Exploration of the Underlying Mechanism of Jiawei Shixiao San in the Treatment of Endometriosis Using Network Pharmacology and Experimental Verification

Exploration of the Underlying Mechanism of Jiawei Shixiao San in the Treatment of Endometriosis Using Network Pharmacology and Experimental Verification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploration of the Underlying Mechanism of Jiawei Shixiao San in the Treatment of Endometriosis Using Network Pharmacology and Experimental Verification Chuchu Zhao, Huifen Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5736127/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aim Shixiao San, composed of Pollen Typha and Faeces Trogopterori, is a traditional Chinese medicine used for the the treatment of endometriosis. Jiawei Shixiao San is a modified formula derived from Shixiao San, adding with Sanguis Draconis, Lignum Sappan, Fritillariae thunbergii bulbus, Semen Coicis, and Radix Notoginseng. The purpose of this study was to elucidate the mechanism of Jiawei Shixiao San in combating endometriosis at a systemic level through network pharmacology and experimental verification. Methods The targets of Jiawei Shixiao San and endometriosis were retrieved from multiple databases. An herbs-compounds-genes network and a protein-protein interaction network were constructed to analyze target interactions. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted using the DAVID database. Finally, CCK-8 assays and flow cytometry analyses were performed to evaluate the anti-endometriosis effects of the active compounds. Results A total of 157 common targets associated with herbs and endometriosis were identified for analysis. The key targets included AKT1, IL-6, TNF, ESR1, TP53, VEGFA, EGFR, and SRC. The potential active compounds identified were quercetin, pelargonidin, isorhamnetin, and kaempferol. Enrichment analysis suggested that Jiawei Shixiao San may treat endometriosis through the PI3K-Akt signaling pathway, resistance to EGFR tyrosine kinase inhibitors, endocrine resistance, and the AGE-RAGE signaling pathway. Further experimental validation demonstrated that kaempferol could inhibit the proliferation of endometrial stromal cells and induce apoptosis. Conclusion In summary, we conducted network pharmacology analysis combined with experimental verification to provide compelling evidence that Jiawei Shixiao San may treat endometriosis by regulating cell proliferation and apoptosis, as well as by modifying inflammation-related genes and signaling pathways. Jiawei Shixiao San endometriosis network pharmacology kaempferol inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Endometriosis (EMS) is a chronic inflammatory disorder characterized by endometrium like tissue growing outside the uterine cavity, primarily in the ovary [ 1 ], affecting 10%-15% women of reproductive age [ 2 ]. Notable symptoms of endometriosis are chronic pelvic pain, dysmenorrhea, menorrhagia, dyspareunia and infertility, which cause serious negative physical and psychological effects [ 3 ]. The current clinical management of endometriosis is mainly based on surgery and medical therapies which are not definitely curative [ 4 – 6 ]. Surgery can alleviate symptoms, remove lesions and improve conception, but recurrences are common [ 4 – 6 ]. The pharmacological therapy, mainly hormonal therapy, may ameliorate pain and reduce post-surgical disease recurrence, however, hormonal therapy usually need a long-term regimen and has many side effects [ 7 – 8 ]. In order to better treat endometriosis or relieve pain, it is particularly important to seek novel alternative treatment methods. In Asian communities, traditional Chinese medicine (TCM) has already been practiced for the prevention, treatment, and cure of diseases for thousands of years and is attracting more and more attention in modern medical medicine due to its good clinical effect and lower toxicity [ 9 ]. In the traditional Chinese medicine theory, clinical manifestations of endometriosis can be attributed to Zheng Jia, which is mainly related to blood stasis and obstruction in lower abdomen causing pelvic pain, abdominal mass, vaginal bleeding and other symptoms [ 10 , 11 ]. Based on the pathogenesis of Zheng Jia, the core therapeutic methods are activating blood circulation and dissipating blood stasis, which are called as “huoxue huayu” in traditional Chinese medicine [ 12 ]. Shixiao san composed of two herbal materials, Pollen Typha (Puhuang) and Faeces Trogopterori (Wulingzhi), is one of the famous Huoxue Huayu recipes recorded in many ancient documents like the Complete Collection of Prescriptions (Taiping Huimin Heji Ju Fang), and has been widely used to treat gynecological disease for centuries [ 13 , 14 ]. According to practical knowledge and clinical experience, the Jiawei Shixiao San (JSX) was prescribed by a TCM physician in Lishui Central Hospital. On the basis of Shixiao san, the JSX added with Sanguis Draconis (xuejie), Lignum Sappan (Sumu), Fritillariae thunbergii bulbus (Zhebeimu) and Semen Coicis (Yiyiren), Radix Notoginseng (Sanqi). Chinese herbal medicines contain multiple compounds and can interact with large number of targets, which affect body function through various mechanisms [ 7 ]. Therefore, it is difficult to precisely identify the functional compounds and clarify the therapeutic mechanisms. Network pharmacology is an burgeoning method based on a large number of database resources and construction of “compounds-genes-pathways” interaction network [ 8 ]. It contributes to precisely identify functional herbal compounds and observe the synergistic multi-compounds, helping to form a holistic understanding of the mechanism of TCM treatment, supporting the experience-based TCM prescription with fundamental evidence [ 8 ]. For this article, network pharmacology was employed to systematically investigate the effective bioactive compounds. Then, the herbs and disease overlapping targets were analysed for the Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) pathway analysis. Furthermore, the hub targets of the common targets were screened through the protein-protein interaction (PPI) network in the STRING database. Finally, immortalized endometrial stromal cell line hEM15A were used to verify the effect of JSX on EMS. Materials and Methods 2.1. Network pharmacology analysis 2.1.1. Collection of active ingredients The active chemical components of puhuang, yiyiren, sanqi and zhebeimu were retrieved from the TCMSP database (Traditional Chinese Medicine Systems Pharmacology, ( http://lsp.nwu.edu.cn/tcmsp.php ) [ 15 ]. In our study, molecules with oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were selected as key active compounds for further analysis [ 16 , 17 ]. The active chemical components of Wulingzhi, Xuejie and Sumu were retrieved from BATMAN-TCM (the Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine, http://bionet.ncpsb.org.cn/batman-tcm/ ) [ 18 ], ETCM (The Encyclopedia of Traditional Chinese Medicine, http://www.tcmip.cn/ETCM/index.php ) [ 19 ]. We downloaded the SDF files of chemical components from the PubChem database and input them into the Swiss ADME network tool ( http://www.swissadme.ch/ ) to pick out efficient compounds according to pharmacokinetics and druglikeness. In our study, molecules with high gatrointestinal absorption and good druglikeness, which is determined by more than three results of five important characteristics (Lipinski, Ghose, Veber, Egan and Muegge filter) are yes, were selected as active compounds [ 20 ]. In order to collect potential targets as many as possible, we retrieved literatures to supplement more active ingredients. 