Exploring the efficacy and mechanism of Bailing capsule to improve polycystic ovary syndrome in mice based on intestinal-derived LPS-TLR4 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with reproductive dysfunction and metabolic abnormalities, particularly characterized by insulin resistance and chronic low-grade inflammation. Multiple clinical studies have clearly demonstrated the significant efficacy and safety of the combination of Bailing capsules (BL) in the treatment of PCOS, but its pharmacological effects and mechanisms still require further study. AIM OF THE STUDY: To evaluate the effect of BL on improving PCOS in mice and explore the mechanism. METHODS: In this study, Dehydroepiandrosterone (DHEA) injection was administered alone and in combination with a high-fat and high-sugar diet to induce PCOS-like mouse. They were randomly divided into five groups: normal group (N), PCOS group (P), Bailing capsule low-dose group (BL-L), Bailing capsule high-dose group (BL-H) and Metformin + Daine-35 group (M + D). Firstly, the effects of BL on ovarian lesions, serum hormone levels, HOMA-IR, intestinal barrier function, inflammation levels, along with the expression of IRS1, PI3K, AKT, TLR4, Myd88, NF-κB p65, TNF-α, IL-6, and Occludin of the ovary, liver and colon were investigated. Finally, the composition of the gut microbiome of fecal was tested. RESULTS: The administration of BL significantly reduced body weight, improved hormone levels, improved IR, and attenuated pathological damage to ovarian tissues, up-regulated the expression of IRS1, PI3K, and AKT in liver. It also decreased serum LPS, TNF-α, and IL-6 levels, while downregulating the expression of Myd88, TLR4, and NF-κB p65. Additionally, BL improved intestinal barrier damage and upregulated the expression of Occludin. Interestingly, the abundance of norank_f__Muribaculacea and Lactobacillus was down-regulated, while the abundance of Akkermansia was significantly up-regulated. CONCLUSION: The results of the study showed that BL exerts a treatment PCOS effect, which may be related to the modulation of the gut microbiota, the improvement of insulin resistance and the intestinal-derived LPS-TLR4 inflammatory pathway. Our research will provide a theoretical basis for the clinical treatment of PCOS.

Spirulina versus metformin for controlling some insulin signaling pathway genes in induced polycystic ovary syndrome rat model.

Polycystic ovary syndrome (PCOS) is a prevalent endocrinologic and gynecologic disorder that affects women of reproductive age; besides, insulin resistance (IR) occurs in 50-70 % of PCOS cases. Metformin (Met) is commonly prescribed for IR management; however, it does not affect IR with some gastrointestinal symptoms. Spirulina platensis (SP) is a blue-green alga that may increase insulin sensitivity. Therefore, our study aims to evaluate SP as an alternative treatment to Met for improving glucose homeostasis by assessing the expression of 11 crucial genes involved in the insulin signaling pathway. After induction of the PCOS model using dehydroepiandrosterone (DHEA) (60 mg/kg bwt) for 30 consecutive days, rats were allocated into six groups. Relative liver weight, glutamic pyruvic transaminase (GPT) serum levels, glutamic-oxaloacetic transaminase (GOT), and insulin were determined. Furthermore, the gene expression of Ins1, Irs1, Pik3ca, Prkcz, Foxo1, Srebf1, Ppargc1a, Pklr, Gk, G6pc, and Pepck in the rat's liver tissue was determined using qRT-PCR. Treatment of the PCOS control group with Met or SP revealed a decrease in all these parameters compared with the PCOS model. Additionally, we found a statistically significant difference in the expression of both the Gk and Prkcz genes. To summarize our study results, SP or Met supplementation to PCOS rats had almost the same effect on assessed relative liver weight, GOT, GPT, and insulin levels compared with PCOS control rats. If further studies confirm and detect more impact of SP on IR in PCOS, SP could be used instead of Met since the latter causes many side effects.

Ghrelin Alleviates Inflammation, Insulin Resistance, and Reproductive Abnormalities in Mice with Polycystic Ovary Syndrome via the TLR4-NF-κB Signaling Pathway.

