Efeitos do extrato de mirtilo em modelo de hipertensão arterial pulmonar induzida por monocrotalina

Introdução: A hipertensão arterial pulmonar (HAP) é uma doença caracterizada por disfunção endotelial progressiva e remodelamento vascular, levando ao aumento da resistência vascular pulmonar e à insuficiência do ventrículo direito (VD). Diversos elementos contribuem para a sua fisiopatologia, como o desequilíbrio entre vasodilatadores e vasoconstritores, maior ativação simpática e, como um dos elementos centrais, o estabelecimento do quadro de estresse oxidativo. Nesse contexto, o uso de antioxidantes naturais pode auxiliar a restaurar a homeostase redox e proteger os tecidos diretamente afetados na patologia, pulmões e VD. Assim, o mirtilo (blueberry - BB), um dos alimentos com maior capacidade antioxidante e tendo apresentado benefícios em outras condições patológicas, surge como uma alternativa de intervenção na HAP. Objetivos: Esse trabalho teve como finalidade avaliar os efeitos do extrato de mirtilo sobre os pulmões e VD em um modelo in vivo de HAP induzida por monocrotalina, verificando sua atuação sobre parâmetros morfo-funcionais, bem como sua modulação do estado redox. Além disso, para avaliar o papel do extrato sobre possíveis vias envolvidas nas alterações do equilíbrio redox e na morte/sobrevivência de cardiomiócitos do VD, o desenvolvimento de um modelo in vitro de lesão de cardiomiócitos induzida por noradrenalina também foi alvo desse estudo. Material e Métodos: Ratos Wistar machos (200 ± 20 gramas, n = 72) foram divididos em oito grupos: Grupo Controle (CTR); Grupo Controle BB 50 mg/kg/dia (CTR BB50); Grupo Controle BB 100 mg/kg/dia (CTR BB100); Grupo Controle BB 200 mg/kg/dia (CTR BB200); Grupo Monocrotalina (MCT); Grupo Monocrotalina BB 50 mg/kg/dia (MCT BB50); Grupo Monocrotalina BB 100 mg/kg/dia (MCT BB100) e Grupo Monocrotalina BB 200 mg/kg/dia (MCT BB200). A HAP foi induzida por dose única de monocrotalina (60 mg / kg, intraperitoneal). Os animais receberam extrato de mirtilo oralmente, por gavagem, nas doses de 50, 100 e 200 mg/kg/dia. O tratamento com extrato de mirtilo durou 5 semanas (2 semanas anterior à indução por MCT e 3 semanas pós-indução). Ao 35º dia do protocolo experimental, os animais foram submetidos às análises morfométricas, ecocardiográficas e hemodinâmicas. Em seguida, os animais sofreram eutanásia, com coleta dos pulmões e coração para posteriores avaliações bioquímicas do equilíbrio redox. Cardiomiócitos (células H9c2) foram cultivados em quatro condições: meio de cultura com ausência de noradrenalina (NE) e extrato de mirtilo (BBE): grupo Controle; meio na presença de 50 μg/ml de extrato de mirtilo: grupo BBE; meio na presença de 100 μM de noradrenalina: grupo NE; e meio na presença de 50 μg/ml de extrato de mirtilo e 100 μM de noradrenalina: grupo BBE+NE. Foram avaliadas a viabilidade celular, a ativação de caspases, o nível de estresse oxidativo e a expressão proteica de enzimas antioxidantes e proteínas sinalizadoras de vias redox e de morte/sobrevivência. Resultados: O tratamento com extrato de mirtilo levou à melhora de parâmetros funcionais afetados pela HAP: aumentou a razão E/A pelo fluxo da tricúspide, a razão AT/ET pelo fluxo da artéria pulmonar, o TAPSE e o débito cardíaco do VD, ao passo que reduziu os diâmetros finais diastólico e sistólico do VD. A intervenção com o extrato também promoveu melhoras hemodinâmicas, evidenciadas pela atenuação dos valores das pressões sistólica do VD e média da artéria pulmonar, bem como reduziu a derivada pressão/tempo de contratilidade do VD. Essas alterações benéficas do extrato parecem ter sido consequência da retomada do equilíbrio redox. No tecido pulmonar, o tratamento diminuiu a produção de espécies reativas de oxigênio (EROs), reduziu a atividade de NADPH oxidases e a expressão da enzima xantina oxidase, reduziu o dano oxidativo a lipídios, levou ao aumento da atividade das enzimas superóxido dismutase (SOD) e glutationa peroxidase (GPx) e elevou o conteúdo de sulfidrilas. Além disso, o extrato