RESUMO
Prenatal exposure to glucocorticoids (GC) is a central topic of interest in medicine since GCs are essential for the maturation of fetal organs and intrauterine growth. Synthetic glucocorticoids, which are used in obstetric practice, exert beneficial effects on the fetus, but have also been reported to lead to intrauterine growth retardation (IUGR). In this study, a model of growth restriction in mice was established through maternal administration of dexamethasone during late gestation. We hypothesised that GC overexposure may adversely affect placental angiogenesis and fetal and placental growth. Female BALB/c mice were randomly assigned to control or dexamethasone treatment, either left to give birth or euthanised on days 15, 16, 17 and 18 of gestation followed by collection of maternal and fetal tissue. The IUGR rate increased to 100% in the dexamethasone group (8 mg/kg body weight on gestational days 14 and 15) and pups had clinical features of symmetrical IUGR at birth. Dexamethasone administration significantly decreased maternal body weight gain and serum corticosterone levels. Moreover, prenatal dexamethasone treatment not only induced fetal growth retardation but also decreased placental weight. In IUGR placentas, VEGFA protein levels and mRNA expression of VEGF receptors were reduced and NOS activity was lower. Maternal dexamethasone administration also reduced placental expression of the GC receptor, αGR. We demonstrated that maternal dexamethasone administration causes fetal and placental growth restriction. Furthermore, we propose that the growth retardation induced by prenatal GC overexposure may be caused, at least partially, by an altered placental angiogenic profile.
Assuntos
Dexametasona , Retardo do Crescimento Fetal/metabolismo , Placenta/metabolismo , Placentação , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Retardo do Crescimento Fetal/induzido quimicamente , Retardo do Crescimento Fetal/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Placenta/fisiopatologia , Gravidez , Receptores de Glucocorticoides/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 µg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 µM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5ß1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 µM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 µM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5ß1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.
Assuntos
Bovinos , Endocanabinoides/metabolismo , Fibronectinas/farmacologia , Preservação do Sêmen/veterinária , Capacitação Espermática/efeitos dos fármacos , Animais , Criopreservação/veterinária , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocanabinoides/genética , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Masculino , Óxido Nítrico , Motilidade dos EspermatozoidesRESUMO
The endocannabinoid system consists in a family of lipids that binds to and activates cannabinoid receptors. There are two receptors so far described, the cannabinoid receptor 1 (CB1) and 2 (CB2). In the context of pregnancy, the endocannabinoid system was shown participates in different key aspects of reproductive events. B-lymphocytes are pleiotropic cells belonging to the adaptive arm of the immune system. Besides immunoglobulin production, B-lymphocytes were recently shown to be actively involved in antigen presentation as well as cytokine production, thus playing a central role in immunity. In this study we first aimed to characterize the expression of CB1 and CB2 receptors in B cells during pregnancy and then analyze the impact of their activation in term of cytokine production by B cells from pregnant and non-pregnant mice. We observed that the expression of CB1 and CB2 receptors in B-lymphocytes is differentially regulated during pregnancy. While CB2 expression is down regulated CB1 is augmented in B-lymphocytes of pregnant mice. Additionally, the treatment of activated B-lymphocytes with specific CB1 and CB2 agonists, showed a different response in term of cytokine production. Particularly, CB1 against boosted the production of the anti-inflammatory cytokine IL-10 by activated B-lymphocytes from pregnant mice.