BACKGROUND: Polycystic ovary syndrome (PCOS) commonly impacts fertile females with potentially severe effects on fertility and metabolism. Blood ghrelin levels are lower in PCOS patients, and exogenous supplements have been proposed for their potential to trigger anti-inflammatory effects at the cellular level. This study aimed to investigate whether pretreatment with ghrelin reduced inflammation, insulin resistance, and reproductive abnormalities in PCOS and the underlying mechanism of this disorder. METHODS: Ghrelin supplementation was first tested in an inflammation model using human ovarian granulosa cells (KGN cells) that were built by treated with Lipolyaccharide. KGN cells were pretreated with ghrelin and exposed to lipopolysaccharide (LPS). Inflammatory gene expression and cytokine production were analyzed by Enzyme-linked immunosorbent assay (ELISA). Based on these results, the PCOS mice model was built with Dehydroepiandrosterone (DHEA) and a high-fat diet. The mRNA and protein expressions of inflammatory factors including Toll-like receptor 4 (TLR4), nuclear factor kappa-B-p65 (NF-κB-p65), Phospho-NF-κB-p65 (p-NF-κB-p65) and myeloid differentiation factor 88 (MYD88) related to the TLR4/NF-κB signaling pathway were evaluated in KGN cells and mouse ovarian tissues using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and western blot, respectively. Lipid metabolism was quantified via an automated biochemical analyzer. RESULTS: The mRNA and protein expressions of interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and tumor necrosis factor alpha (TNF-α) in ghrelin pretreated KGN cells were lower than the LPS group (p < 0.05). Protein expression was reduced for TLR4, NF-κB-p65, and MYD88 within KGN cells of ghrelin groups compared to the LPS group (p < 0.05). Ghrelin treatment restored the estrous cycle and slowed weight gain and abdominal fat weight of PCOS mice (p < 0.05). Ghrelin treatment decreased the serum concentrations of testosterone, luteinizing hormone, insulin, IL-6, IL-1ß, and TNF-α compared to the PCOS group (p < 0.05). Estradiol concentrations of mice treated with ghrelin were higher than the PCOS group (p < 0.05). The concentrations of low and high-density lipoprotein, triglyceride, and cholesterol in mice treated with ghrelin were lower than in the PCOS mice (p < 0.05). Inflammatory gene expression for IL-6, IL-1ß, TNF-α, TLR4, NF-κB-p65, and MYD88 decreased in the ovarian tissues of ghrelin-treated mice compared to the PCOS group (p < 0.05), along with reduced protein expression of TLR4, p-NF-κB-p65, and MYD88 (p < 0.05). CONCLUSIONS: In the present study, ghrelin treatment effectively reduced inflammation in vitro, and attenuated insulin resistance and reproductive abnormalities in PCOS mice through the TLR4/NF-κB signaling pathway, highlighting potential therapeutic avenues for future PCOS treatments and research directions.

Apelin receptor modulation mitigates letrozole-induced polycystic ovarian pathogenesis in mice.

AIMS: Polycystic ovarian syndrome (PCOS) is one of the most common (about 5-20%) reproductive disorders in women of reproductive age; it is characterized by polycystic ovaries, hyperandrogenism, and oligo/ anovulation. The levels and expression of ovarian adipokines are deregulated in the PCOS. Apelin is an adipokine that acts through its receptor (APJ) and is known to express in the various tissues including the ovary. It has also been suggested that apelin and APJ could be targeted as therapeutic adjuncts for the management of PCOS. However, no study has been conducted on the management of PCOS by targeting the apelin system. Thus, we aimed to evaluate its impact on combating PCOS-associated ovarian pathogenesis. METHODS: The current work employed a letrozole-induced-hyperandrogenism PCOS-like mice model to investigate the effects of apelin13 and APJ, antagonist ML221. The PCOS model was induced by oral administration of letrozole (1 mg/kg) for 21 days. A total of four experimental groups were made, control, PCOS control, PCOS + aplein13, and PCOS + ML221. The treatment of apelin13 and ML221 was given from day 22 for two weeks. KEY FINDINGS: The letrozole-induced PCOS-like features such as hyperandrogenism, cystic follicle, decreased corpus luteum, elevated levels of LH/FSH ratio, and up-regulation of ovarian AR expression were ameliorated by apelin13 and ML221 treatment. However, the PCOS-augmented oxidative stress and apoptosis were suppressed by apelin 13 treatments only. ML221 treatment still showed elevated oxidative stress and stimulated apoptosis as reflected by decreased antioxidant enzymes and increased active caspase3 and Bax expression. The expression of ERs was elevated in all groups except control. Furthermore, the PCOS model showed elevated expression of APJ and apelin13 treatment down-regulated its own receptor. Overall, observing the ovarian histology, corpus luteum formation, and decreased androgen levels by both apelin13 and ML221 showed ameliorative effects on the cystic ovary. SIGNIFICANCE: Despite the similar morphological observation of ovarian histology, apelin13 and ML221 exhibited opposite effects on oxidative stress and apoptosis. Therefore, apelin13 (which down-regulates APJ) and ML221 (an APJ antagonist) may have suppressed APJ signalling, which would account for our findings on the mitigation of polycystic ovarian syndrome. In conclusion, both apelin13 and ML221 mediated mitigation have different mechanisms, which need further investigation.

Acetate ameliorates ovarian mitochondrial dysfunction in letrozole-induced polycystic ovarian syndrome rat model by improving mitofusin-2.