retomou a expressão do fator 2 relacionado ao fator nuclear eritróide 2 (Nrf2) e diminuiu a razão da expressão proteica dos receptores de endotelina ETA/ETB. A intervenção com o mirtilo diminui a hipertrofia do VD, avaliada pelos índices de massa do VD/massa corporal, massa do VD/massa do ventrículo esquerdo e massa do VD/comprimento da tíbia. De forma semelhante à descrita para o tecido pulmonar, o extrato de mirtilo gerou retomada do equilíbrio redox no VD, ao ocasionar diminuição da atividade de NADPH oxidases, redução da lipoperoxidação e estimular maior atividade da enzima catalase. Os efeitos do extrato de mirtilo, quando avaliados sobre cardiomiócitos cultivados na presença de noradrenalina, incluem aumento da viabilidade celular e diminuição da atividade e expressão de caspases. Esses resultados foram associados à prevenção do quadro de estresse oxidativo induzido por noradrenalina, uma vez que verificamos diminuição da produção de EROs, da expressão da isoforma Nox2 das NADPH oxidases e do marcador de oxidação de ácidos graxos 4-hidroxinonenal (4-HNE). Também observamos aumento das expressões das enzimas SOD, GPx e catalase quando do tratamento com o extrato. Em relação às vias de sinalização, a noradrenalina levou ao aumento da expressão da proteína quinase B (Akt), Forkhead box O3a (FoxO3a) e da proteína quinase alfa ativada por AMP (AMPKα), bem como à diminuição da expressão proteica do transdutor de sinal e ativador da transcrição 3 (STAT3). O extrato atenuou o aumento da expressão de Akt, aumentou ainda mais a expressão FoxO3a e restaurou a expressão de STAT3. Além disso, extrato aumentou a expressão das proteínas mTOR e p70S6. Conclusão: O presente estudo demonstrou que o tratamento com o extrato de mirtilo na HAP promove benefícios morfométricos, funcionais e hemodinâmicos cardiopulmonares. Particularmente, esses efeitos estão relacionados com a melhora do estado redox no tecido pulmonar e VD. O extrato de mirtilo ocasiona alterações positivas no equilíbrio redox através de sua atuação sobre diferentes vias de sinalização, que envolvem também a regulação de mecanismos de morte e sobrevivência celular de cardiomiócitos. Assim, o extrato de mirtilo surge como uma possível intervenção dietoterápica no tratamento da HAP.Introduction: Pulmonary arterial hypertension (PAH) is a disease characterized by progressive endothelial dysfunction and vascular remodeling, leading to increased pulmonary vascular resistance and right ventricular (RV) insufficiency. Several elements contribute to its pathophysiology, such as the imbalance between vasodilators and vasoconstrictors, higher sympathetic activation and, as one of its central elements, the establishment of oxidative stress. In this context, the use of natural antioxidants can help restore redox homeostasis and protect the tissues directly affected by the pathology, lungs and RV. Thus, blueberry (BB), one of the foods with higher antioxidant capacity and having shown benefits in other pathological conditions, appears as an alternative intervention in PAH. Objectives: The aim of this study was to evaluate the role of blueberry extract on the lungs and RV in an in vivo model of monocrotaline-induced PAH, verifying its effects on morphofunctional parameters, as well as its modulation of the redox state. Furthermore, we aimed to evaluate the role of the extract on possible pathways involved in redox balance changes and cardiomyocytes death/survival in the RV. For that, the development of an in vitro model of norepinephrine-induced cardiomyocyte injury was also subject of this study. Material and Methods: Male Wistar rats (200 ± 20 grams, n = 72) were divided into eight groups: Control Group (CTR); Control Group BB 50 mg/kg/day (CTR BB50); Control Group BB 100 mg/kg/day (CTR BB100); Control Group BB 200 mg/kg/day (CTR BB200); Monocrotaline Group (MCT); Monocrotaline BB Group 50 mg/kg/day (MCT BB50); Monocrotaline BB Group 100 mg/kg/day (MCT BB100) and Monocrotaline BB Group 200 mg/kg/day (MCT BB200). PAH was induced by a single dose of monocrotaline (60 mg/kg, intraperitoneal). The animals received blueberry extract orally by gavage at the doses of 50, 100 and 200 mg/kg/day. Blueberry treatment lasted 5 weeks (2 weeks prior to MCT induction and 3 weeks post-induction). On the 35th day of the experimental protocol, the animals were submitted to morphometric, echocardiographic and hemodynamic analyses. Then, the animals were euthanized, with lung and heart harvesting for further biochemical redox balance assessments. Cardiomyocytes (H9c2 cells) were incubated under four conditions: culture medium in the absence of norepinephrine (NE) and blueberry extract (BBE): Control group; culture medium in the presence of 50 μg/ml blueberry extract: BBE group; culture medium in the presence of 100 μM noradrenaline: NE group; and culture medium in the presence of 50 μg/ml blueberry extract and 100 μM norepinephrine: BBE + NE group. Cell viability, caspases activation, oxidative stress level and protein expression of antioxidant enzymes and redox and death/survival signaling proteins were evaluated. Results: Treatment with blueberry extract improved functional parameters affected by PAH: increased E/A ratio for tricuspid flow, AT/ET ratio for pulmonary artery flow, RV TAPSE, and RV cardiac output, while reduced the final diastolic and systolic diameters of the RV. Intervention with the extract also promoted hemodynamic improvements, evidenced by the attenuation of the RV systolic pressure and mean pulmonary artery pressure, as well as reduced the RV positive pressure/time derivative. These beneficial alterations of the extract seem to have been a consequence of the redox balance recovery. In lung tissue, treatment decreased the production of reactive oxygen species (ROS), reduced NADPH oxidase activity and xanthine oxidase expression, reduced oxidative damage to lipids, led to increased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and increased sulfhydryl content. In addition, the extract increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and decreased the protein expression ratio of ETA/ETB endothelin receptors. Blueberry intervention decreased RV hypertrophy, as assessed by RV mass/body mass, RV mass/left ventricle mass and RV mass / tibial length indices. Like that described for lung tissue, blueberry extract induced redox balance recovery in the RV, causing decreased NADPH oxidase activity, reduced lipoperoxidation and stimulated catalase enzyme activity. The effects of blueberry extract when evaluated on cardiomyocytes cultured in the presence of norepinephrine include increased cell viability and decreased activity and expression of caspases. These results were associated with the prevention of norepinephrine-induced oxidative stress, since we observed a decrease in ROS production, NADPH oxidase isoform Nox2 expression and 4-hydroxynonenal fatty acid oxidation marker. We also observed increased expression of SOD, GPx and catalase enzymes when cells were treated with the extract. Regarding signaling pathways, norepinephrine led to increased expression of Protein kinase B (Akt), Forkhead box O3a (FoxO3a) and AMP-activated protein kinase alpha (AMPKα), as well as decreased protein expression of the Signal transducer and activator of transcription 3 (STAT3). The extract attenuated the increase in Akt expression, further increased FoxO3a expression and restored STAT3 expression. In addition, blueberry extract increased expression of Mammalian target of rapamycin (mTOR) and p70S6 proteins. Conclusion: The present study demonstrated that treatment with blueberry extract in PAH promotes cardiopulmonary morphometric, functional and hemodynamic benefits. Particularly, these effects are related to the improvement of redox state in lung tissue and RV. Blueberry extract causes positive changes in redox balance through its action on different signaling pathways, which also involve the regulation of cell death and survival mechanisms of cardiomyocytes. Thus, blueberry extract appears as a possible dietary intervention in the treatment of PAH

