Adrenalectomy attenuates hyperalgesia but does not regulate muscle wasting in a female rat model of fibromyalgia.

Although it is well established that fibromyalgia (FM) syndrome is characterized by chronic diffuse musculoskeletal hyperalgesia, very little is known about the effect of this pathology on muscle tissue plasticity. Therefore, the present study aimed to characterize the putative alterations in skeletal muscle mass in female rats subjected to a FM model by inducing chronic diffuse hyperalgesia (CDH) through double injections of acidic saline (pH 4.0) into the left gastrocnemius muscle at 5-day intervals. To determine protein turnover, the total proteolysis, proteolytic system activities and protein synthesis were evaluated in oxidative soleus muscles of pH 7.2 (control) and pH 4.0 groups at 7 days after CDH induction. All animals underwent behavioural analyses of mechanical hyperalgesia, strength and motor performance. Our results demonstrated that, in addition to hyperalgesia, rats injected with acidic saline exhibited skeletal muscle loss, as evidenced by a decrease in the soleus fibre cross-sectional area. This muscle loss was associated with increased proteasomal proteolysis and expression of the atrophy-related gene (muscle RING-finger protein-1), as well as reduced protein synthesis and decreased protein kinase B/S6 pathway activity. Although the plasma corticosterone concentration did not differ between the control and pH 4.0 groups, the removal of the adrenal glands attenuated hyperalgesia, but it did not prevent the increase in muscle protein loss in acidic saline-injected animals. The data suggests that the stress-related hypothalamic-pituitary-adrenal axis is involved in the development of hyperalgesia, but is not responsible for muscle atrophy observed in the FM model induced by intramuscular administration of acidic saline. Although the mechanisms involved in the attenuation of hyperalgesia in rats injected with acidic saline and subjected to adrenalectomy still need to be elucidated, the results found in this study suggest that glucocorticoids may not represent an effective therapeutic approach to alleviate FM symptoms.

Non-canonical Ca2+- Akt signaling pathway mediates the antiproteolytic effects induced by oxytocin receptor stimulation in skeletal muscle.

Although it has been previously demonstrated that oxytocin (OXT) receptor stimulation can control skeletal muscle mass in vivo, the intracellular mechanisms that mediate this effect are still poorly understood. Thus, rat oxidative skeletal muscles were isolated and incubated with OXT or WAY-267,464, a non-peptide selective OXT receptor (OXTR) agonist, in the presence or absence of atosiban (ATB), an OXTR antagonist, and overall proteolysis was evaluated. The results indicated that both OXT and WAY-267,464 suppressed muscle proteolysis, and this effect was blocked by the addition of ATB. Furthermore, the WAY-induced anti-catabolic action on protein metabolism did not involve the coupling between OXTR and Gαi since it was insensitive to pertussis toxin (PTX). The decrease in overall proteolysis induced by WAY was probably due to the inhibition of the autophagic/lysosomal system, as estimated by the decrease in LC3 (an autophagic/lysosomal marker), and was accompanied by an increase in the content of Ca2+-dependent protein kinase (PKC)-phosphorylated substrates, pSer473-Akt, and pSer256-FoxO1. Most of these effects were blocked by the inhibition of inositol triphosphate receptors (IP3R), which mediate Ca2+ release from the sarcoplasmic reticulum to the cytoplasm, and triciribine, an Akt inhibitor. Taken together, these findings indicate that the stimulation of OXTR directly induces skeletal muscle protein-sparing effects through a Gαq/IP3R/Ca2+-dependent pathway and crosstalk with Akt/FoxO1 signaling, which consequently decreases the expression of genes related to atrophy, such as LC3, as well as muscle proteolysis.

Antioxidants and cardioprotective effects of ethyl acetate fraction of Canavalia rosea leaves in myocardial ischemia-reperfusion injury.

Different degrees in the biological activities of Canavalia rosea had been previously reported . In this study, our group assessed the cardioprotective effects of the ethyl acetate fraction (EAcF) of the Canavalia rosea leaves. Firstly, it was confirmed, by in vitro approach, that the EAcF has high antioxidant properties due to the presence of important secondary metabolites, as flavonoids. In order to explore their potential protector against cardiovascular disorders, hearts were previously perfused with EAcF (300 µg.mL-1) and submitted to the global ischemia followed by reperfusion in Langendorff system. The present findings have demonstrated that EAcF restored the left ventricular developed pressure and decreased the arrhythmias severity index. Furthermore, EAcF significantly increased the glutathiones peroxidase activity with decreased malondialdehyde and creatine kinase levels. EAcF was effective upon neither the superoxide dismutase, glutationes reductase nor the catalase activities. In addition, the Western blot analysis revealed that ischemia-reperfusion injury significantly upregulates caspase 3 protein expression, while EAcF abolishes this effect. These results provide evidence that the EAcF reestablishes the cardiac contractility and prevents arrhythmias; it is suggested that EAcF could be used to reduce injury caused by cardiac reperfusion. However more clinical studies should be performed, before applying it in the clinic.

Oxytocin induces anti-catabolic and anabolic effects on protein metabolism in the female rat oxidative skeletal muscle.

