Bierhaus A, Chevion S, Chevion M, Hofmann M, Quehenberger P, Illmer T, Luther T, Berentshtein E, Tritschler H, Müller M, Wahl P, Ziegler R, Nawroth P P
Department of Internal Medicine, University of Heidelberg, Germany.
Diabetes. 1997 Sep;46(9):1481-90. doi: 10.2337/diab.46.9.1481.
Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.
细胞抗氧化防御机制的耗竭以及晚期糖基化终产物（AGEs）产生氧自由基被认为在糖尿病血管并发症的发病机制中起主要作用。在此我们证明，用AGE白蛋白（500 nmol/l）孵育培养的牛主动脉内皮细胞（BAECs）会导致还原型谷胱甘肽（GSH）和抗坏血酸水平受损。结果，细胞氧化应激增加导致转录因子NF-κB活化，从而促进包括内皮组织因子在内的各种NF-κB调控基因的上调。在AGE白蛋白诱导之前用天然存在的抗氧化剂α-硫辛酸补充细胞抗氧化防御，可完全防止AGE白蛋白依赖性的还原型谷胱甘肽和抗坏血酸耗竭。电泳迁移率变动分析（EMSA）显示，只要在AGE白蛋白刺激前至少30分钟添加α-硫辛酸，AGE白蛋白介导的NF-κB活化也会以时间和剂量依赖性方式降低。抑制作用并非由于与蛋白质-DNA结合的物理相互作用，因为直接加入结合反应中的α-硫辛酸并未阻止重组NF-κB的结合活性。蛋白质印迹进一步证明，α-硫辛酸抑制NF-κB从细胞质释放并转运到细胞核中。因此，α-硫辛酸减少了AGE白蛋白诱导的NF-κB介导的与糖尿病相关的内皮基因的转录和表达，如组织因子和内皮素-1。因此，通过细胞外给予α-硫辛酸补充细胞抗氧化防御机制可在体外减少AGE白蛋白诱导的内皮功能障碍。
