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

为探讨凝血酶对血管内皮细胞活性氧（ROS）产生的影响，采用荧光探针DCFH-DA检测了培养的人脐静脉内皮细胞（HUVEC）细胞内过氧化氢（H2 O2）的含量。1U/ml凝血酶作用HUVEC 24小时，ROS水平增加至对照组的2倍。羰基氰化物间氯苯腙，用于降低线粒体膜质子梯度和噻吩甲酰三氟丙酮，复合物II抑制剂的代理，显着抑制凝血酶处理的HUVEC中的ROS的过度产生。奎纳克林，NADPH氧化酶的抑制剂，也显着抑制ROS的过度产生。与此相反，没有显着的抑制作用，吲哚美辛，环氧合酶的抑制剂，或L-NAME，一氧化氮合酶的抑制剂，观察。复合物I的抑制剂DPI和复合物III的抑制剂myxothiazol也没有表现出对ROS产生的显著影响。共聚焦激光显微镜成像显示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（1 mM）预处理可明显抑制凝血酶处理的HUVEC产生ROS，提示凝血酶可引起HUVEC线粒体呼吸链和NADPH氧化酶产生ROS，进而消耗NO，导致血管内皮细胞损伤。此外，阿司匹林可保护血管内皮细胞免受Trombin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.