2.1.2. Potential medicine targets prediction After getting all the active ingredients, we collected the corresponding targets of each ingredient by the Swiss Target Prediction database ( http://www.swisstargetprediction.ch/ ) with the species limited as “Homo sapiens” [ 21 , 22 ]. Then the herb-compound-target network were built by the Cytoscape 3.8.2 software. 2.1.3. Collection of potential targets in EMS The potential therapeutic targets for EMS were acquired with the keyword “endometriosis” from the following five public databases: GeneCards ( https://www.genecards.org/ ) [ 23 ], OMIM ( http://www.omim.org/ ) [ 24 ], DisGeNET ( https://www.disgenet.org ) [ 25 ], TTD ( http://db.idrblab.net/ttd/ ) [ 26 ], and DrugBank ( https://www.drugbank.ca ) [ 27 ]. Limiting the species to “Homo sapiens”, the UniProt website ( https://www.uniprot.org/ ) were used to convert protein name into gene symbol. Then, the overlapping genes between the medicine-targets and disease-targets, which were the potential therapeutic targets of the 7 herbs against EMS, were identified and visualized through the Venn diagram. 2.1.4. PPI network construction PPI analysis provided protein-protein interaction data and helped to highlight the hub genes. The overlapping genes between the herbs and disease were input into the STRING database ( https://string-db.org/ ) with the species limited to “Homo sapiens”. And The confidence score > 0.4 was selected to predict the protein interactions. PPI network files were then imported into the network visualization program Cytoscape. The importance of the nodes were evaluated by the value of degree, betweenness and closeness, the 8 targets whose these three values were all on the top 10 were selected as the hub genes. 2.1.5. Enrichment analysis GO and KEGG pathway enrichment analyses were performed for the classification of gene functions and obtain significantly enriched biological pathways associated with the common targets between 7 herbal medicine and EMS. The common targets were input into DAVID 6.8 ( https://david.ncifcrf.gov/ ) and restricted the species to “Homo sapiens”. Only functional annotations with P value smaller than 0.05 were considered to be statistically significant. The top 20 significant items were presented via a bubble graph. 2.2. Experimental verification of network analysis 2.2.1. Cell culture Immortalized endometrial stromal cell line hEM15A was purchased from Shanghai Cell Biology Medical Research Institute, Chinese Academy of Sciences. The cells were maintained in DMEM/F-12(Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, San Diego, USA). The cells were incubated in a 37℃ humidified incubator with 5% CO 2 . 2.2.2. Cell treatment and viability assay Kaempferol was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved with DMSO. The hEM15A cells were digested and seeded in 96-well plates for 24h. Subsequently, the cells were treated with kaempferol (0, 10, 20, 40 and 60 µM) for 24 or 48 hours. Then the hEM15A cells were reacted with 10 µL/well CCK-8 solution for 2h. The absorbance was measured at an optical density (OD) of 450 nm using a microplate reader (Bio-Rad Laboratories Inc.,Hercules, CA, USA). 2.2.3. Cell apoptosis assay After treatment with 40 µM kaempferol for 48 hours, the hEM15a cells were collected and double-stained with 5 µL Annexin V–PE and 5 µL 7-AAD (BD, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The apoptosis was analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA). 2.3. Statistical analysis Statistical analysis was performed with Statistical Product and Service Solutions (SPSS) 22.0 statistical software (IBM Corporation, Armonk, NY, USA). All results were continuous variables and normal distribution, shown as mean ± SD. The two-tailed unpaired Student's t-test was used to identify differences between two groups that were statistically significant. P < 0.05 were regarded as the threshold for statistical significance. Results 3.1. Network pharmacology 3.1.1. Active compounds and targets of 7 herbal medicine A total of 46 active ingredients were obtained from 7 herbs, and there were 190 potential targets from puhuang, 232 targets from sanqi, 143 targets from sumu, 72 targets from wuliingzhi, 253 targets from xuejie, 108 targets from yiyiren and 368 targets from zhebeimu. H (Fig. 1 ). Judging form the degree of the network, the 10 top active compounds were as follows: quercetin (degree = 203), pelargonidin (degree = 101), isorhamnetin (degree = 101), Mandenol (degree = 101), kaempferol-3-O-α-L-rhamnosyl(1→2)-β-D-glucoside_qt (degree = 101), (2S)-5-Methoxy-6-Methylflavan-7-Ol (degree = 101), 2,4-Dihydroxy-6-Methoxychalcone (degree = 101), kaempferol (degree = 99), beta-sitosterol (degree = 90), Sandaracopimaric Acid (degree = 85). It indicates that these compounds serve a critical therapeutic role in EMS. After deleting repeated genes, 571 putative targets for 7 herbs and 1056 putative endometriosis related targets were screened out. There were 157 common targets between herbal compounds and EMS filtered as the vital targets for further testing (Fig. 2 ). 3.1.2. Hub genes against EMS in the PPI Network To explore the key hub genes of these 7 herbal medicine as treatment against EMS, the PPI network was constructed by the STRING database and visualized by using Cytoscape. There were 157 nodes and 1980 edges in the PPI network (Fig. 3 ). The degree centrality (DC), betweenness centrality (BC), and the average closeness centrality (CC) were calculated to help detect the key hub genes. There were 8 targets whose DC, BC, and CC values were all among the top 10 highest: AKT1, IL-6, TNF, ESR1, TP53, VEGFA, EGFR and SRC, indicating that the 7 herbal medicine might exert its pharmacological effects mainly through these targets. 3.1.3. Enrichment analysis In order to further study the identified candidate target genes on EMS, the 157 common targets were uploaded to the DAVID database to perform GO enrichment analysis and KEGG pathway enrichment analysis. GO enrichment analysis were conducted from three sides: biological process (BP), molecular function (MF) and cell composition (CC) (Fig. 4 a). In the BP group, the first five items were “signal transduction”, “negative regulation of apoptotic process”, “protein phosphorylation”, “positive regulation of gene expression” and “negative regulation of gene expression”. In the aspect of CC, the targets were primarily enriched in “cytoplasm”, “cytosol”, “plasma membrane”, “integral component of plasma membrane” and “cell surface”. In the MF group, the top five items were “identical protein binding”, “ATP binding”, “protein serine/threonine/tyrosine kinase activity”, “protein kinase activity” and “enzyme binding”. Based on the results of pathway enrichment, the remarkable pathways were as follows: PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, Endocrine resistance, AGE-RAGE signaling pathway, ErbB signaling pathway, VEGF signaling pathway (Fig. 4 b). 