STAT3 expression in porcine ovarian granulosa cells inhibits apoptosis and promotes cell proliferation.It is also involved in inflammation, with increased phosphorylation  levels during inflammation.These findings suggest that up-regulating STAT3 expression or inhibiting its phosphorylation could improve ovarian function. [34]MAPK is a crucial signaling pathway linked to androgen production and insulin resistance in PCOS.Its role in the development and maturation of oocytes, as well as the initiation of ovulation, is of utmost importance.Inhibiting the MAPK pathway pharmacologically leads to a decrease in the expression of mitotic cyclin D2 and a reduction in granulocyte proliferation; lower levels of granulocytes indirectly interfere with follicle development and maturation. [35,36]UN kinase is a significant branch of the mitogen-activated protein kinase signaling pathway.In vivo, high expression of p-c-Jun promotes trophoblast migration and invasion and enhances ovarian function. [37]HRAS belongs to the RAS gene family and acts as an upstream regulator of the RAS/RAF/MEK/ERK/ MAPK pathway.In patients with polycystic ovary syndrome, the expression of serum HRAS correlates with metabolic profiles such as body mass index, fasting glucose levels, and fasting insulin levels [38] .EGFR, a tyrosine kinase-type receptor, plays a role in crucial reproductive processes like embryo implantation and metaphase.It influences the proliferation and apoptosis of ovarian granulosa cells, thereby inducing oocyte division and maturation.40] Based on the core targets, we obtained 17 key elements that are associated with them.These components can serve as the fundamental active ingredients in the treatment of PCOS using the AR pair.Angelica sinensis contains coniferyl ferulate, ferulic acid, levistolide A, ligustilide, sedanolide, Senkyunolide A, and Senkyunolide B as its active ingredients.Radix Rehmanniae, on the other hand, contains Acteoside, Baicalin, Caffeic Acid, Cistanoside F, Geniposide, Isoacteoside, Kaempferol-3-O-Glucuronide, Rehmannioside A, Rehmapicroside, and Reineckiagenin.Through molecular docking analysis, it was found that Reineckiagenin had the highest docking score with each core target.The study also revealed that Reineckiagenin exhibits estrogen-like activity by binding to estrogen receptors ERα and GPR30.It regulates the hypothalamic-pituitary-ovarian axis, inhibits the secretion of luteinising hormone and follicle-stimulating hormone, and suppresses the production of inflammatory mediators such as iNOS, COX-2, and IL-6.Consequently, it generates anti-inflammatory effects by reducing the concentration of C-reactive protein in the blood plasma. [41]o enrichment analysis showed that the treatment of PCOS using AR primarily involves the regulation of nutrients, vascular lesions, and oxidative substance metabolism.Various organelles, hormone levels, and steroid metabolism influence this process.It encompasses multiple pathways associated with inflammation and metabolic processes.This indicates that the therapeutic effects of traditional Chinese medicines on this disease are achieved through multiple components, targets, pathways, and biological mechanisms.
Based on the KEGG enrichment analysis, the AGE-RAGE signaling pathway was identified as the most significant therapeutic pathway.This pathway plays a crucial role in mediating insulin resistance and triggering inflammatory responses in polycystic ovaries.Factors like high glucose and obesity lead to non-enzymatic glycosylation of proteins, lipids, and DNA in the body.This, in turn, increases the production of advanced glycation end products, which bind to the RAGE receptor on cell membranes.The binding of advanced glycation end products to RAGE activates the AGE-RAGE signaling pathway, resulting in insulin resistance, inflammation, oxidative stress, and the development of PCOS. [42,43]his study, even though it potentially identifies the targets and signaling pathways of AR in the treatment of PCOS, has certain limitations due to the fact that TCM has great potential in treating PCOS.Network pharmacology uses these newly discovered chemical components of AR as a research topic.TCM is not just a straight collection of chemical-compounds, but both its structure and chemical constituents are transparent.A variety of ingredients in AR can produce different effects depending on their concentrations, contents, and interactions.Moreover, AR pharmacological effects in the treatment of PCOS cannot be fully revealed by the databases used to compile data on targets, components, etc.A scientific hypothesis cannot be divorced from experimental verification -and network pharmacology research is no exception.To verify the accuracy and reliability of the prediction results of the network pharmacology of AR in the treatment of PCOS, it is necessary to combine computational prediction with experimental confirmation and to conduct biological experiments on the prediction results of the network pharmacology of AR.This provides strong evidence to support the development of new Chinese medical treatments for PCOS and other diseases.

Conclusion
In conclusion, the integration of network pharmacology and molecular docking technology revealed that Angelica sinensis and Radix Rehmanniae primarily exert their therapeutic effects by targeting multiple molecules such as SRC, HSP90AA1, STAT3, MAPK1, JUN, and others.Through their 17 active ingredients, these herbs regulate lipid metabolism, insulin resistance, and androgen levels to treat PCOS.The main mechanism of action involves the modulation of the AGE-RAGE signaling pathway, which is crucial in PCOS treatment.This study provides insights into the potential pharmacological basis and mechanism of action of AR for PCOS treatment.The network pharmacology approach used here offers a comprehensive analysis of the multi-component, multi-target, functional, and signaling pathway aspects of these herbs in the context of PCOS treatment.This lays the groundwork for future in-depth research and clinical evaluations.

Figure 1 .
Figure 1.Flow chart of the study.

Figure 2 .
Figure 2. (A) Venn diagram of AR targets and PCOS disease-related targets.(B) Drug-ingredient-target-disease network diagram.(C) PPI network diagram of candidate genes.(D) Topological analysis diagram of the candidate genes.AR = angelica sinensis -radix rehmanniae, PCOS = polycystic ovary syndrome.
sinensis -radix rehmanniae, PCOS = polycystic ovary syndrome.www.md-journal.comwas used to visualize the top-ranked results.A docking score lower than −4.25 kcal/mol indicates some binding activity, while a score lower than −7.0 kcal/mol indicates strong binding activity.The present results showed that the average docking score was −9.14 kJ/mol, among which the results with docking score ≤ −7.0 kJ/mol accounted for 91.17%, which reflected that the compounds in the AR pairs had good binding ability to the core target according to the scoring results.Heatmaps demonstrated all the scoring results, as in Figure4B.Some molecular docking results were visualized by PyMol 2.4.0, as shown in Figure5.

Figure 3 .
Figure 3. GO enrichment analysis pathway diagram.(A) Enrichment of GO biological process.(B) Enrichment of GO molecular function.(C) Enrichment of GO cellular component.GO = gene ontology.

Table 1 TCM
Active ingredients.