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To investigate the effect of rapamycin on mitochondrial dynamic balance in diabetic rats subjected to cerebral ischemia-reperfusion injury. Male Sprague Dawley (SD) rats (n = 78) were treated with high fat diet combined with streptozotocin injection to construct diabetic model in rats. Transient middle cerebral artery occlusion (MCAO) of 2 hours was induced and the brains were harvested after 1 and 3 days of reperfusion. Rapamycin was injected intraperitoneally for 3 days prior to and immediately after operation, once a day. The neurological function was assessed, infarct volumes were measured and HE staining as well as immunohistochemistry were performed. The protein of hippocampus was extracted and Western blotting were performed to detect the levels of mTOR, mitochondrial dynamin related proteins (DRP1, p-DRP1, OPA1), SIRT3, and Nix/BNIP3L. Diabetic hyperglycemia worsened the neurological function performance (p < 0.01), enlarged infarct size (p < 0.01) and increased ischemic neuronal cell death (p < 0.01). The increased damage was associated with elevations of p-mTOR, p-S6, and p-DRP1; and suppressions of SIRT3 and Nix/BNIP3L. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and reversed the biomarker alterations caused by diabetes. High glucose activated mTOR pathway and caused mitochondrial dynamics toward fission. The protective effect of rapamycin against diabetes-enhanced ischemic brain damage was associated with inhibiting mTOR pathway, redressing mitochondrial dynamic imbalance, and elevating SIRT3 and Nix/BNIP3L expression.
Assuntos
Lesões Encefálicas , Isquemia Encefálica , Diabetes Mellitus Experimental , Traumatismo por Reperfusão , Sirtuína 3 , Ratos , Masculino , Animais , Ratos Sprague-Dawley , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Dinâmica Mitocondrial , Diabetes Mellitus Experimental/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Sirtuína 3/metabolismo , Encéfalo/metabolismo , Lesões Encefálicas/complicações , Isquemia Encefálica/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/complicações , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
Connexin (Cx) 43 is the most widely expressed gap junction protein in follicle granulosa cells and plays an important role in follicle development and growth. The aims of this study were to investigate the effects of LH on the expression of Cx43 and key proteins in the downstream Wnt-ß/catenin signalling pathway and to explore the mechanism underlying the regulation of Cx43 expression in granulosa cells. Primary culture granulosa cells were obtained from 21-day-old Sprague-Dawley rats, and were treated with different concentrations of LH (150, 300 and 600 IU L-1). Cx43 expression in granulosa cells was detected using immunofluorescence. Western blotting was used to detect the expression of Cx43, ß-catenin and Axin2 proteins (Axin2 is a protein that in humans is encoded by the AXIN2 gene, which presumably plays an important role in the regulation of the stability of ß-catenin in the Wnt signaling pathway) in granulosa cells with and without FH535 treatment (a Wnt/ß-catenin signalling pathway inhibitor). Cx43 expression was detected in the cytoplasm and cell membrane of granulosa cells. Treatment with a high concentration of LH (300 IU L-1) increased the expression of ß-catenin and Axin2, as well as that of Cx43. FH535 treatment reduced the LH-induced increases in Cx43, ß-catenin and Axin2. These results indicate that LH upregulates Cx43 expression in granular cells by activating the Wnt/ß-catenin signalling pathway.
Assuntos
Conexina 43/metabolismo , Células da Granulosa/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Feminino , Células da Granulosa/metabolismo , Ratos Sprague-Dawley , Regulação para Cima , beta Catenina/metabolismoRESUMO
Cryopreservation and transplantation of the ovarian tissue is an alternative method by which malignant tumor survivors can recover fertility. Previously, it was reported that follicle stimulating hormone (FSH) promoted the survival and functioning of the ovarian tissue after in vitro cultivation. In this study, the expression of the luteinizing hormone receptor (LHR) was observed on the granule cell membrane after luteinizing hormone (LH) (0.3 IU/mL) was supplied as an exogenous hormone into the cultivation medium during ovarian vitrification in the postnatal period (PND) (1, 7, 14, 21, 28, 42, and 56 days PND). The expression of vascular endothelial growth factor (VEGF) and Connexins (Cx), and the recovery of ovarian functions were then assessed in mice models. The results showed that LH increased the production of normal follicles, and upregulated the expression of VEGF, Cx37, and Cx43 in vitrified ovaries. LH administration also shortened the recovery time of the estrus cycle in mice models. Additionally, no difference was observed in the rate of pregnancy and size of the first litter between the experimental and control groups. In conclusion, LH could promote the survival and functioning of the ovaries by upregulating the expression of VEGF, Cx43, and Cx37 during ovarian cryopreservation and transplantation.
