Curcumin Mitigates Polycystic Ovary Syndrome in Mice by Suppressing TLR4/MyD88/NF-κB Signaling Pathway Activation and Reducing Intestinal Mucosal Permeability

Curcumin Mitigates Polycystic Ovary Syndrome in Mice by Suppressing TLR4/MyD88/NF-κB Signaling Pathway Activation and Reducing Intestinal Mucosal Permeability | 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 Article Curcumin Mitigates Polycystic Ovary Syndrome in Mice by Suppressing TLR4/MyD88/NF-κB Signaling Pathway Activation and Reducing Intestinal Mucosal Permeability zhen Wang, Qin Yang, Qin Wan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4625677/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Polycystic ovary syndrome (PCOS) stands as a prevalent endocrine-metabolic disorder impairing female fertility, often intertwined with alterations in intestinal mucosal barrier integrity and chronic inflammation. Curcumin, renowned for its multifaceted biological activities including anti-inflammatory, anti-tumor, and antioxidant properties, emerges as a potential therapeutic agent for PCOS. In this study, we investigated the effects and underlying mechanisms of curcumin in mitigating PCOS in mice. The PCOS mouse model was induced by administering dehydroepiandrosterone (DHEA) dissolved in soybean oil (6mg/100g BW) for 20 days, followed by a 45-day intervention period with curcumin. Subsequent euthanasia enabled examination of relevant indicators. Evaluation encompassed ovarian and colonic morphology, intestinal mucosal barrier function, and the TLR4/MyD88/NF-κB signaling pathway in each experimental group. Our findings indicate that curcumin holds promise in alleviating PCOS symptoms. By inhibiting the activation of the TLR4/MyD88/NF-κB signaling pathway and reducing intestinal mucosal permeability, curcumin demonstrates potential as a clinical intervention for this disorder. These findings highlight the therapeutic potential of curcumin in managing PCOS and warrant further clinical investigation to validate its efficacy. Biological sciences/Immunology Biological sciences/Molecular biology Biological sciences/Plant sciences Curcumin PCOS TLR4/MyD88/NF-κB intestinal mucosal permeability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Polycystic ovary syndrome (PCOS) stands out as one of the prevalent endocrine-metabolic disorders significantly impacting female fertility, exhibiting a reported global prevalence ranging from 4–21% 1 . Characterized by ovulation dysfunction and hyperandrogenism, PCOS patients commonly manifest various metabolic disturbances, including insulin resistance and hyperinsulinemia 2 , 3 . Moreover, PCOS is implicated in potential complications such as type 2 diabetes (T2DM), metabolic syndrome, cardiovascular diseases, endometrial cancer, and chronic inflammatory conditions, severely compromising reproductive health and quality of life 4 , 5 . Despite extensive research, the precise etiology of PCOS remains elusive, with a paucity of clinically effective pharmaceutical interventions, thus necessitating the exploration of novel therapeutic strategies. The pathogenesis of PCOS is multifaceted, believed to be influenced by both genetic predispositions and environmental factors 6 . Emerging evidence underscores the pivotal role of gut microbiota in the progression and pathogenesis of PCOS 7 . Dysbiosis of the intestinal microbiota can compromise intestinal mucosal integrity, leading to heightened intestinal mucosal permeability 8 , 9 , 10 . Notably, the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa B (NF-κB) pathway has been implicated in PCOS pathogenesis 11 , 12 . Activation of TLR4 by lipopolysaccharides (LPS) from Gram-negative gut bacteria triggers downstream MyD88-dependent NF-κB signaling, fostering a pro-inflammatory milieu 13 , 14 . Activated NF-κB orchestrates the release of pro-inflammatory cytokines, culminating in low-grade systemic and ovarian inflammation, perturbed follicular development, and eventual PCOS pathogenesis 12 , 15 , 16 . Curcumin, a polyphenolic lipophilic compound derived from turmeric, is reputed for its diverse biological activities, including anti-inflammatory, anti-tumor, and antioxidant properties 17 , 18 . Research indicates that curcumin holds promise in delaying the onset and progression of T2DM by ameliorating intestinal permeability, fortifying intestinal barrier function, diminishing circulating LPS levels, and mitigating systemic low-grade inflammation. Given the chronic inflammatory nature of PCOS, whether curcumin could impede PCOS progression by bolstering intestinal barrier function and quelling systemic low-grade inflammation warrants investigation 19 . In this study, leveraging a murine PCOS model, we sought to elucidate the effects and underlying mechanisms of curcumin on PCOS pathogenesis. Our findings hold potential for unraveling the intricate interplay between PCOS, intestinal mucosal barrier integrity, inflammation, and paving the way for novel clinical interventions. 2. Methods 2.1. Animal Experiments The experiments were approved by the Ethics Committee of Southwest Medical University (NO. KY2020031), and were reported in accordance with the ARRIVE guidelines. All methods were performed in accordance with relevant guidelines and regulations. In this experiment, 21-day-old female C57BL/6J mice were purchased from Chengdu Dossy Experimental Animals Co., Ltd., Chengdu, China, and kept in specialized cages with a controlled temperature (25 ± 1℃) in a 12-hour light/dark cycle room. All mice were randomly divided into 3 groups (15 mice/group): control group (CON), model group (MOD), and curcumin treatment group (CUR). Mice in the MOD group and the CUR group were injected subcutaneously with dehydroepiandrosterone (DHEA) (6mg/100g BW) dissolved in soybean oil for 20 days to induce the PCOS mode, while the CON group was injected daily with the same amount of soybean oil 20 . After 20 days, the CUR group received Curcumin (200 mg/kg) was dissolved in 1% carboxymethyl cellulose (CMC) for 45 days. At the same time, the CON group and the Mod group were administered equal dosage of 1% CMC as a control. After 45 days of intervention, mice in each group were anesthetized with ketamine (20 mg/kg) and xylazine (3 mg/kg), and the eyeball blood and related specimens were collected immediately for further analysis. 2.2. Vaginal smear From the first day of modeling to the last day of treatment, vaginal smears were collected at AM 9:00 every day, and the estrous cycle of all mice was evaluated by Wright-Giemsa staining microscopy. The estrous cycle of normal mice should cycle in the order of proestrus, estrus, postestrus and interestrus. The normal estrous cycle is generally 4–5 days 21 . If the duration of the estrus cycle is prolonged and the cycle is disordered, it is considered that there is estrous cycle disorder 22 . 2.3. Hematoxylin-eosin (HE) staining After the mice were sacrificed, the isolated ovary and colon tissues were immediately fixed in 4% paraformaldehyde, and then the tissues were dehydrated and embedded in paraffin to make paraffin sections. To evaluate ovarian and colon damage, sections were HE stained and morphological changes were observed using an Olympus light microscope (Melville, NY). For stained ovarian sections, the number of cystic follicle and corpora luteum was counted by two individuals blinded to the source of the sections. According to the previous literature, cystic follicles refer to follicles without oocytes, which mainly appear as large fluid-filled cysts with a thin granulosa cell layer and a thick theca cell layer 23 . 