Efeitos do trapidil sobre o estado redox e remodelamento do ventrículo direito no modelo de hipertensão arterial pulmonar induzida por monocrotalina

A hipertensão arterial pulmonar (HAP) é uma doença progressiva caracterizada por aumento da resistência vascular pulmonar e consequente aumento da pós-carga do ventrículo direito, levando à insuficiência ventricular direita e morte. Distúrbios entre vasodilatadores e vasoconstritores são consequências agravadas por desequilíbrios redox presentes na HAP. O Trapidil é uma droga antiplaquetária, antimitogênica e inibidora de fosfodiesterase usada atualmente na clínica para prevenir a restenose. Estudos recentes também descrevem a capacidade do fármaco de melhorar o equilíbrio redox, diminuindo a peroxidação lipídica e formação de superóxido. Nesse sentido, o objetivo desse trabalho foi verificar os efeitos do Trapidil no modelo de hipertensão arterial pulmonar induzida por monocrotalina, avaliando o estado redox e o remodelamento cardíaco. Para isso, foram utilizados ratos Wistar com 5 semanas de idade divididos em quatro grupos: Controle, Controle + Trapidil, Monocrotalina e Monocrotalina + Trapidil. A HAP foi induzida com uma injeção intraperitoneal de monocrotalina 60 mg/kg no dia 0. O tratamento com o Trapidil começou no dia 7 (5 mg/kg/dia – Experimento 1 ou 8 mg/kg/dia – Experimento 2) até o dia 14, quando os animais foram sacrificados logo após ecocardiografia e cateterismo do ventrículo direito. O coração foi isolado, separado em ventrículo direito e esquerdo (VD e VE), pesado e armazenado para análises de expressão proteica (SERCA, Fosfolambam, p-Fosfolambam e RyR) e bioquímica do estado redox. Observamos aumento da congestão hepática e da hipertrofia do VD nos animais que receberam monocrotalina. O Trapidil (5 mg/kg/dia) foi capaz de reduzir a congestão hepática, entretanto não reduziu a hipertrofia do VD. A pressão sistólica e as derivadas pressão/tempo positiva e negativa se mostraram aumentadas nos animais com indução da HAP, enquanto que os animais que receberam o fármaco (8 mg/kg/dia) apresentaram derivadas pressão/volume positivas com valores não diferentes do grupo Controle. Houve aumento dos diâmetros diastólico e sistólico do VD nos grupos que receberam monocrotalina, indicando dilatação da câmara. Já o tratamento com Trapidil (5 mg/kg/dia) reduziu esses diâmetros. Funcionalmente, observamos diminuição da fração de encurtamento, razão pico E/A e TAPSE nos grupos que receberam monocrotalina, sendo que o fármaco não modificou esses parâmetros. A análise do estado redox demonstrou maior lipoperoxidação e atividade da glutationa peroxidase nos grupos monocrotalina, já o tratamento com Trapidil (8 mg/kg/dia) reduziu a atividade de NADPH oxidases e aumentou a razão GSH/glutationas totais. A expressão proteica de Fosfolambam no VD aparece diminuída nos grupos monocrotalina; a expressão proteica de SERCA e RyR aparece aumentada nos grupos que receberam Trapidil (8 mg/kg/dia). Os dados observados sugerem que o Trapidil induz uma melhora do remodelamento cardíaco direito em modelo de HAP induzida por monocrotalina. A melhora do balanço redox e maior expressão de SERCA e RyR sem aumento da derivada pressão/volume positiva indica que os animais tratados com o fármaco podem estar mais preparados para enfrentar a progressão da doença.Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary vascular resistance and consequent increased afterload of the right ventricle, leading to right ventricular failure and death. PAH disorders between vasodilators and vasoconstrictors are consequences aggravated by redox imbalances present in the disease. Trapidil is an antiplatelet, phosphodiesterase’s inhibitory and antimitogenic drug currently used in the clinic to prevent restenosis. Recent studies also describe the ability of Trapidil to enhance the redox balance by decreasing lipid peroxidation and superoxide formation. In this way, the objective of this study was to assess the effects of Trapidil in pulmonary arterial hypertension model induced by monocrotaline, evaluating the redox state and cardiac remodeling. Male Wistar rats (5 weeks old) were divided into four groups: Control; Control + Trapidil; Monocrotaline; Monocrotaline + Trapidil. PAH was induced with an intraperitoneal injection of monocrotaline 60mg/kg on day 0. Treatment with Trapidil started on day 7 (5 mg / kg / day - Experiment 1 or 8 mg / kg / day - Experiment 2) until day 14, when the animals were sacrificed after echocardiography and catheterization of the right ventricle. The heart was isolated, separated into right and left ventricle (RV and LV), weighed and stored for protein expression analysis (SERCA, phospholamban, p-phospholamban and RyR) and biochemistry analysis of redox balance. We observed increased hepatic congestion and RV hypertrophy in animals that received monocrotaline. Trapidil (5 mg/kg/day) was able to reduce the hepatic congestion, though not reduced RV’s hypertrophy. The systolic pressure and positive and negative pressure/time derivatives have shown increased in animals with induced PAH, whereas animals given the drug (8 mg/kg/day) positive pressure/time derivative values were not different compared to Control group. We also observed an increase of RV’s diastolic and systolic diameters in the groups that received monocrotaline, indicating dilatation of the chamber, as treatment with Trapidil (5mg/ kg/day) reduced these diameters. Functionally, we observed a decrease of fractional shortening, peak E/A ratio and TAPSE in the groups that received monocrotaline, whereas Trapidil did not modify these parameters. The analysis of the redox state showed higher lipid peroxidation and glutathione peroxidase activity in groups with induced PAH. Treatment with Trapidil (8 mg/kg/day) reduced the activity of NADPH oxidases and increased the GSH/total glutathiones ratio. Protein expression of phospholamban in RV appears diminished in monocrotaline groups; protein expression of RyR and SERCA appears enhanced in the groups treated with Trapidil (8 mg/kg/day). The observed data suggest that the Trapidil induces an improvement in right ventricle remodeling in the model of PAH induced by monocrotaline. The improvement in the redox balance and increased expression of RyR and SERCA without increasing the positive pressure/volume derivative indicates that animals treated with the Trapidil may be more prepared to face the progression of the disease