Assuntos
Criopreservação , Hormônio Luteinizante/metabolismo , Ovário/fisiologia , Ovário/transplante , Animais , Feminino , Masculino , Camundongos , Ovário/citologia , Gravidez , TransplanteRESUMO
Pre-ischemic hyperglycemia increases the occurrence of post-ischemic seizures both in experimental and clinical settings. The underlying mechanisms are not fully delineated; however, activation of mammalian target of rapamycin (mTOR) has been shown to be engaged in the pathogenesis of epilepsy, in which seizures are a regular occurrence. Therefore, we wanted to explore specifically the capacity of an mTOR inhibitor, rapamycin, in preventing post-ischemic seizures in hyperglycemic rats and to explore the underlying molecular mechanisms. The results showed that none of the rats in the sham control, EG ischemic, or within 3â¯h of I/R in hyperglycemic ischemic groups experienced seizures. Generalized tonic-clonic seizures were observed in all 8/8 of hyperglycemic ischemic rats at 16â¯h of I/R. Treatment with rapamycin successfully blocked post-ischemic seizures in 7/8 hyperglycemic ischemic animals. Rapamycin also lessened the neuronal death extraordinarily in hyperglycemic ischemic animals as revealed by histopathological studies. Protein analysis revealed that transient ischemia resulted in increases in p-mTOR and p-S6, especially in the hippocampi of the hyperglycemic ischemic rats. Rapamycin treatment completely blocked mTOR activation. Furthermore, hyperglycemic ischemia induced a much prominent rise of p-ERK1/2 both in the cortex and the hippocampi compared with EG counterparts; whereas rapamycin suppressed it. We conclude that the development of post-ischemic seizures in the hyperglycemic animals may be associated with activations of mTOR and ERK1/2 pathways and that rapamycin treatment inhibited the post-ischemic seizures effectively by suppressing the mTOR and ERK1/2 signaling.
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Isquemia Encefálica/tratamento farmacológico , Hiperglicemia/tratamento farmacológico , Sistema de Sinalização das MAP Quinases , Convulsões/prevenção & controle , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Anticonvulsivantes/farmacologia , Morte Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Hiperglicemia/metabolismo , Masculino , Neurônios/metabolismo , Fosforilação , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Background: Alzheimer's disease (AD) is an age-related neurodegenerative disease that is clinically characterized by progressive cognitive decline. Glucagon-like peptide-1 (GLP-1) is a hormone that belongs to the incretin family and is released in response to nutrient intake. It plays a role in maintaining metabolic homeostasis and has been suggested to be involved in maintaining the brain microenvironment. However, the role of GLP-1 in AD pathogenesis has not been fully illustrated. Objective: This study aims to investigate the clinical relevance of GLP-1 in AD and the effects of GLP-1 in amyloid-ß (Aß) metabolism in vitro. Methods: In this study, 39 AD patients and 120 cognitively intact controls were included. Plasma levels of GLP-1 were measured using ELISA. SH-SY5Y cells overexpressing human amyloid precursor protein (APP) were treated with GLP-1. Western blot analysis was used to assess the effects of GLP-1 on the metabolism of Aß. Results: Plasma GLP-1 levels were decreased with aging. Plasma GLP-1 levels were lower in AD patients in comparison with healthy older adults. Plasma GLP-1 levels were positively associated with Mini-Mental State Examination scores but negatively associated with plasma pTau181 levels. GLP-1 dose-dependently increased the area fraction of mitochondrial staining in vitro. Furthermore, GLP-1 dose-dependently promoted the α-cleavage of APP, thus reducing the generation of Aß. Conclusions: GLP-1 has neuroprotective effects in AD, and therefore the decrease in GLP-1 levels during aging might contribute to the development of AD.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Biomarcadores , Peptídeo 1 Semelhante ao Glucagon , Humanos , Peptídeo 1 Semelhante ao Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Doença de Alzheimer/sangue , Masculino , Idoso , Feminino , Biomarcadores/sangue , Peptídeos beta-Amiloides/sangue , Cognição/fisiologia , Idoso de 80 Anos ou mais , Precursor de Proteína beta-Amiloide/sangue , Pessoa de Meia-Idade , Linhagem Celular Tumoral , Proteínas tau/sangue , Testes de Estado Mental e Demência , Envelhecimento/sangueRESUMO