Androgen excess and metabolic abnormality largely contribute to the pathogenesis of polycystic ovarian syndrome (PCOS), which primarily precipitates ovarian dysfunction and infertility in reproductive-age women. Impaired mitochondrial function and epigenetic alteration have been linked to the development of PCOS. However, it is unknown whether acetate would exert a therapeutic effect on ovarian mitochondrial dysfunction in PCOS. Herein, the study hypothesized that acetate reverses ovarian mitochondrial dysfunction in experimental PCOS rat model, possibly through modulation of mitofusin-2 (MFn2). Eight-week-old female Wistar rats were randomized into four groups (n = 5). Induction of PCOS was performed by 1 mg/kg letrozole (p.o.), administered for 21 days. Thereafter, the rats were treated with acetate (200 mg/kg; p.o.) for 6 weeks. The PCOS rats demonstrated androgen excess, multiple ovarian cysts, elevated anti-mullerian hormone and leptin and decreased SHBG, adiponectin and 17-ß estradiol with corresponding increase in ovarian transforming growth factor-ß1. Additionally, inflammation (tumor growth factor and nuclear factor-kB), elevated caspase-6, decreased hypoxia-inducible factor-1α and elevated histone deacetylase-2 (HDAC2) were observed in the ovaries of PCOS rats, while mitochondrial abnormality with evidence of decreased adenosine triphosphate synthase and MFn2 was observed in rats with PCOS. Treatment with acetate reversed the alterations. The present results collectively suggest that acetate ameliorates ovarian mitochondrial abnormality, a beneficial effect that is accompanied by MFn2 with consequent normalization of reproductive-endocrine profile and ovarian function. Perhaps, the present data provide hope for PCOS individuals that suffer infertility.

Mitigation of letrozole induced polycystic ovarian syndrome associated inflammatory response and endocrinal dysfunction by Vitex negundo seeds.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder in women that necessitates effective and safe treatment alternatives. This study aimed to evaluate the therapeutic efficacy of Vitex negundo seed in a letrozole-induced PCOS rat model. RESULTS: Findings of the present study demonstrated that administration of hydro-ethanolic extract of Vitex negundo (VNE) effectively restored endocrino-metabolic imbalances associated with PCOS, along with correction of antioxidant enzymes level, proinflammatory cytokines, and apoptotic bio-markers. LC-MS analysis confirmed the presence of cinnamic acid, plumbagin and nigundin B as the prominent phytochemicals in VNE. The observed beneficial effects could be attributed to the active compounds in Vitex negundo extract, which exhibited hypoglycemic, hypolipidemic, and catabolic effects on body weight. Additionally, the extract contributed to hormonal balance regulation by modulating the steroidogenic enzymes, specifically by tuning gonadotropins level and correcting the LH:FSH ratio, through the modulation of ERα signalling and downregulation of NR3C4 expression. The antioxidant properties of phytochemicals in Vitex negundo seed were apparent through the correction of SOD and catalase activity. While it's anti-inflammatory and antiapoptotic action were associated with the regulation of mRNA expression of TNF-α, IL-6, BAX, Bcl2. Molecular docking study further indicated the molecular interaction of above mentioned active phytocompounds of VNE with ERα, NR3C4 and with TNFα that plays a critical mechanistic gateway to the regulation of hormone signalling as well as synchronizing the inflammation cascade. Furthermore, the histomorphological improvement of the ovaries supported the ameliorative action of Vitex negundo extract in the letrozole-induced PCOS model. CONCLUSIONS: This study indicates the potential of Vitex negundo seed as a multifaceted therapeutic option for PCOS. VNE offers a holistic strategy for PCOS with antiandrogenic, anti-inflammatory, and antioxidant properties, driven by its major compounds like cinnamic acid, plumbagine, and nigundin B.

CPEB1 induces autophagy and promotes apoptosis in ovarian granulosa cells of polycystic ovary syndrome.

Inflammatory damage in ovarian granulosa cells (GCs) is a key mechanism in polycystic ovary syndrome (PCOS), cytoplasmic polyadenylation element binding protein-1 (CPEB1) is important in inflammatory regulation, however, its role in PCOS is unclear. We aim to research the mechanism of CPEB1 in ovarian GCs in PCOS using dehydroepiandrosterone (DHEA)-induced PCOS rat models and testosterone-incubated GC models. The pathophysiology in PCOS rats was analyzed. Quantitative-realtime-PCR, TUNEL, immunohistochemistry, and Western blot were applied for quantification. Additionally, cell counting kit-8, flow cytometry, immunofluorescence, Western blot, and Monodansylcadaverine staining were performed. We found that PCOS rat models exhibited a disrupted estrus cycle, elevated serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), increased LH/FSH ratio, and heightened ovarian index. Furthermore, reduced corpus luteum and increased follicular cysts were observed in ovarian tissue. In ovarian tissue, autophagy and apoptosis were activated and CPEB1 was overexpressed. In vitro, CPEB1 overexpression inhibited cell viability and sirtuin-1 (SIRT1), activated tumor necrosis factor-α, and interleukin-6 levels, as well as apoptosis and autophagy; however, CPEB1 knockdown had the opposite effect. In conclusion, overexpression of CPEB1 activated autophagy and apoptosis of ovarian GCs in PCOS.