3.2. In vitro experiment identification 3.2.1. Cell proliferation after kaempferol treatment We performed the CCK-8 assay to detect the response of the hEM15A cells to kaempferol treatment. Results indicated that, after treated for 48h, when the concentration of kaempferol is higher than 40 µM, the viability of hEM15A cells were markedly decreased compared with DMSO group. However, when treated only for 24 hours, kaempferol had no significant effect on the cell proliferation (Fig. 5 ). 3.2.2. Cell apoptosis after kaempferol treatment To determine whether the underlying mechanism of kaempferol suppressed cell proliferation was concerned with apoptosis, we used an Annexin V–PE/7-AAD assay to investigate the apoptotic rate of hEM15A cells. The results showed that the cells apoptotic rate of the kaempferol treatment group (40 µM) was obviously higher than the control group after treated for 48h ( P <0.05) (Fig. 6 ). Discussion The specific mechanism of endometriosis is still incompletely understood up to now, but endometriosis is always considered as a chronic, estrogen-dependent, inflammatory disease [ 2 ]. Endometriosis can lead to a series of symptoms such as dysmenorrhea, pelvic pain, dyspareunia and infertility, and for women have no fertility requirement, reliefing the pelvic pain is their most urgent demands [ 5 ]. TCM is commonly used as a complementary and alternative treatment in the treatment of EMs, especially to relieve endometriosis-associated pain [ 28 ]. Shixiao San is one of the famous analgesic and Huoxue Huayu Chinese herbal formulas and it has been widely used to treat gynecological disorders [ 29 ]. JSX is a new formula originated from classic Shixiao San formula. In our previous clinical application, we have achieved a good result in relieving dysmenorrhea, but the detailed mechanism of JSX on endometriosis is currently unknown. In this study, the network pharmacology approach was applied to illustrate the synergistic molecular mechanism of JSX in treating endometriosis. In the present study, we identified 46 active compounds and 571 targets of JSX, 1056 targets of endometriosis, and our Venn diagram indicated that JSX and endometriosis had 157 common targets. Furthermore, the degrees for all of the 157 common targets were calculated using the Cytoscape software. The PPI network indicated that AKT1, IL-6, TNF, ESR1, TP53, VEGFA, EGFR and SRC may play key roles in the effect of JSX against endometriosis. The results of the GO enrichment analysis indicated that JSX might treat endometriosis via regulating cell apoptotic process and protein phosphorylation. Combined with the results of the top genes in the PPI network, KEGG pathway enrichment analysis manifested that JSX might treat endometriosis through PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, Endocrine resistance, AGE-RAGE signaling pathway, ErbB signaling pathway and VEGF signaling pathway. Protein kinase B (AKT), a versatile serine–threonine kinase, emerging as one of the most critical regulators in many signaling pathways, helps to maintain the normal physiological cell functions, such as cell cycle control, apoptosis, differentiation and metabolic processes [ 30 ]. In mammals, there are three Akt isozymes AKT1, AKT2, and AKT3 [ 30 ]. AKT1 is a crucial regulatory kinase that transmits signals via the PI3K/AKT cell-signaling cascade, which regulates cell growth and survival [ 31 ]. AKT1 is elevated phosphorylation and overactive in most human tumors and is linked to poor clinical prognosis [ 30 ]. In endometriosis, compared to control endometrium, AKT1 phosphorylation was highly elevated in minimal-mild endometriosis, while moderate in later stages [ 32 ]. A large amount of clinical trials have focused on inhibiting or reducing AKT1 hyper-activity as a promising treatment for cancer therapies [ 33 ]. However, whether targeting the AKT1 pathway can help to treat endometriosis is still need further research. IL-6, tumor necrotic factor alpha (TNFα) and vascular endothelial growth factor (VEGF) are pro-inflammatory cytokines which have crucial role in the pathogenesis and pain of endometriosis, and are early diagnostic markers and promising treatment targets for endometriosis [ 34 ]. IL6, mainly secreted from macrophages and endometriotic cells, is considered to be the promoter of many biological activities including pro- and anti-inflammatory, making IL-6 a tool to relieve the pelvic pain of endometriosis [ 35 ]. Serum and peritoneal fluid levels of interleukin-6 were higher in the endometriosis patients and were correlated with the stage of endometriosis [ 36 ]. The herbs-compounds-genes network was established to search for the active ingredints. The results showed that sanqi and yiyiren were the herbs related with most common ingredints. And 10 key compounds were obtained as follows: quercetin, pelargonidin, isorhamnetin, Mandenol, kaempferol-3-O-α-L-rhamnosyl(1→2)-β-D-glucoside_qt, (2S)-5-Methoxy-6-Methylflavan-7-Ol, 2,4-Dihydroxy-6-Methoxychalcone, kaempferol, beta-sitosterol and Sandaracopimaric Acid. Quercetin and kaempferol are members of the general flavonoids, widely distributed in a variety of plants like Allium, Malus, Delphinium, and Camellia [ 37 , 38 ]. Many previous studies had already demonstrated that Quercetin has the ability to inhibit cell proliferation, induce cell apoptosis, anti-inflammation in endometriosis cell lines and autoimplanted mouse models [ 38 – 39 ]. However, up to date, the association between kaempferol and endometriosis is still unknown. In the present study, we showed that, after the exposure of kaempferol, the proliferation rate of hEM15A cells was inhibited and the apoptosis was enhanced. Kaempferol has recently been reported to have anti-oxidant, and anti-inflammatory bioactive functions, and have protective effects on many organs such as liver, colon, ovary,and stomach by regulating the cell cycle arrest, proliferation, migration, and the tight junction proteins [ 40 – 43 ]. The consumption of kaempferol-rich food has an essential anticancer role in the developing of skin, liver, and colon cancer but do not cause apparent toxicity to normal cells, indicating that kaempferol could be developed as a high safety and low side efects anticancer agent [ 44 ]. Luo and his colleagues found that, in ovarian cancer cell lines, kaempferol could suppress cell proliferation and cause apoptosis, and inhibited Akt phosphorylation [ 43 , 45 ]. In breast cancer cell lines, kaempferol also effectively inhibits the proliferation and induces apoptosis through downregulation of the expressions of cancer progression proteins and decrease the anti-inflammatory proteins level [ 46 , 47 ]. In Pancreatic cancer cell lines, appropriate doses (48µM) of kaempferol may potentiate the efficacy of Erlotinib in inhibiting cell proliferation and promoting cell apoptosis via synergistically inhibiting the expressions of p-EGFR and p-AKT [ 48 ]. Another study found that both quercetin and kaempferol may block the activation of AKT and decrease the phospho-AKT levels in 3T3-L1 Preadipocytes [ 49 ]. Another researchers provided molecular evidence that kaempferol inhibited the invasion and migration of hepatocellular carcinoma cells by reducing the activity and protein expression of MMP-9 and suppressing the phosphorylation of the Akt expression [ 50 ]. In this study, the results was in good concordance with the analysis presented above, but whether kaempferol affected the proliferation and apoptosis through regulating phospho-AKT levels and PI3K-Akt signaling pathway should be studied further. To sum up, network pharmacology indicated that the mechanism of JSX in treating endometriosis was the result of multi-mechanism and multi-pathway synergy, might be through modulation of the inflammation and/or the endocrine system, and through PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, AGE-RAGE signaling pathway, ErbB signaling pathway and VEGF signaling pathway. 