Riluzole stimulates BDNF release from human platelets

Brain-derived neurotrophic factor (BDNF) has several functions in the central nervous system, where it contributes to brain development and its functionality through affecting neuronal survival and activity and also modulating neurotransmitter levels. This neurotrophin is also found in the serum, but its origin and peripheral function remain unknown. Although the source of circulating BDNF is uncertain, it is stored in platelets and can be released through pharmacological treatment. Decreased levels of BDNF in the serum have been related to the pathophysiology of depression, and this relationship is reinforced by the reversal of this condition by treatment with antidepressants. Recently, riluzole has been proposed for the treatment of depression because it has the ability to lower extracellular glutamate levels and increase BDNF expression; and both mechanisms could be associated with its antidepressant action. Considering that riluzole enhances BDNF levels in the serum of patients, we investigated if treatment with this drug could stimulate the release of this neurotrophin from human platelets obtained from healthy subjects. When platelets were incubated with riluzole for 4 h, the basal value of BDNF (92.9 ± 11.1 pg 10−6 platelets) was significantly increased ( < 0.05, = 27). This stimulatory effect was achieved at low concentrations of riluzole (from 10 M) and was not observed when platelets were incubated with the drug for 24 h. The direct action of riluzole evoking BDNF release from human platelets at therapeutic concentrations is important and may contribute to the understanding of its mechanisms of action in the treatment of depression

Development of a standardized physical fitness test for SWAT officers

Discusses the benefits of having a standardized, proven test of the physical fitness of SWAT team members

Profile of pterostilbene-induced redox homeostasis modulation in cardiac myoblasts and heart tissue