Dehydroepiandrosterone with a high-fat diet treatment at inducing polycystic ovary syndrome in rat model.

OBJECTIVE: This study aimed to evaluate the effects of dehydroepiandrosterone (DHEA) and DHEA combined with a high-fat diet (HFD) treatment of reproductive and endocrine metabolism in rats and then identify an ideal model of polycystic ovary syndrome (PCOS). METHODS: Three-week-old female Sprague-Dawley rats were injected subcutaneously with DHEA or oil, fed with or without a HFD, for 21 days, during which body weight, feed intake, and estrous cycle monitoring were carried out. Fasting blood glucose was measured, and serum fasting insulin, testosterone, dihydrotestosterone (DHT), estradiol, progesterone, luteinizing hormone (LH), anti-Müllerian hormone (AMH), and follicle-stimulating hormone (FSH) were estimated by ELISA. Serum total cholesterol (TC), total triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by colorimetric assay. Whereas, histologic changes in rat ovaries were evaluated by H&E staining. Ovarian steroid hormone synthases and their protein levels (StAR, 3ß-HSD2, 17ß-HSD1, CYP11A1, CYP17A1, and CYP19A1) were examined by Western blotting. RESULTS: Both DHEA and DHEA + HFD-treated rats lost a regular estrous cycle; had polycystic ovarian changes, significantly higher serum fasting insulin and testosterone levels; and increased ovarian StAR, 3ß-HSD2, and CYP11A1 protein levels. Additionally, rats in the DHEA + HFD-treated group were obese; had elevated fasting blood glucose, TG, DHT, AMH levels and LH:FSH ratios; increased ovarian 17ß-HSD1 protein levels. CONCLUSION: DHEA combined with HFD treatment is more effective at inducing PCOS than DHEA alone. The reproductive and endocrine metabolic aspects of this method are more consistent with the clinical characteristics of PCOS patients.

Plastic-related endocrine disrupting chemicals significantly related to the increased risk of estrogen-dependent diseases in women.

OBJECTIVE: To evaluate the association between exposure to plastic-related endocrine-disrupting chemicals (EDCs), specifically Bisphenol A (BPA), Phthalates, Cadmium, and Lead, and the risk of estrogen-dependent diseases (EDDs) such as polycystic ovary syndrome (PCOS), endometriosis, or endometrial cancer by conducting a meta-analysis of relevant studies. METHODS: PubMed, Web of Science, and Cochrane Library databases were used for literature retrieval of articles published until the 21st of April 2023. Literature that evaluated the association between BPA, phthalates, cadmium, and/or lead exposure and the risk of PCOS, endometriosis, or endometrial cancer development or exacerbation were included in our analysis. STATA/MP 17.0 was used for all statistical analyses. RESULTS: Overall, 22 articles were included in our meta-analysis with a total of 83,641 subjects all of whom were females aged between 18 and 83 years old. The overall effect size of each study was as follows: endometriosis risk in relation to BPA exposure ES 1.82 (95% CI; 1.50, 2.20). BPA and PCOS risk ES 1.61 (95% CI; 1.39, 1.85). Phthalate metabolites and endometriosis risk; MBP ES 1.07 (95% CI; 0.86, 1.33), MEP ES 1.05 (95% CI; 0.87, 1.28), MEHP ES 1.15 (95% CI; 0.67, 1.98), MBzP ES 0.97 (95% CI; 0.63, 1.49), MEOHP ES 1.87 (95% CI; 1.21, 2.87), and MEHHP ES 1.98 (95% CI; 1.32, 2.98). Cadmium exposure and endometrial cancer risk ES 1.14 (95% CI; 0.92, 1.41). Cadmium exposure and the risk of endometriosis ES 2.54 (95% CI; 1.71, 3.77). Lead exposure and the risk of endometriosis ES 1.74 (95% CI; 1.13, 2.69). CONCLUSION: Increased serum, urinary, or dietary concentration of MBzP and MEHP in women is significantly associated with endometriosis risk. Increased cadmium concentration is associated with endometrial cancer risk.

The effect of adipose-derived mesenchymal stem cell transplantation on ovarian mitochondrial dysfunction in letrozole-induced polycystic ovary syndrome in rats: the role of PI3K-AKT signaling pathway.