2.4. Insulin tolerance tests (ITT) An insulin tolerance tests (ITT) was performed immediately after 45 days of curcumin treatment. The basal blood glucose levels of mice in each group were measured after the mice were fasted for 6 hours. Then, intraperitoneal insulin (1 IU/kg body weight) was injected immediately, and blood glucose was measured 30, 60, and 90 minutes after insulin injection. GraphPad Prism software was used to calculate the total area under glucose response curve (AUC). 2.5. Enzyme linked immunosorbent assay In order to detect the levels of testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH) in plasma, as well as the levels of different inflammatory factors, including TNF-α (tumor necrosis factor-alpha, TNF-α), IL-6 (interleukin-6, IL-6), IL-10 and IL-17A in ovaries and plasma, ELISA was used according to the manufacturers' instructions (Jiangsu Enzyme Immune Industrial Co., Ltd, Yancheng, China). 2.6. Western blot analysis The total protein concentration of frozen ovarian tissue and colon tissue was determined using BCA protein assay kit. Total proteins were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was then blocked with 5% nonfat milk powder for 2h. Then, the PVDF membrane with primary antibody added was incubated at 4℃ overnight and washed three times with TBST. Primary antibodies include TLR-4, MyD88, NF-κB p65, occludin, and ZO-1. The first three primary antibodies were purchased from Wuhan Sanying Biotechnology Co., LTD., and the last two were purchased from Beijing Bioss Biotechnology Co., LTD. Subsequently, the PVDF membrane was incubated with the appropriate HRP-conjugated secondary antibody for 2h at room temperature and washed again with TBST three times. Chemiluminescence method was used for color development, GAPDH was used as the internal reference, and Image J software was used to analyze the band gray value. The blot of each protein was obtained using a gel imaging system. 2.7 Immunohistochemistry Paraffin slices were followed by antigen unmasking process and were treated with 3% H2O2. Subsequently, the tissues were covered with 10% bovine serum albumin for 60 min to block nonspecific binding. The slices were incubated with anti-occludin (13409-1-AP, Proteintech) and anti-ZO-1 (21773-1-AP, Proteintech) at 4°C overnight. The second day, the slices were incubated with corresponding secondary antibody (HRP labeled) at room temperature for 60 min. Finally, slices were stained with DAB and were evaluated under a light microscope at 200× magnification. 2.8. Plasma LPS analysis Limulus amebocyte lysate kit was used to detect the plasma LPS levels of mice in each group (Xiamen Bioendo Technology Co., Ltd, Xiamen, China). 50 µL of diluted plasma (1:4 diluted with endotoxin-free water) was dispensed to each well in a 96-well plate according to the manufacturer's instructions. Subsequently, 50 µL/ well of limulus amebocyte lysate reagent was added to each well. The plates supplemented with reagents were incubated for 30 min at 37°C. After incubation, 100 µL of chromogenic substrate warmed to 37 ° C was added to each well. Then, the plates were incubated again at 37°C for 6 min. The reaction was stopped by adding 100µL of 25% glacial acetic acid solution. Finally, the optical density of the microplate at 545 nm was measured with a reader (Thermo Scientific, USA). 2.9. Statistical analysis All experimental data were analyzed using GraphPad Prism software 6.01 (GraphPad Software Inc., CA, USA). The results were expressed as mean ± SEM. Based on the data conforming to a Gaussian distribution and variance equality, the significance difference between the two groups was analyzed by the independent t -test. Results of P < 0.05 were considered significant. 3. Results 3.1. Routine parameters of mice in diverse groups There were no significant differences in body weights among 3 groups at the beginning of the study. After 45 days of treatment, the body weight of mice in CUR group was significantly lower than that in MOD group ( P = 0.0001). This result indicated that curcumin was effective in reducing weight in patients with PCOS. T and LH levels were notably increased in MOD group compared with CON group (T, P = 0.0002; LH, P = 0.0019). Compared with the MOD group, T and LH levels were significantly lower in CUR (T, P = 0.0103; LH, P = 0.0228). Similarly, the LH/FSH ratio of MOD group was significantly higher than that of CON group ( P = 0.0072), and the LH/FSH ratio of CUR group was notably lower than that of MOD group ( P = 0.0384). FSH levels were significantly lower in MOD group compared with CON group ( P = 0.0214), and there were no differences between MOD and CUR groups. In addition, compared with the CON group, the E2 level in the MOD group was obviously lower ( P = 0.0140). CUR intervention promoted an increase in E2 level compared with MOD group ( P = 0.0498)(Table 1 ). Table 1 Routine parameters of mice with diverse groups in PCOS. Measurements CON MOD CUR Unpaired t tests a b Body Weight, g 20.78 ± 0.34 22.76 ± 0.29 21.73 ± 0.28 0.0001 0.0160 T, ng/ml 0.48 ± 0.06 1.30 ± 0.12 0.85 ± 0.06 0.0002 0.0103 FSH, mIU/ml 11.41 ± 1.97 5.34 ± 0.80 8.02 ± 0.94 0.0214 0.0622 LH, mIU/ml 5.95 ± 0.33 8.39 ± 0.42 6.72 ± 0.42 0.0019 0.0228 LH/FSH, 0.59 ± 0.11 1.75 ± 0.31 0.91 ± 0.15 0.0072 0.0384 E2, µmol/l 51.99 ± 1.25 41.64 ± 3.06 49.11 ± 1.07 0.0140 0.0498 a:CON vs. MOD, b:MOD vs. CUR 3.2. Curcumin intervention improved the estrous cycles of PCOS in mice. Wright-Giemsa staining was performed on vaginal smears to evaluate differences in estrous cycles of mice in each group (Fig. 1 ).The CON group showed a regular estrous cycle, lasting 4–5 days. However, as the modeling progressed, the estrous cycle of MOD group became disordered and stopped during estrus. Interestingly, after curcumin intervention, the estrous cycle of mice in the CUR group gradually returned to normal, suggesting that curcumin can improve the estrous cycle disorder of PCOS. 3.3. Curcumin ameliorated ovarian histopathological injury in PCOS. HE staining was used to assess the alteration of ovarian pathology of mice in each group. Compared with the CON group, the number of cystic follicles in the MOD group was notably increased ( P = 0.0040), but the corpus luteum was reduced or disappeared( P = 0.0248), indicating that the ovarian function of mice in the MOD group was impaired. Intriguingly, curcumin intervention reduced the number of cystic follicles ( P = 0.0179) and increased the formation of corpus luteum( P = 0.0442), indicating that curcumin helps improve the pathological changes in ovarian tissue (Fig. 2 ). 