AIMS: Although it is well established that skeletal muscle contains oxytocin (OT) receptors and OT-knockout mice show premature development of sarcopenia, the role of OT in controlling skeletal muscle mass is still unknown. Therefore, the present work aimed to determine OT's effects on skeletal muscle protein metabolism. MAIN METHODS: Total proteolysis, proteolytic system activities and protein synthesis were assessed in isolated soleus muscle from prepubertal female rats. Through in vivo experiments, rats received 3-day OT treatment (3UI.kg-1.day-1, i.p.) or saline, and muscles were harvested for mass-gain assessment. KEY FINDINGS: In vitro OT receptor stimulation reduced total proteolysis, specifically through attenuation of the lysosomal and proteasomal proteolytic systems, and in parallel activated the Akt/FoxO1 signaling and suppressed atrogenes (e.g., MuRF-1 and atrogin-1) expression induced by motor denervation. On the other hand, the protein synthesis was not altered by in vitro treatment with the OT receptor-selective agonist. Although short-term OT treatment did not change the atrogene mRNA levels, the protein synthesis was stimulated, resulting in soleus mass gain, probably through an indirect effect. SIGNIFICANCE: Taken together, these data show for the first time that OT directly inhibits the proteolytic activities of the lysosomal and proteasomal systems in rat oxidative skeletal muscle by suppressing atrogene expression via stimulation of Akt/FoxO signaling. Moreover, the data obtained from in vivo experiments suggest OT's ability to control rat oxidative skeletal muscle mass.

Short-term hiit does not promote oxidative stress or muscle damage / Hiit de corto plazo no promueve estrés oxidativo o daños musculares / Hiit de curto prazo não promove estresse oxidativo ou danos musculares

ABSTRACT Introduction: High intensity interval training (HIIT) is a method that is widely used today. Objective: The present study aimed to evaluate the effects of HIIT on markers of oxidative stress and muscle damage in rats. Methods: The sample consisted of 60-day-old Wistar rats, divided into two groups: a control group (n=8) and an HIIT group (n=8). The training consisted of fourteen 20-second swimming sessions (loaded with weights equivalent to 14% of their body weight) with 10-second intervals between each session, performed for 12 consecutive days. Results: HIIT induced a reduction (−17.75%) in thiobarbituric acid reactive substances (an oxidative stress marker) in hepatic tissue (p=0.0482). There was also a reduction (−31.80%) in the HIIT group in the level of superoxide dismutase enzyme activity in the liver (p=0.0375). However, there were no differences between the groups in catalase, glutathione peroxidase, glutathione reductase, the total content of SH sulfhydryls, hydroperoxides, or carbonylated proteins in the hepatic tissue. No significant differences were found in any of these markers in the gastrocnemius muscle. The muscle damage markers creatinine kinase and lactate dehydrogenase were also similar between the groups in the gastrocnemius. Conclusion: The conclusion was that that short-term HIIT does not cause oxidative stress or muscle damage. Level of evidence I; High-quality randomized clinical trial with or without statistically significant difference, but with narrow confidence intervals.

RESUMEN Introducción: El entrenamiento en intervalos de alta intensidad (HIIT) es un método muy utilizado actualmente. Objetivo: El presente estudio tuvo como objetivo evaluar los efectos del HIIT en corto plazo sobre marcadores de estrés oxidativo y daño muscular en ratones. Métodos: La muestra consistió en ratones Wistar con 60 días de edad, divididos en dos grupos: grupo control (n = 8) y grupo HIIT (n = 8). El entrenamiento consistió en catorce sesiones de natación de 20 segundos (con cargas equivalentes a 14% del peso corporal) con intervalos de 10 segundos entre cada sesión, realizadas durante 12 días consecutivos. Resultados: El HIIT indujo una reducción (-17,75%) de las sustancias reactivas al ácido tiobarbitúrico (un marcador de estrés oxidativo) en el tejido hepático (p = 0,0482). También hubo reducción (~31,80%) en el grupo HIIT en el nivel de enzima superóxido dismutasa en el hígado (p=0,0375). Sin embargo, no hubo diferencias entre los grupos con relación a catalasa, glutatión peroxidasa, glutatión reductasa, tenor total de sulfhidrilos SH, hidroperóxidos o proteínas carboniladas en el tejido hepático. No fue encontrada ninguna diferencia significativa en ninguno de esos marcadores en el músculo gastrocnemio. Los marcadores de lesión muscular, creatinina quinasa y lactato deshidrogenasa también fueron similares entre los grupos en el gastrocnemio. Conclusión: Fue posible concluir que el HIIT de corta duración no causa estrés oxidativo o daño muscular. Nivel de evidencia I; Estudio clínico aleatorizado de alta calidad con o sin diferencia estadísticamente significativa, pero con intervalos de información estrechos.

RESUMO Introdução: O treinamento intervalado de alta intensidade (HIIT) é um método muito utilizado atualmente. Objetivo: O presente estudo objetivou avaliar os efeitos do HIIT em curto prazo sobre marcadores de estresse oxidativo e dano muscular em ratos. Métodos: A amostra consistiu em ratos Wistar com 60 dias de idade, divididos em dois grupos: grupo controle (n = 8) e grupo HIIT (n = 8). O treinamento consistiu em quatorze sessões de natação de 20 segundos (com cargas equivalentes a 14% do peso corporal) com intervalos de 10 segundos entre cada sessão, realizadas por 12 dias consecutivos. Resultados: O HIIT induziu uma redução (-17,75%) das substâncias reativas ao ácido tiobarbitúrico (um marcador de estresse oxidativo) no tecido hepático (p = 0,0482). Houve também redução (-31,80%) no grupo HIIT no nível de enzima superóxido dismutase no fígado (p = 0,0375). No entanto, não houve diferenças entre os grupos com relação a catalase, glutationa peroxidase, glutationa redutase, teor total de sulfidrilas SH, hidroperóxidos ou proteínas carboniladas no tecido hepático. Nenhuma diferença significativa foi encontrada em qualquer um desses mascadores no músculo gastrocnêmio. Os marcadores de lesão muscular, creatinina quinase e lactato desidrogenase, também foram semelhantes entre os grupos no gastrocnêmio. Conclusão: Foi possível concluir que o HIIT de curta duração não causa estresse oxidativo ou dano muscular. Nível de evidência I; Estudo clínico randomizado de alta qualidade com ou sem diferença estatisticamente significante, mas com intervalos de informação estreitos.