Background and objective: PCOS is a common metabolic disorder in women of reproductive age, which pathogenesis is very complex. The role of ferroptosis in PCOS is a novel finding, and the mechanistic studies are not clear. Metformin is a commonly used drug of PCOS but few studies on whether metformin can improve the follicle development and ovarian function in PCOS. We aims to use PCOS mouse model to study the effect of metformin on PCOS based on the ovarian function and explored the regulation of metformin in PCOS mice by intervening in ferroptosis pathway. Materials and methods: C57 BL/6J female mice aged 4-5 weeks were purchased and gavaged with letrozole (1 mg/kg/day) combined with high-fat diet for 21days to establish PCOS model, and control group was set up. After modeling, the mice were divided into PCOS model group and metformin treatment group (Met) (n=6).The Met group were gavaged metformin (200 mg/kg/day) for 28 days. The body weight, estrous cycle, glucose tolerance test (OGTT)and insulin resistance test (ITT) were monitored. Then, The mice were euthanized to collect serum and ovaries. Elisa was used to detect changes in related serum hormones (E2, LH, FSH, TP). Ovaries used for molecular biology experiments to detect changes in GPX4, SIRT3, AMPK/p-AMPK, and mTOR/p-mTOR by Western blot and qPCR. Results: Compared with the model group mice, body weight was significantly reduced, and their estrous cycle was restored in Met group. The results of OGTT and ITT showed an improvment of glucose tolerance and insulin resistance. Morphological results showed that after metformin treatment, polycystic lesions in ovaries were reduced, the ovarian function was restored, and the expressions of SIRT3 and GPX4 were elevated. WB results demonstrated that the expressions of p-mTOR and p-AMPK in ovaries were significantly reduced in Model group, but reversed in MET group. Conclusion: Our study confirmed metformin could not only improve body weight and metabolism disorders, but also improve ovarian dysfunction in PCOS mice.In addition, we explored metformin could regulate ferroptosis to improve PCOS via the SIRT3/AMPK/mTOR pathway. Our study complements the mechanisms by which metformin improves PCOS.
Assuntos
Ferroptose , Resistência à Insulina , Metformina , Síndrome do Ovário Policístico , Sirtuína 3 , Humanos , Feminino , Camundongos , Animais , Síndrome do Ovário Policístico/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Proteínas Quinases Ativadas por AMP/metabolismo , Peso Corporal , Serina-Treonina Quinases TORRESUMO
INTRODUCTION: Electronic cigarette use has become increasingly popular, with potential consequences for reproductive health. We aimed to investigate the effects of different components of e-liquid on the ovary and compare the impact of low nicotine concentration e-liquids (LN e-liquids) and high nicotine concentration e-liquids (HN e-liquids) on ovarian toxicity. METHODS: A total of 378 rat ovaries were divided into seven groups, including control (no intervention), nicotine (0.05 mg/mL), flavoring (0.25 µL/mL), propylene glycol (PG) (2.5 µL/mL), vegetable glycerin (VG) (2.0 µL/mL), LN e-liquid (0.05 mg nicotine + 0.25 µL flavoring + 2.5 µL PG + 2.0 µL VG + 0.25 µL distilled water/mL medium) and HN e-liquid groups (0.05 mg nicotine + 0.05 µL flavoring + 0.5 µL PG + 0.4 µL VG + 0.05 µL distilled water/mL medium). After three hours of in vitro culture, ovarian morphology, oxidation levels [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA)], and apoptosis levels [factor related apoptosis (Fas), Cyt-c, Caspase-9, Caspase-3] were analyzed. RESULTS: Our findings indicate that nicotine has limited impact on the ovary, while flavoring, PG, and VG all cause ovarian damage including morphological damage, disruption of oxidative balance and promotion of apoptosis, with VG having the most significant effect. Moreover, LN e-liquids may lead to more severe ovarian damage than HN e-liquids at an equal intake of total nicotine. CONCLUSIONS: Our study highlights that in e-liquid formula, nicotine has a limited effect on the ovaries, but flavoring, PG, and VG all cause damage to the ovaries, with VG the most damaging. At a consistent level of total nicotine intake, e-liquids with low nicotine concentrations cause more damage to the ovaries than those with high nicotine concentrations. These findings contribute to a better understanding of the impact of e-liquids on ovarian health and have important implications for public health policy.