3.4. Curcumin improved the mucosal barrier of colon HE staining of colonic tissue in each group showed that the pathological injury of colon tissue in MOD group was obvious, manifested as colon mucosal erosion, disordered and sparse villus structure, and discontinuous brush border, while curcumin intervention attenuated mucosal erosion, villus structure disorder and brush border discontinuity, indicating that curcumin helps improve the pathological changes in colon tissue (Fig. 3 ). Compared with the CON group, colon tissue ZO-1 ( P = 0.0042) and occludin ( P = 0.0026) were significantly decreased in the MOD group, Meanwhile, the abnormal decrease in ZO-1 ( P = 0.0088) and Occludin ( P = 0.0369) in PCOS in the CUR group was increased, indicating that curcumin has the ability to increase the level of ZO-1 and Occludin in colonic tissue, thereby ameliorating the intestinal mucosal barrier (Fig. 3 ). 3.5Curcumin improved intestinal barrier function To determine the effects of curcumin on intestinal barrier integrity, expression of tight junction proteins such as occludin and ZO-1 were analyzed using immunohistochemistry. Compared with the CON group, occludin ( P < 0.001) and ZO-1 ( P < 0.001) were significantly decreased in the MOD group. Compared with the MOD group, occludin ( P = 0.0046) and ZO-1 ( P = 0.0197) expression were increased in the CUR group (Fig. 4 ). These results suggested that curcumin may improve intestinal barrier function by enhancing the expression of occludin and ZO-1 proteins. 3.6. Curcumin improved insulin resistance in PCOS According to area under the curve AUC of insulin tolerance tests, the Insulin sensitivity of the MOD group was significantly lower than that of the CON group (P = 0.0032). Compared with the MOD group, insulin sensitivity was increased in the CUR group ( P = 0.0134) (Fig. 5 ). 3.6. Curcumin reduced inflammation of plasma and ovary in PCOS. According to the results of experimental analysis, we found that the levels of IL-17A (plasma: P = 0.0066, ovary: P = 0.0189), IL-6 (plasma: P = 0.0177, ovary: P = 0.0163), and TNF-α (plasma: P = 0.0066, ovary: P = 0.0343) in plasma and ovary of the MOD group were significantly higher than those in the CON group, and the level of IL-10 was significantly lower. Notably, the levels of IL-17A (plasma: P = 0.0362, ovary: P = 0.0379), IL-6 (plasma: P = 0.0344, ovary: P = 0.0313), and TNF-α (plasma: P = 0.0070, ovary: P = 0.0398) were significantly decreased and the level of IL-10 was significantly increased after Curcumin intervention. In summary, curcumin reduced inflammation in the plasma and ovaries of PCOS patients by suppressing pro-inflammatory cytokines and enhancing anti-inflammatory IL-10 (Fig. 6 ). 3.7. Curcumin attenuated metabolic endotoxemia by decreasing LPS and TLR4/MyD88/NF-κB signaling pathway Plasma-translocated LPS derived from Gram-negative bacteria was detected with Limulus reagent. Compared with the CON group, plasma LPS in the MOD group was significantly increased ( P = 0.0033). However, plasma LPS was significantly lower in the CUR group compared with MOD ( P = 0.0110), indicating the ability of curcumin to attenuate endotoxemia in PCOS. In addition, the relative expression of TLR4, MyD88 and NF-κB p65 was significantly increased in the MOD group ( P = 0.0135, P = 0.0014, P = 0.0015) but reduced by curcumin administration ( P = 0.0360, P = 0.0040, P = 0.0046) (Fig. 6 ). 4. Discussion In this study, we explored the therapeutic efficacy of curcumin in PCOS treatment. By employing various conventional indicators such as pathological examination, weight loss, improvement of insulin resistance (IR), and normalization of abnormal hormone levels, we established that curcumin possesses the potential to ameliorate PCOS symptoms, consistent with prior research findings 24 , 25 , 26 , 27 , 28 , 29 . Our intervention demonstrated promising preventive and therapeutic effects, suggesting a viable avenue for PCOS management. Further investigations unveiled a potential correlation between the improvement of PCOS phenotype and the reduction of intestinal mucosal permeability, along with the inhibition of the TLR4/MyD88/NF-κB signaling pathway. Evidence from multiple studies has underscored the involvement of chronic inflammation in the etiology and progression of PCOS, characterized by elevated pro-inflammatory cytokines and reduced anti-inflammatory cytokines 30 , 32 . Notably, aberrant levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-17A in plasma and follicular fluid have been associated with poor oocyte quality and compromised reproductive outcomes 30 , 31 , 33 . Our study revealed that curcumin treatment significantly decreased the levels of these pro-inflammatory cytokines while concurrently increasing the levels of anti-inflammatory cytokine IL-10, suggesting its potential in attenuating ovarian inflammation by modulating cytokine profiles. Emerging research on the intestinal microbiota has implicated gut dysbiosis in PCOS pathogenesis, leading to compromised intestinal mucosal barrier integrity and heightened mucosal permeability 6 , 34 , 35 . This disruption allows the translocation of lipopolysaccharide (LPS), derived from Gram-negative bacteria, into the bloodstream, triggering systemic low-grade inflammation and subsequent ovarian inflammation 11 , 12 , 36 . Our findings demonstrated marked colonic tissue pathology in the PCOS model group, accompanied by decreased expression of tight junction proteins (ZO-1, Occludin) and elevated plasma LPS levels. Remarkably, curcumin intervention ameliorated colonic pathological damage, increased tight junction protein expression, and reduced plasma LPS levels, suggesting its potential in restoring intestinal mucosal barrier function and mitigating endotoxemia in PCOS. Activation of the TLR4/MyD88/NF-κB signaling pathway is pivotal in mediating inflammatory responses, particularly in PCOS 13 , 37 , 38 , where it exacerbates insulin resistance 15 , 16 . Our study demonstrated that curcumin intervention downregulated the expression levels of TLR4, MyD88, and NF-κB p65 in ovarian tissue, indicating its potential in attenuating the inflammatory cascade and improving insulin resistance in PCOS. Conclusion This study elucidates the role of intestinal mucosal barrier damage and systemic inflammatory response in PCOS pathophysiology, implicating the activation of the TLR4/MyD88/NF-κB signaling pathway. Curcumin emerges as a potential therapeutic intervention, capable of mitigating ovarian tissue damage, restoring intestinal mucosal integrity, reducing mucosal permeability, and alleviating systemic and ovarian inflammation by inhibiting the TLR4/MyD88/NF-κB signaling pathway. These findings underscore curcumin's potential as a promising intervention for the prevention and treatment of PCOS. Declarations Conflict of interest The authors declare no conflict of interest. Funding This study was supported by Key Research and Development Project of Leshan Science and Technology Bureau (No.21SZD109). Author Contribution WQ, and YQ designed and wrote the paper. YQ, and WZ performed research. All authors have read and approved the final manuscript. Data Availability Data is provided within the manuscript or supplementary information files. References Wang T, et al. Dietary alpha-Linolenic Acid-Rich Flaxseed Oil Exerts Beneficial Effects on Polycystic Ovary Syndrome Through Sex Steroid Hormones-Microbiota-Inflammation Axis in Rats. Front. Endocrinol. (Lausanne). 