Assuntos
Linfócitos B/imunologia , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Interleucina-10/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Gravidez , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genéticaRESUMO
INTRODUCTION: Symptomatic or asymptomatic Shiga toxin producing Escherichia coli (STEC) infections during early pregnancy may cause maternal or fetal damage mediated by Shiga toxin type 2 (Stx2). The aim of this study is to elucidate the mechanisms responsible for early pregnancy loss in rats treated with Stx2. METHODS: Sprague Dawley pregnant rats were intraperitoneally injected at day 8 of gestation with a sublethal dose (0.5 ng of Stx2/g of total body weight, 250 µl) of purified Stx2. Control rats were injected with the same volume of PBS. The expression of globotriaosylceramide (Gb3) glycosphingolipid receptor for Stx2 was evaluated by thin-layer chromatography (TLC). Regions of hypoxia in decidual tissue were determined by pimonidazole immunohistochemistry and vascular endothelial growth factor (VEGF) expression by Western blot and immunohistochemistry. Tumor necrosis factor-alpha (TNF-α) levels in serum and decidual tissue were evaluated by ELISA. Serum progesterone levels were determined by RIA. RESULTS: Decidual tissue from both, control and Stx2-treated rats showed similar expression of Gb3 receptor. Intrauterine growth restriction was observed in Stx2-treated rats, associated with hypoxia and an increase of decidual TNF-α levels. Decrease of serum progesterone levels and decidual VEGF expression were also demonstrated. DISCUSSION: Our findings indicate that Stx2 reaches the uteroplacental unit, binds Gb3 and triggers damage in decidual tissue. Poor oxygen supply accompanied with damage in the uteroplacental unit and inflammation could be responsible for the early pregnancy loss. Decrease in the pregnancy protective factors, serum progesterone and local VEGF, may contribute to the pregnancy loss.
Assuntos
Aborto Espontâneo/patologia , Hipóxia/patologia , Inflamação/patologia , Toxina Shiga II , Aborto Espontâneo/induzido quimicamente , Aborto Espontâneo/metabolismo , Animais , Feminino , Hipóxia/metabolismo , Inflamação/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Triexosilceramidas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND AND PURPOSE: Uveitis is a prevalent intraocular inflammatory disease and one of the most damaging ocular conditions. Pretreatment with melatonin prevented ocular inflammation induced by an intravitreal injection of bacterial LPS in the Syrian hamster. Here, we have assessed the anti-inflammatory effects of melatonin administered after the onset of ocular inflammation. EXPERIMENTAL APPROACH: The eyes of male Syrian hamsters were intravitreally injected with vehicle or LPS. Melatonin was injected i.p. every 24 h, starting 12 or 24 h after the LPS injection. A clinical evaluation (with a score index based on clinical symptoms), the number of infiltrating cells, protein concentration and PGE2 and PGF2α levels in the aqueous humour, as well as retinal NOS activity, lipid peroxidation and TNF-α levels were assessed. Retinal function was assessed by scotopic electroretinography, and light microscopy and immunohistochemistry were used to evaluate the state of the retinal structure. KEY RESULTS: Both treatment regimens with melatonin decreased clinical symptoms, reduced the leakage of cells and proteins, and decreased PG levels in aqueous humour from eyes injected with LPS. In addition, melatonin treatment blocked the decrease in scotopic electroretinogram a- and b-wave amplitude, protected the retinal structure and reduced the increase in NOS activity, lipid peroxidation and TNF-α levels, induced by LPS. CONCLUSIONS AND IMPLICATIONS: These results indicate that treatment with melatonin, starting after the onset of uveitis, attenuated ocular inflammation induced by LPS in the Syrian hamster and support the use of melatonin as a therapeutic resource for uveitis treatment.
Assuntos
Antioxidantes/farmacologia , Humor Aquoso/efeitos dos fármacos , Melatonina/farmacologia , Retina/efeitos dos fármacos , Uveíte/metabolismo , Animais , Humor Aquoso/metabolismo , Cricetinae , Dinoprosta/imunologia , Dinoprosta/metabolismo , Dinoprostona/imunologia , Dinoprostona/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Imuno-Histoquímica , Injeções Intravítreas , Peroxidação de Lipídeos/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Masculino , Mesocricetus , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Retina/imunologia , Retina/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Uveíte/induzido quimicamente , Uveíte/imunologiaRESUMO
Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.
