RESUMO
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.
Assuntos
Síndrome do Ovário Policístico , Animais , Feminino , Ratos , Apoptose , Autofagia , Hormônio Foliculoestimulante Humano , Células da Granulosa , Hormônio Luteinizante , Fatores de Poliadenilação e Clivagem de mRNA/genética , Síndrome do Ovário Policístico/induzido quimicamente , Testosterona , Fatores de TranscriçãoRESUMO
The pathologic mechanism of polycystic ovary syndrome (PCOS) is related to increased autophagy of granulosa cells. Both berberine and metformin have been shown to improve PCOS, but whether the combination of berberine and metformin can better improve PCOS by inhibiting autophagy remains unclear. PCOS models were constructed by injecting dehydroepiandrosterone into rats, and berberine, metformin or berberine combined with metformin was administered to rats after modeling. Rats' body weight and ovarian weight were measured before and after modeling. Histopathological examination of ovarian tissue and estrous cycle analysis of rats were performed. Insulin resistance, hormone levels, oxidative stress, and lipid metabolism in PCOS rats were assessed. Expression of the AMPK/AKT/mTOR pathway and autophagy-related proteins was analyzed by Western blot assays. Granulosa cells were isolated from rat ovarian tissue and identified by immunofluorescence staining followed by transmission electron microscopy analysis. Berberine combined with metformin reduced the body weight and ovarian weight of PCOS rats, increased the number of primordial and primary follicles, decreased the number of secondary and atretic follicles, normalized the estrous cycle, and improved insulin resistance, androgen biosynthesis, oxidative stress and lipid metabolism disorders, and increased estrogen production. In addition, berberine combined with metformin reduced the number of autophagosomes in granulosa cells, which may be related to AMPK/AKT/mTOR pathway activation, decreased Beclin1 and LC3II/LC3I levels, and increased p62 expression. Berberine combined with metformin could inhibit autophagy by activating the AMPK/AKT/mTOR pathway in PCOS, indicating that berberine combined with metformin is a potential treatment strategy for PCOS.
Assuntos
Autofagia , Berberina , Metformina , Síndrome do Ovário Policístico , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Feminino , Animais , Metformina/farmacologia , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/patologia , Autofagia/efeitos dos fármacos , Berberina/farmacologia , Ratos , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Ratos Sprague-Dawley , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Resistência à Insulina , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Quimioterapia Combinada , Estresse Oxidativo/efeitos dos fármacosRESUMO
OBJECTIVE: GuizhiFulingWan (GFW) has been reported to be effective against polycystic ovary syndrome (PCOS) by possessing oxidative stress and inflammation which related to PI3K/AKT/NF-κB, Nrf2/HO-1 pathway. This study aims to probe the effects and mechanisms of GFW combined with rosiglitazone on PCOS via PI3K/AKT/NF-κB and Nrf2/HO-1 pathways. METHODS: A rat PCOS model established by dehydroepiandrosterone (DHEA) injection. The experiment was allocated to control, DHEA, GFW, rosiglitazone, GFW + rosiglitazone groups. Treatment for 30 days, we monitored weight and ovarian weight of rats. Fasting blood glucose (FBG), fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR), lipid metabolism indexes, estrous cycle and sex hormone-, inflammation-, oxidative stress-related factors were examined. Hematoxylin&eosin staining assessed ovarian tissue pathological changes. Western blot determined PI3K/AKT/NF-κB, Nrf2/HO-1 pathways-related markers. RESULTS: GFW and rosiglitazone treatment suppressed body weight and ovarian weight in PCOS rats. They also decreased FBG, FINS, HOMA-IR while inhibited total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and enhanced high-density lipoprotein (HDL). They ameliorated estrous cycle, ovarian histological changes and follicular development. They restrained testosterone (T), luteinizing hormone (LH) and accelerated estradiol (E2), progesterone (P), follicle stimulating hormone (FSH). They inhibited glutathione peroxidase (GSH-Px), malondialdehyde (MDA), superoxide dismutase (SOD) in serum while increased GSH-Px, SOD and decrease MDA in ovarian tissues. They reduced C-reactive protein, interleukin-18 (IL-18), tumor necrosis factor-α (TNF-α), IL-6, IL-1ß levels. GFW and rosiglitazone co-intervention regulated PI3K/AKT/NF-κB and Nrf2/HO-1 pathways in PCOS rats. CONCLUSION: GFW alleviated ovarian dysfunction in PCOS rats, which may be related to the PI3K/AKT/NF-κB, Nrf2/HO-1 pathways.