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Ovarian tissue transplantation is now considered as a procedure to preserve the fertility of young women patients undergoing cancer therapy. The present study investigated the effects and mechanism of human menopausal gonadotrophin (HMG) intervention on vascular remoulding in ovarian heterotopic autotransplantation. Ovaries of 8-week-old mice were cultured in vitro with different concentrations of HMG for 3h for measuring the expression of vascular endothelial growth factor (VEGF). The cultured ovaries were implanted under the kidney capsule and removed 24, 36, 48 h or 1 month after transplantation. Revascularization, fluid exudation and the number of surviving ovarian follicles were observed. The results showed that VEGF was increased 1.6-6.5 times in the HMG intervention groups. Revascularization appeared 24-36 h after transplantation and was earlier than that of the control. Fluid exudation increased incrementally with increasing HMG concentrations. The total number of surviving ovarian follicles was increased by 1.2-1.5 times in the HMG 0.15 IU/ml group as compared with the other groups 1 month after transplantation. It is concluded that intervention with HMG in vitro before transplantation could improve the blood supply reconstruction and survival of the autotransplanted ovarian follicles, which might be associated with increased VEGF expression.
Assuntos
Menotropinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/irrigação sanguínea , Ovário/transplante , Transplantes , Animais , Antígenos CD34/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Ciclo Estral , Feminino , Camundongos , Camundongos Endogâmicos ICR , Modelos Animais , Neovascularização Fisiológica/fisiologia , Técnicas de Cultura de Órgãos , Folículo Ovariano/fisiologia , Ovário/metabolismo , Transplante Autólogo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Background: Hyperglycemia is one of the major risk factors for stroke and stroke recurrence, leading to aggravated neuronal damage after cerebral ischemia/reperfusion (I/R). ERK1/2 signaling pathway plays a vital role in cerebral ischemic injury. However, the role of the ERK1/2 pathway in hyperglycemia-aggravated ischemic brain damage is not clear. Methods: Streptozotocin (STZ; 50 mg/kg)-induced diabetes (blood glucose ≥12 mmol/L) or control groups in adult Sprague-Dawley rats were further subdivided into I/R (carotid artery/vein clamping), I/R + PD98059 (I/R plus ERK1/2 inhibitor), and Sham-operated groups (n = 10 each). Neurobehavioral status (Neurological behavior scores) and the volume of the cerebral infarction (TTC staining); brain mitochondrial potential (JCI ratio test) and cell apoptosis (TUNEL assay); RAS protein expression, phosphorylated/total ERK1/2 and Drp-1 (Dynamic-related protein 1) protein levels (Western blotting); mitochondrial fusion-related proteins mitofusin-1/2 (Mfn1/2), optic atrophy (OPA-1) and mitochondrial fission 1 (Fis1), and autophagy-associated proteins Beclin-1, LC3-I/II and P62 (Western blotting and immunohistochemistry) were analyzed. Results: The I/R + PD98059 group demonstrated better neurobehavior on the 1st (p < 0.05) and the 3rd day (p < 0.01) than the I/R group. Compared to the Sham group, cerebral ischemia/reperfusion brought about neuronal damage in the I/R group (p <0.01). However, treatment with PD98059 showed an improved situation with faster recovery of mitochondrial potential and less apoptosis of neuronal cells in the I/R + PD98059 group (p < 0.01). The I/R group had a higher-level expression of RAS and phosphorylated ERK1/2 and Drp-1 than the diabetes mellitus (DM) group (p < 0.01). The PD98059 treated group showed decreased expression of p-ERK1/2, p-Drp-1, Fis1, and Beclin-1, LC3-I/II and P62, but increased Mfn1/2 and OPA-1 than the I/R group (p < 0.01). Conclusion: Hyperglycemia worsens cerebral ischemia/reperfusion-induced neuronal damage via ERK1/2 activated cell autophagy and mitochondrial fission.