Strength Training Reduces Cardiac and Renal Oxidative Stress in Rats with Renovascular Hypertension. / Treino de Força Reduz Stress Oxidativo Cardíaco e Renal em Ratos com Hipertensão Renovascular.

BACKGROUND: Strength training has beneficial effects on kidney disease, in addition to helping improve antioxidant defenses in healthy animals. OBJECTIVE: To verify if strength training reduces oxidative damage to the heart and contralateral kidney caused by the renovascular hypertension induction surgery, as well as to evaluate alterations in the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) endogenous antioxidant enzymes. METHODS: Eighteen male rats were divided into three groups (n=6/group): sham, hypertensive, and trained hypertensive. The animals were induced to renovascular hypertension through left renal artery ligation. Strength training was initiated four weeks after the induction of renovascular hypertension, continued for a 12-weeks period, and was performed at 70% of 1RM. After the training period, the animals were euthanized and the right kidney and heart were removed for quantitation of hydroperoxides, malondialdehyde and sulfhydryl groups, which are markers of oxidative damage. In addition, the activity of SOD, CAT, and GPx antioxidant enzymes was also measured. The adopted significance level was 5% (p < 0.05). RESULTS: After strength training, a reduction in oxidative damage to lipids and proteins was observed, as could be seen by reducing hydroperoxides and total sulfhydryl levels, respectively. Furthermore, an increased activity of superoxide dismutase, catalase, and glutathione peroxidase antioxidant enzymes was observed. CONCLUSION: Strength training is able to potentially reduce oxidative damage by increasing the activity of antioxidant enzymes. (Arq Bras Cardiol. 2021; 116(1):4-11).

FUNDAMENTO: O treino de força tem efeitos benéficos em doenças renais, além de ajudar a melhorar a defesa antioxidante em animais saudáveis. OBJETIVO: Verificar se o treino de força reduz o dano oxidativo ao coração e rim contralateral para cirurgia de indução de hipertensão renovascular, bem como avaliar as alterações na atividade das enzimas antioxidantes endógenas superóxido dismutase (SOD), catalase (CAT) e glutationa peroxidase (GPx). MÉTODOS: Dezoito ratos machos foram divididos em três grupos (n=6/grupo): placebo, hipertenso e hipertenso treinado. Os animais foram induzidos a hipertensão renovascular através da ligação da artéria renal esquerda. O treino de força foi iniciado quatro semanas após a indução da hipertensão renovascular, teve 12 semanas de duração e foi realizada a 70% de 1RM. Depois do período de treino, os animais foram submetidos a eutanásia e o rim esquerdo e o coração foram retirados para realizar a quantificação de peróxidos de hidrogênio, malondialdeído e grupos sulfidrílicos, que são marcadores de danos oxidativos. Além disso, foram medidas as atividades das enzimas antioxidantes superóxido dismutase, catalase e glutationa peroxidase. O nível de significância adotado foi de 5% (p < 0,05). RESULTADOS: Depois do treino de força, houve redução de danos oxidativos a lipídios e proteínas, como pode-se observar pela redução de peróxidos de hidrogênio e níveis sulfidrílicos totais, respectivamente. Além disso, houve um aumento nas atividades das enzimas antioxidantes superóxido dismutase, catalase e glutationa peroxidase. CONCLUSÃO: O treino de força tem o potencial de reduzir danos oxidativos, aumentando a atividades de enzimas antioxidantes. (Arq Bras Cardiol. 2021; 116(1):4-11).

Resistance training improves cardiac function and cardiovascular autonomic control in doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX) is an anticancer chemotherapy drug that is widely used in clinical practice. It is well documented that DOX impairs baroreflex responsiveness and left ventricular function and enhances sympathetic activity, cardiac sympathetic afferent reflexes and oxidative stress, which contribute to hemodynamic deterioration. Because resistance training (RT)-induced cardioprotection has been observed in other animal models, the objective of this study was to assess the effects of RT during DOX treatment on hemodynamics, arterial baroreflex, cardiac autonomic tone, left ventricular function and oxidative stress in rats with DOX-induced cardiotoxicity. Male Wistar rats were submitted to a RT protocol (3 sets of 10 repetitions, 40% of one-repetition maximum (1RM) of intensity, 3 times per week, for 8 weeks). The rats were separated into 3 groups: sedentary control, DOX sedentary (2.5 mg/kg of DOX intraperitoneal injection, once a week, for 6 weeks) and DOX + RT. After training or time control, the animals were anesthetized and 2 catheters were implanted for hemodynamic, arterial baroreflex and cardiac autonomic tone. Another group of animals was used to evaluate left ventricular function. We found that RT in DOX-treated rats decreased diastolic arterial pressure, heart rate, sympathetic tone and oxidative stress. In addition, RT increased arterial baroreflex sensitivity, vagal tone and left ventricular developed pressure in rats with DOX-induced cardiotoxicity. In summary, RT is a useful non-pharmacological strategy to attenuate DOX-induced cardiotoxicity.

