γ-分泌酶抑制剂对血管内皮细胞抗过氧化氢损伤的保护作用及其机制  

The protective effects and mechanism of DAPT against H2O2-induced vascular endothelial cells injury

作　　者：杨阳[1] 梁振兴[1] 王宁[1] 段维勋[1] 金振晓[1] 易定华[1] 

机构地区：[1]第四军医大学西京医院心血管外科,西安710032

出　　处：《中华实验外科杂志》2013年第5期887-890,共4页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家十二五科技支撑计划资助项目（2011BAI11B20）;国家自然科学基金资助项目（81102687、81070198）;陕西省社会发展推动基金资助项目（2012JQ4001）

摘　　要：目的探讨Notch信号特异性阻断剂γ-分泌酶抑制剂DAPT对血管内皮细胞抗过氧化氢（H2O2）损伤的保护作用及机制。方法分别用不同浓度H2O2（100、200、400μmoL／L）处理体外培养的人脐静脉内皮细胞（HUVECs）2、4、8h，噻唑蓝（MTT）比色法测定内皮细胞活力；Western blot法检测Notch信号通路受体Notchl、线粒体凋亡通路相关蛋白bax及抗凋亡蛋白B淋巴细胞／白血病-2（bcl-2）的表达。H2O2（300μmol／L）建立HUVECs损伤模型，不同浓度DAPT（40、20、10μmol／L）与H2O2共同处理HUVECs2、4、6h，分别检测细胞存活率、黏附能力、凋亡率；Western blot法检测过氧化氢和DAPT共处理的内皮细胞中Notchl、bcl-2和bax蛋白的表达。结果经不同浓度的H2O2（100、200、400μmol／L）处理内皮细胞2、4、6h后，内皮细胞的存活率明显降低（P〈0．01）；Notchl和bax的表达升高，抗凋亡蛋白bcl-2的表达降低。DAPT能明显提高H2O2损伤的内皮细胞存活率和黏附能力，降低凋亡率（P〈0．01）；经DAPT和H2O2共处理的内皮细胞与过氧化氢损伤组比较，Notchl和bax蛋白的表达显著降低，但是bcl-2蛋白的表达显著升高（P〈0．01），其中DAPT浓度为10μmol／L时效果最明显。结论低浓度DAPT对内皮细胞抗过氧化氢损伤有保护作用，可能与其抗氧化能力和抑制Notch信号通路有关。Objective To investigatetheprotective effects and mechanism of γ-secretase inhibitor DAPT, which is a specific blocker of the Notch signaling pathway, against H2O2-induced vascular endothelial cells injury. Methods The cultured human umbilical vein endothelial cells （HUVECs） were treated with DAPT （10, 20, 40 μmol/L）for 2, 4 and 6 h, methyl thiazol tctrazolium （MTT） method was used to detect the survival rate of cells; Western blotting was used to detect the expression of Notehl, bax and B lymphocytcs/leukemia-2 （bel-2） protein. The injury of HUVECs was induced by H2O2 （300 μ mol/L） treatment, and serial concentrations of DAPT （10, 20, 40 μmol/L）were applied together for 2, 4 and 6 h, MTT method was used to detect the survival ratios, Adhesion assay was used to determinate the adhesive rate of ceils; TdT-mediated dUTP nick end labeling （TUNEL） was used to detect the apoptotic rate of cells; Western bolt was used to detect the expression of Notchl, bcl-2 and bax protein. Results Aftertreatment with H2O2 （100, 200, 400 μmol/L） for 2, 4 and 6 h, the survival ratio of HUVECs decreased significantly. Western blotting showed the expression of Notchl and Baxincreased significantly, but the expression of bcl-2 decreased significantly, after being treated by H2O2 （P 〈 0. 01 ）. The survival ratio, adhesion capacity were improved significantly by DAPT treatment, and the apoptotic rate was decreased significantly （P 〈 0. 01 ）. In addition, the expression of Notchl and bax was decreased significantly, but the expression of bcl-2 was increased significantly （P 〈 0. 01 ）. These protective effects were much more significant when concentration of DAPT was 10 μmol/L compared with other concentrations observed. Conclu- sion Low concentrations of DAPT treatment can protect against H2O2 induced injuries on culturedhuman umbilical vein endothelial cells via its antioxidant capacity and Notch signaling pathway.

关 键 词：人脐静脉内皮细胞 凋亡 Γ-分泌酶抑制剂 NOTCH信号通路 

分 类 号：R285[医药卫生—中药学]