同型半胱氨酸硫内酯损伤血管内皮细胞的机制研究

Homocysteine thiolactone damages cultured endothelial cells

目的:在体外培养的内皮细胞中探讨同型半胱氨酸硫内酯(HTL)致血管内皮细胞损伤作用及其机制。方法:体外培养的人脐静脉内皮细胞,与不同浓度的HTL孵育,用ELISA检测内皮细胞中肿瘤坏死因子α(TNF-α)和可溶性细胞间黏附分子(sICAM)-1的浓度,荧光显微镜检测活性氧簇(ROS)的含量、核转录因子κB(NF-κB)的激活及IκBα蛋白表达,同时检测内皮细胞活力、乳酸脱氢酶(LDH)漏出量与一氧化氮(NO)水平。结果:HTL(1 mmol/L)孵育内皮细胞3 h后显著增加细胞ROS含量,刺激NF-κB转入胞核而活化,孵育细胞24 h后明显升高细胞上清液中sICAM-1和TNF-α浓度;降低细胞活力和NO的水平,增加LDH的漏出量。抗氧化剂NAC、NADPH氧化酶抑制剂Apocynin和NF-κB抑制剂PDTC可显著抑制HTL所致ROS含量的增加以及NF-κB激活,降低HTL刺激的培养液中sICAM-1和TNF-α的浓度,增加培养液中NO水平。结论:HTL诱导的血管内皮细胞功能损伤的机制可能与诱导氧化应激以及NF-κB活化有关。

AIM：To approach the mechanisms of homocysteine thiolactone（HTL）-induced damage in endothelial cells.METHODS： Human umbilical vein endothelial cells（HUVECs） were incubated with HTL.The concentrations of soluble intercellular adhesion molecule（sICAM）-1 and TNF-α in the conditioned medium were measured by ELISA.The activity of NF-κB and the level of ROS were determined by fluorescence microscopy.Cell viability,activity of lactate dehydrogenase（LDH） and content of nitric oxide（NO） in the medium were also detected.RESULTS： Exposure of HUVECs to HTL at concentration of 1 mmol/L for 3 h potentiated the activity of NF-κB and increased the level of ROS.Incubation of HUVECs with HTL（1 mmol/ L for 24 h） markedly decreased the cell viability and NO content,and increased the level of LDH,sICAM-1 and TNF-α in the culture medium.Pretreatment with NAC,apocynin or PDTC markedly inhibited the increased activity of NF-κB and decreased the levels of ROS,TNF-α,sICAM-1,NO and LDH in a dose-dependent manner.CONCLUSION： The dysfunction of endothelial cells induced by homocysteine thiolactone in vitro may be related to the oxidative stress and the activation of NF-κB.