Treino de Força Reduz Stress Oxidativo Cardíaco e Renal em Ratos com Hipertensão Renovascular / Strength Training Reduces Cardiac and Renal Oxidative Stress in Rats with Renovascular Hypertension

Resumo Fundamento O treino de força tem efeitos benéficos em doenças renais, além de ajudar a melhorar a defesa antioxidante em animais saudáveis. Objetivo Verificar se o treino de força reduz o dano oxidativo ao coração e rim contralateral para cirurgia de indução de hipertensão renovascular, bem como avaliar as alterações na atividade das enzimas antioxidantes endógenas superóxido dismutase (SOD), catalase (CAT) e glutationa peroxidase (GPx). Métodos Dezoito ratos machos foram divididos em três grupos (n=6/grupo): placebo, hipertenso e hipertenso treinado. Os animais foram induzidos a hipertensão renovascular através da ligação da artéria renal esquerda. O treino de força foi iniciado quatro semanas após a indução da hipertensão renovascular, teve 12 semanas de duração e foi realizada a 70% de 1RM. Depois do período de treino, os animais foram submetidos a eutanásia e o rim esquerdo e o coração foram retirados para realizar a quantificação de peróxidos de hidrogênio, malondialdeído e grupos sulfidrílicos, que são marcadores de danos oxidativos. Além disso, foram medidas as atividades das enzimas antioxidantes superóxido dismutase, catalase e glutationa peroxidase. O nível de significância adotado foi de 5% (p < 0,05). Resultados Depois do treino de força, houve redução de danos oxidativos a lipídios e proteínas, como pode-se observar pela redução de peróxidos de hidrogênio e níveis sulfidrílicos totais, respectivamente. Além disso, houve um aumento nas atividades das enzimas antioxidantes superóxido dismutase, catalase e glutationa peroxidase. Conclusão O treino de força tem o potencial de reduzir danos oxidativos, aumentando a atividades de enzimas antioxidantes. (Arq Bras Cardiol. 2021; 116(1):4-11)

Abstract Background Strength training has beneficial effects on kidney disease, in addition to helping improve antioxidant defenses in healthy animals. Objective To verify if strength training reduces oxidative damage to the heart and contralateral kidney caused by the renovascular hypertension induction surgery, as well as to evaluate alterations in the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) endogenous antioxidant enzymes. Methods Eighteen male rats were divided into three groups (n=6/group): sham, hypertensive, and trained hypertensive. The animals were induced to renovascular hypertension through left renal artery ligation. Strength training was initiated four weeks after the induction of renovascular hypertension, continued for a 12-weeks period, and was performed at 70% of 1RM. After the training period, the animals were euthanized and the right kidney and heart were removed for quantitation of hydroperoxides, malondialdehyde and sulfhydryl groups, which are markers of oxidative damage. In addition, the activity of SOD, CAT, and GPx antioxidant enzymes was also measured. The adopted significance level was 5% (p < 0.05). Results After strength training, a reduction in oxidative damage to lipids and proteins was observed, as could be seen by reducing hydroperoxides and total sulfhydryl levels, respectively. Furthermore, an increased activity of superoxide dismutase, catalase, and glutathione peroxidase antioxidant enzymes was observed. Conclusion Strength training is able to potentially reduce oxidative damage by increasing the activity of antioxidant enzymes. (Arq Bras Cardiol. 2021; 116(1):4-11)

Inclusion complex with ß-cyclodextrin is a key determining factor for the cardioprotection induced by usnic acid.

Ischemia-reperfusion (I/R) injury causes oxidative stress, leading to severe cardiac dysfunction. Thus, biologically active compounds with antioxidant properties may be viewed as a promising therapeutic strategy against oxidative-related cardiac disorders. Usnic acid (UA), a natural antioxidant, was complexed with ß-cyclodextrin (ßCD) to improve its bioavailability. Wistar male rats were orally treated with the free form of UA (50 mg/kg) or the inclusion complex UA/ßCD (50 mg/kg) for seven consecutive days. Afterward, hearts were subjected to I/R injury, and the cardiac contractility, rhythmicity, infarct size, and antioxidant enzyme activities were evaluated. Here, we show that neither UA nor UA/ßCD treatments developed signs of toxicity. After I/R injury, animals treated with UA/ßCD showed improved post-ischemic cardiac functional recovery while the release of cell injury biomarkers decreased. Following reduced cardiac damage, a lower incidence of ventricular arrhythmias and smaller myocardial infarct size were associated with reduced lipid peroxidation, along with preserved activity of antioxidant enzymes compared to untreated rats. Surprisingly, uncomplexed UA did not protect hearts against IR injury. Altogether, our results indicate that the inclusion complex UA/ßCD is a critical determining factor responsible for the cardioprotection action of UA, suggesting the involvement of an antioxidant-dependent mechanisms. Moreover, our findings support that UA/ßCD is a structurally engineered compound with active cardioprotective properties.

Topical application of (S)-(-)-limonene is as effective as phonophoresis for improving oxidative parameters of injured skeletal muscle in rats.