2020; 11:284. DOI: 10.3389/fendo.2020.00284 . Mirza FG, et al. Polycystic Ovarian Syndrome (PCOS): Does the Challenge End at Conception?. Int. J. Environ. Res. Public. 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Biochem. 2018; 47(4): 1497–508. DOI: 10.1159/000490866 . Makkar S, et al. Hyaluronic Acid Binding to TLR4 Promotes Proliferation and Blocks Apoptosis in Colon Cancer. Mol. Cancer. Ther. 2019;18(12): 2446–56. DOI: 10.1158/1535-7163.MCT-18-1225 . Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile1Fig.S1originalbandsofWesternblotforZO1andoccludinincolontissue.pdf Supplementaryfile2Fig.S2originalbandsofWesternblotforTLR4MyD88andNFBp65inovarytissues.pdf Cite Share Download PDF Status: Published Journal Publication published 02 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 20 Aug, 2024 Reviews received at journal 02 Aug, 2024 Reviews received at journal 30 Jul, 2024 Reviewers agreed at journal 23 Jul, 2024 Reviewers agreed at journal 23 Jul, 2024 Reviewers invited by journal 23 Jul, 2024 Editor assigned by journal 23 Jul, 2024 Editor invited by journal 23 Jul, 2024 Submission checks completed at journal 08 Jul, 2024 First submitted to journal 23 Jun, 2024 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-4625677","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":333607439,"identity":"2448e7a7-4ce7-4d9a-9659-b4bff144e2b1","order_by":0,"name":"zhen Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYFACxgYwZQBkPUioqCFNC7PBgzPHSLAMqIVN8mELM2GV8u2H2yR+7qi1N2fvPVaR2MDGwN/enYDf8DOJbZK9Z44zW/acS7uRuEOGQeLM2Q34tUgwtt3gbTvGZnAjx+xG4hk2oEgufi3yMxjbbv5tO8ZjcP+NWUFiGzNhLQw3GNtu87bVSBjc4DFjIEoL0C/tv2XbDhgYnMkxlkg4c4yHoF/k248/NnzbVmdvcPyM4ccfFTVy/O29BBwGAYfhLB5ilINAHbEKR8EoGAWjYCQCAPmYSxnNHK2GAAAAAElFTkSuQmCC","orcid":"","institution":"Leshan People’s Hospital","correspondingAuthor":true,"prefix":"","firstName":"zhen","middleName":"","lastName":"Wang","suffix":""},{"id":333607440,"identity":"497a644b-2476-4656-9b7b-eb99f095718d","order_by":1,"name":"Qin Yang","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Yang","suffix":""},{"id":333607441,"identity":"0e4487bc-1dba-4618-bf45-7a53fee788b5","order_by":2,"name":"Qin Wan","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Wan","suffix":""}],"badges":[],"createdAt":"2024-06-23 14:47:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4625677/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4625677/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-81034-5","type":"published","date":"2024-12-02T15:58:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61481534,"identity":"f5ad6165-421a-4df3-8e2e-f63984f0b9c6","added_by":"auto","created_at":"2024-07-31 08:55:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":685551,"visible":true,"origin":"","legend":"\u003cp\u003eEstrous cycle changes in each group of mice. (A) Vaginal smears of proestrus stage. (B) Vaginal smears of estrous stage. (C) Vaginal smears of metestrus stage. (D) Vaginal smears of diestrus stage. (E-G) Representative estrous cycles of diverse groups, 1: diestrus stage, 2: proestrus stage, 3: estrus stage, 4: metestrus stage. Original magnification (×100).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/ab6b823b4d41ad2d86e1743e.png"},{"id":61483134,"identity":"c0e13996-c608-4f67-96f2-8538e2f7a527","added_by":"auto","created_at":"2024-07-31 09:11:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":733394,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of curcumin on ovarian tissue morphology in PCOS with Hematoxylin-eosin (H\u0026amp;E) staining. A: CON, B: MOD, C: CUR. D: Changes in the number of cystic follicles. E: Changes in the number of corpus luteum. GCL: granular cell layer, L:luteum. Original magnification (40×).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/1f3df476ec0da008ced4dc31.png"},{"id":61481537,"identity":"4cffe674-f15d-467e-8738-136aa6ad1a33","added_by":"auto","created_at":"2024-07-31 08:55:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":493488,"visible":true,"origin":"","legend":"\u003cp\u003echanges of Pathology for colon tissue and intestinal mucosal barrier in each group. A: Colon tissue in CON group with Hematoxylin-eosin (H\u0026amp;E) staining, B: Colon tissue in MOD group with H\u0026amp;E staining, C: Colon tissue in CUR group with H\u0026amp;E staining, D: Western blot bands of ZO-1 and occludin in colonic tissues, E: occludin expression levels, F: ZO-1 expression levels. Original magnification (200×).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/aec73bc253aee6ae68fc3d32.png"},{"id":61482300,"identity":"ca422fcb-3411-4350-ba78-fae94d69334e","added_by":"auto","created_at":"2024-07-31 09:03:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":401782,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin enhanced the expression of occludin and ZO-1 proteins in each group. A: occludin was assessed in CON group by immunohistochemistry, B: occludin was assessed in MOD group by immunohistochemistry, C: occludin was assessed in CUR group by immunohistochemistry, D: ZO-1 was assessed in CON group by immunohistochemistry, E: ZO-1 was assessed in MOD group by immunohistochemistry, F: ZO-1 was assessed in CUR group by immunohistochemistry, G: occludin expression levels, H: ZO-1 expression levels. Original magnification (200×). * P＜0.05，** P＜0.01.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/811f0e7cc25e660d003929b0.png"},{"id":61481531,"identity":"98ad8770-c280-40de-b87a-5bc5d006a877","added_by":"auto","created_at":"2024-07-31 08:55:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":102841,"visible":true,"origin":"","legend":"\u003cp\u003eInsulin tolerance test. A. blood glucose levels, B. AUC of glucose. * \u003cem\u003eP\u003c/em\u003e＜0.05，**\u003cem\u003e P\u003c/em\u003e＜0.01.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/307a138be4874899920e50f2.png"},{"id":61483132,"identity":"e3ec7aa3-ae5a-445d-8324-6b88d58f4932","added_by":"auto","created_at":"2024-07-31 09:11:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":120332,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of plasma or ovarian inflammatory in diverse groups of mice. A-D:\u003c/p\u003e\n\u003cp\u003ePlasma in each group was collected respectively for detection of IL-17A (A), IL-6 (B), IL-10 (C), TNF-α (D). E-H: Ovary in each group was collected respectively for detection of IL-17A (E), IL-6 (F), IL-10 (G), TNF-α (H). I: Plasma LPS levels. J: Western blot bands of TLR-4, MyD88 and NF-κB p65 in ovary tissues. K: TLR4 expression levels. L: MyD88 expression levels. M: NF-κB p65 expression levels. Data are expressed as mean±SEM. * \u003cem\u003eP\u003c/em\u003e＜0.05，** \u003cem\u003eP\u003c/em\u003e＜0.01，*** \u003cem\u003eP\u003c/em\u003e＜0.001，**** \u003cem\u003eP\u003c/em\u003e＜0.0001，NS not significant.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/d3e190c7ebb3d8619a9454f3.png"},{"id":70964871,"identity":"3c75402f-cb3c-4961-b85f-53590f18646e","added_by":"auto","created_at":"2024-12-09 16:16:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3436284,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/3e85f7fc-cdf3-4015-b932-d57ed75614c5.pdf"},{"id":61482297,"identity":"31343711-9b90-4475-9e0b-bb3fe0253167","added_by":"auto","created_at":"2024-07-31 09:03:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":53819,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1Fig.S1originalbandsofWesternblotforZO1andoccludinincolontissue.