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Inhibition of Cell Growth and VEGF Expression in Ovarian Cancer Cells by Flavonoids. Nutrition and Cancer. 2008; 60:800–809. Jianjun Pei, Anna Chen, Linguo Zhao, Fuliang Cao, Gang Ding, Wei Xiao. One-Pot Synthesis of Hyperoside by a Three-Enzyme Cascade Using a UDP-Galactose Regeneration System. J Agric Food Chem. 2017 Jul 26;65(29):6042-6048. Luo H, Rankin GO, Li Z, Depriest L, Chen YC. Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway. Food Chem. 2011 Sep 15;128(2):513-519. Li Zhu, Lijun Xue. Kaempferol suppresses proliferation and induces cell cycle arrest, apoptosis, and DNA damage in breast cancer cells. Oncol Res. 2019 Jun 21;27(6):629-634. Geum-A Lee, Kyung-Chul Choi, Kyung-A Hwang. Treatment with Phytoestrogens Reversed Triclosan and Bisphenol A-Induced Anti-Apoptosis in Breast Cancer Cells. Biomol. Ther. 2018, 26, 503–511. Zhengguang Zhang, Yuanyuan Guo, Meijuan Chen, Feiyan Chen, Bing Liu, Cunsi Shen. Kaempferol potentiates the sensitivity of pancreatic cancer cells to erlotinib via inhibition of the PI3K/AKT signaling pathway and epidermal growth factor receptor. Inflammopharmacology. 2021 Oct;29(5):1587-1601. William Y Boadi, Andrew Lo. Effects of Quercetin, Kaempferol, and Exogenous Glutathione on Phospho- and Total-AKT in 3T3-L1 Preadipocytes. J Diet Suppl. 2018 Nov 2;15(6):814-826. Po-Chung Ju, Yung-Chuan Ho, Pei-Ni Chen, Hsiang-Lin Lee, Szu-Yu Lai, Shun-Fa Yang, et al . Kaempferol inhibits the cell migration of human hepatocellular carcinoma cells by suppressing MMP-9 and Akt signaling. Environ Toxicol. 2021 Oct;36(10):1981-1989. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5736127","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":395891327,"identity":"63b7c651-d86a-40ce-b52d-ddc6de436aa1","order_by":0,"name":"Chuchu Zhao","email":"","orcid":"","institution":"Lishui Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chuchu","middleName":"","lastName":"Zhao","suffix":""},{"id":395891329,"identity":"11eb6ba0-92e3-4974-8b55-19478687553e","order_by":1,"name":"Huifen Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYJACZgYGCX4gdfhBQoWEnDyxWiQbGNjSDD6csTA2bCBOCwNQC4+B5My2ikSGAwSUGxw/e/h1YZuFBL90W4Ix7zyJBMYG5oePbuDTciYvzXpmm4SE5JzDBx7zbpPIY2dgMzbOwaflQI6ZMW+bRJ3BjTSgLdskihkbeNik8Wo5/wasRcL+Ro6BNO8cicSGA4S03MgxfgzSYiCRA/R+AxFaJG+8MWPmOSchIXEjDRjIxySMDZsJ+IXvfI7xZ56yOgn+GcnAqKypk5Nnb374GJ8WhQMMbBKoQsx4lIOAfAMD8wcCakbBKBgFo2CkAwAXYEpH4w5eZAAAAABJRU5ErkJggg==","orcid":"","institution":"Lishui Central Hospital","correspondingAuthor":true,"prefix":"","firstName":"Huifen","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-12-30 14:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5736127/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5736127/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72733359,"identity":"8656a1af-8347-48e8-a74c-a1e43d8f56fc","added_by":"auto","created_at":"2025-01-01 07:37:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2001711,"visible":true,"origin":"","legend":"\u003cp\u003eThe herbs-compounds-genes network constructed by the Cytoscape 3.8.2 software. The pink circles represent the herbs, the hexagons represent the active compounds, and the blue rhombus represent the predicted targets. The edges symbolize the interactions between compounds and targets, and the node size is proportional to the degree of interaction.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5736127/v1/dd160c3037ac7c7409ef7d00.png"},{"id":72733761,"identity":"ef9512e3-1c21-4747-b25e-e04d149cbc8b","added_by":"auto","created_at":"2025-01-01 07:45:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":344573,"visible":true,"origin":"","legend":"\u003cp\u003eThe 157 common targets of the putative 7 herbal medicine targets and the EMS targets.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5736127/v1/9dd8ffc628fa53cc11bec0bb.png"},{"id":72733361,"identity":"06f4d5af-5191-469c-b88d-025e7670798d","added_by":"auto","created_at":"2025-01-01 07:37:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2510256,"visible":true,"origin":"","legend":"\u003cp\u003eThe PPI network of 157 common nodes constructed by the Cytoscape 3.8.2 software. The node size and the color changes from light to dark reflect the importance in the network.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5736127/v1/47c0a73523d049cb35eb4ccb.png"},{"id":72733364,"identity":"2ea337a5-2d12-431e-acb0-688027347b87","added_by":"auto","created_at":"2025-01-01 07:37:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4394827,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG enrichment analysis of the 157 anti-EMS targets. (a) The top 10 remarkably results of the GO enrichment analysis. The ordinate stands for three domains enriched GO of the targets: biological process (BP), cellular component (CC), and molecular function (MF), and the abscissa represents the values of fold change. (b)The top 20 KEGG pathway of the 157 anti-EMS targets. Count is the enriched gene number in a certain pathway, fold enrichment is the percentage of the enriched gene number to the total gene number in each pathway.\u003c/p\u003e","description":"","filename":"Fig4a.png","url":"https://assets-eu.researchsquare.com/files/rs-5736127/v1/2d386456694b8ccab4b14bea.png"},{"id":72733385,"identity":"e4393edb-777b-4be9-9c98-054b4ef79c5f","added_by":"auto","created_at":"2025-01-01 07:37:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":287413,"visible":true,"origin":"","legend":"\u003cp\u003eKaempferol treatment on proliferation of hEM15A cells. Cells were incubated with increasing doses of kaempferol (10-60 μM) for 24-48 h. Data are mean ± SD from triplicate experiments. Compared with control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5736127/v1/7afdc80c9e70120cdc7ea115.png"},{"id":72733371,"identity":"1c02760f-f8a3-4d35-b545-b26cb998e41b","added_by":"auto","created_at":"2025-01-01 07:37:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":277283,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of kaempferol on the apoptosis of hEM15A cells. Kaempferol group: hEM15A cells were treated with 40 μM kaempferol for 48 h. Control group: hEM15A cellswere treated with DMSO for 48 h. Apoptosis percentage was analyzed by Flow cytometry analysis. Data are mean ± SD from triplicate experiments. Compared with control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5736127/v1/f671f4c653d98fb417ab3b51.png"},{"id":72916826,"identity":"f3ab474c-896a-45f0-a628-ba383919f33e","added_by":"auto","created_at":"2025-01-03 