OBJECTIVE: The present study aimed to elucidate how mesenchymal stem cells (MSCs) application could efficiently attenuate pathological changes of letrozole-induced poly cystic ovary syndrome (PCOS) by modulating mitochondrial dynamic via PI3K-AKT pathway. METHODS: Thirty-two female rats were randomly divided into four experimental groups: Sham, PCOS, PCOS + MSCs, and PCOS + MSCs + LY294002. The Sham group received 0.5% w/v carboxymethyl cellulose (CMC); the PCOS group received letrozole (1 mg/kg, daily) in 0.5% CMC for 21 days. Animals in the PCOS + MSCs group received 1 × 106 MSCs/rat (i.p,) on the 22th day of the study. In the PCOS + MSCs + LY294002 group, rats received LY294002 (PI3K-AKT inhibitor) 40 min before MSC transplantation. Mitochondrial dynamic gene expression, mitochondrial membrane potential (MMP), citrate synthase (CS) activity, oxidative stress, inflammation, ovarian histological parameters, serum hormone levels, homeostatic model assessment for insulin resistance (HOMA-IR), insulin and glucose concentrations, p-PI3K and p-AKT protein levels were evaluated at the end of the experiment. RESULTS: PCOS rats showed a significant disruption of mitochondrial dynamics and histological changes, lower MMP, CS, ovary super oxide dismutase (SOD) and estrogen level. They also had a notable rise in insulin and glucose concentrations, HOMA-IR, testosterone level, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, ovarian malondialdehyde (MDA) content as well as a notable decrease in p-PI3K and p-AKT protein levels compared to the Sham group. In the PCOS + MSCs group, the transplantation of MSCs could improve the above parameters. Administration of LY294002 (PI3K-AKT pathway inhibitor) deteriorated mitochondrial dynamic markers, oxidative stress status, inflammation markers, hormonal levels, glucose, and insulin levels and follicular development compared to the PCOS + MSCs group. CONCLUSIONS: This study demonstrated that the protective effects of MSC transplantation in regulating mitochondrial dynamics, promoting mitochondrial biogenesis, competing with redox status and inflammation response were mainly mediated through the PI3K-AKT pathway in the PCOS model.

Evaluating the potential of daily intake of polystyrene microplastics via drinking water in inducing PCOS and its ovarian fibrosis progression using female zebrafish.

Polystyrene microplastics, extensively considered endocrine disrupting chemicals, disturb the reproductive system of living organisms. Polycystic ovary syndrome (PCOS), the reproductive endocrinopathy, is longstanding concern due to its eternal impacts as reproductive disorder and infertility. Despite several reports in reproductive and endocrine toxicity, there is inadequate literature regarding the daily intake of polystyrene-microplastics via drinking water in causing PCOS and leading to ovarian fibrosis in long-term. The present study investigated whether daily consumption of polystyrene-microplastics at doses equivalent to human exposure can cause PCOS and progress to ovarian fibrosis, using female zebrafish as model. Resembling letrozole-PCOS zebrafish model, daily intake of polystyrene-microplastics displayed hallmark PCOS pathophysiology; like excess body weight and %Gonadosomatic index, decreased Follicle Stimulating Hormone and ß-estradiol, increased Luteinising Hormone, brain and ovarian Testosterone (39.3% and 75% respectively). Correspondingly, ovarian histology revealed more developing (stage I and II) oocytes and less mature oocytes alongwith cystic lesions; like follicular membrane disorganization, zona pellucida invagination, theca hypertrophy, basophilic granular accumulation and oocyte buddings. Lipid deposition in intestinal and ovarian tissues was evidenced and increased fasting blood glucose manifesting insulin resistance. The expression of PCOS biomarkers (tox3, dennd1a, fem1a) was significantly disturbed. Polystyrene microplastics played vital role in inducing PCOS further enhancing oxidative stress, which positively influences inflammation and aggravate ovarian mitophagy, shedding light on its ability to harshen PCOS into ovarian fibrosis, which is characterized by collagen deposition and upregulation of pro-fibrogenic biomarker genes. These findings illustrate the potential of daily microplastics intake via drinking water in triggering PCOS and its progression to ovarian fibrosis.

Exploring the molecular mechanisms by which per- and polyfluoroalkyl substances induce polycystic ovary syndrome through in silico toxicogenomic data mining.

The pathogeny of polycystic ovary syndrome (PCOS) is intricate, with endocrine disruptors (EDCs) being acknowledged as significant environmental factors. Research has shown a link between exposure to per- and polyfluoroalkyl substances (PFAS) and the development and progression of PCOS, although the precise mechanism is not fully understood. This study utilized toxicogenomics and comparative toxicogenomics databases to analyze data and investigate how PFAS mixtures may contribute to the development of PCOS. The results indicated that 74 genes are associated with both PFAS exposure and PCOS progression. Enrichment analysis suggested that cell cycle regulation and steroid hormone synthesis may be crucial pathways through which PFAS mixtures participate in the development of PCOS, involving important genes such as CCNB1 and SRD5A1. Furthermore, the study identified transcription factors (TFs) and miRNAs that may be involved in the onset and progression of PCOS, constructing regulatory networks encompassing TFs-mRNA interactions and miRNA-mRNA relationships to elucidate their regulatory roles in gene expression. By utilizing data mining techniques based on toxicogenomic databases, this study provides relatively comprehensive insights into the association between exposure factors and diseases compared to traditional toxicology studies. These findings offer new perspectives for further in vivo or in vitro investigations and contribute to understanding the pathogenesis of PCOS, thereby providing valuable references for identifying clinical treatment targets.

