Nicorandil prevents endothelial dysfunction due to antioxidative effects via normalisation of NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats

Serizawa et al. Cardiovascular Diabetology 2011, 10:105 http://www.cardiab.com/content/10/1/105 CARDIO VASCULAR DIABETOLOGY ORIGINAL INVESTIGATION Open Access Nicorandil prevents endothelial dysfunction due to antioxidative effects via normalisation of NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats Ken-ichi Serizawa†, Kenji Yogo†, Ken Aizawa†, Yoshihito Tashiro† and Nobuhiko Ishizuka* Abstract Background: Nicorandil, an anti-angina agent, reportedly improves outcomes even in angina patients with diabetes. However, the precise mechanism underlying the beneficial effect of nicorandil on diabetic patients has not been examined. We investigated the protective effect of nicorandil on endothelial function in diabetic rats because endothelial dysfunction is a major risk factor for cardiovascular disease in diabetes. Methods: Male Sprague-Dawley rats (6 weeks old) were intraperitoneally injected with streptozotocin (STZ, 40 mg/ kg, once a day for 3 days) to induce diabetes. Nicorandil (15 mg/kg/day) and tempol (20 mg/kg/day, superoxide dismutase mimetic) were administered in drinking water for one week, starting 3 weeks after STZ injection. Endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anaesthetised rats. Cultured human coronary artery endothelial cells (HCAECs) were treated with high glucose (35.6 mM, 24 h) and reactive oxygen species (ROS) production with or without L-NAME (300 μM), apocynin (100 μM) or nicorandil (100 μM) was measured using fluorescent probes. Results: Endothelial function as evaluated by FMD was significantly reduced in diabetic as compared with normal rats (diabetes, 9.7 ± 1.4%; normal, 19.5 ± 1.7%; n = 6-7). There was a 2.4-fold increase in p47phox expression, a subunit of NADPH oxidase, and a 1.8-fold increase in total eNOS expression in diabetic rat femoral arteries. Nicorandil and tempol significantly improved FMD in diabetic rats (nicorandil, 17.7 ± 2.6%; tempol, 13.3 ± 1.4%; n = 6). Nicorandil significantly inhibited the increased expressions of p47phox and total eNOS in diabetic rat femoral arteries. Furthermore, nicorandil significantly inhibited the decreased expression of GTP cyclohydrolase I and the decreased dimer/monomer ratio of eNOS. ROS production in HCAECs was increased by high-glucose treatment, which was prevented by L-NAME and nicorandil suggesting that eNOS itself might serve as a superoxide source under high-glucose conditions and that nicorandil might prevent ROS production from eNOS. Conclusions: These results suggest that nicorandil improved diabetes-induced endothelial dysfunction through antioxidative effects by inhibiting NADPH oxidase and eNOS uncoupling. Keywords: Endothelial dysfunction, Diabetes, Nicorandil, Reactive oxidative species, eNOS, NADPH oxidase * Correspondence: † Contributed equally Product Research Department, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka 412-8513 Japan © 2011 Serizawa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Serizawa et al. Cardiovascular Diabetology 2011, 10:105 http://www.cardiab.com/content/10/1/105 Background Diabetes mellitus is regarded as an independent major risk factor for the development of cardiovascular disease, since long-term survival and freedom from cardiac events were reduced in diabetic coronary angioplasty patients [1-3]. Endothelial dysfunction plays a central role in diabetic vascular diseases [4]. A common mechanism underlying this endothelial dysfunction could involve increased production of reactive oxygen species (ROS) in vascular tissue [5]. High glucose greatly increases endothelial superoxide production [6], leading to an eNOS uncoupling state, followed by reduction of NO production and increased ROS production [7-11] which act to quench NO. Reduced NO availability will lead to attenuation of its beneficial vascular effects such as vasodilation, regulation of vascular smooth muscle proliferation, and expression of cellular adhesion molecules involved in the initiation of atherosclerotic plaque formation [12]. Therefore, increased ROS production in diabetes has been speculated to reduce endothelial NO availability, leading to endothelial dysfunction [13,14]. Nicorandil, an anti-angina agent with ATP-sensitive potassium channel opening and nitrate-like activity, reportedly improves prognosis in patients with angina pectoris via preconditioning effects [15], and also exerted endothelial protective effects in both clinical settings and animal studies. Long-term administration of nicorandil significantly improved endothelial function in patients with ischemic heart disease or with cardiovascular risk factors, as evaluated by measurement of flow-mediated dilation (FMD) in forearm arteries [16,17]. In the swine heart, nicorandil reduced myocardial no-reflow after ischemia reperfusion by protecting endothelial function [18]. In human umbilical vein endothelial cells, nicorandil inhibited apoptosis induced by serum starvation by inhibiting ROS production [19]. Furthermore, nicorandil protected from diabetic through inhibition of the production of ROS stimulated by high glucose [20]. Therefore, we hypothesised that nicorandil can prevent diabetic endothelial dysfunction. In the present study, we investigated the protective effect of nicorandil on endothelial function in streptozotocin (STZ)-induced diabetic rats by measuring FMD in femoral arteries using a high-resolution ultrasound system under in vivo conditions in which blood flow, many humoral factors and nerve activity were maintained. The mechanism underlying the protective action of nicorandil was also investigated in relation to ROS production in the endothelium both in vivo and in vitro. Methods Animals Male Sprague-Dawley rats (Charles River Japan, Yokohama, Japan, 6 weeks old, 200-240 g) were used in all experiments. All rats were fed ordinary laboratory chow Page 2 of 10 and allowed free access to water under a constant light and dark cycle of 12 h. Diabetes was induced by intraperitoneal administration of STZ (40 mg/kg) once a day for 3 days. One week after STZ administration, glucose concentrations were measured. Diabetes was considered to have been induced when the glucose level was higher than 250 mg/dL. Nicorandil (15 mg/kg/day) and tempol (20 mg/kg/day) were administered in drinking water for one week, starting 3 weeks after STZ administration. All animal procedures were conducted in accordance with Chugai Pharmaceutical’s ethical guidelines for animal care, and all experimental protocols were approved by the Animal Care Committee of the institution and conformed to the Guide for the Care and Use of Laboratory Animals published by the US Nationa (...truncated)