16:01:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11006130,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5736127/v1/1932d4cf-a622-47e8-a02f-b801c13cd9d8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploration of the Underlying Mechanism of Jiawei Shixiao San in the Treatment of Endometriosis Using Network Pharmacology and Experimental Verification","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis (EMS) is a chronic inflammatory disorder characterized by endometrium like tissue growing outside the uterine cavity, primarily in the ovary [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], affecting 10%-15% women of reproductive age [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notable symptoms of endometriosis are chronic pelvic pain, dysmenorrhea, menorrhagia, dyspareunia and infertility, which cause serious negative physical and psychological effects [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The current clinical management of endometriosis is mainly based on surgery and medical therapies which are not definitely curative [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Surgery can alleviate symptoms, remove lesions and improve conception, but recurrences are common [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The pharmacological therapy, mainly hormonal therapy, may ameliorate pain and reduce post-surgical disease recurrence, however, hormonal therapy usually need a long-term regimen and has many side effects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In order to better treat endometriosis or relieve pain, it is particularly important to seek novel alternative treatment methods.\u003c/p\u003e \u003cp\u003eIn Asian communities, traditional Chinese medicine (TCM) has already been practiced for the prevention, treatment, and cure of diseases for thousands of years and is attracting more and more attention in modern medical medicine due to its good clinical effect and lower toxicity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the traditional Chinese medicine theory, clinical manifestations of endometriosis can be attributed to Zheng Jia, which is mainly related to blood stasis and obstruction in lower abdomen causing pelvic pain, abdominal mass, vaginal bleeding and other symptoms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Based on the pathogenesis of Zheng Jia, the core therapeutic methods are activating blood circulation and dissipating blood stasis, which are called as \u0026ldquo;huoxue huayu\u0026rdquo; in traditional Chinese medicine [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Shixiao san composed of two herbal materials, Pollen Typha (Puhuang) and Faeces Trogopterori (Wulingzhi), is one of the famous Huoxue Huayu recipes recorded in many ancient documents like the Complete Collection of Prescriptions (Taiping Huimin Heji Ju Fang), and has been widely used to treat gynecological disease for centuries [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. According to practical knowledge and clinical experience, the Jiawei Shixiao San (JSX) was prescribed by a TCM physician in Lishui Central Hospital. On the basis of Shixiao san, the JSX added with Sanguis Draconis (xuejie), Lignum Sappan (Sumu), Fritillariae thunbergii bulbus (Zhebeimu) and Semen Coicis (Yiyiren), Radix Notoginseng (Sanqi).\u003c/p\u003e \u003cp\u003eChinese herbal medicines contain multiple compounds and can interact with large number of targets, which affect body function through various mechanisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, it is difficult to precisely identify the functional compounds and clarify the therapeutic mechanisms. Network pharmacology is an burgeoning method based on a large number of database resources and construction of \u0026ldquo;compounds-genes-pathways\u0026rdquo; interaction network [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It contributes to precisely identify functional herbal compounds and observe the synergistic multi-compounds, helping to form a holistic understanding of the mechanism of TCM treatment, supporting the experience-based TCM prescription with fundamental evidence [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor this article, network pharmacology was employed to systematically investigate the effective bioactive compounds. Then, the herbs and disease overlapping targets were analysed for the Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) pathway analysis. Furthermore, the hub targets of the common targets were screened through the protein-protein interaction (PPI) network in the STRING database. Finally, immortalized endometrial stromal cell line hEM15A were used to verify the effect of JSX on EMS.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Network pharmacology analysis\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Collection of active ingredients\u003c/h2\u003e \u003cp\u003eThe active chemical components of puhuang, yiyiren, sanqi and zhebeimu were retrieved from the TCMSP database (Traditional Chinese Medicine Systems Pharmacology, (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://lsp.nwu.edu.cn/tcmsp.php\u003c/span\u003e\u003cspan address=\"http://lsp.nwu.edu.cn/tcmsp.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In our study, molecules with oral bioavailability (OB)\u0026thinsp;\u0026ge;\u0026thinsp;30% and drug-likeness (DL)\u0026thinsp;\u0026ge;\u0026thinsp;0.18 were selected as key active compounds for further analysis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The active chemical components of Wulingzhi, Xuejie and Sumu were retrieved from BATMAN-TCM (the Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bionet.ncpsb.org.cn/batman-tcm/\u003c/span\u003e\u003cspan address=\"http://bionet.ncpsb.org.cn/batman-tcm/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], ETCM (The Encyclopedia of Traditional Chinese Medicine, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.tcmip.cn/ETCM/index.php\u003c/span\u003e\u003cspan address=\"http://www.tcmip.cn/ETCM/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We downloaded the SDF files of chemical components from the PubChem database and input them into the Swiss ADME network tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch/\u003c/span\u003e\u003cspan address=\"http://www.swissadme.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to pick out efficient compounds according to pharmacokinetics and druglikeness. In our study, molecules with high gatrointestinal absorption and good druglikeness, which is determined by more than three results of five important characteristics (Lipinski, Ghose, Veber, Egan and Muegge filter) are yes, were selected as active compounds [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In order to collect potential targets as many as possible, we retrieved literatures to supplement more active ingredients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Potential medicine targets prediction\u003c/h2\u003e \u003cp\u003eAfter getting all the active ingredients, we collected the corresponding targets of each ingredient by the Swiss Target Prediction database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the species limited as \u0026ldquo;Homo sapiens\u0026rdquo; [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Then the herb-compound-target network were built by the Cytoscape 3.8.2 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Collection of potential targets in EMS\u003c/h2\u003e \u003cp\u003eThe potential therapeutic targets for EMS were acquired with the keyword \u0026ldquo;endometriosis\u0026rdquo; from the following five public databases: GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.omim.org/\u003c/span\u003e\u003cspan address=\"http://www.omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], DisGeNET (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.disgenet.org\u003c/span\u003e\u003cspan address=\"https://www.disgenet.