The therapeutic effect of anti-CD19 antibody on DHEA-induced PCOS mice.

Immune dysregulation has been summarized as a critical factor in the occurrence and development of Polycystic ovary syndrome (PCOS), but potential mediators and mechanisms remain unclear. Our previous study showed that CD19+ B cells were involved in the pathogenesis of dehydroepiandrosterone (DHEA)-induced PCOS mice. Here, we studied the therapeutic potential of anti-CD19 antibody (aCD19 Ab) on DHEA-induced PCOS mice. The results showed that aCD19 Ab treatment improved ovarian pathological structure and function of PCOS mice, manifested by an increased number of corpus luteum, a decreased number of cystic follicles and atretic follicles, and regular estrus cycles. The aCD19 Ab treatment reduced the proportion of splenic CD21+ CD23low marginal zone B cells as well as the level of serum IgM and decreased the percentage of peripheral blood and splenic neutrophils. In particular, aCD19 Ab treatment reduced the apoptosis of granulosa cells and macrophage infiltration in ovarian secondary follicles of PCOS mice, as well as the expression of TNF-α in ovarian tissue and serum TNF-α levels. Moreover, we confirmed that TNF-α induced the apoptosis of human ovarian granulosa tumor cell line cells in vitro. Thus, our work demonstrates that aCD19 Ab treatment improves ovarian pathological phenotype and function by reducing local and systemic inflammation in PCOS mice, which may provide a novel insight into PCOS therapy.

Letrozole induced a polycystic ovary syndrome model in zebrafish by interfering with the hypothalamic-pituitary-gonadal axis.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disease in women of childbearing age, with an incidence of 5-10%. This study compared the traits of zebrafish with three diagnostic criteria for human PCOS, and the diagnostic criteria for zebrafish PCOS were proposed: decreased fecundity, elevated testosterone (T) or 11-ketotestosterone (11-KT) levels and increased cortical-alveolar oocyte (CO) ratio, enhancing the zebrafish PCOS model's accuracy. According to the mammalian PCOS classification, the type of zebrafsh PCOS is divided into four phenotypes (A, B, C and D), but the four phenotypes of zebrafish PCOS are not fully covered in the existing studies (A and D). In this study, we successfully induced phenotype B zebrafish PCOS model using the aromatase inhibitor, letrozole (LET). That is, wild-type female zebrafish were exposed to 1000 µg/L LET for 30 days. Reproductive tests showed decreased fecundity in female zebrafish exposed to LET (Control: 132.63, 146.00, 173.00; LET: 29.20, 90.00, 82.71). Hormone analysis showed that female zebrafish exposed to LET had significantly lower 17ß-estradiol/testosterone (E2/T) ratios, indicating elevated T levels. Meanwhile, levels of 11-KT in the ovaries exposed to LET were significantly up-regulated (Control: 0.0076 pg/µg; LET: 0.0138 pg/µg). Pathological sections of the ovary showed fewer CO in the LET-exposed group (Control: 16.27%; LET: 8.38%). In summary, the zebrafish PCOS model summarized and studied in this study provide a reliable and economical tool for the screening of therapeutic drugs, as well as for the etiology research and treatment strategies of PCOS.

The significant improvement in ovarian PCOS syndrome using hydralazine and alendronate aromatase inhibitor FDA-approved drugs in Wistar rat models.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility. The aim of this study was to investigate the therapeutic potential of vitamin C, glutamine, mesalazine, hydralazine, and alendronate as new drug candidates for the treatment of letrozole-induced PCOS in female Wistar rats. PCOS was induced in rats by intramuscular injection of estradiol valerate (2 mg/kg body weight for 28 days). The rats then received normal saline (PCOS group), letrozole (0.5 mg/kg), vitamin C (100 mg/kg), glutamine (1000 mg/kg), mesalazine (200 mg/kg), hydralazine (30 mg/kg), and alendronate (17.5 mg/kg). Serum testosterone, LH, FSH, estradiol and progesterone levels were determined by ELISA method. H&E staining was used for histological analysis in the ovarian tissues. The groups treated with hydralazine and alendronate, show a significant decrease in testosterone, LH hormone, cystic and atretic follicles, and a significant increase in the number of single layer, multilayer, antral, graafian follicles and the volume of corpus luteum as compared to the PCOS group. Hydrolazine and alendronate appear to be effective in restoring folliculogenesis and increasing ovulation in PCOS rat. So that the natural process of ovulation and the improvement of the histology of polycystic ovaries and its shift towards healthy and active ovaries were observed. This finding supports the potential beneficial effect of hydrolazine and alendronate on improving PCOS complication.

Myricetin ameliorates polycystic ovary syndrome in mice by brown adipose tissue activation.