Assuntos
NF-kappa B , Síndrome do Ovário Policístico , Feminino , Humanos , Animais , Ratos , Síndrome do Ovário Policístico/tratamento farmacológico , Fator 2 Relacionado a NF-E2 , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Rosiglitazona/farmacologia , Rosiglitazona/uso terapêutico , DesidroepiandrosteronaRESUMO
OBJECTIVE: Premature ovarian failure (POF), also known as primary ovarian insufficiency, is a major cause of infertility in female worldwide. Excessive apoptosis and impaired autophagy in ovarian granulosa cells are the main pathological mechanisms of POF. The total flavonoids from semen cuscutae (TFSC) are often used in the treatment of gynecological endocrine disorders. In addition, low intensity pulsed ultrasound (LIPUS) is report as an effective method to improve ovarian function. This study aims to investigate the protective effect of POF by the combined use of TFSC and LIPUS. METHODS: POF rats model and granulosa cell model were successfully induced by tripterygium glycosides and cyclophosphamide, respectively. After that, model rats and cells received TFSC plus LIPUS administration. Then ovarian histomorphology, senescence, estrus cycle, and serum sex hormone levels were detected in rats. Ovarian tissue and granulosa cells autophagy and apoptosis levels were also assessed. RESULTS: Disturbed sex hormone levels, atrophied and senescent ovaries, and abnormal estrous cycle were found in POF rats. Meanwhile, cell autophagy was inhibited and cell apoptosis was activated in POF ovarian tissue and granulosa cells. However, TFSC combined with LIPUS improved these changes, and this combination treatment exhibited synergistic effects. The abnormal expression of the cell apoptosis-, autophagy-, and PI3K/AKT/mTOR signaling pathway-related proteins were also improved by combination treatment. CONCLUSION: The study found that the combination of TFSC and LIPUS can alleviate POF by modulating cell autophagy and apoptosis. The findings may provide a viable scientific basis for POF treatment.
Assuntos
Medicamentos de Ervas Chinesas , Flavonoides , Insuficiência Ovariana Primária , Sêmen , Ondas Ultrassônicas , Animais , Feminino , Humanos , Ratos , Apoptose , Hormônios Esteroides Gonadais/efeitos adversos , Células da Granulosa/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Insuficiência Ovariana Primária/terapiaRESUMO
BACKGROUND: Polycystic ovarian syndrome (PCOS) is the most common endocrine disease in women of childbearing age which is often associated with abnormal proliferation or apoptosis of granulosa cells (GCs). Studies proved that long non-coding RNA SNHG12 (lncRNA SNHG12) is significantly increased in ovarian cancer and cervical cancer patients and cells. The inhibition of lncRNA SNHG12 restrains the proliferation, migration, and invasion in tumor cells. OBJECTIVE: This study explores the role of lncRNA SNHG12 in the apoptosis of GCs in PCOS and the underlying regulated mechanism. METHODS: In this study, the injection of dehydroepiandrosterone (DHEA) successfully induced the PCOS model in SD rats. The human granulosa-like tumor cell line KGN was incubated with insulin to assess the effects of lncRNA SNHG12 on GC proliferation and apoptosis. RESULTS: Overexpression of lncRNA SNHG12 influenced the body weight, ovary weight, gonadal hormone, and pathological changes, restrained the expressions of microRNA (miR)-129 and miR-125b, while downregulation of lncRNA SNHG12 exerted the opposite effects in PCOS rats. After silencing lncRNA SNHG12 in cells, the cell viability and proliferation were lessened whereas apoptosis of cells was increased. A loss-of-functions test was implemented by co-transfecting miR-129 and miR-125b inhibitors into lncRNA SNHG12-knocking down cells to analyze the effects on cell viability and apoptosis. Next, the existence of binding sites of SNHG12 and miR-129/miR-125b was proved based on the pull-down assay. CONCLUSION: lncRNA SNHG12 might be a potential regulatory factor for the development of PCOS by sponging miR-129 and miR-125b in GCs.
Assuntos
MicroRNAs , Síndrome do Ovário Policístico , RNA Longo não Codificante , Humanos , Feminino , Ratos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Síndrome do Ovário Policístico/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ratos Sprague-Dawley , Células da Granulosa/metabolismo , Proliferação de Células , Apoptose/genéticaRESUMO
INTRODUCTION: Polycystic ovarian syndrome (PCOS) is a common endocrinopathy in women. MicroRNAs (miRNAs) have been proven to play a crucial role in balancing the proliferation and apoptosis of granulosa cells (GCs) in PCOS. MATERIAL AND METHODS: The miRNA of PCOS was screened by bioinformatics analysis, and microRNA 646 (miR-646) was found to be involved in insulin-related pathways by enrichment analysis. The cell counting kit-8 (CCK-8), cell colony formation, and the 5-ethynyl-2'-deoxyuridine (EdU) assays were used to explore the effect of miR-646 on proliferation of GCs, flow cytometry was used to measure the cell cycle and apoptosis, and Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to explore the biological mechanism of miR-646. The human ovarian granulosa cells KGN were selected by measuring the miR-646 and via insulin-like growth factor 1 (IGF-1) levels and used for cell transfection. RESULTS: Overexpressed miR-646 inhibited KGN cell proliferation, and silenced miR-646 advanced it. Most cells were arrested in the S phase of cell cycle with overexpressed-miR-646, while after silencing miR-646, cells were arrested in the G2/M phase. And the miR-646 mimic raised apoptosis in KGN cells. Also, a dual-luciferase reporter proved the regulation effect of miR-646 on IGF-1, miR-646 mimic inhibited IGF-1, and miR-646 inhibitor advanced IGF-1. The cyclin D1, cyclin-dependent kinase 2 (CDK2), and B-cell CLL/lymphoma 2 (Bcl-2) levels were inhibited with overexpressed-miR-646, while silenced-miR-646 promoted their expression, and the bcl-2-like protein 4 (Bax) level was the opposite. This study found that silenced-IGF1 antagonized the promotive effect of the miR-646 inhibitor on cell proliferation. CONCLUSIONS: MiR-646 inhibitor treatment can promote the proliferation of GCs by regulating the cell cycle and inhibiting apoptosis, while silenced-IGF-1 antagonizes it.