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], TTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"http://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and DrugBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.drugbank.ca\u003c/span\u003e\u003cspan address=\"https://www.drugbank.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Limiting the species to \u0026ldquo;Homo sapiens\u0026rdquo;, the UniProt website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to convert protein name into gene symbol. Then, the overlapping genes between the medicine-targets and disease-targets, which were the potential therapeutic targets of the 7 herbs against EMS, were identified and visualized through the Venn diagram.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.1.4. PPI network construction\u003c/h2\u003e \u003cp\u003ePPI analysis provided protein-protein interaction data and helped to highlight the hub genes. The overlapping genes between the herbs and disease were input into the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the species limited to \u0026ldquo;Homo sapiens\u0026rdquo;. And The confidence score\u0026thinsp;\u0026gt;\u0026thinsp;0.4 was selected to predict the protein interactions. PPI network files were then imported into the network visualization program Cytoscape. The importance of the nodes were evaluated by the value of degree, betweenness and closeness, the 8 targets whose these three values were all on the top 10 were selected as the hub genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.1.5. Enrichment analysis\u003c/h2\u003e \u003cp\u003eGO and KEGG pathway enrichment analyses were performed for the classification of gene functions and obtain significantly enriched biological pathways associated with the common targets between 7 herbal medicine and EMS. The common targets were input into DAVID 6.8 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and restricted the species to \u0026ldquo;Homo sapiens\u0026rdquo;. Only functional annotations with \u003cem\u003eP\u003c/em\u003e value smaller than 0.05 were considered to be statistically significant. The top 20 significant items were presented via a bubble graph.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental verification of network analysis\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Cell culture\u003c/h2\u003e \u003cp\u003eImmortalized endometrial stromal cell line hEM15A was purchased from Shanghai Cell Biology Medical Research Institute, Chinese Academy of Sciences. The cells were maintained in DMEM/F-12(Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, San Diego, USA). The cells were incubated in a 37℃ humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Cell treatment and viability assay\u003c/h2\u003e \u003cp\u003eKaempferol was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved with DMSO. The hEM15A cells were digested and seeded in 96-well plates for 24h. Subsequently, the cells were treated with kaempferol (0, 10, 20, 40 and 60 \u0026micro;M) for 24 or 48 hours. Then the hEM15A cells were reacted with 10 \u0026micro;L/well CCK-8 solution for 2h. The absorbance was measured at an optical density (OD) of 450 nm using a microplate reader (Bio-Rad Laboratories Inc.,Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Cell apoptosis assay\u003c/h2\u003e \u003cp\u003eAfter treatment with 40 \u0026micro;M kaempferol for 48 hours, the hEM15a cells were collected and double-stained with 5 \u0026micro;L Annexin V\u0026ndash;PE and 5 \u0026micro;L 7-AAD (BD, Franklin Lakes, NJ, USA) according to the manufacturer\u0026rsquo;s instructions. The apoptosis was analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with Statistical Product and Service Solutions (SPSS) 22.0 statistical software (IBM Corporation, Armonk, NY, USA). All results were continuous variables and normal distribution, shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The two-tailed unpaired Student's t-test was used to identify differences between two groups that were statistically significant. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were regarded as the threshold for statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Network pharmacology\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. Active compounds and targets of 7 herbal medicine\u003c/h2\u003e \u003cp\u003eA total of 46 active ingredients were obtained from 7 herbs, and there were 190 potential targets from puhuang, 232 targets from sanqi, 143 targets from sumu, 72 targets from wuliingzhi, 253 targets from xuejie, 108 targets from yiyiren and 368 targets from zhebeimu. H (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Judging form the degree of the network, the 10 top active compounds were as follows: quercetin (degree\u0026thinsp;=\u0026thinsp;203), pelargonidin (degree\u0026thinsp;=\u0026thinsp;101), isorhamnetin (degree\u0026thinsp;=\u0026thinsp;101), Mandenol (degree\u0026thinsp;=\u0026thinsp;101), kaempferol-3-O-α-L-rhamnosyl(1\u0026rarr;2)-β-D-glucoside_qt (degree\u0026thinsp;=\u0026thinsp;101), (2S)-5-Methoxy-6-Methylflavan-7-Ol (degree\u0026thinsp;=\u0026thinsp;101), 2,4-Dihydroxy-6-Methoxychalcone (degree\u0026thinsp;=\u0026thinsp;101), kaempferol (degree\u0026thinsp;=\u0026thinsp;99), beta-sitosterol (degree\u0026thinsp;=\u0026thinsp;90), Sandaracopimaric Acid (degree\u0026thinsp;=\u0026thinsp;85). It indicates that these compounds serve a critical therapeutic role in EMS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter deleting repeated genes, 571 putative targets for 7 herbs and 1056 putative endometriosis related targets were screened out. There were 157 common targets between herbal compounds and EMS filtered as the vital targets for further testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. Hub genes against EMS in the PPI Network\u003c/h2\u003e \u003cp\u003eTo explore the key hub genes of these 7 herbal medicine as treatment against EMS, the PPI network was constructed by the STRING database and visualized by using Cytoscape. There were 157 nodes and 1980 edges in the PPI network (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The degree centrality (DC), betweenness centrality (BC), and the average closeness centrality (CC) were calculated to help detect the key hub genes. There were 8 targets whose DC, BC, and CC values were all among the top 10 highest: AKT1, IL-6, TNF, ESR1, TP53, VEGFA, EGFR and SRC, indicating that the 7 herbal medicine might exert its pharmacological effects mainly through these targets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3. Enrichment analysis\u003c/h2\u003e \u003cp\u003eIn order to further study the identified candidate target genes on EMS, the 157 common targets were uploaded to the DAVID database to perform GO