The aim of this study is to investigate the effects of limonene, alone or associated with therapeutic ultrasound, on oxidative stress following skeletal muscle injury. Thirty male Wistar rats were divided into 5 groups: CTR-control, MI-muscle injury without treatment, TPU-therapeutic pulsed ultrasound alone, TPU + LIM-phonophoresis with 5% limonene, and LIM-5% limonene applied topically. Muscle injury was induced by a mechanical abrupt impact over gastrocnemius muscle. The animals were treated in the following intervals: 2, 12, 24, 48, 72, and 96 h after injury. Blood and gastrocnemius samples were collected 98 h after lesion for data analysis. Creatine kinase (CK) and lactate dehydrogenase (LDH) activity, lipid peroxidation (TBARS) levels, catalase (CAT), and superoxide dismutase (SOD) activity were assessed. CK (p = 0.01), SOD activity (p < 0.01), and TBARS levels (p < 0.01) were increased after injury. There was no effect on LDH levels in any group. Phonophoresis (TABRS p < 0.01; SOD p = 0.01), TPU alone (TBARS p < 0.01; SOD p = 0.01), and LIM alone (TBARS p < 0.01; SOD p < 0.01) reduced TBARS levels and SOD activity after muscle injury. There was no change for CAT activity after injury. Only phonophoresis reduced CK activity after injury (p < 0.01). There was no difference between phonophoresis, TPU alone and LIM alone groups for TBARS, SOD, CAT, and LDH. Limonene alone and TPU alone were effective in reducing oxidative stress parameters after skeletal muscle injury. Only phonophoresis decreased CK activity. Skeletal muscle injury increases reactive oxidative species (ROS) levels and muscle proteins activity as creatine kinase (CK) and lactate dehydrogenase (LDH). Five percent limonene, alone or associated with therapeutic pulsed ultrasound, exhibited reduction of CK, superoxide dismutase (SOD) and catalase (CAT) activity, and lipid peroxidation markers (TBARS). Graphical abstract.

NOX-dependent reactive oxygen species production underlies arrhythmias susceptibility in dexamethasone-treated rats.

Dexamethasone is the most clinically used glucocorticoid with an established role in the treatment of a wide spectrum of inflammatory-related diseases. While the therapeutic actions are well known, dexamethasone treatment causes a number of cardiovascular side effects, which are complex, frequent and, in some cases, clinically unnoticeable. Here, we investigated whether a therapeutic regimen of dexamethasone affects cardiac arrhythmogenesis, focusing on the contribution of Nox-derived reactive oxygen species (ROS). Male Wistar rats were treated with dexamethasone (2 mg/kg, i.p.) for 7 days. Afterward, hemodynamic measurements, autonomic modulation, left ventricular function, cardiac fibrosis, reactive oxygen species (ROS) generation, Nox protein expression, superoxide dismutase (SOD) and catalase activities, and arrhythmias incidence were evaluated. Here, we show that dexamethasone increases blood pressure, associated with enhanced cardiac and vascular sympathetic modulation. Moreover, a marked increase in the cardiac ROS generation was observed, whereas the enhanced SOD activity did not prevent the higher levels of lipid peroxidation in the dexamethasone group. On the other hand, increased cardiac Nox 4 expression and hydrogen peroxide decomposition rate was observed in dexamethasone-treated rats, while Nox 2 remained unchanged. Interestingly, although preserved ventricular contractility and ß-adrenergic responsiveness, we found that dexamethasone-treated rats displayed greater interstitial and perivascular fibrosis than control. Surprisingly, despite the absence of arrhythmias at basal condition, we demonstrated, by in vivo and ex vivo approaches, that dexamethasone-treated rats are more susceptible to develop harmful forms of ventricular arrhythmias when challenged with pharmacological drugs or burst pacing-induced arrhythmias. Notably, concomitant treatment with apocynin, an inhibitor of NADPH oxidase, prevented these ectopic ventricular events. Together, our results reveal that hearts become arrhythmogenic during dexamethasone treatment, uncovering the pivotal role of ROS-generating NADPH oxidases for arrhythmias vulnerability.

Effects of high doses of glucocorticoids on insulin-mediated vasodilation in the mesenteric artery of rats.

Several pathological conditions predict the use of glucocorticoids for the management of the inflammatory response; however, chronic or high dose glucocorticoid treatment is associated with hyperglycemia, hyperlipidemia, and insulin resistance and can be considered a risk factor for cardiovascular disease. Therefore, we investigated the mechanisms involved in the vascular responsiveness and inflammatory profile of mesenteric arteries of rats treated with high doses of glucocorticoids. Wistar rats were divided into a control (CO) group and a dexamethasone (DEX) group, that received dexamethasone for 7 days (2mg/kg/day, i.p.). Blood samples were used to assess the lipid profile and insulin tolerance. Vascular reactivity to Phenylephrine (Phe) and insulin, and O2•-production were evaluated. The intracellular insulin signaling pathway PI3K/AKT/eNOS and MAPK/ET-1 were investigated. Regarding the vascular inflammatory profile, TNF-α, IL-6, IL-1ß and IL-18 were assessed. Dexamethasone-treated rats had decreased insulin tolerance test and endothelium-dependent vasodilation induced by insulin. eNOS inhibition caused vasoconstriction in the DEX group, which was abolished by the ET-A antagonist. Insulin-mediated relaxation in the DEX group was restored in the presence of the O2.- scavenger TIRON. Nevertheless, in the DEX group there was an increase in Phe-induced vasoconstriction. In addition, the intracellular insulin signaling pathway PI3K/AKT/eNOS was impaired, decreasing NO bioavailability. Regarding superoxide anion generation, there was an increase in the DEX group, and all measured proinflammatory cytokines were also augmented in the DEX group. In addition, the DEX-group presented an increase in low-density lipoprotein cholesterol (LDL-c) and total cholesterol (TC) and reduced high-density lipoprotein cholesterol (HDL-c) levels. In summary, treatment with high doses of dexamethasone promoted changes in insulin-induced vasodilation, through the reduction of NO bioavailability and an increase in vasoconstriction via ET-1 associated with generation of O2•- and proinflammatory cytokines.

d-Limonene Ameliorates Myocardial Infarction Injury by Reducing Reactive Oxygen Species and Cell Apoptosis in a Murine Model.