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Isquemia Encefálica , Hiperglicemia , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Animais , Autofagia , Proteína Beclina-1/metabolismo , Encéfalo/metabolismo , Hiperglicemia/complicações , Sistema de Sinalização das MAP Quinases , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/metabolismo , EstreptozocinaRESUMO
INTRODUCTION: Electronic cigarettes (e-cigarettes) have recently become popular as an alternative to conventional cigarettes. The aim of this study is to investigate the effects of e-cigarette refill liquid (e-liquid) on follicular development and estrogen secretion in rats and whether it is related to the Hippo signaling pathway, a pathway that can regulate follicle growth. METHODS: Ovaries from 21- and 35-day-old rats were divided into three groups: control (no intervention), 0.05 mg, and 0.5 mg (e-liquids containing 0.5 mg and 5 mg of nicotine/kg). The rates were cultured for three hours in vitro. At the end of culture, HE staining was performed to observe the follicle morphology and calculate the percentage of normal follicles, and the expression of Yes-associated protein (YAP, target factors of the Hippo signaling pathway) and CYP19 (aromatase, a key enzyme in estrogen synthesis) were observed by immunohistochemistry. Western blotting was performed to detect the expression levels of CYP19, YAP, phosphorylated YAP (PYAP), large tumor suppressor 2 (LATS2, factors upstream of YAP in the Hippo signaling pathway), and phosphorylated LATS2 (PLATS2). Estrogen concentrations were determined using ELISA. RESULTS: HE staining showed that the percentage of normal follicles decreased, and immunohistochemistry showed that the expression of CYP19 and YAP significantly decreased after e-liquid intervention. ELISA showed that the estrogen concentration in the ovaries decreased after e-liquid intervention. Western blot results indicated that CYP19, LATS2, and YAP expression, decreased after e-liquid intervention, but PLATS2 and PYAP expression increased. CONCLUSIONS: We found that the e-liquids may impair the development of rat ovarian follicles and reduce estrogen secretion through Hippo signaling pathway.
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Chloroform-induced olfactory mucosal degeneration has been reported in adult rats following gavage. We used fixed-point chloroform infusions on different postnatal days (PNDs) to investigate the effects of early olfactory bilateral deprivation on the main olfactory bulbs in Sprague Dawley rats. The experimental groups included rats infused with chloroform (5 µl) or saline (sham, 5 µl) on PNDs 3 and 8, and rats not receiving infusions (control) (n = 6 in all groups). Rats receiving chloroform on PND 3 showed significant hypoevolutism when compared to those in other groups (P < 0.05). There was a complete disappearance and a significant reduction in the size of olfactory glomeruli in the PND 3 and 8 groups, respectively, when compared to the respective sham groups. Rats receiving chloroform on PND 3 had significant memory impairment (P < 0.01) and increased levels of learned helplessness (P < 0.05), as measured using the Morris water maze and tail suspension tests, respectively. GABAA receptor alpha5 subunit (α5GABAAR) expression in hippocampal neurons was significantly lower in rats receiving chloroform on PND 3 than in rats in other groups (P < 0.01), as measured using immunohistochemistry and polymerase chain reaction. There was thus a critical period for the preservation of regenerative ability in olfactory receptor neurons, during which damage and olfactory deprivation led to altered rhinencephalon structure and disappearance of olfactory glomeruli, which induced hypoevolutism. Olfactory deprivation after the critical period had no significant effect on olfactory receptor neuron regeneration, leading to reduced developmental and behavioral effects in Sprague Dawley rats.
