Giardino I, Edelstein D, Brownlee M
Department of Medicine, Albert Einstein College of Medicine, New York, USA.
J Clin Invest. 1996 Mar 15;97(6):1422-8. doi: 10.1172/JCI118563.
Hyperglycemia rapidly induces an increase in intracellular advanced glycation end products (AGEs) in bovine endothelial cells, causing an alteration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee. 1994. J. Clin. Invest. 94:110-117). Because sugar or sugar-adduct autoxidation is critical for AGE formation in vitro, we evaluated the role of reactive oxygen species (ROS) in intracellular AGE formation, using bovine aortic endothelial cells. 30 mM glucose increased intracellular ROS formation by 250% and lipid peroxidation by 330%, while not affecting ROS in the media. In cells depleted of glutathione, intracellular AGE accumulation increased linearly with ROS generation as measured by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 AU* mm/5x10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Deferoxamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hyperglycemia-induced formation of both ROS and AGE. To differentiate an effect of ROS generation on AGE formation from an effect of more distal oxidative processes, GM7373 endothelial cell lines were generated that stably expressed the peroxidation-suppressing proto-oncogene bcl-2. bcl-2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generation of intracellular AGEs, and that inhibition of oxidant pathways prevents intracellular AGE formation.
高血糖迅速诱导牛内皮细胞内细胞内晚期糖基化终产物（AGEs）增加，导致碱性成纤维细胞生长因子（bFGF）活性改变（贾迪诺，I.，D.埃德尔斯坦，和M.布朗利。1994年。《临床研究杂志》94:110 - 117）。由于糖或糖加合物的自氧化在体外AGE形成中至关重要，我们使用牛主动脉内皮细胞评估了活性氧（ROS）在细胞内AGE形成中的作用。30 mM葡萄糖使细胞内ROS形成增加250%，脂质过氧化增加330%，而不影响培养基中的ROS。在用谷胱甘肽耗尽的细胞中，通过免疫印迹和荧光探针DCFH测量，细胞内AGE积累随ROS产生呈线性增加（AGE 0.258 - 3.531 AU*mm/5x10(4)细胞，DCF 57 - 149平均AU，r = 0.998，P < 0.002）。去铁胺、α-生育酚和二甲基亚砜均抑制高血糖诱导的ROS和AGE形成。为了区分ROS产生对AGE形成的影响与更远端氧化过程的影响，构建了稳定表达抑制过氧化原癌基因bcl-2的GM7373内皮细胞系。bcl-2对高血糖诱导的细胞内ROS形成没有影响。相反，bcl-2表达降低了脂质过氧化（3小时时降低100%，168小时时降低29%）和AGE形成（168小时时降低55%）。这些数据表明，依赖ROS的过程在细胞内AGE的产生中起核心作用，并且抑制氧化途径可防止细胞内AGE形成。
