Melatonin prevents overproduction of reactive oxygen species and vascular dysfunction induced by cyclophosphamide.

AIMS: Overproduction of reactive oxygen species (ROS) is a pathologic hallmark of cyclophosphamide toxicity. For this reason, antioxidant compounds emerge as promising tools for preventing tissue damage induced by cyclophosphamide. We hypothesized that melatonin would display cytoprotective action in the vasculature by preventing cyclophosphamide-induced oxidative stress. MATERIALS AND METHODS: Male C57BL/6 mice (22-25 g) were injected with a single dose of cyclophosphamide (300 mg/kg; i.p.). Mice were pretreated or not with melatonin (10 mg/kg/day, i.p.), given during 4 days before cyclophosphamide injection. Functional (vascular reactivity) and oxidative/inflammatory patterns were evaluated at 24 h in resistance arteries. The antioxidant action of melatonin was assessed in vitro in cultured vascular smooth muscle cells (VSMCs) of mesenteric arteries. KEY FINDINGS: Cyclophosphamide induced ROS generation in both mesenteric arterial bed (MAB) and cultured VSMCs, and this was normalized by melatonin. Cyclophosphamide-induced ROS generation and lipoperoxidation in the bladder and kidney was also prevented by melatonin. Increased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were detected in the MAB of cyclophosphamide-treated mice, all of which were prevented by melatonin. Functional assays using second-order mesenteric arteries of cyclophosphamide-treated mice revealed a decrease in vascular contractility. Melatonin prevented vascular hypocontractility in the cyclophosphamide group. Melatonin partially prevented the decrease in myeloperoxidase (MPO) and N-acetyl-beta-D-glucosaminidase (NAG) activities in the MAB of the cyclophosphamide group. SIGNIFICANCE: Melatonin may constitute a novel and promising therapeutic approach for management of the toxic effects induced by cyclophosphamide in the vasculature.

The Labdane Ent-3-Acetoxy-Labda-8(17), 13-Dien-15-Oic Decreases Blood Pressure In Hypertensive Rats / O Labdano Ácido Ent-3-Acetóxi-Labda-8(17), 13-Dieno-15-Óico Reduz Pressão Arterial Em Ratos Hipertensos

Abstract Background: Labdane-type diterpenes induce lower blood pressure via relaxation of vascular smooth muscle; however, there are no studies describing the effects of labdanes in hypertensive rats. Objective: The present study was designed to investigate the cardiovascular actions of the labdane-type diterpene ent-3-acetoxy-labda-8(17), 13-dien-15-oic acid (labda-15-oic acid) in two-kidney 1 clip (2K-1C) renal hypertension. Methods: Vascular reactivity experiments were performed in aortic rings isolated from 2K-1C and normotensive (2K) male Wistar rats. Nitrate/nitrite (NOx) measurement was performed in aortas by colorimetric assay. Blood pressure measurements were performed in conscious rats. Results: Labda-15-oic acid (0.1-300 µmol/l) and forskolin (0.1 nmol/l - 1 µmol/l) relaxed endothelium-intact and endothelium-denuded aortas from both 2K-1C and 2K rats. Labda-15-oic acid was more effective at inducing relaxation in endothelium-intact aortas from 2K pre-contracted with phenylephrine when compared to the endothelium-denuded ones. Forskolin was more potent than labda-15-oic acid at inducing vascular relaxation in arteries from both 2K and 2K-1C rats. Labda-15-oic acid-induced increase in NOx levels was lower in arteries from 2K-1C rats when compared to 2K rats. Intravenous administration of labda-15-oic acid (0.3-3 mg/kg) or forskolin (0.1-1 mg/kg) induced hypotension in conscious 2K-1C and 2K rats. Conclusion: The present findings show that labda-15-oic acid induces vascular relaxation and hypotension in hypertensive rats.

Resumo Fundamento: Diterpenos do tipo labdano induzem uma queda da pressão arterial por meio do relaxamento do músculo liso vascular; todavia, não há estudos que descrevam os efeitos de labdanos em ratos hipertensos. Objetivo: O presente estudo foi desenvolvido para investigar as ações cardiovasculares do labdano ácido ent-3-acetóxi-labda-8(17),13-dieno-15-óico (labda-15-óico) na hipertensão renal dois rins-1 clipe (2R-1C). Métodos: Foram feitos experimentos de reatividade vascular em anéis aórticos isolados de ratos machos 2R-1C e normotensos (2R). A medição de Nitrato/Nitrito (NOx) foi feita nas aortas por meio de ensaio colorimétrico. As medidas de pressão arterial foram feitas em ratos conscientes. Resultados: O ácido labda-15-óico (0,1 - 300 µmol/l) e a forscolina (0,1 nmol/l - 1 µmol/l) relaxaram as aortas com endotélio intacto e as aortas sem endotélio dos ratos 2R-1C e 2R. O labda-15-óico mostrou-se mais eficaz na indução do relaxamento em aortas com endotélio intacto de 2R pré-contraídas com fenilefrina em comparação àquelas sem endotélio. A forscolina mostrou-se mais potente do que o ácido labda-15-óico na indução do relaxamento vascular nas artérias tanto de ratos 2R-1C quanto de ratos 2R. O aumento dos níveis de NOx induzido pelo ácido labda-15-óico foi menor nas artérias de ratos 2R-1C em comparação a ratos 2R. A administração intravenosa de ácido labda-15-óico (0,3-3 mg/kg) ou forscolina (0,1-1 mg/kg) induziu hipertensão em ratos 2R-1C e 2R conscientes. Conclusão: Os presentes resultados mostram que o labda-15-óico induz relaxamento vascular e hipotensão em ratos hipertensos.

