Efeito redox ativo da vitamina D em coração, fígado e cérebro de ratos

A vitamina D é um pró-hormônio que, além de desempenhar um papel essencial na homeostase do cálcio e do metabolismo mineral ósseo, recentemente tem sido relacionada a uma grande variedade de funções biológicas, tais como a regulação da proliferação e da diferenciação celular, a modulação imunológica, o desenvolvimento cerebral e a redução do risco de desenvolvimento de alterações cardiovasculares. Por outro lado, existe atualmente um descontrole na utilização de suplementação vitamínica, incluindo de vitamina D, o que pode levar a intoxicações, cujos efeitos celulares ainda são pouco estudados. Nesse sentido, o objetivo deste estudo foi verificar o efeito de diferentes doses e concentrações de colecalciferol e calcitriol em estudos in vivo e in vitro, respectivamente, sobre parâmetros de estresse oxidativo em coração, fígado e cérebro de ratos. Inicialmente, verificamos se a administração sistêmica de vitamina D, por 21 dias, afeta as defesas antioxidantes e os parâmetros de dano oxidativo em ratos Wistar. Foi utilizada suplementação de vitamina D3 em uma dose limítrofe e uma dose suprafisiológica (5.000 ou 30.000 UI/Kg/dia, respectivamente). Nossos dados demonstram que a suplementação de vitamina D3 possui efeitos tóxicos quando administrada em doses suprafisiológicas, e parece ser segura quando em doses limítrofes. O músculo cardíaco apresentou apenas uma redução dos produtos de lipoperoxidação em ratos tratados com a dose de 30.000 UI/Kg/dia, enquanto a menor dose não alterou nenhum dos parâmetros avaliados. O fígado apresentou um aumento nos níveis de carbonilas em ambas às concentrações, bem como um desequilíbrio na atividade antioxidante enzimática em animais tratados com a dose mais alta. O cérebro, especialmente o córtex, foi afetado em animais tratados com a dose de 30.000 UI/Kg/dia, apresentando um desequilíbrio nas defesas antioxidantes, apesar de termos verificado uma redução na oxidação de lipídeos e proteínas em córtex e hipocampo. Nosso próximo objetivo foi detectar a contribuição do 1α,25(OH)2D, o metabólito mais ativo da vitamina D, sobre as alterações causadas pela administração da vitamina D no status redox dos mesmos tecidos. Foi avaliado o efeito da incubação, à 37ºC em banho metabólico, de fatias de coração, fígado, córtex cerebral e hipocampo provenientes de ratos saudáveis, em meio contendo calcitriol (1α,25(OH)2D) nas concentrações de 50-1.000 nM. Nossos resultados mostraram, que mesmo altas concentrações de 1α,25(OH)2D protegem o tecido cardíaco do dano oxidativo e incrementam o status antioxidante, enquanto o fígado foi pouco afetado. O cérebro sofreu um significante desequilíbrio nos parâmetros de estado redox, evidenciadas por alterações oxidativas em lipídeos e na modulação da atividade de enzimas antioxidantes. Nossos dados permitem concluir que a 1α,25(OH)2D modula o estado redox de forma diferente no cérebro, fígado e coração. Em adição, os efeitos verificados in vivo, por meio da administração da vitamina D3, são conflitantes quando comparados com os dados obtidos pela ação direta do calcitriol, indicando que possivelmente os efeitos sistêmicos da vitamina D não são atribuídos apenas ao 1α,25(OH)2D, e podem ser o resultado de um efeito combinado dos metabólitos da vitamina D. Os achados aqui expostos trazem um importante alerta em relação à suplementação com vitamina D, que em altas doses pode causar alterações oxidativas em cérebro e fígado.Vitamin D is a prohormone that plays an essential role in calcium homeostasis and bone mineral metabolism, however, is now recognized as part of a wide variety of biological functions, such as regulating cell differentiation and proliferation, modulating immune system and brain development, as well as reducing the risk of cardiovascular diseases. Nowadays, the use of vitamin supplementation is unmanageability, which can lead to poisoning, whose cellular effects are still poorly understood. Accordingly, the objective of this study was to investigate the effect of different doses and concentrations of cholecalciferol and calcitriol (1α,25(OH)2D) in experimental models in vivo and in vitro, respectively, on parameters of oxidative stress in heart, liver, and brain of rats. Initially, we determine whether systemic administration of vitamin D, for 21 days, affects antioxidant defenses and oxidative damage parameters in treated-animal tissues. We used vitamin D3 supplementation in a borderline dose and in a supraphysiological doses (5,000 or 30,000 IU/kg/day, respectively). Our data demonstrate that supplementation with vitamin D3 have toxic effects when given in supraphysiological doses, and appears to be safe when the lowest doses were adopted. The cardiac muscle showed only a reduction in lipid peroxidation products, in rats treated with a dose of 30,000 IU/kg/day, while the administration of 5,000 IU/kg/day did not change any of the parameters evaluated. The liver showed increased levels of carbonyls at both concentrations, as well as an imbalance in the antioxidant enzymes network, in animals treated with the highest dose. The brain, especially the cortex, was affected in animals treated with the dose of 30000 IU/Kg/day, an imbalance in the antioxidant defenses was found, although we have observed a reduction in oxidation of lipids and proteins in cortex and hippocampus. Our next goal was to detect the contribution of1α,25(OH)2D, the more active metabolite of vitamin D, on the metabolic changes caused by vitamin D administration on the redox status of heart, liver, and brain. We evaluated the effect of incubation (37 ° C in a metabolic water bath) of tissue slices from heart, liver, cerebral cortex, and hippocampus obtained from healthy rats in a medium containing calcitriol at a final concentrations of 50-1000 nM. Our results showed that even high concentrations of 1α,25(OH)2D protected cardiac muscle from oxidative damage and increased the antioxidant status, while the liver was unaffected. The brain has suffered a significant imbalance in the redox state parameters, evidenced by oxidative changes in lipids and the modulations of antioxidant enzymes activities. In conclusion, our data shows that 1α,25(OH)2D modulates the redox state differently in the brain, liver and heart. In addition, the effects observed in vivo, by administration of vitamin D3, are conflicting when compared with results obtained by the direct action of the vitamin most active metabolite, calcitriol, possibly indicating that the systemic effects of vitamin D are not allocated only to calcitriol, and may be the result of the combined effect of vitamin D metabolites. The findings shown here bring us an important warning, regarding to vitamin D supplementation, which in high doses can cause oxidative changes in brain and liver