Myocardial infarction (MI) leads to high mortality, and pharmacological or percutaneous primary interventions do not significantly inhibit ischemia/reperfusion injuries, particularly those caused by oxidative stress. Recently, research groups have evaluated several naturally occurring antioxidant compounds for possible use as therapeutic alternatives to traditional treatments. Studies have demonstrated that d-limonene (DL), a monoterpene of citrus fruits, possesses antioxidant and cardiovascular properties. Thus, this work sought to elucidate the mechanisms of protection of DL in an isoproterenol-induced murine MI model. It was observed that DL (10 µmol) attenuated 40% of the ST elevation, reduced the infarct area, prevented histological alterations, abolished completely oxidative stress damage, restored superoxide dismutase activity, and suppressed pro-apoptotic enzymes. In conclusion, the present study demonstrated that DL produces cardioprotective effects from isoproterenol-induced myocardial infarction in Swiss mice through suppression of apoptosis.

Resistance exercise mediates remote ischemic preconditioning by limiting cardiac eNOS uncoupling.

BACKGROUND: Currently viewed as a complementary non-pharmacological intervention for preventing cardiac disorders, long-term aerobic training produces cardioprotection through remote ischemic preconditioning (RIPC) mechanisms. However, RIPC triggered by acute exercise remains poorly understood. Although resistance exercise (RE) has been highly recommended by several public health guidelines, there is no evidence showing that RE mediates RIPC. Hence, we investigated whether RE induces cardiac RIPC through nitric oxide synthase (NOS)-dependent mechanism. METHODS AND RESULTS: Acute RE at 40% of the maximal load augmented systemic nitrite levels, associated with increased cardiac eNOS phosphorylation, without affecting nNOS activity. Using an experimental model of myocardial infarction (MI) through ischemia-reperfusion (IR), RE fully prevented the loss of cardiac contractility and the extent of MI size compared to non-exercised (NE) rats. Moreover, RE mitigated aberrant ST-segment and reduced life-threatening arrhythmias induced by IR. Importantly, inhibition of NOS abolished the RE-mediated cardioprotection. After IR, NE rats showed increased cardiac eNOS activity, associated with reduced dimer/monomer ratio. Supporting the pivotal role of eNOS coupling during MI, non-exercised rats displayed a marked generation of reactive oxygen species (ROS) and oxidative-induced carbonylation of proteins, whereas RE prevented these responses. We validated our data demonstrating a restoration of physiological ROS levels in NE + IR cardiac sections treated with BH4, a cofactor oxidatively depleted during eNOS uncoupling, while cardiac ROS generation from exercised rats remained unchanged, suggesting no physiological needs of supplemental eNOS cofactors. CONCLUSION: Together, our findings strongly indicate that RE mediates RIPC by limiting eNOS uncoupling and mitigates myocardial IR injury.

α-Terpineol reduces cancer pain via modulation of oxidative stress and inhibition of iNOS.

α-Terpineol (TP) is present in a wide range of essential oils of the genus Eucalyptus, with recognized potential for a range of biological effects, such as analgesic. Hence, our study aimed to investigate the effect of TP on cancer pain induced by sarcoma 180 in Swiss mice. Our results showed that TP reduced significantly mechanical hyperalgesia and spontaneous and palpation-induced nociception, improved paw use without reducing tumor growth and grip strength. Importantly, no evident biochemical and hematological toxicity was oberved. Furthermore, TP increased the tissue antioxidant capacity due to ferric-reducing antioxidant power (FRAP) and glutathione (GSH). TP also reduced inducible nitric oxide synthase (iNOS) immunocontent in the tumors. Molecular docking estimated that TP binds within the same range of iNOS regions (other iNOS inhibitors), such as N-Nitroarginine methyl ester (L-NAME). These data provide strong evidence that TP may be an interesting candidate for the development of new safe analgesic drugs that are effective for cancer pain control.

Myrtenol protects against myocardial ischemia-reperfusion injury through antioxidant and anti-apoptotic dependent mechanisms.

Myrtenol is a monoterpene with multiple pharmacological activities. However, although monoterpenes have been proposed to play beneficial roles in a variety of cardiac disorders, pharmacological actions of myrtenol in the heart are not yet reported. Hence, the aim of this study was to evaluate whether myrtenol promotes cardioprotection against myocardial ischemia-reperfusion (IR) injury, and the mechanisms involved in these effects. Male Wistar rats were orally treated for seven consecutive days with myrtenol (50 mg/kg) or N-acetyl cysteine (1.200 mg/kg, NAC). Afterward, hearts were subjected to myocardial IR injury. Here, we show that the severe impairment of contractile performance induced by IR was significantly prevented by myrtenol or NAC. Moreover, myrtenol abolished aberrant electrocardiographic waveform (ST-segment elevation), as well as reduced life-threatening arrhythmias and infarct size induced by IR injury. Importantly, myrtenol fully prevented the massive increase of cardiac reactive oxygen species generation and oxidative stress damage. Accordingly, myrtenol restored the impairment of endogenous antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase and reductase) activities and balance of pro- and anti-apoptotic pathways (Bax and Bcl-2), associated with decreased apoptotic cells. Taken together, our data show that myrtenol promotes cardioprotection against IR injury through attenuation of oxidative stress and inhibition of pro-apoptotic pathway.