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/1a1041cc60bb40c7b91cb998.pdf"},{"id":61483825,"identity":"262fd07f-7f64-4638-8e8e-2fdbca3bf488","added_by":"auto","created_at":"2024-07-31 09:19:44","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":85986,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2Fig.S2originalbandsofWesternblotforTLR4MyD88andNFBp65inovarytissues.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4625677/v1/300281cbc3836def735166d9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Curcumin Mitigates Polycystic Ovary Syndrome in Mice by Suppressing TLR4/MyD88/NF-κB Signaling Pathway Activation and Reducing Intestinal Mucosal Permeability","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePolycystic ovary syndrome (PCOS) stands out as one of the prevalent endocrine-metabolic disorders significantly impacting female fertility, exhibiting a reported global prevalence ranging from 4\u0026ndash;21%\u003csup\u003e1\u003c/sup\u003e. Characterized by ovulation dysfunction and hyperandrogenism, PCOS patients commonly manifest various metabolic disturbances, including insulin resistance and hyperinsulinemia\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Moreover, PCOS is implicated in potential complications such as type 2 diabetes (T2DM), metabolic syndrome, cardiovascular diseases, endometrial cancer, and chronic inflammatory conditions, severely compromising reproductive health and quality of life\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Despite extensive research, the precise etiology of PCOS remains elusive, with a paucity of clinically effective pharmaceutical interventions, thus necessitating the exploration of novel therapeutic strategies.\u003c/p\u003e \u003cp\u003eThe pathogenesis of PCOS is multifaceted, believed to be influenced by both genetic predispositions and environmental factors\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Emerging evidence underscores the pivotal role of gut microbiota in the progression and pathogenesis of PCOS\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Dysbiosis of the intestinal microbiota can compromise intestinal mucosal integrity, leading to heightened intestinal mucosal permeability\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Notably, the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa B (NF-κB) pathway has been implicated in PCOS pathogenesis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Activation of TLR4 by lipopolysaccharides (LPS) from Gram-negative gut bacteria triggers downstream MyD88-dependent NF-κB signaling, fostering a pro-inflammatory milieu\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Activated NF-κB orchestrates the release of pro-inflammatory cytokines, culminating in low-grade systemic and ovarian inflammation, perturbed follicular development, and eventual PCOS pathogenesis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurcumin, a polyphenolic lipophilic compound derived from turmeric, is reputed for its diverse biological activities, including anti-inflammatory, anti-tumor, and antioxidant properties\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Research indicates that curcumin holds promise in delaying the onset and progression of T2DM by ameliorating intestinal permeability, fortifying intestinal barrier function, diminishing circulating LPS levels, and mitigating systemic low-grade inflammation. Given the chronic inflammatory nature of PCOS, whether curcumin could impede PCOS progression by bolstering intestinal barrier function and quelling systemic low-grade inflammation warrants investigation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, leveraging a murine PCOS model, we sought to elucidate the effects and underlying mechanisms of curcumin on PCOS pathogenesis. Our findings hold potential for unraveling the intricate interplay between PCOS, intestinal mucosal barrier integrity, inflammation, and paving the way for novel clinical interventions.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animal Experiments\u003c/h2\u003e \u003cp\u003eThe experiments were approved by the Ethics Committee of Southwest Medical University (NO. KY2020031), and were reported in accordance with the ARRIVE guidelines. All methods were performed in accordance with relevant guidelines and regulations. In this experiment, 21-day-old female C57BL/6J mice were purchased from Chengdu Dossy Experimental Animals Co., Ltd., Chengdu, China, and kept in specialized cages with a controlled temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃) in a 12-hour light/dark cycle room.\u003c/p\u003e \u003cp\u003eAll mice were randomly divided into 3 groups (15 mice/group): control group (CON), model group (MOD), and curcumin treatment group (CUR). Mice in the MOD group and the CUR group were injected subcutaneously with dehydroepiandrosterone (DHEA) (6mg/100g BW) dissolved in soybean oil for 20 days to induce the PCOS mode, while the CON group was injected daily with the same amount of soybean oil \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter 20 days, the CUR group received Curcumin (200 mg/kg) was dissolved in 1% carboxymethyl cellulose (CMC) for 45 days. At the same time, the CON group and the Mod group were administered equal dosage of 1% CMC as a control. After 45 days of intervention, mice in each group were anesthetized with ketamine (20 mg/kg) and xylazine (3 mg/kg), and the eyeball blood and related specimens were collected immediately for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Vaginal smear\u003c/h2\u003e \u003cp\u003eFrom the first day of modeling to the last day of treatment, vaginal smears were collected at AM 9:00 every day, and the estrous cycle of all mice was evaluated by Wright-Giemsa staining microscopy. The estrous cycle of normal mice should cycle in the order of proestrus, estrus, postestrus and interestrus. The normal estrous cycle is generally 4\u0026ndash;5 days\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. If the duration of the estrus cycle is prolonged and the cycle is disordered, it is considered that there is estrous cycle disorder \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Hematoxylin-eosin (HE) staining\u003c/h2\u003e \u003cp\u003eAfter the mice were sacrificed, the isolated ovary and colon tissues were immediately fixed in 4% paraformaldehyde, and then the tissues were dehydrated and embedded in paraffin to make paraffin sections. To evaluate ovarian and colon damage, sections were HE stained and morphological changes were observed using an Olympus light microscope (Melville, NY). For stained ovarian sections, the number of cystic follicle and corpora luteum was counted by two individuals blinded to the source of the sections. According to the previous literature, cystic follicles refer to follicles without oocytes, which mainly appear as large fluid-filled cysts with a thin granulosa cell layer and a thick theca cell layer\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Insulin tolerance tests (ITT)\u003c/h2\u003e \u003cp\u003eAn insulin tolerance tests (ITT) was