In brief: Brown adipose tissue impaired in polycystic ovary syndrome (PCOS) plays a crucial role in the treatment of PCOS. This study shows that myricetin potently improves PCOS by activating brown adipose tissue (BAT). Abstract: PCOS is a complex endocrine disease characterized by hyperandrogenism, anovulation and polycystic ovary, and is often accompanied by metabolic disorder such as insulin resistance. BAT has been considered as a promising target for the treatment of obesity and other metabolic disease. In this study, we showed that 3 weeks of myricetin (a compound from natural product) treatment improved metabolic capacity and insulin sensitivity by activating BAT in dehydroepiandrosterone (DHEA)-induced PCOS mice. Furthermore, increased number of corpus luteum and decreased cystic formation were observed in PCOS mice. With the hormone levels such as luteinizing hormone (LH) were reversed, estrous cycle was also normalized after myricetin treatment. Eventually, myricetin markedly improved reproductive defects in PCOS mice. In short, our results suggest that myricetin treatment dramatically ameliorates ovarian dysfunction and metabolic disturbances in PCOS and provides a novel perspective for the treatment of PCOS.

Effects of melatonin and metformin on the ovaries of rats with polycystic ovary syndrome.

OBJECTIVE: To study the combined and isolated effects of melatonin and metformin in the ovarian tissue of rats with PCOS. DESIGN: Experimental study using a rat model of PCOS induced by continuous light exposure. INTERVENTION(S): Forty adult female rats were divided into 5 groups: physiological estrus phase (Sham); permanente estrus with PCOS induced by continuous lighting exposure for 60 consecutive days (control); with PCOS treated with melatonin; with PCOS treated with metformin; with PCOS treated with melatonin + metformin. After 60 days of treatments, all rats were killed, and ovaries were collected and processed for paraffin-embedding. Formalin-fixed paraffin-embedded sections were stained with hematoxylin and eosin or subjected to immunohistochemistry for proliferation (Ki-67) and apoptosis (cleaved caspase 3) detection markers. SETTING: Federal University of São Paulo, Brazil. ANIMALS: Forty adult female Wistar rats (Rattus norvegicus albinus). MAIN OUTCOME MEASURE(S): Number of corpus luteum and ovarian cysts, number of ovarian follicles (primary and antral follicles), number of interstitial cells, percentage of ovarian follicles (primary and antral follicles), and of interstitial cells immunostained to cleaved caspase-3 and Ki-67. RESULTS: Absence of corpus luteum, a higher number of cysts, and increased nuclear volume and area of interstitial cells, along with a decrease in primary and antral follicle numbers, were noticed in the control group compared with the Sham group. Melatonin and metformin treatments attenuated these effects, although the combined treatment did not mitigate the increased number of cysts and ovaries induced by PCOS. An increase in theca interna cell apoptosis was observed in the control group, whereas melatonina and metformin treatments reduced it significantly. A higher percentage of caspase-3-immunostained granulosa cells was noted in the Sham and all treated groups compared with the control group; no aditive effects on ovarian cell apoptosis were observed in the combined treatment. The percentage of Ki-67- immunostained granulosa cells was significantly higher in the control group compared with the Sham group. However, the combined treatment, not melatonin and metformin alone, mitigated this effect. A higher percentage of Ki-67-immunostained interstitial cells was observed in all treated groups compared with the Sham and control groups, whereas no additive effects in that immunoreactivity were observed in the combined treatment. CONCLUSIONS: Melatonin and metformin may improve ovarian function in rats with PCOS. The combined melatonin and metformin treatment is more effective in attenuating excessive granulosa cell proliferation, but it is not more effective in improving ovarian function than these drugs applied alone in rats with PCOS.

Gonadotropin upregulates intraovarian calpains-1 and -2 during ovarian follicular recruitment in the SD rat model.

Calpain role has been shown in the cumulus cell-oocyte complexes and, corpus luteum. We investigated the association of calpains-1 and -2 in ovarian folliculogenesis using the Sprague-Dawley (SD) rat model and steroidogenesis in the human granulosa cells (hGCs). We induced PCOS in 42-day-old SD rats by letrozole oral gavage for 21 days. Premature ovarian failure (POF) was induced in 21-day-old SD rats by 4-vinylcyclohexene diepoxide (VCD). Ovulation and ovarian hyperstimulatory (OHS) syndrome were induced by pregnant mare gonadotropin (PMSG) + human chorionic gonadotropin (hCG) treatments in 21 days SD rats, respectively. Steroidogenesis is stimulated in human granulosa cells (hGCs) by forskolin and the response of 17-beta-estradiol (E2) on calpains expression was checked in hGCs. The protein expression by immunoblotting and activity by biochemical assay of calpains-1 and -2 showed an oscillating pattern in the ovarian cycle. PMSG-induced follicular recruitment showed upregulation of calpains-1 and -2, but with no change during ovarian function cessation (POF). Upregulated calpain-2 expression and calpain activity was found in the hCG +PMSG-induced ovulation. Letrozole-induced PCOS showed downregulation of calpain-1, but upregulation of calpain-2. PMSG+hCG-induced OHS led to the upregulation of calpain-1. Letrozole and metformin separately increased the expression level of calpains-1 and -2 in the hGCs during luteinization. In conclusion, the expression levels of calpains -1 and -2 are increased with ovarian follicular recruitment by PMSG and calpain-1 is decreased in the PCOS condition, and letrozole and metformin upregulate the expression of calpains-1 and -2 during luteinization in the hGCs possibly via E2 action.