Assuntos
MicroRNAs , Síndrome do Ovário Policístico , Humanos , Feminino , Fator de Crescimento Insulin-Like I/metabolismo , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Células da Granulosa/metabolismo , Proliferação de Células/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/farmacologiaRESUMO
Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder. Sitagliptin (Sit) and rosiglitazone (Ros) are widely used to treat PCOS, but the mechanism is unclear. This study explored the mechanism that Sit and Ros inhibited autophagy and inflammation in PCOS. In this study, 50 female SD rats were divided into 5 groups (n = 10): control, PCOS, Sit, Ros, and Sit+Ros group. The body weight and ovarian weight were measured 2 h after the last administration, and fasting blood glucose, insulin levels were determined. Lipid metabolism and pathological changes were detected by an automatic biochemical analyzer and HE staining. Sex hormone, oxidative stress and inflammatory levels were detected by ELISA. PCR detected IL-18, TNF-α, IL-6, IL-1ß, ATG3, and ATG12 mRNA. The PI3K/AKT/mTOR, TLR4/NF-κB pathway and autophagy-related proteins were detected by western blot. Finally, the number of autophagolysosomes was detected by transmission electron microscopy. Sit or Ros alone reduced body weight, ovarian weight, fasting blood glucose, and insulin levels in PCOS rats. It also improved lipid metabolism, sex hormone levels, oxidative stress and pathological changes, restored the estrous cycle, and corpus luteum quantity. In addition, it could reduce the levels of IL-18, TNF-α, IL-6, IL-1ß, ATG3, and ATG12 mRNA, inhibit the expression of Beclin1, LC3, PI3K/AKT/mTOR, and TLR4/NF-κB pathway proteins. The Sit+Ros group was more effective than single administration. In conclusion, Sit+Ros inhibited the PI3K/AKT/mTOR, TLR4/NF-κB pathways, thereby inhibiting the autophagy and inflammation of PCOS rats, which will provide a theoretical basis for PCOS.
Assuntos
Insulinas , Síndrome do Ovário Policístico , Animais , Feminino , Ratos , Autofagia , Glicemia , Peso Corporal , Hormônios Esteroides Gonadais , Inflamação , Interleucina-18 , Interleucina-6 , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Síndrome do Ovário Policístico/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , RNA Mensageiro , Rosiglitazona/farmacologia , Receptor 4 Toll-Like , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Increasing evidence indicates that circular RNAs (CircRNAs) have an important role in human diseases, including polycystic ovary syndrome (PCOS). Recently, circ_0043533, a novel circRNA, was proposed to be involved in the progression of PCOS. However, its role in PCOS has not been explored. In this study, the expression levels of circ_0043533 and miR-1179 in ovarian granulosa cells (OGCs) were examined by qRT-PCR analysis. Moreover, knockdown of circ_0043533 in OGC lines COV434 and KGN, respectively, the cell viability, proliferation, apoptosis, and cycle-related markers of insulin-triggered OGCs were examined by CCK-8, EdU staining, flow cytometry, and western blot assays, respectively. The interaction between circ_0043533 and miR-1179 was examined by bioinformatics, dual-luciferase assay, and RNA immunoprecipitation. Besides, effects of the miR-1179 inhibitor on cell viability and apoptosis in OGC lines with circ_0043533 knockdown were also evaluated. OGCs and insulin-treated OGCs exhibited higher circ_0043533 levels in comparison to the IOSE80 cells. Additionally, knockdown of circ_0043533 remarkably inhibited the cell viability and proliferation and promoted the apoptosis of insulin-treated COV434 and KGN cells, respectively. Meanwhile, circ_0043533 knockdown could down-regulate the Bcl-2, CDK2, and Cyclin D1 expressions, and up-regulate the Bax levels. Furthermore, we demonstrated that circ_0043533 acted as a sponge to absorb miR-1179. Interestingly, miR-1179 inhibition remarkably attenuated the effect of circ_0043533 silence on cell proliferation and apoptosis in insulin-treated COV434 and KGN cells. Taken together, this study revealed that circ_0043533 knockdown restrained the malignant progression of PCOS via targeting miR-1179. Our data suggested that circ_0043533 would serve as a novel therapeutic target for PCOS.