Effects of a Single Bout of Resistance Exercise in Different Volumes on Endothelium Adaptations in Healthy Animals.

BACKGROUND:: Resistance exercise (RE) has been recommended for patients with cardiovascular diseases. Recently, a few studies have demonstrated that the intensity of a single bout of RE has an effect on endothelial adaptations to exercise. However, there is no data about the effects of different volumes of RE on endothelium function. OBJECTIVE:: The aim of the study was to evaluate the effects of different volumes of RE in a single bout on endothelium-dependent vasodilatation and nitric oxide (NO) synthesis in the mesenteric artery of healthy animals. METHODS:: Male Wistar rats were divided into three groups: Control (Ct); low-volume RE (LV, 5 sets x 10 repetitions) and high-volume RE (HV, 15 sets x 10 repetitions). The established intensity was 70% of the maximal repetition test. After the exercise protocol, rings of mesenteric artery were used for assessment of vascular reactivity, and other mesenteric arteries were prepared for detection of measure NO production by DAF-FM fluorescence. Insulin responsiveness on NO synthesis was evaluated by stimulating the vascular rings with insulin (10 nM). RESULTS:: The maximal relaxation response to insulin increased in the HV group only as compared with the Ct group. Moreover, the inhibition of nitric oxide synthesis (L-NAME) completely abolished the insulin-induced vasorelaxation in exercised rats. NO production showed a volume-dependent increase in the endothelial and smooth muscle layer. In endothelial layer, only Ct and LV groups showed a significant increase in NO synthesis when compared to their respective group under basal condition. On the other hand, in smooth muscle layer, NO fluorescence increased in all groups when compared to their respective group under basal condition. CONCLUSIONS:: Our results suggest that a single bout of RE promotes vascular endothelium changes in a volume-dependent manner. The 15 sets x 10 repetitions exercise plan induced the greatest levels of NO synthesis. FUNDAMENTOS:: O exercício resistido (ER) tem sido recomendado para pacientes com doenças cardiovasculares. Recentemente, alguns estudos demonstraram que a intensidade de uma sessão de ER exerce um efeito sobre a disfunção endotelial. No entanto, não há dados sobre os efeitos de diferentes volumes de ER sobre a função endotelial. OBJETIVO:: O objetivo deste estudo foi avaliar os efeitos de diferentes volumes de ER, realizados em uma única sessão, sobre a vasodilatação dependente do endotélio e síntese de óxido nítrico (NO) em artéria mesentérica de animais saudáveis. MÉTODOS:: Ratos Wistar machos foram divididos em três grupos: Controle (Ct); baixo volume (BV, 5 séries x 10 repetições) e alto volume de ER (AV, 15 séries x 10 repetições). Foi estabelecida a intensidade de 70% do teste de repetição máxima. Após o protocolo de exercício, anéis de artéria mesentérica foram utilizados na avaliação da reatividade vascular, e outras artérias mesentéricas foram preparadas para a detecção da produção de NO por fluorescência com para do DAF-FM. A resposta à insulina pela síntese de NO foi avaliada estimulando-se os anéis vasculares com insulina (10nM). RESULTADOS:: A resposta máxima do relaxamento induzido por insulina foi aumentada somente no grupo AV em comparação ao grupo Ct. Além disso, a inibição da síntese do NO (L-NAME), aboliu completamente o relaxamento vascular induzido por insulina em ratos exercitados. A produção de NO mostrou um aumento dependente do volume no endotélio e no músculo liso. No endotélio, apenas os grupos Ct e BV mostraram aumento significativo na síntese de NO quando comparado aos seus respectivos grupos sob condição basal. No entanto, no músculo liso, a fluorescência foi aumentada em todos os grupos quando comparados aos seus respectivos grupos sob a condição basal. CONCLUSÕES:: Nossos resultados sugerem que uma única sessão de ER foi capaz de promover adaptações no endotélio vascular. Além disso, nós observamos que este efeito é volume-dependente e o volume de 15 séries x10 repetições induziu o maior aumento na síntese de NO.

Cardioprotective Action of Ginkgo biloba Extract against Sustained ß-Adrenergic Stimulation Occurs via Activation of M2/NO Pathway.