enrichment analysis and KEGG pathway enrichment analysis. GO enrichment analysis were conducted from three sides: biological process (BP), molecular function (MF) and cell composition (CC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In the BP group, the first five items were \u0026ldquo;signal transduction\u0026rdquo;, \u0026ldquo;negative regulation of apoptotic process\u0026rdquo;, \u0026ldquo;protein phosphorylation\u0026rdquo;, \u0026ldquo;positive regulation of gene expression\u0026rdquo; and \u0026ldquo;negative regulation of gene expression\u0026rdquo;. In the aspect of CC, the targets were primarily enriched in \u0026ldquo;cytoplasm\u0026rdquo;, \u0026ldquo;cytosol\u0026rdquo;, \u0026ldquo;plasma membrane\u0026rdquo;, \u0026ldquo;integral component of plasma membrane\u0026rdquo; and \u0026ldquo;cell surface\u0026rdquo;. In the MF group, the top five items were \u0026ldquo;identical protein binding\u0026rdquo;, \u0026ldquo;ATP binding\u0026rdquo;, \u0026ldquo;protein serine/threonine/tyrosine kinase activity\u0026rdquo;, \u0026ldquo;protein kinase activity\u0026rdquo; and \u0026ldquo;enzyme binding\u0026rdquo;. Based on the results of pathway enrichment, the remarkable pathways were as follows: PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, Endocrine resistance, AGE-RAGE signaling pathway, ErbB signaling pathway, VEGF signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. In vitro experiment identification\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Cell proliferation after kaempferol treatment\u003c/h2\u003e \u003cp\u003eWe performed the CCK-8 assay to detect the response of the hEM15A cells to kaempferol treatment. Results indicated that, after treated for 48h, when the concentration of kaempferol is higher than 40 \u0026micro;M, the viability of hEM15A cells were markedly decreased compared with DMSO group. However, when treated only for 24 hours, kaempferol had no significant effect on the cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Cell apoptosis after kaempferol treatment\u003c/h2\u003e \u003cp\u003eTo determine whether the underlying mechanism of kaempferol suppressed cell proliferation was concerned with apoptosis, we used an Annexin V\u0026ndash;PE/7-AAD assay to investigate the apoptotic rate of hEM15A cells. The results showed that the cells apoptotic rate of the kaempferol treatment group (40 \u0026micro;M) was obviously higher than the control group after treated for 48h (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe specific mechanism of endometriosis is still incompletely understood up to now, but endometriosis is always considered as a chronic, estrogen-dependent, inflammatory disease [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Endometriosis can lead to a series of symptoms such as dysmenorrhea, pelvic pain, dyspareunia and infertility, and for women have no fertility requirement, reliefing the pelvic pain is their most urgent demands [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. TCM is commonly used as a complementary and alternative treatment in the treatment of EMs, especially to relieve endometriosis-associated pain [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Shixiao San is one of the famous analgesic and Huoxue Huayu Chinese herbal formulas and it has been widely used to treat gynecological disorders [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. JSX is a new formula originated from classic Shixiao San formula. In our previous clinical application, we have achieved a good result in relieving dysmenorrhea, but the detailed mechanism of JSX on endometriosis is currently unknown. In this study, the network pharmacology approach was applied to illustrate the synergistic molecular mechanism of JSX in treating endometriosis.\u003c/p\u003e \u003cp\u003eIn the present study, we identified 46 active compounds and 571 targets of JSX, 1056 targets of endometriosis, and our Venn diagram indicated that JSX and endometriosis had 157 common targets. Furthermore, the degrees for all of the 157 common targets were calculated using the Cytoscape software. The PPI network indicated that AKT1, IL-6, TNF, ESR1, TP53, VEGFA, EGFR and SRC may play key roles in the effect of JSX against endometriosis. The results of the GO enrichment analysis indicated that JSX might treat endometriosis via regulating cell apoptotic process and protein phosphorylation. Combined with the results of the top genes in the PPI network, KEGG pathway enrichment analysis manifested that JSX might treat endometriosis through PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, Endocrine resistance, AGE-RAGE signaling pathway, ErbB signaling pathway and VEGF signaling pathway.\u003c/p\u003e \u003cp\u003eProtein kinase B (AKT), a versatile serine\u0026ndash;threonine kinase, emerging as one of the most critical regulators in many signaling pathways, helps to maintain the normal physiological cell functions, such as cell cycle control, apoptosis, differentiation and metabolic processes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In mammals, there are three Akt isozymes AKT1, AKT2, and AKT3 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. AKT1 is a crucial regulatory kinase that transmits signals via the PI3K/AKT cell-signaling cascade, which regulates cell growth and survival [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. AKT1 is elevated phosphorylation and overactive in most human tumors and is linked to poor clinical prognosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In endometriosis, compared to control endometrium, AKT1 phosphorylation was highly elevated in minimal-mild endometriosis, while moderate in later stages [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A large amount of clinical trials have focused on inhibiting or reducing AKT1 hyper-activity as a promising treatment for cancer therapies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, whether targeting the AKT1 pathway can help to treat endometriosis is still need further research. IL-6, tumor necrotic factor alpha (TNFα) and vascular endothelial growth factor (VEGF) are pro-inflammatory cytokines which have crucial role in the pathogenesis and pain of endometriosis, and are early diagnostic markers and promising treatment targets for endometriosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. IL6, mainly secreted from macrophages and endometriotic cells, is considered to be the promoter of many biological activities including pro- and anti-inflammatory, making IL-6 a tool to relieve the pelvic pain of endometriosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Serum and peritoneal fluid levels of interleukin-6 were higher in the endometriosis patients and were correlated with the stage of endometriosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe herbs-compounds-genes network was established to search for the active ingredints. The results showed that sanqi and yiyiren were the herbs related with most common ingredints. And 10 key compounds were obtained as follows: quercetin, pelargonidin, isorhamnetin, Mandenol, kaempferol-3-O-α-L-rhamnosyl(1\u0026rarr;2)-β-D-glucoside_qt, (2S)-5-Methoxy-6-Methylflavan-7-Ol, 2,4-Dihydroxy-6-Methoxychalcone, kaempferol, beta-sitosterol and Sandaracopimaric Acid. Quercetin and kaempferol are members of the general flavonoids, widely distributed in a variety of plants like Allium, Malus, Delphinium, and Camellia [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Many previous studies had already demonstrated that Quercetin has the ability to inhibit cell proliferation, induce cell apoptosis, anti-inflammation in endometriosis cell lines and autoimplanted mouse models [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, up to date, the association between kaempferol and endometriosis is still unknown.