氰酸盐诱导人脐静脉内皮细胞氧化应激损伤

Cyanate Induces Oxidative Stress Injury in Human Umbilical Vein Endothelial Cells

该研究探讨尿素水解产物氰酸盐(cyanate)对体外培养的血管内皮细胞氧化应激和功能障碍的作用。培养人脐静脉内皮细胞系(human umbilical vein endothelial cells,HUVECs),通过CCK8法检测氰酸盐对内皮细胞活力的影响;采用DCFH-DA法检测ROS水平;用比色法测定NO水平;分别用细胞免疫荧光和Western blot检测ICAM-1(intercelluar adhesion molecule-1)、e NOS(endothelial nitric oxide synthase)表达。氰酸盐呈浓度依赖性影响内皮细胞活力,与对照组相比,当氰酸盐浓度为1.00 mmol/L时,细胞活力受到明显抑制(P<0.05);与正常组和阴性对照组(甘露醇组)相比,氰酸盐诱导内皮细胞内ROS水平明显升高;NO水平明显减少(P<0.05)。免疫荧光结果显示,氰酸盐作用内皮细胞24 h后,ICAM-1荧光明显增强,e NOS明显减弱(P<0.05)。Western blot结果显示,内皮细胞内ICAM-1水平随氰酸盐浓度升高和负荷时间延长上调,而e NOS水平下调(P<0.05)。氰酸盐诱导血管内皮细胞氧化应激产生和功能障碍。

The study was to explore the mechanism of oxidative stress and dysfunction in human umbilical vein endothelial cells（HUVECs） treated with cyanate in vitro. The vitality of the cells treated with cyanate of different concentrations were measured by the CCK8 method. ROS level was detected by DCFH-DA assay. The NO level was detected by a commercial NO assay kit. The levels of intercelluar adhesion molecule-1（ICAM-1） and endothelial nitric oxide synthase（e NOS） proteins were observed by immunofluorescence. Western blot analysis was used to assess the protein levels of ICAM-1 and e NOS. The activities of HUVECs in culture was inhibited by cyanate in a does dependent manner（P0.05）. Compared with the normal group and the control（mannitol） group, ROS levels were greatly increased after cyanate loading 24 h（P0.05）. NO level was decreased significantly at 24 h after cyanate treatment（P0.05）. Immunofluorescence showed high positive immunostaining of ICAM-1 after 1.00 mmol/L cyanate treatment 24 h（P0.05）. However, the immunostaining of e NOS was lowered（P0.05）. The level of ICAM-1 was remarkably increased, and e NOS was clearly reduced. Cyanate can induce in HUVECs oxidative stress and dysfunction.