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产生活性氧物种(ROS)的影响，我们用荧光探针(DCFH-DA)分析了培养的人脐静脉内皮细胞(HUVEC)内过氧化氢的含量。在HUVEC中，1U/ml凝血酶作用24小时后，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乙酮和复合体II抑制剂噻吩甲酰三氟丙酮可显著抑制凝血酶处理的HUVEC中ROS的过量产生。NADPH氧化酶的抑制剂奎纳克林也显著抑制了ROS的过量产生。环氧合酶抑制剂消炎痛和一氧化氮合酶抑制剂L-NAME均未见明显的抑制作用。复合体I的抑制剂DPI和复合体III的抑制剂Myxthiiazol对ROS的产生也没有显著影响。CON聚焦激光显微成像显示DCFH-DA的绿色荧光与MitoTracker Red的红色荧光共存，表明线粒体产生过氧化氢。阿司匹林(1 MM)可显著抑制凝血酶诱导的血管内皮细胞ROS的产生，提示凝血酶可引起线粒体呼吸链ROS和NADPH氧化酶的过度产生，进而导致血管内皮细胞NO的消耗，导致血管内皮细胞的损伤。此外，阿司匹林还可保护血管内皮细胞，使其免受转氨酶诱导的ROS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.