Cerebral ketone body oxidation is facilitated by a high fat diet enriched with advanced glycation end products in normal and diabetic rats

Diabetesmellitus(DM)causesimportantmodificationsintheavailabilityanduseofdifferentenergysubstratesinvariousorgansandtissues.Similarly,dietarymanipulationssuchashighfatdietsalsoaffectsystemicenergymetabolism.However,howthebrainadaptstothesesituationsremainsunclear.Toinvestigatetheseissues,controlandalloxan-inducedtypeIdiabeticratswerefedeitherastandardorahighfatdietenrichedwithadvancedglycationendproducts(AGEs)(HAGEdiet).TheHAGEdietincreasedtheirlevelsofbloodketonebodies,andthiseffectwasexacerbatedbyDMinduction.Todeterminetheeffectsofdietand/orDMinductiononkeycerebralbioenergeticparameters,bothketonebodies(b-hydroxybutyricacid)andlactateoxidationweremeasured.Inparallel,theexpressionofMonocarboxylateTransporter1(MCT1)and2(MCT2)isoformsinhippocampalandcorticalslicesfromratssubmittedtothesedietswasassessed.KetonebodyoxidationincreasedwhilelactateoxidationdecreasedinhippocampalandcorticalslicesinbothcontrolanddiabeticratsfedaHAGEdiet.Inparallel,theexpressionofbothMCT1andMCT2increasedonlyinthecerebralcortexindiabeticratsfedaHAGEdiet.TheseresultssuggestashiftinthepreferentialcerebralenergysubstrateutilizationinfavorofketonebodiesinanimalsfedaHAGEdiet,aneffectthat,inDManimals,isaccompaniedbytheenhancedexpressionoftherelatedtransporters

Can glutathione be a biomarker for suicide risk in women 18 months postpartum?