performed immediately after 45 days of curcumin treatment. The basal blood glucose levels of mice in each group were measured after the mice were fasted for 6 hours. Then, intraperitoneal insulin (1 IU/kg body weight) was injected immediately, and blood glucose was measured 30, 60, and 90 minutes after insulin injection. GraphPad Prism software was used to calculate the total area under glucose response curve (AUC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Enzyme linked immunosorbent assay\u003c/h2\u003e \u003cp\u003eIn order to detect the levels of testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH) in plasma, as well as the levels of different inflammatory factors, including TNF-α (tumor necrosis factor-alpha, TNF-α), IL-6 (interleukin-6, IL-6), IL-10 and IL-17A in ovaries and plasma, ELISA was used according to the manufacturers' instructions (Jiangsu Enzyme Immune Industrial Co., Ltd, Yancheng, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Western blot analysis\u003c/h2\u003e \u003cp\u003eThe total protein concentration of frozen ovarian tissue and colon tissue was determined using BCA protein assay kit. Total proteins were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was then blocked with 5% nonfat milk powder for 2h. Then, the PVDF membrane with primary antibody added was incubated at 4℃ overnight and washed three times with TBST. Primary antibodies include TLR-4, MyD88, NF-κB p65, occludin, and ZO-1. The first three primary antibodies were purchased from Wuhan Sanying Biotechnology Co., LTD., and the last two were purchased from Beijing Bioss Biotechnology Co., LTD. Subsequently, the PVDF membrane was incubated with the appropriate HRP-conjugated secondary antibody for 2h at room temperature and washed again with TBST three times. Chemiluminescence method was used for color development, GAPDH was used as the internal reference, and Image J software was used to analyze the band gray value. The blot of each protein was obtained using a gel imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin slices were followed by antigen unmasking process and were treated with 3% H2O2. Subsequently, the tissues were covered with 10% bovine serum albumin for 60 min to block nonspecific binding. The slices were incubated with anti-occludin (13409-1-AP, Proteintech) and anti-ZO-1 (21773-1-AP, Proteintech) at 4\u0026deg;C overnight. The second day, the slices were incubated with corresponding secondary antibody (HRP labeled) at room temperature for 60 min. Finally, slices were stained with DAB and were evaluated under a light microscope at 200\u0026times; magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Plasma LPS analysis\u003c/h2\u003e \u003cp\u003eLimulus amebocyte lysate kit was used to detect the plasma LPS levels of mice in each group (Xiamen Bioendo Technology Co., Ltd, Xiamen, China). 50 \u0026micro;L of diluted plasma (1:4 diluted with endotoxin-free water) was dispensed to each well in a 96-well plate according to the manufacturer's instructions. Subsequently, 50 \u0026micro;L/ well of limulus amebocyte lysate reagent was added to each well. The plates supplemented with reagents were incubated for 30 min at 37\u0026deg;C. After incubation, 100 \u0026micro;L of chromogenic substrate warmed to 37 \u0026deg; C was added to each well. Then, the plates were incubated again at 37\u0026deg;C for 6 min. The reaction was stopped by adding 100\u0026micro;L of 25% glacial acetic acid solution. Finally, the optical density of the microplate at 545 nm was measured with a reader (Thermo Scientific, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were analyzed using GraphPad Prism software 6.01 (GraphPad Software Inc., CA, USA). The results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Based on the data conforming to a Gaussian distribution and variance equality, the significance difference between the two groups was analyzed by the independent \u003cem\u003et\u003c/em\u003e-test. Results of \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Routine parameters of mice in diverse groups\u003c/h2\u003e \u003cp\u003eThere were no significant differences in body weights among 3 groups at the beginning of the study. After 45 days of treatment, the body weight of mice in CUR group was significantly lower than that in MOD group (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0001). This result indicated that curcumin was effective in reducing weight in patients with PCOS. T and LH levels were notably increased in MOD group compared with CON group (T, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0002; LH, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0019). Compared with the MOD group, T and LH levels were significantly lower in CUR (T, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0103; LH, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0228). Similarly, the LH/FSH ratio of MOD group was significantly higher than that of CON group (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0072), and the LH/FSH ratio of CUR group was notably lower than that of MOD group (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0384). FSH levels were significantly lower in MOD group compared with CON group (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0214), and there were no differences between MOD and CUR groups. In addition, compared with the CON group, the E2 level in the MOD group was obviously lower (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0140). CUR intervention promoted an increase in E2 level compared with MOD group (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0498)(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRoutine parameters of mice with diverse groups in PCOS.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMeasurements\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMOD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCUR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eUnpaired \u003cem\u003et\u003c/em\u003e tests\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody Weight, g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e22.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e21.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT, ng/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0103\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFSH, mIU/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0622\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLH, mIU/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0228\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLH/FSH,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0072\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0384\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE2, \u0026micro;mol/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e51.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e41.64\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e49.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0498\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003ea:CON vs. MOD, b:MOD vs. CUR\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Curcumin intervention improved the estrous cycles of PCOS in mice.