Weidig P, McMaster D, Bayraktutan U
Department of Medicine, Institute of Clinical Science Block B, Queen's University Belfast, Belfast BT12 6BJ, UK.
Diabetes Obes Metab. 2004 Nov;6(6):432-41. doi: 10.1111/j.1462-8902.2004.00364.x.
Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O(2)(-)) are associated with the pathogenesis of endothelial cell dysfunction in diabetes mellitus. This study was designed to investigate the underlying causes of oxidative stress in coronary microvascular endothelial cells (CMECs) exposed to hyperglycaemia.
CMECs were cultured under normal (5.5 mmol/l) or high glucose (22 mmol/l) concentrations for 7 days. The activity and expression (protein level) of endothelial NO synthase (eNOS), inducible NOS (iNOS), NAD(p)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were investigated by specific activity assays and Western analyses, respectively, while the effects of hyperglycaemia on nitrite and O(2)(-) generation were investigated by Griess reaction and cytochrome C reduction assay, respectively.
Hyperglycaemia did not alter eNOS or iNOS protein expressions and overall nitrite generation, an index of NO production. However, it significantly reduced the levels of intracellular antioxidant glutathione by 50% (p < 0.05) and increased the protein expressions and activities of p22-phox, a membrane-bound component of pro-oxidant NAD(p)H oxidase and antioxidant enzymes (p < 0.05). Free radical scavengers, namely, Tiron and mercaptopropionylglycine (MPG) (0.1-1 micromol/l) reduced hyperglycaemia-induced antioxidant enzyme activity and increased glutathione and nitrite generation to the levels observed in CMEC cultured in normoglycaemic medium (p < 0.01). The differences in enzyme activity and expressions were independent of the increased osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments.
These results suggest that hyperglycaemia-induced oxidative stress may arise in CMEC as a result of enhanced pro-oxidant enzyme activity and diminished generation of antioxidant glutathione. By increasing the antioxidant enzyme capacity, CMEC may protect themselves against free radical-induced cell damage in diabetic conditions.
过量的自由基如一氧化氮（NO）和超氧阴离子（O₂⁻）与糖尿病患者内皮细胞功能障碍的发病机制有关。本研究旨在探讨暴露于高血糖环境下的冠状动脉微血管内皮细胞（CMECs）氧化应激的潜在原因。
将CMECs在正常（5.5 mmol/L）或高糖（22 mmol/L）浓度下培养7天。分别通过特定活性测定和蛋白质印迹分析来研究内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）、NAD（P）H氧化酶和抗氧化酶（即超氧化物歧化酶（SOD）、过氧化氢酶和谷胱甘肽过氧化物酶（GPx））的活性和表达（蛋白质水平），同时分别通过格里斯反应和细胞色素C还原试验来研究高血糖对亚硝酸盐和O₂⁻生成的影响。
高血糖并未改变eNOS或iNOS的蛋白质表达以及总的亚硝酸盐生成量（NO产生的一个指标）。然而，它使细胞内抗氧化剂谷胱甘肽水平显著降低了50%（p < 0.05），并增加了促氧化剂NAD（P）H氧化酶的膜结合成分p22 - phox以及抗氧化酶的蛋白质表达和活性（p < 0.05）。自由基清除剂，即钛铁试剂和巯基丙酰甘氨酸（MPG）（0.1 - 1 μmol/L）降低了高血糖诱导的抗氧化酶活性，并使谷胱甘肽和亚硝酸盐生成增加至在正常血糖培养基中培养的CMECs中观察到的水平（p < 0.01）。如在平行实验中使用等摩尔浓度的甘露醇所研究的那样，酶活性和表达的差异与高糖水平产生的渗透压升高无关。
这些结果表明，高血糖诱导的氧化应激可能在CMECs中由于促氧化酶活性增强和抗氧化剂谷胱甘肽生成减少而产生。通过增加抗氧化酶能力，CMECs可能在糖尿病状态下保护自身免受自由基诱导的细胞损伤。