Assuntos
Apoptose/efeitos dos fármacos , Linfoma de Células T/metabolismo , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Proteína Quinase C/genética , Tiroxina/farmacologia , Animais , Anexina A5 , Linhagem Celular Tumoral , Proliferação de Células , Corantes , Fragmentação do DNA/efeitos dos fármacos , Regulação da Expressão Gênica , Glutationa/metabolismo , Linfoma de Células T/genética , Linfoma de Células T/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Propídio , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Quinase C/metabolismo , Proteólise/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
INTRODUCTION: The decidual reaction and the formation of new vessels in the uterus are two crucial processes during embryo implantation. Previously, we observed that lysophosphatidic acid (LPA) increases cyclooxygenase-2 derived - prostaglandin E2 production during implantation in the rat uterus and that it augments the expression of decidualization (IGFBP-1) and vascularization (IL-10) markers. Both cyclooxygenase and nitric oxide synthase (NOS) are known enzymes involved in these processes. Thus, we became interested in studying which factors contribute to LPA receptor-specific role during the decidual and the vascular reaction at implantation. METHODS: We adopted a pharmacological approach in vitro incubating the uterus from rats on day 5 of gestation (day of implantation) with LPA, DGPP (a highly selective antagonist of LPA3, an LPA receptor) and cyclooxygenase and NOS selective and non-selective inhibitors. We determined NOS activity, prostaglandin E2 production and IGFBP-1 and IL-10 expression to evaluate decidualization and vascularization. RESULTS: We observed that LPA augmented the activity of the inducible NOS isoform through LPA1/LPA3. Inducible NOS activity participated in the induction of cyclooxygenase-2/prostaglandin E2 increase stimulated by LPA. Also, cyclooxygenase-2 derived prostaglandins mediated LPA-stimulatory action on NOS activity. Both cyclooxygenase-2 and inducible NOS mediated LPA effect on IGFBP-1 and IL-10 expression. CONCLUSIONS: These results suggest the participation of LPA/LPA3 in the production of crucial molecules involved in vascularization and decidualization, two main processes that prepare the uterine milieu for embryo invasion during implantation.
Assuntos
Decídua/irrigação sanguínea , Implantação do Embrião , Lisofosfolipídeos/metabolismo , Placentação , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Útero/irrigação sanguínea , Animais , Biomarcadores/metabolismo , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Decídua/citologia , Decídua/efeitos dos fármacos , Decídua/metabolismo , Dinoprostona/metabolismo , Implantação do Embrião/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Lisofosfolipídeos/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Gravidez , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Útero/citologia , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
Anandamide (AEA) is a lipid mediator that participates in the regulation of several reproductive functions. This study investigated the endocannabinoid system in normal (NP) and preeclamptic (PE) placentas, and analyzed the potential functional role of AEA in the regulation of nitric oxide synthesis. The protein expression and localization of NAPE-PLD, FAAH and CB1 receptor were analyzed in normal and preeclamptic pregnancies using immunoblotting and immunohistochemistry. NAPE-PLD expression was shown to be significantly higher (p < 0.05) in PE tissues than in NP. In contrast, a decrease in FAAH protein (p < 0.001) was detected in placentas collected from women with preeclampsia. Both enzymes were mainly located in the syncytiotrophoblasts from normal and preeclamptic tissues. No differences were seen in CB1 receptor from both groups of placental villous. Exogenous and endogenous AEA significantly increased NOS activity. Although pre-incubation with AM251 (CB1 antagonist) had no effect, co-incubation with both AEA and AM251 diminished NOS activity from normal term placentas. We observed increased NOS activity in placental villous from women with preeclampsia compared with normotensive pregnant women. Furthermore, NOS activity from preeclamptic tissues was diminished by co-treatment with AM251, illustrating that the NO levels could be modulated by AEA. These data suggest that AEA may be one of the factors involved in the regulation of NOS activity in normal and preeclamptic placental villous. Interestingly, the differential expression of NAPE-PLD and FAAH suggests that AEA could play an important role in the pathophysiology of PE.