\u003c/p\u003e \u003cp\u003eIn the present study, we showed that, after the exposure of kaempferol, the proliferation rate of hEM15A cells was inhibited and the apoptosis was enhanced. Kaempferol has recently been reported to have anti-oxidant, and anti-inflammatory bioactive functions, and have protective effects on many organs such as liver, colon, ovary,and stomach by regulating the cell cycle arrest, proliferation, migration, and the tight junction proteins [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The consumption of kaempferol-rich food has an essential anticancer role in the developing of skin, liver, and colon cancer but do not cause apparent toxicity to normal cells, indicating that kaempferol could be developed as a high safety and low side efects anticancer agent [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Luo and his colleagues found that, in ovarian cancer cell lines, kaempferol could suppress cell proliferation and cause apoptosis, and inhibited Akt phosphorylation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In breast cancer cell lines, kaempferol also effectively inhibits the proliferation and induces apoptosis through downregulation of the expressions of cancer progression proteins and decrease the anti-inflammatory proteins level [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In Pancreatic cancer cell lines, appropriate doses (48\u0026micro;M) of kaempferol may potentiate the efficacy of Erlotinib in inhibiting cell proliferation and promoting cell apoptosis via synergistically inhibiting the expressions of p-EGFR and p-AKT [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Another study found that both quercetin and kaempferol may block the activation of AKT and decrease the phospho-AKT levels in 3T3-L1 Preadipocytes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Another researchers provided molecular evidence that kaempferol inhibited the invasion and migration of hepatocellular carcinoma cells by reducing the activity and protein expression of MMP-9 and suppressing the phosphorylation of the Akt expression [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In this study, the results was in good concordance with the analysis presented above, but whether kaempferol affected the proliferation and apoptosis through regulating phospho-AKT levels and PI3K-Akt signaling pathway should be studied further.\u003c/p\u003e \u003cp\u003eTo sum up, network pharmacology indicated that the mechanism of JSX in treating endometriosis was the result of multi-mechanism and multi-pathway synergy, might be through modulation of the inflammation and/or the endocrine system, and through PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, AGE-RAGE signaling pathway, ErbB signaling pathway and VEGF signaling pathway. Moreover, according to the experiment results, we speculate that the primary active ingredients of JSX, particularly kaempferol, may treat endometriosis by influencing cell viability and apoptosis. Nevertheless, more in vitro and in vivo experimental validation is needed to be conducted in-depth studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the self-raised project of Lishui Science and Technology Grant (2019SJZC28) and grants from Lishui Central Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChuchu Zhao, Guiying Zhang, Congcong Wu:\u0026nbsp;project development,\u0026nbsp;data\u0026nbsp;collection,\u0026nbsp;and manuscript writing.\u0026nbsp;Hongyou Zhou, Xiaomin Wu and Huifen Yang:\u0026nbsp;Project development, Data analysis, and Manuscript editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZondervan KT, Becker CM, Missmer SA. 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Environ Toxicol. 2021 Oct;36(10):1981-1989.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Jiawei Shixiao San, endometriosis, network pharmacology, kaempferol, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-5736127/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5736127/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eShixiao San, composed of Pollen Typha and Faeces Trogopterori, is a traditional Chinese medicine used for the the treatment of endometriosis. Jiawei Shixiao San is a modified formula derived from Shixiao San, adding with Sanguis Draconis, Lignum Sappan, Fritillariae thunbergii bulbus, Semen Coicis, and Radix Notoginseng. The purpose of this study was to elucidate the mechanism of Jiawei Shixiao San in combating endometriosis at a systemic level through network pharmacology and experimental verification.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe targets of Jiawei Shixiao San and endometriosis were retrieved from multiple databases. An herbs-compounds-genes network and a protein-protein interaction network were constructed to analyze target interactions. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted using the DAVID database. Finally, CCK-8 assays and flow cytometry analyses were performed to evaluate the anti-endometriosis effects of the active compounds.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 157 common targets associated with herbs and endometriosis were identified for analysis. The key targets included AKT1, IL-6, TNF, ESR1, TP53, VEGFA, EGFR, and SRC. The potential active compounds identified were quercetin, pelargonidin, isorhamnetin, and kaempferol. Enrichment analysis suggested that Jiawei Shixiao San may treat endometriosis through the PI3K-Akt signaling pathway, resistance to EGFR tyrosine kinase inhibitors, endocrine resistance, and the AGE-RAGE signaling pathway. Further experimental validation demonstrated that kaempferol could inhibit the proliferation of endometrial stromal cells and induce apoptosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn summary, we conducted network pharmacology analysis combined with experimental verification to provide compelling evidence that Jiawei Shixiao San may treat endometriosis by regulating cell proliferation and apoptosis, as well as by modifying inflammation-related genes and signaling pathways.\u003c/p\u003e","manuscriptTitle":"Exploration of the Underlying Mechanism of Jiawei Shixiao San in the Treatment of Endometriosis Using Network Pharmacology and Experimental Verification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-01 07:37:42","doi":"10.21203/rs.3.rs-5736127/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fec34753-8287-400d-95d8-a1d909afc3e4","owner":[],"postedDate":"January 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-03T15:53:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-01 07:37:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5736127","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5736127","identity":"rs-5736127","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}