Thrombin-induoed vascular injury through the overproduction of reactive oxygen species

为了阐明凝血酶对血管内皮细胞活性氧（ROS）产生的影响，我们利用荧光探针（DCFH-DA）分析了培养的人脐静脉内皮细胞（HUVEC）细胞内过氧化氢的含量。在HUVEC中，通过1U/ml凝血酶处理24小时，ROS增加到对照的两倍。降低线粒体膜质子梯度的羰基氰化物间氯苯肼和复合物II抑制剂壬基三氟丙酮均能显著抑制凝血酶处理的HUVEC中ROS的过量产生。作为NADPH氧化酶的抑制剂，Quinacrine也能显著抑制ROS的过量产生。相反，吲哚美辛（一种环氧化酶抑制剂）和L-NAME（一种no合成酶抑制剂）没有明显的抑制作用。配合物I的抑制剂DPI和配合物III的抑制剂myxothiazol对ROS的产生也没有明显的影响。激光共焦显微镜成像显示，DCFH-DA的绿色荧光与Mitotracker red的红色荧光共定位，表明线粒体产生过氧化氢。阿司匹林（1mM）预处理可显著抑制凝血酶处理的HUVEC中ROS的产生。上述结果表明，凝血酶可引起线粒体呼吸链ROS和NADPH氧化酶的过量产生，进而引起血管内皮细胞NO的消耗，导致血管内皮细胞损伤。此外，阿司匹林可以保护血管内皮细胞免受凝血蛋白诱导的ROS产生的损伤。

To clarify the effect of thrombin on the production of reactive oxygen species (ROS) in vasular endothelial cells, we analyzed intracellular amount of hydrogen peroxide in cultured human umbilical vein endothelial cells (HUVEC) using the fluorescence probe (DCFH-DA). In HUVEC, ROS was increased up to two-folds of control by the treatment of 1U/ml thrombin for 24 hours. Carbonyl cyanide m-chlorophenylhyazone, an agent for decreasing the mitochondrial membrane proton gradient and thenoyltrifluoroacetone, a complex II inhibitor, markedly suppressed the overproduction of ROS in thrombin-treated HUVEC. Quinacrine, an inhibitor for NADPH oxidase, also significantly suppressed the overproduction of ROS. In contrast, no significant inhibitory effects of indomethacin, an inhibitor for cyclooxygenase, or of L-NAME, an inhibitor for NO synthase, were observed. DPI, an inhibitor for complex I, and myxothiazol, an inhibitor for complex III, also did not exhibit a significant effect on the production of ROS. Con focal laser microscopy imaging showed that green fluoresscence of DCFH-DA colocalized with red fluorescence of Mitotracker Red, indicating hydrogen peroxide production from mitochondria. Pre-treatment of aspirin (1mM) markedly suppressed the ROS production in thrombin-treated HUVEC.These findings showed that thrombin could cause the overproduction of ROS from mitochondrial respiratory chain and NADPH oxidase, and subsequently the consumption of NO in vascular endothelial cells, leading to injury of vscular endothelial cells. In addition, aspirin may protect vascular endothelial cells from damage by trombin-induced ROS poduction.