Assuntos
Endocanabinoides/metabolismo , Óxido Nítrico/biossíntese , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Receptores de Canabinoides/metabolismo , Adulto , Amidoidrolases/metabolismo , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Feminino , Humanos , Óxido Nítrico Sintase/metabolismo , Fosfolipase D/metabolismo , Placenta/efeitos dos fármacos , Placenta/patologia , Alcamidas Poli-Insaturadas/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Pré-Eclâmpsia/patologia , Gravidez , Distribuição Tecidual , Adulto JovemRESUMO
BACKGROUND AND PURPOSE: Maternal infections are one of the main causes of adverse developmental outcomes including embryonic resorption and preterm labour. In this study a mouse model of inflammation-associated preterm delivery was developed, and used to study the relationship between nitric oxide (NO) and prostaglandins (PGs). EXPERIMENTAL APPROACH: The murine model of preterm labour was achieved by assaying different doses of bacterial lipopolysaccharides (LPS). Once established, it was used to analyse uterine levels of prostaglandins E(2) and F(2α) (by radioimmunoassay), cyclooxygenases (COX) and NOS proteins (by Western blot) and NO synthase (NOS) activity. Effects of inhibitors of COX and NOS on LPS-induced preterm labour were also studied. In vitro assays with a nitric oxide donor (SNAP) were performed to analyse the modulation of prostaglandin production by NO. KEY RESULTS: Lipopolysaccharide increased uterine NO and PG synthesis and induced preterm delivery. Co-administration of meloxicam, a cyclooxygenase-2 inhibitor, or aminoguanidine, an inducible NOS inhibitor, prevented LPS-induced preterm delivery and blocked the increase in PGs and NO. Notably, the levels of NO were found to determine its effect on PG synthesis; low concentrations of NO reduced PG synthesis whereas high concentrations augmented them. CONCLUSIONS AND IMPLICATIONS: An infection-associated model of preterm labour showed that preterm delivery can be prevented by decreasing PG or NO production. NO was found to have a dual effect on PG synthesis depending on its concentration. These data contribute to the understanding of the interaction between NO and PGs in pregnancy and parturition, and could help to improve neonatal outcomes.
Assuntos
Dinoprosta/biossíntese , Dinoprostona/biossíntese , Óxido Nítrico/metabolismo , Trabalho de Parto Prematuro/fisiopatologia , Animais , Western Blotting , Inibidores de Ciclo-Oxigenase 2/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Guanidinas/farmacologia , Inflamação/fisiopatologia , Lipopolissacarídeos/toxicidade , Meloxicam , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase/metabolismo , Trabalho de Parto Prematuro/etiologia , Gravidez , Radioimunoensaio , Tiazinas/farmacologia , Tiazóis/farmacologia , Útero/metabolismoRESUMO
In women, the association between chronic marijuana smoking and early miscarriage has long been known. Anandamide, a major endocannabinoid, mimics some of the psychotropic, hypnotic and analgesic effects of Delta(9)-tetrahydrocannabinol, the psychoactive component of marijuana. The uterus contains the highest concentrations of anandamide yet discovered in mammalian tissues and this suggests that it might play a role in reproduction. The production of small amounts of nitric oxide (NO) regulates various physiological events including implantation and myometrial relaxation, but in an inflammatory setting such as sepsis, NO has toxic effects as it is a free radical. The results presented in this study indicate that anandamide modulates NO production induced by lipopolysaccharide (LPS) in an in-vitro murine model. It was shown that LPS-induced NO synthesis and tissue damage were mediated by anandamide, as a cannabinoid receptor type I antagonist could block the effect of LPS (P < 0.001). This endotoxin inhibited anandamide uterine degradation (P < 0.05) and increased the expression of one of its synthesizing enzymes (P < 0.05). Contrary to the known anti-inflammatory and protective effects, in this model anandamide seems to act as a pro-inflammatory molecule modulating the production of NO induced by LPS. This proinflammatory effect of anandamide may be implicated in pathological reproductive events such as septic abortion.