Assuntos
Hipocampo/metabolismo , Transtornos do Olfato/patologia , Mucosa Olfatória/patologia , Neurônios Receptores Olfatórios/patologia , Receptores de GABA-A/metabolismo , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Clorofórmio/toxicidade , Depressão/etiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Elevação dos Membros Posteriores , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos do Olfato/induzido quimicamente , Mucosa Olfatória/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Privação Sensorial , Solventes/toxicidadeRESUMO
Hyperglycemia is known to exacerbate neuronal death resulted from cerebral ischemia. The mechanisms are not fully understood. The mammalian target of rapamycin (mTOR) pathway regulates cell growth, division and apoptosis. Recent studies suggest that activation of mTOR may mediate ischemic brain damage. The objective of the present experiment is to explore whether mTOR mediates ischemic brain damage in acute hyperglycemic animals. Rats were subjected to 10 min of forebrain ischemia under euglycemic, hyperglycemic and rapamycin-treated hyperglycemic conditions. The rat brain samples were collected from the cortex and hippocampi after 3h and 16h of reperfusion. The results showed that hyperglycemia significantly increased neuronal death in the cortex and hippocampus and the exacerbation effect of hyperglycemia was associated with further activation of mTOR under control and/or ischemic conditions. Inhibition of mTOR with rapamycin ameliorated the damage and suppressed hyperglycemia-elevated p-MTOR, p-P70S6K and p-S6. In addition, hyperglycemia per se increased the levels of cytosolic cytochrome c and autophagy marker LC3-II, while rapamycin alleviated these alterations. It is concluded that activation of mTOR signaling may play a detrimental role in mediating the aggravating effect of hyperglycemia on cerebral ischemia.
Assuntos
Isquemia Encefálica/prevenção & controle , Hiperglicemia/induzido quimicamente , Transdução de Sinais/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Hiperglicemia/complicações , Imunossupressores/farmacologia , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genéticaRESUMO
BACKGROUD: Ovarian transplantation is a useful method for preserving the fertility of young women with cancer who undergo radiotherapy and chemotherapy. Follicle-stimulating hormone (FSH) is use to protect transplanted ovarian tissues from ischemia injury through promoting revascularization after transplantation, but the side effect of high level FSH is ovarian overstimulation leading to substantial follicular loss. In this study, we investigated the optimal usage of FSH on revascularization in the in vitro cultured ovarian tissues before and after transplantation. RESULTS: FSH mainly exhibited an additive response in the gene and protein expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and follicle stimulating hormone receptor (FSHR) with its raised concentrations (0.15 IU/ml, 0.30 IU/ml and 0.60 IU/ml) and prolonged treatment (3 h, 6 h, 12 h, 24 h). The concentrations with 0.60 IU/ml FSH could obviously promoted the expression of VEGF, bFGF and FSHR, but under this concentration FSH could also overstimulated the ovarian tissue leading to follicular loss. With the increase of culture time, the gene and protein expression of VEGF and bFGF both were up-regulated in all of the FSH added groups, but FSHR expression decreased when culture time exceeded 12 h. So we chose 0.30 IU/ml FSH added concentration and 6 h culture time as the FSH usage condition in functional revascularization verification experiment, and found that under this condition FSH promoted 2.5 times increase of vascular density in treated group than in control group after ovarian tissues transplantation. CONCLUSION: Ovarian intervention with 0.30 IU/ml FSH for 6 h is an optimal FSH usage condition which could accelerate the revascularization in the allotransplanted ovarian tissue and can not produce ovarian overstimulation.