This study was designed to investigate the effect of pterostilbene (PTS) on cardiac oxidative stress in vitro, as this is a simple and promising methodology to study cardiac disease. Cardiac myoblasts (H9c2 cells) and homogenised cardiac tissue were incubated with the PTS and cyclodextrin (PTS ? HPbCD) complex for 1 and 24 h, respectively, at concentrations of 50 lM for the cells and 25 and 50 lM for cardiac tissue. The PTS ? HPbCD complex was used to increase the solubility of PTS in water. After the pretreatment period, cardiomyoblasts were challenged with hydrogen peroxide (6.67 lM) for 10 min, while cardiac tissue was submitted to a hydroxyl radical generator system (30 min). Cellular viability, oxidative stress biomarkers (e.g. total reactive oxygen species (ROS), carbonyl assay and lipoperoxidation) and the antioxidant response (e.g. sulfhydryl and the antioxidant enzyme activities of superoxide dismutase, catalase and glutathione peroxidase) were evaluated. In cardiomyoblasts, the PTS ? HPbCD complex (50 lM) increased cellular viability. Moreover, the PTS ? HPbCD complex also significantly increased sulfhydryl levels in the cells submitted to an oxidative challenge. In cardiac tissue, lipid peroxidation, carbonyls and ROS levels were significantly increased in the groups submitted to oxidative damage, while the PTS ? HPbCD complex significantly reduced ROS levels in these groups. In addition, the PTS ? HPbCD complex also provoked increased catalase activity in both experimental protocols. These data suggest that the PTS ? HPbCD complex may play a cardioprotective role through a reduction of ROS levels associated with an improved antioxidant response

The brief methylprednisolone administration is crucial to mitigate cardiac dysfunction after myocardial infarction

Acute myocardial infarction (AMI) is one of the major causes of heart failure and mortality. Glucocorticoids administration post-infarction has long been proposed, but it has shown conflicting results so far. This controversy may be associated with the glucocorticoid type and the period when it is administered. To elucidate these, the present aims to evaluate if the brief methylprednisolone acetate administration is determinant for heart adaptation after AMI. Male Wistar rats were divided into 3 groups: sham-operated (SHAM); infarcted (AMI); infarcted treated with methylprednisolone acetate (AMI+M). Immediately after surgery, the AMI+M group received a single dose of methylprednisolone acetate (40 mg/kg i.m.). After 56 days, the cardiac function was assessed and lungs, liver and heart were collected to determine rates of hypertrophy and congestion. Heart was used for oxidative stress and metalloproteinase activity analyses. Methylprednisolone acetate attenuated matrix metalloproteinase-2 activity, cardiac dilatation, and prevented the onset of pulmonary congestion, as well as avoided cardiac hypertrophy. Our data indicate that administration of methylprednisolone acetate shortly after AMI may be a therapeutic alternative for attenuation of detrimental ventricular remodeling

A multi-site campaign to measure solar-like oscillations in Procyon. II. Mode frequencies

We have analyzed data from a multi-site campaign to observe oscillations in the F5 star Procyon. The data consist of high-precision velocities that we obtained over more than three weeks with eleven telescopes. A new method for adjusting the data weights allows us to suppress the sidelobes in the power spectrum. Stacking the power spectrum in a so-called echelle diagram reveals two clear ridges that we identify with even and odd values of the angular degree (l=0 and 2, and l=1 and 3, respectively). We interpret a strong, narrow peak at 446 muHz that lies close to the l=1 ridge as a mode with mixed character. We show that the frequencies of the ridge centroids and their separations are useful diagnostics for asteroseismology. In particular, variations in the large separation appear to indicate a glitch in the sound-speed profile at an acoustic depth of about 1000 s. We list frequencies for 55 modes extracted from the data spanning 20 radial orders, a range comparable to the best solar data, which will provide valuable constraints for theoretical models. A preliminary comparison with published models shows that the offset between observed and calculated frequencies for the radial modes is very different for Procyon than for the Sun and other cool stars. We find the mean lifetime of the modes in Procyon to be 1.29 +0.55/-0.49 days, which is significantly shorter than the 2-4 days seen in the Sun.Comment: accepted for publication in Ap