Acute Ethanol Intake Induces NAD(P)H Oxidase Activation and Rhoa Translocation in Resistance Arteries / O consumo agudo de etanol induz a ativação da NAD(P)H oxidase e translocação da RhoA em artérias de resistência

Abstract Background: The mechanism underlying the vascular dysfunction induced by ethanol is not totally understood. Identification of biochemical/molecular mechanisms that could explain such effects is warranted. Objective: To investigate whether acute ethanol intake activates the vascular RhoA/Rho kinase pathway in resistance arteries and the role of NAD(P)H oxidase-derived reactive oxygen species (ROS) on such response. We also evaluated the requirement of p47phox translocation for ethanol-induced NAD(P)H oxidase activation. Methods: Male Wistar rats were orally treated with ethanol (1g/kg, p.o. gavage) or water (control). Some rats were treated with vitamin C (250 mg/kg, p.o. gavage, 5 days) before administration of water or ethanol. The mesenteric arterial bed (MAB) was collected 30 min after ethanol administration. Results: Vitamin C prevented ethanol-induced increase in superoxide anion (O2-) generation and lipoperoxidation in the MAB. Catalase and superoxide dismutase activities and the reduced glutathione, nitrate and hydrogen peroxide (H2O2) levels were not affected by ethanol. Vitamin C and 4-methylpyrazole prevented the increase on O2- generation induced by ethanol in cultured MAB vascular smooth muscle cells. Ethanol had no effect on phosphorylation levels of protein kinase B (Akt) and eNOS (Ser1177 or Thr495 residues) or MAB vascular reactivity. Vitamin C prevented ethanol-induced increase in the membrane: cytosol fraction ratio of p47phox and RhoA expression in the rat MAB. Conclusion: Acute ethanol intake induces activation of the RhoA/Rho kinase pathway by a mechanism that involves ROS generation. In resistance arteries, ethanol activates NAD(P)H oxidase by inducing p47phox translocation by a redox-sensitive mechanism.

Resumo Fundamento: O mecanismo da disfunção vascular induzido pelo consumo de etanol não é totalmente compreendido. Justifica-se, assim a identificação de mecanismos bioquímicos e moleculares que poderiam explicar tais efeitos. Objetivos: Investigar se a ingestão aguda de etanol ativa a via vascular RhoA/Rho quinase em artérias de resistência e o papel das espécies reativas de oxigênio (ERO) derivadas da NAD(P)H oxidase nessa resposta. Nós também avaliamos se ocorreu translocação da p47phox e ativação da NAD(P)H oxidase após o consumo agudo de etanol. Métodos: Ratos Wistar machos foram tratados com etanol via oral (1g/kg, p.o. gavagem) ou água (controle). Alguns ratos foram tratados com vitamina C (250 mg/kg, p.o. gavagem, 5 dias) antes de água ou etanol. O leito arterial mesentérico (LAM) foi coleado 30 min após a administração de etanol. Resultados: A vitamina C preveniu o aumento da geração de ânion superóxido (O2 -) e lipoperoxidação no LAM induzidos pelo etanol. A atividade da catalase (CAT), da superóxido dismutase (SOD) e os níveis de glutationa reduzida(GSH), nitrato e peróxido de hidrogênio (H2O2) não foram afetados após a ingestão aguda de etanol. A vitamina C e o 4-metilpirazol preveniram o aumento na geração de O2 - induzido pelo etanol em cultura de células do músculo liso vascular (CMLV). O etanol não afetou a fosforilação da proteína quinase B (Akt) e nem da óxido nítrico sintase endotelial (eNOS) (nos resíduos de Ser1177 ou Thr495) ou a reatividade vascular do LAM. A vitamina C preveniu o aumento da razão membrana:citosol da p47phox e a expressão da RhoA no LAM de rato induzido pelo etanol. Conclusão: A ingestão aguda de etanol induz a ativação da via RhoA/Rho quinase por um mecanismo que envolve a geração de ERO. Nas artérias de resistência, o etanol ativa NAD(P)H oxidase induzindo a translocação da p47phox por um mecanismo redox-sensível.