Background: Suicide risk is prominent among the problems affecting populations, mainly due to the broad family, psychosocial and economic impact. Most individuals at suicidal risk have some mental disorder. There is considerable evidence that psychiatric disorders are accompanied by the activation of neuro-immune and neuro-oxidative pathways. The aim of the study is to evaluate the serum levels of oxidative stress biomarkers in women at risk of suicide after 18 months of postpartum. Methods: This is a case-control study, nested within a cohort study. From this cohort, 45 women [15 without mood disorders and 30 with mood disorders (Major depression and Bipolar disorder)] were selected at 18 months postpartum, the depression and suicide risk were assessed using the MiniInternational Neuropsychiatric Interview Plus (MINI-Plus) instrument, module A and C, respectively. Blood was collected and stored for later analysis of the reactive species (DCFH), superoxide dismutase (SOD), and glutathione reduced (GSH). For data analysis, the SPSS program was used. To compare the nominal covariates with the outcome GSH levels, the Student’s t-test or analysis of variance (ANOVA) was used. Spearman’s correlation was performed for analysis between the quantitative covariates and the outcome. To analyze the interaction between the factors, multiple linear regression was performed. Bonferroni analysis was used as an additional/secondary result to visualize differences in glutathione levels according to risk severity. After the adjusted analysis, p-values < 0.05 were considered statistically significant. Results: The percentage of suicide risk observed in our sample of women at 18 months postpartum was 24.4% (n = 11). After adjusting for the independent variables, only the presence of suicide risk remained associated with the outcome (β = 0.173; p = 0.007), low levels of GSH at 18 months after postpartum. Likewise, we verified the difference in GSH levels according to the degree of suicide risk, observing a significant association between the differences in glutathione means in the group of women with moderate to high risk compared to the reference group (no suicide risk) (p = 0.009). Conclusion: Our findings suggest that GSH may be a potential biomarker or etiologic factor in women at moderate to high risk of suicide

Avaliação dos efeitos da homocisteína em tecidos cardíacos e cerebral (ex vivo) e em cultura de astrócitos adultos : possível papel protetor da vitamina D

As hiperhomocisteinemias (HHcy), leve e moderada são consideradas um fator de risco para doenças cardiovasculares e cerebrais, entretanto os mecanismos e as complicações decorrentes dessa condição ainda não estão bem estabelecidos. Ela ocorre em 5-10% da população geral e em 40% dos pacientes com doenças vascular periférica e doenças cerebrovasculares. Estudos recentes têm demonstrado que a vitamina D (calcitriol) possui efeitos protetores em diversos modelos experimentais que enfatizam suas possíveis ações antioxidantes. O objetivo principal dessa tese de doutorado foi estabelecer um protocolo experimental com diferentes concentrações de homocisteína em fatias de cortex cerebral e coração e em cultura de astrócitos de ratos adultos. Seguindo esses modelos experimentais, investigamos alguns parâmetros bioquímicos em córtex cerebral e coração de ratos. Posteriormente foi analisado o efeito protetor do calcitriol. No primeiro capítulo da presente tese, observamos que a incubação de 30 μM de Hcy por 30 min e 60 min em fatias de coração, alterou a função e a permeabilidade mitocondrial, o estado redox e a atividade das enzimas da cadeia respiratória; o calcitriol foi capaz de prevenir a maioria dos efeitos da Hcy. No segundo capítulo, vimos que em fatias de córtex cerebral a Hcy prejudica o metabolismo energético, aumentando a morte neuronal e induzindo estresse oxidativo. Todavia, o calcitriol atenuou esses efeitos deletérios induzidos pela Hcy através da ativação do receptor de vitamina D. No último capítulo desta tese, realizamos um estudo em cultura primária de astrócitos corticais de ratos Wistar adultos. Nossos resultados demonstram que a Hcy ativa a via do fator nuclear kappa B (NFκB), inibindo a expressão de heme oxigenase 1 (HO-1), promovendo alterações morfológicas, aumentando a resposta inflamatória e diminuindo as defesas antioxidantes e a atividade da Na+, K+ - ATPase. Em resumo, em todos modelos experimentais estudados nesta tese, a Hcy, mesmo em concentrações leves e moderadas causou alterações na homeostasia celular. A vitamina D preveniu parte destes efeitos, tornando-se um possível ferramenta terapêutica no intuito de atenuar os efeitos da Hcy.Hyperhomocysteinemia (HHcy), mild and moderate are a risk factor for cardiovascular and cerebral diseases, but the mechanisms and complications of this condition are not yet well established. It occurs in 5-10% of the general population and 40% of patients with peripheral vascular and cerebrovascular disease. Recent studies have shown that vitamin D (calcitriol) has protective effects in various experimental models which emphasize their potential antioxidant actions. The main objective of this PhD thesis was to establish an experimental with different concentrations of homocysteine in slices of cerebral cortex and heart in adult rat astrocyte cultures. Following this experimental model, we investigated some biochemical parameters in the cerebral cortex and heart of rats. It was subsequently examined the protective effect of calcitriol. In the first chapter of this thesis, we found that incubation of 30 μM of Hcy for 30 min and 60 min in heart slices change the function and mitochondrial permeability, redox state and activity of the enzymes of the respiratory chain; calcitriol was able to prevent most of the effects of Hcy. In the second chapter, we demonstrated that Hcy in the cerebral cortex slices impairs energy metabolism, increasing neuronal death and inducing oxidative stress. However, calcitriol attenuated these Hcy-induced deleterious by activation of vitamin receptor D. In the last chapter of this thesis, we conducted a study in primary culture of cortical astrocytes. Our results demonstrate that the Hcy active the pathway of nuclear factor kappa B (NFκB) inhibiting heme oxygenase expression 1 (HO-1), promoting morphological changes, increasing the inflammatory response and decreased antioxidant defenses and activity of the Na +, K + - ATPase. In summary, in all experimental models studied in this PhD thesis, Hcy, even in mild and moderate concentrations caused deleterious actions in cellular homeostasis. Vitamin D warned of these effects, becoming a potential therapeutic target in order to attenuate the effects of Hcy