\u003c/h2\u003e \u003cp\u003eWright-Giemsa staining was performed on vaginal smears to evaluate differences in estrous cycles of mice in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).The CON group showed a regular estrous cycle, lasting 4\u0026ndash;5 days. However, as the modeling progressed, the estrous cycle of MOD group became disordered and stopped during estrus. Interestingly, after curcumin intervention, the estrous cycle of mice in the CUR group gradually returned to normal, suggesting that curcumin can improve the estrous cycle disorder of PCOS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Curcumin ameliorated ovarian histopathological injury in PCOS.\u003c/h2\u003e \u003cp\u003eHE staining was used to assess the alteration of ovarian pathology of mice in each group. Compared with the CON group, the number of cystic follicles in the MOD group was notably increased (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0040), but the corpus luteum was reduced or disappeared(\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0248), indicating that the ovarian function of mice in the MOD group was impaired. Intriguingly, curcumin intervention reduced the number of cystic follicles (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0179) and increased the formation of corpus luteum(\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0442), indicating that curcumin helps improve the pathological changes in ovarian tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Curcumin improved the mucosal barrier of colon\u003c/h2\u003e \u003cp\u003eHE staining of colonic tissue in each group showed that the pathological injury of colon tissue in MOD group was obvious, manifested as colon mucosal erosion, disordered and sparse villus structure, and discontinuous brush border, while curcumin intervention attenuated mucosal erosion, villus structure disorder and brush border discontinuity, indicating that curcumin helps improve the pathological changes in colon tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with the CON group, colon tissue ZO-1 (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0042) and occludin (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0026) were significantly decreased in the MOD group, Meanwhile, the abnormal decrease in ZO-1 (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0088) and Occludin (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0369) in PCOS in the CUR group was increased, indicating that curcumin has the ability to increase the level of ZO-1 and Occludin in colonic tissue, thereby ameliorating the intestinal mucosal barrier (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5Curcumin improved intestinal barrier function\u003c/h2\u003e \u003cp\u003eTo determine the effects of curcumin on intestinal barrier integrity, expression of tight junction proteins such as occludin and ZO-1 were analyzed using immunohistochemistry. Compared with the CON group, occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and ZO-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were significantly decreased in the MOD group. Compared with the MOD group, occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0046) and ZO-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0197) expression were increased in the CUR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results suggested that curcumin may improve intestinal barrier function by enhancing the expression of occludin and ZO-1 proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Curcumin improved insulin resistance in PCOS\u003c/h2\u003e \u003cp\u003eAccording to area under the curve AUC of insulin tolerance tests, the Insulin sensitivity of the MOD group was significantly lower than that of the CON group \u003cem\u003e(P\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0032). Compared with the MOD group, insulin sensitivity was increased in the CUR group (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0134) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Curcumin reduced inflammation of plasma and ovary in PCOS.\u003c/h2\u003e \u003cp\u003eAccording to the results of experimental analysis, we found that the levels of IL-17A (plasma: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0066, ovary: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0189), IL-6 (plasma: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0177, ovary: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0163), and TNF-α (plasma: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0066, ovary: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0343) in plasma and ovary of the MOD group were significantly higher than those in the CON group, and the level of IL-10 was significantly lower. Notably, the levels of IL-17A (plasma: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0362, ovary: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0379), IL-6 (plasma: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0344, ovary: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0313), and TNF-α (plasma: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0070, ovary: \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0398) were significantly decreased and the level of IL-10 was significantly increased after Curcumin intervention. In summary, curcumin reduced inflammation in the plasma and ovaries of PCOS patients by suppressing pro-inflammatory cytokines and enhancing anti-inflammatory IL-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Curcumin attenuated metabolic endotoxemia by decreasing LPS and TLR4/MyD88/NF-κB signaling pathway\u003c/h2\u003e \u003cp\u003ePlasma-translocated LPS derived from Gram-negative bacteria was detected with Limulus reagent. Compared with the CON group, plasma LPS in the MOD group was significantly increased (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0033). However, plasma LPS was significantly lower in the CUR group compared with MOD (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0110), indicating the ability of curcumin to attenuate endotoxemia in PCOS. In addition, the relative expression of TLR4, MyD88 and NF-κB p65 was significantly increased in the MOD group (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0135, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0014, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0015) but reduced by curcumin administration (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0360, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0040, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0046) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we explored the therapeutic efficacy of curcumin in PCOS treatment. By employing various conventional indicators such as pathological examination, weight loss, improvement of insulin resistance (IR), and normalization of abnormal hormone levels, we established that curcumin possesses the potential to ameliorate PCOS symptoms, consistent with prior research findings\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Our intervention demonstrated promising preventive and therapeutic effects, suggesting a viable avenue for PCOS management. Further investigations unveiled a potential correlation between the improvement of PCOS phenotype and the reduction of intestinal mucosal permeability, along with the inhibition of the TLR4/MyD88/NF-κB signaling pathway.