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Hormônio Foliculoestimulante/farmacologia , Neovascularização Fisiológica , Transplante de Órgãos , Ovário/irrigação sanguínea , Ovário/transplante , Animais , Biomarcadores , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Imuno-Histoquímica , Camundongos , Ovário/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Transplante Homólogo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The objectives of the present study are to investigate the activation of mTOR and ERK1/2 signaling after cerebral ischemia in diabetic rats and to examine the neuroprotective effects of rapamycin. Ten minutes transient global cerebral ischemia was induced in straptozotocin-induced diabetic hyperglycemic rats and non-diabetic, euglycemic rats. Brain samples were harvested after 16 h of reperfusion. Rapamycin or vehicle was injected 1 month prior to the induction of ischemia. The results showed that diabetes increased ischemic neuronal cell death and associated with elevations of p-P70S6K and Ras/ERK1/2 and suppression of p-AMPKα. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and suppressed phosphorylation of P70S6K and ERK1/2. It is concluded that diabetes activates mTOR and ERK1/2 signaling pathways in rats subjected to transient cerebral ischemia and inhibition of mTOR by rapamycin reduces ischemic brain damage and suppresses the mTOR and ERK1/2 signaling in diabetic settings.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia/efeitos dos fármacos , Western Blotting , Isquemia Encefálica/sangue , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Marcação In Situ das Extremidades Cortadas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Ratos , Ratos Wistar , Serina-Treonina Quinases TOR/genéticaRESUMO
Ovarian follicular damages were caused by cryoinjury during the process of ovarian vitrification and ischemia/reperfusion during the process of ovarian transplantation. And appropriate FSH plays an important role in antiapoptosis during ovarian follicle development. Therefore, in this study, 0.3 IU/mL FSH was administered into medium during mouse ovarian cryopreservation by vitrification to ascertain the function of FSH on ovarian vitrification and avascular transplantation. The results suggested that the expressions of Cx37, Cx43, apoptotic molecular caspase-3, and angiogenesis molecular VEGF were confirmed using immunohistochemistry, western blotting, and real-time PCR, and the results suggested that the treatment with FSH remarkably increased the number of morphologically normal follicles in vitrified/warmed ovaries by upregulating the expression of Cx37, Cx43, VEGF, and VEGF receptor 2, but downregulating the expression of caspase-3. In addition, the vitrified/warmed ovaries were transplanted, and the related fertility was analyzed, and the results suggested that the fertility, neoangiogenesis, and follicle reserve were remarkably increased in the FSH administrated group. Taken together, administration of 0.3 IU/mL FSH during ovarian cryopreservation by vitrification can maintain ovarian survival during ovarian vitrification and increases the blood supply with avascular transplantation via upregulation of Cx43, Cx37, and VEGF/VEGFR2, as well as through its antiapoptotic effects.
Assuntos
Caspase 3/biossíntese , Conexina 43/biossíntese , Conexinas/biossíntese , Folículo Ovariano/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Conexina 43/genética , Conexinas/genética , Feminino , Hormônio Foliculoestimulante/administração & dosagem , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Vitrificação/efeitos dos fármacos , Proteína alfa-4 de Junções ComunicantesRESUMO
BACKGROUND: Our previous studies suggested that low-dose gossypol combined with steroid hormones has a reversible antifertility role in adult male rats, and the course of treatment was shorter than that of either gossypol or steroid hormones alone. This result suggested that low-dose gossypol and steroid hormones have a drug synergistic effect on antifertility. The aim of the study was to find the target organs of the antifertility synergistic effect of the combined regimen. METHODS: Thirty-two adult male rats were divided into four groups randomly: group GH, rats were fed orally with gossypol acetic acid (GA, 12.5 mg×kg(-1)×d(-1)) and desogestrel (DSG, 0.125 mg×kg(-1)×d(-1))/ethinylestradiol (EE, 0.025 mg×kg(-1)×d(-1))/testosterone undecanoate (TU, 100 mg×kg(-1)×d(-1)); group G, a single dose of GA (12.5 mg×kg(-1)×d(-1)) was given; group H, the same dosage of DSG/EE/TU as in group GH were administered; group C, rats were treated with vehicle (1% methyl cellulose) as control. Testes and epididymis were removed at 8 weeks post-treatment for evaluating their weight, volumes, volume fraction, and total volume of testicular tissue structures and the seminiferous tubule diameter using stereological assay. Sperm cell numbers and the motility of epididymal sperm were quantitated by flow cytometry and morphological methods. RESULTS: Compared with group C, spermatogenesis was normal in group G and suppressed in groups H and GH. Similar changes of testicular tissue structures and sperm number were found in groups H and GH. The decreases of epididymal sperm number and motility in group GH were greater than that of the low-dose gossypol or steroid hormones alone group. CONCLUSIONS: The suppression of spermatogenesis was induced by steroid hormones in the combined regimen, and the epididymis was the target organ of low-dose gossypol. Combined use of low-dose gossypol and steroid hormones played a comprehensive antifertility role in their synergistic effect on reducing the number and motility of epididymal sperm.