Homocysteine induces glial reactivity in adult rat astrocyte cultures

Astrocytes are dynamic glial cells associated to neurotransmitter systems, metabolic functions, antioxidant defense, and inflammatory response, maintaining the brain homeostasis. Elevated concentrations of homocysteine (Hcy) are involved in the pathogenesis of age-related neurodegenerative disorders, such as Parkinson and Alzheimer diseases. In line with this, our hypothesis was that Hcy could promote glial reactivity in a model of cortical primary astrocyte cultures from adult Wistar rats. Thus, cortical astrocytes were incubated with different concentrations of Hcy (10, 30, and 100 μM) during 24 h. After the treatment, we analyzed cell viability, morphological parameters, antioxidant defenses, and inflammatory response. Hcy did not induce any alteration in cell viability; however, it was able to induce cytoskeleton rearrangement. The treatment with Hcy also promoted a significant decrease in the activities of Na+, K+ ATPase, superoxide dismutase (SOD), and glutathione peroxidase (GPx), as well as in the glutathione (GSH) content. Additionally, Hcy induced an increase in the pro-inflammatory cytokine release. In an attempt to elucidate the putative mechanisms involved in the Hcy-induced glial reactivity, we measured the nuclear factor kappa B (NFκB) transcriptional activity and heme oxygenase 1 (HO-1) expression, which were activated and inhibited by Hcy, respectively. In summary, our findings provide important evidences that Hcy modulates critical astrocyte parameters from adult rats, which might be associated to the aging process

1,25-dihydroxyvitamin D3 exerts neuroprotective effects in an ex vivo model of mild hyperhomocysteinemia

Elevated plasma homocysteine (Hcy) levels have been detected in patients with various neurodegenerative conditions. Studies of brain tissue have revealed that hyperhomocysteinemia may impair energy metabolism, resulting in neuronal damage. In addition, new evidence has indicated that vitamin D plays crucial roles in brain development, brain metabolism and neuroprotection. The aim of this study was to investigate the neuroprotective effects of 1,25-dihydroxivitamin D3 (calcitriol) in cerebral cortex slices that were incubated with a mild concentration of Hcy. Cerebral cortex slices from adult rats were first pre-treated for 30 min with one of three different concentrations of calcitriol (50 nM, 100 nM and 250 nM), followed by Hcy for 1 h to promote cellular dysfunction. Hcy caused changes in bioenergetics parameters (e.g., respiratory chain enzymes) and mitochondrial functions by inducing changes in mitochondrial mass and swelling. Here, we used flow cytometry to analyze neurons that were double-labelled with Propidium Iodide (PI) and found that Hcy induced an increase in NeuN+/PI cells but did not affect GFAP+/Pi cells. Hcy also induced oxidative stress by increasing reactive oxygen species generation, lipid peroxidation and protein damage and reducing the activity of antioxidant enzymes (e.g., SOD, CAT and GPx). Calcitriol (50 nM) prevented these alterations by increasing the level of the vitamin D receptor. Our findings suggest that using calcitriol may be a therapeutic strategy for treating the cerebral complications caused by Hcy