DAPT在ox-LDL损伤HUVECs过程中的细胞保护作用

目的:探究γ-分泌酶抑制剂N-N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)在氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)损伤人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)模型中的细胞保护作用及其对Notch信号通路的调控。方法:体外培养HUVECs,用oxLDL处理HUVECs构建细胞损伤模型。实验分为对照组、ox-LDL处理组、DAPT处理组和DAPT+ox-LDL处理组。用倒置相差显微镜观察不同处理方法下细胞的形态变化;CCK-8法检测细胞存活率;Western blot法检测蛋白Notch1、Notch4和Jagged1的表达情况。结果:体外培养HUVECs,倒置相差显微镜下发现ox-LDL处理组细胞死亡和碎片增多,经DAPT预处理后,ox-LDL作用造成的细胞损伤死亡较少,细胞碎片较少。通过CCK-8法检测发现ox-LDL处理组细胞存活率降低,DAPT处理组细胞存活率升高,DAPT预处理后ox-LDL造成存活率降低的幅度变小。在ox-LDL作用下,Notch1和Jagged1蛋白表达量降低,Notch4表达量升高;而DAPT作用下Notch1和Jagged1表达量升高,Notch4表达量降低;ox-LDL与DAPT共同作用时蛋白接近正常水平。结论:ox-LDL对HUVECs具有损伤作用;DAPT减轻ox-LDL对HUVECs造成的损伤;DAPT保护HUVECs免受ox-LDL损伤的作用与Notch信号通路有关。

DAPT attenuates ox-LDL-induced human umbilical vein endothelial cell injury

AIM: To investigate the effect of N-N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) on the Notch signaling pathway in a model of oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVEC) damage.METHODS: HUVECs were divided into control group, ox-LDL group, DAPT group and ox-LDL+DAPT group. The morphological changes of the HUVECs with different treatments were observed under light microscope. The viability of the HUVECs was measured by CCK-8 assay. The protein expression levels of Notch1, Notch4 and Jagged1 were determined by Western blot.RESULTS: ox-LDL induced great damage to the HUVECs, evidenced by increased cell death and debris in the culture. However, the cell damage was abolished by adding DAPT into the culture. The viability of the HUVECs was increased by co-treatment with DAPT and ox-LDL. ox-LDL treatment significantly decreased the protein expression levels of Notch1 and Jagged1, and elevated Notch4. However, these changes were totally reversed by DAPT. None of these proteins showed significant change in the HUVECs co-treated with DAPT and ox-LDL as compared with control group.CONCLUSION: ox-LDL is able to induce HUVEC damage in vitro. DAPT attenuates ox-LDL-induced damage in the HUVECs by regulating the Notch signaling pathway.