Abstract
Background3',4'-Dihydroxyflavonol (DiOHF) is an effective antioxidant that acutely preserves nitric oxide (NO) activity in the presence of elevated reactive oxygen species (ROS). We hypothesized that DiOHF treatment (7 days, 1 mg/kg per day s.c.) would improve relaxation in mesenteric arteries from diabetic rats where endothelial dysfunction is associated with elevated oxidant stress.Methodology/Principal FindingsIn mesenteric arteries from diabetic rats there was an increase in ROS, measured by L-012 and 2',7'-dichlorodihydrofluorescein diacetate fluorescence. NADPH oxidase-derived superoxide levels, assayed by lucigenin chemiluminescence, were also significantly increased in diabetic mesenteric arteries (diabetes, 4892±946 counts/mg versus normal 2486±344 counts/mg, n = 7–10, p<0.01) associated with an increase in Nox2 expression but DiOHF (2094±300 counts/mg, n = 10, p<0.001) reversed that effect. Acetylcholine (ACh)-induced relaxation of mesenteric arteries was assessed using wire myography (pEC50 = 7.94±0.13 n = 12). Diabetes significantly reduced the sensitivity to ACh and treatment with DiOHF prevented endothelial dysfunction (pEC50, diabetic 6.86±0.12 versus diabetic+DiOHF, 7.49±0.13, n = 11, p<0.01). The contribution of NO versus endothelium-derived hyperpolarizing factor (EDHF) to ACh-induced relaxation was assessed by evaluating responses in the presence of TRAM-34+apamin+iberiotoxin or N-nitro-L-arginine+ODQ respectively. Diabetes impaired the contribution of both NO (maximum relaxation, Rmax diabetic 24±7 versus normal, 68±10, n = 9–10, p<0.01) and EDHF (pEC50, diabetic 6.63±0.15 versus normal, 7.14±0.12, n = 10–11, p<0.01) to endothelium-dependent relaxation. DiOHF treatment did not significantly affect the EDHF contribution but enhanced NO-mediated relaxation (Rmax 69±6, n = 11, p<0.01). Western blotting demonstrated that diabetes also decreased expression and increased uncoupling of endothelial NO synthase (eNOS). Treatment of the diabetic rats with DiOHF significantly reduced vascular ROS and restored NO-mediated endothelium-dependent relaxation. Treatment of the diabetic rats with DiOHF also increased eNOS expression, both in total and as a dimer.Conclusions/SignificanceDiOHF improves NO activity in diabetes by reducing Nox2-dependent superoxide production and preventing eNOS uncoupling to improve endothelial function.
Highlights
Endothelial dysfunction, characterized by the impairment of endothelium-dependent relaxation, is recognised as a critical and initiating factor in the development of diabetes-induced vascular complications [1,2]
Body weights and blood glucose The body weight gained, blood glucose and HbA1c levels of the rats are shown in Table 1. 8 weeks after treatment with streptozotocin or vehicle, the body weight gained in normal rats was significantly greater than in diabetic rats (Table 1)
This study demonstrates that treatment of type 1 diabetic rats with the synthetic flavonol DiOHF (1mg/kg, per day) for 7 days reduces the levels of vascular oxidative stress and improves endothelium-dependent relaxation in mesenteric arteries
Summary
Endothelial dysfunction, characterized by the impairment of endothelium-dependent relaxation, is recognised as a critical and initiating factor in the development of diabetes-induced vascular complications [1,2]. Clinical trials with antioxidants have failed to clearly demonstrate any beneficial effect on vascular function [9,10]. More effective antioxidants, acting perhaps by targeting the specific sources of reactive oxygen species (ROS) might prove more beneficial than direct scavenging strategies [8,11,12]. Potential targets for pharmacological therapies include NADPH oxidase, endothelial nitric oxide synthase (eNOS) and mitochondria all of which have been reported to be sources of increased ROS in diabetes [2,13]
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