\u003c/p\u003e \u003cp\u003eEvidence from multiple studies has underscored the involvement of chronic inflammation in the etiology and progression of PCOS, characterized by elevated pro-inflammatory cytokines and reduced anti-inflammatory cytokines\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Notably, aberrant levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-17A in plasma and follicular fluid have been associated with poor oocyte quality and compromised reproductive outcomes\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our study revealed that curcumin treatment significantly decreased the levels of these pro-inflammatory cytokines while concurrently increasing the levels of anti-inflammatory cytokine IL-10, suggesting its potential in attenuating ovarian inflammation by modulating cytokine profiles.\u003c/p\u003e \u003cp\u003eEmerging research on the intestinal microbiota has implicated gut dysbiosis in PCOS pathogenesis, leading to compromised intestinal mucosal barrier integrity and heightened mucosal permeability \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This disruption allows the translocation of lipopolysaccharide (LPS), derived from Gram-negative bacteria, into the bloodstream, triggering systemic low-grade inflammation and subsequent ovarian inflammation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our findings demonstrated marked colonic tissue pathology in the PCOS model group, accompanied by decreased expression of tight junction proteins (ZO-1, Occludin) and elevated plasma LPS levels. Remarkably, curcumin intervention ameliorated colonic pathological damage, increased tight junction protein expression, and reduced plasma LPS levels, suggesting its potential in restoring intestinal mucosal barrier function and mitigating endotoxemia in PCOS.\u003c/p\u003e \u003cp\u003eActivation of the TLR4/MyD88/NF-κB signaling pathway is pivotal in mediating inflammatory responses, particularly in PCOS\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, where it exacerbates insulin resistance\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Our study demonstrated that curcumin intervention downregulated the expression levels of TLR4, MyD88, and NF-κB p65 in ovarian tissue, indicating its potential in attenuating the inflammatory cascade and improving insulin resistance in PCOS.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThis study elucidates the role of intestinal mucosal barrier damage and systemic inflammatory response in PCOS pathophysiology, implicating the activation of the TLR4/MyD88/NF-κB signaling pathway. Curcumin emerges as a potential therapeutic intervention, capable of mitigating ovarian tissue damage, restoring intestinal mucosal integrity, reducing mucosal permeability, and alleviating systemic and ovarian inflammation by inhibiting the TLR4/MyD88/NF-κB signaling pathway. These findings underscore curcumin's potential as a promising intervention for the prevention and treatment of PCOS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by Key Research and Development Project of Leshan Science and Technology Bureau (No.21SZD109).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWQ, and YQ designed and wrote the paper. YQ, and WZ performed research. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang T, et al. 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DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1535-7163.MCT-18-1225\u003c/span\u003e\u003cspan address=\"10.1158/1535-7163.MCT-18-1225\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Curcumin, PCOS, TLR4/MyD88/NF-κB, intestinal mucosal permeability","lastPublishedDoi":"10.21203/rs.3.rs-4625677/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4625677/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolycystic ovary syndrome (PCOS) stands as a prevalent endocrine-metabolic disorder impairing female fertility, often intertwined with alterations in intestinal mucosal barrier integrity and chronic inflammation. Curcumin, renowned for its multifaceted biological activities including anti-inflammatory, anti-tumor, and antioxidant properties, emerges as a potential therapeutic agent for PCOS. In this study, we investigated the effects and underlying mechanisms of curcumin in mitigating PCOS in mice. The PCOS mouse model was induced by administering dehydroepiandrosterone (DHEA) dissolved in soybean oil (6mg/100g BW) for 20 days, followed by a 45-day intervention period with curcumin. Subsequent euthanasia enabled examination of relevant indicators. Evaluation encompassed ovarian and colonic morphology, intestinal mucosal barrier function, and the TLR4/MyD88/NF-κB signaling pathway in each experimental group. Our findings indicate that curcumin holds promise in alleviating PCOS symptoms. By inhibiting the activation of the TLR4/MyD88/NF-κB signaling pathway and reducing intestinal mucosal permeability, curcumin demonstrates potential as a clinical intervention for this disorder. These findings highlight the therapeutic potential of curcumin in managing PCOS and warrant further clinical investigation to validate its efficacy.\u003c/p\u003e","manuscriptTitle":"Curcumin Mitigates Polycystic Ovary Syndrome in Mice by Suppressing TLR4/MyD88/NF-κB Signaling Pathway Activation and Reducing Intestinal Mucosal Permeability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 08:55:39","doi":"10.21203/rs.3.rs-4625677/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-20T08:52:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-02T09:01:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-30T08:21:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136062749053949930391892134674061914696","date":"2024-07-23T18:08:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115552447380499291417844429087558597496","date":"2024-07-23T12:12:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-23T12:00:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-23T07:05:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-23T05:20:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-08T05:16:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-23T14:45:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7840e9e1-2d56-4136-952c-2c8f81c4feeb","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35325908,"name":"Biological sciences/Immunology"},{"id":35325909,"name":"Biological sciences/Molecular biology"},{"id":35325910,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2024-12-09T16:06:03+00:00","versionOfRecord":{"articleIdentity":"rs-4625677","link":"https://doi.org/10.1038/s41598-024-81034-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-12-02 15:58:03","publishedOnDateReadable":"December 2nd, 2024"},"versionCreatedAt":"2024-07-31 08:55:39","video":"","vorDoi":"10.1038/s41598-024-81034-5","vorDoiUrl":"https://doi.org/10.1038/s41598-024-81034-5","workflowStages":[]},"version":"v1","identity":"rs-4625677","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4625677","identity":"rs-4625677","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}