Comparative effects of the antioxidant glutathione with metformin and Diane-35 on hormonal, metabolic, and inflammatory indicators in a DHEA-induced PCOS rat model.

OBJECTIVE: Comparison of hormonal, metabolic and inflammatory markers of glutathione with metformin and Diane-35 in a rat model of PCOS induced by dehydroepiandrosterone. METHODS: Twenty-five female rats were randomized into four groups. Group 1 was administered a subcutaneous dose of 0.2 ml saline/day. Group 2 was given 0.2 ml of 1% carboxymethyl cellulose (CMC)/day orally for 28 days. A PCOS model was established with DHEA in rats. Group 3 was given 4.5 mg/kg/day of Diane-35 orally dissolved in 1% CMC for 28 days. Group 4 was given 300 mg/kg/day of metformin orally dissolved in 1 ml of saline for 28 days, and Group 5 was administered 100 mg/kg of glutathione intraperitoneally on days 35, 42, and 49. On day 56, the rats were sacrificed. Serum markers and follicle count were examined. RESULTS: Serum IL-6, hs-CRP, insulin, testosterone, SHBG, and MDA values were significantly lower in the glutathione group than in the PCOS group (p = 0.0006, p = 0.023, p = 0.0082, p = 0.0007, p = 0.0048, and p < 0.0001, respectively).The number of all follicles was similar between the control and glutathione groups (p < 0.05). When we compared the other groups with the PCOS group, the number of primary, secondary, atretic, and cystic follicles was significantly lower in the metformin and glutathione groups. The number of primordial and antral follicles was significantly higher than in the PCOS group. CONCLUSIONS: Glutathione plays anti-inflammatory and antioxidant roles, similar to metformin, by lowering serum IL-6, insulin, testosterone, CRP, and MDA levels; decreasing atretic/cystic follicle count; and improving antral follicle count and folliculogenesis in PCOS patients.

Troxerutin dampened hypothalamic neuroinflammation via microglial IL-22/IL-22R1/IRF3 activation in dihydrotestosterone-induced polycystic ovary syndrome rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common reproductive-endocrine condition in premenopausal women. Troxerutin, a common clinical anti-coagulant agent, was shown to work as a strong IL-22 boosting agent counteracting the hyperactivated gonadotrophin releasing hormone (GnRH) neurons and heightened GnRH release, the neuroendocrine origin of PCOS with unknown mechanism in rats. Exploring the off-label use of troxerutin medication for PCOS is thus sorely needed. METHODS: Serum IL-22 content and hypothalamic IL-22 protein were detected. Inflammatory factor levels in hypothalamo-pituitary were evaluated. Immunofluorescence staining was employed to determine the activation and M1/M2-prone polarization of microglia in arcuate hypothalamus and median eminence. RNA-sequencing and transcriptome analysis were applied to explore the potential driver of microglia M2-polarization in response to IL-22 bolstering effect. The function of microglial IL-22/IL-22R1/IRF3 system was further verified using in vivo knockdown of IL-22R1 and a potent IRF3 inhibitor in BV2 microglial cell lines in vitro. RESULTS: Troxerutin augmented serum IL-22 content, and its consequent spillover into the hypothalamus led to the direct activation of IL-22R1/IRF3 system on microglia, thereby promoted microglia M2 polarization in arcuate hypothalamus and median eminence, dampened hypothalamic neuroinflammation, inhibited hyperactive GnRH and rescued a breadth of PCOS-like traits in dihydrotestosterone (DHT) rats. The salutary effects of troxerutin treatment on hypothalamic neuroinflammation, microglial M1/2 polarization, GnRH secretion and numerous PCOS-like features were blocked by in vivo knockdown of IL-22R1. Moreover, evidence in vitro illustrated that IL-22 supplement to BV-2 microglia cell lines promoted M2 polarization, overproduction of anti-inflammatory marker and limitation of pro-inflammatory factors, whereas these IL-22 effects were blunted by geldanamycin, a potent IRF3 inhibitor. CONCLUSION: Here, the present study reported the potential off-label use of troxerutin medication, a common clinical anti-coagulant agent and an endogenous IL-22 enhancer, for multiple purposes in PCOS. The rational underlying the application of troxerutin as a therapeutic choice in PCOS derived from its activity as an IL-22 memetic agent targeting the neuro-endocrine origin of PCOS, and its promotive impact on microglia M2 polarization via activating microglial IL-22R1/IRF3 system in the arcuate hypothalamus and median eminence of DHT female rats.