Ginkgo biloba is the most popular phytotherapic agent used worldwide for treatment of several human disorders. However, the mechanisms involved in the protective actions of Ginkgo biloba on cardiovascular diseases remain poorly elucidated. Taking into account recent studies showing beneficial actions of cholinergic signaling in the heart and the cholinergic hypothesis of Ginkgo biloba-mediated neuroprotection, we aimed to investigate whether Ginkgo biloba extract (GBE) promotes cardioprotection via activation of cholinergic signaling in a model of isoproterenol-induced cardiac hypertrophy. Here, we show that GBE treatment (100 mg/kg/day for 8 days, v.o.) reestablished the autonomic imbalance and baroreflex dysfunction caused by chronic ß-adrenergic receptor stimulation (ß-AR, 4.5 mg/kg/day for 8 days, i.p.). Moreover, GBE prevented the upregulation of muscarinic receptors (M2) and downregulation of ß1-AR in isoproterenol treated-hearts. Additionally, we demonstrated that GBE prevents the impaired endothelial nitric oxide synthase activity in the heart. GBE also prevented the pathological cardiac remodeling, electrocardiographic changes and impaired left ventricular contractility that are typical of cardiac hypertrophy. To further investigate the mechanisms involved in GBE cardioprotection in vivo, we performed in vitro studies. By using neonatal cardiomyocyte culture we demonstrated that the antihypertrophic action of GBE was fully abolished by muscarinic receptor antagonist or NOS inhibition. Altogether, our data support the notion that antihypertrophic effect of GBE occurs via activation of M2/NO pathway uncovering a new mechanism involved in the cardioprotective action of Ginkgo biloba.

Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase.

B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B1- (B1R-/-), B2- (B2R-/-) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R-/-). For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R-/- presented similar level of vascular dysfunction as found in B1R-/- or B2R-/- mice. In accordance, aortic rings from B1R-/- or B2R-/- mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R-/- and B2R-/- mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R-/- or B2R-/- mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B1- and B2-kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.

Effects of a Single Bout of Resistance Exercise in Different Volumes on Endothelium Adaptations in Healthy Animals / Efeitos de uma Sessão de Exercício Resistido em Diferentes Volumes sobre Adaptações Endoteliais em Animais Saudáveis

Abstract Background: Resistance exercise (RE) has been recommended for patients with cardiovascular diseases. Recently, a few studies have demonstrated that the intensity of a single bout of RE has an effect on endothelial adaptations to exercise. However, there is no data about the effects of different volumes of RE on endothelium function. Objective: The aim of the study was to evaluate the effects of different volumes of RE in a single bout on endothelium-dependent vasodilatation and nitric oxide (NO) synthesis in the mesenteric artery of healthy animals. Methods: Male Wistar rats were divided into three groups: Control (Ct); low-volume RE (LV, 5 sets x 10 repetitions) and high-volume RE (HV, 15 sets x 10 repetitions). The established intensity was 70% of the maximal repetition test. After the exercise protocol, rings of mesenteric artery were used for assessment of vascular reactivity, and other mesenteric arteries were prepared for detection of measure NO production by DAF-FM fluorescence. Insulin responsiveness on NO synthesis was evaluated by stimulating the vascular rings with insulin (10 nM). Results: The maximal relaxation response to insulin increased in the HV group only as compared with the Ct group. Moreover, the inhibition of nitric oxide synthesis (L-NAME) completely abolished the insulin-induced vasorelaxation in exercised rats. NO production showed a volume-dependent increase in the endothelial and smooth muscle layer. In endothelial layer, only Ct and LV groups showed a significant increase in NO synthesis when compared to their respective group under basal condition. On the other hand, in smooth muscle layer, NO fluorescence increased in all groups when compared to their respective group under basal condition. Conclusions: Our results suggest that a single bout of RE promotes vascular endothelium changes in a volume-dependent manner. The 15 sets x 10 repetitions exercise plan induced the greatest levels of NO synthesis.

Resumo Fundamentos: O exercício resistido (ER) tem sido recomendado para pacientes com doenças cardiovasculares. Recentemente, alguns estudos demonstraram que a intensidade de uma sessão de ER exerce um efeito sobre a disfunção endotelial. No entanto, não há dados sobre os efeitos de diferentes volumes de ER sobre a função endotelial. Objetivo: O objetivo deste estudo foi avaliar os efeitos de diferentes volumes de ER, realizados em uma única sessão, sobre a vasodilatação dependente do endotélio e síntese de óxido nítrico (NO) em artéria mesentérica de animais saudáveis. Métodos: Ratos Wistar machos foram divididos em três grupos: Controle (Ct); baixo volume (BV, 5 séries x 10 repetições) e alto volume de ER (AV, 15 séries x 10 repetições). Foi estabelecida a intensidade de 70% do teste de repetição máxima. Após o protocolo de exercício, anéis de artéria mesentérica foram utilizados na avaliação da reatividade vascular, e outras artérias mesentéricas foram preparadas para a detecção da produção de NO por fluorescência com para do DAF-FM. A resposta à insulina pela síntese de NO foi avaliada estimulando-se os anéis vasculares com insulina (10nM). Resultados: A resposta máxima do relaxamento induzido por insulina foi aumentada somente no grupo AV em comparação ao grupo Ct. Além disso, a inibição da síntese do NO (L-NAME), aboliu completamente o relaxamento vascular induzido por insulina em ratos exercitados. A produção de NO mostrou um aumento dependente do volume no endotélio e no músculo liso. No endotélio, apenas os grupos Ct e BV mostraram aumento significativo na síntese de NO quando comparado aos seus respectivos grupos sob condição basal. No entanto, no músculo liso, a fluorescência foi aumentada em todos os grupos quando comparados aos seus respectivos grupos sob a condição basal. Conclusões: Nossos resultados sugerem que uma única sessão de ER foi capaz de promover adaptações no endotélio vascular. Além disso, nós observamos que este efeito é volume-dependente e o volume de 15 séries x10 repetições induziu o maior aumento na síntese de NO.