DNA甲基化调控p16表达在替格瑞洛改善血管内皮功能中的作用及机制  

The role and mechanism of ticagrelor on the vascular endothelial function through DNMT1/p16 signaling pathwuay

作　　者：朱伯谦 宋兵战[1] 陈凯[1] 姜旭 王振兴[1] ZHU Boqian;SONG Bingzhan;CHEN Kai;JIANG Xu;WANG Zhenxing(Department of Cardiology,Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing,210029,China)

机构地区：[1]南京中医药大学附属医院心内科,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2023年第2期169-178,共10页Journal of Nanjing Medical University(Natural Sciences)

基　　金：白求恩·医学科学研究基金项目(AX084ES);国家自然科学基金青年项目(81900237);江苏省自然科学基金青年项目(BK20191093)。

摘　　要：目的:探讨替格瑞洛对氧化型低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导内皮损伤的影响及机制。方法:采用ELISA法检测人血清内皮素-1、一氧化氮含量,CCK-8法、EdU法、Transwell法检测细胞活力、增殖及迁移能力,流式细胞术检测细胞凋亡,real-time PCR法、Western blot法检测DNA甲基化转移酶1(DNA Methyltransferase 1,DNMT1)、p16表达,慢病毒过表达载体构建p16的过表达系统,慢病毒敲除载体构建DNMT1和p16的敲除系统,甲基化特异性PCR法测定p16启动子区甲基化水平。结果:替格瑞洛促进ox-LDL诱导的人脐静脉内皮细胞(human umbilical endothelial cell,HUVEC)活力、增殖及迁移能力,并显著抑制ox-LDL诱导的细胞凋亡;替格瑞洛抑制ox-LDL诱导的HUVEC中p16表达,而过表达p16可逆转替格瑞洛对ox-LDL诱导HUVEC损伤的保护作用;DNMT1通过影响p16启动子区的甲基化水平抑制p16表达。结论:替格瑞洛可通过DNMT1介导的DNA甲基化调控p16表达,进而减轻ox-LDL诱导的HUVEC损伤,改善血管内皮功能。Objective:To investigate the effect and mechanism of ticagrelor on endothelial injury induced by ox-LDL.Methods:ELISA method was used to detect the serum ET-1 and NO contents.Cell viability,proliferation and migration ability were determined by CCK-8,EdU kit and Transwell assay respectively.Annexin V-PI assay was used to detect cell apoptosis.The levels of DNMT1 and p16 were detected by Real-time PCR and Western blot.The lentiviral overexpression vector pLVX-puro-EGFP was used to construct the p16 overexpression system.The lentiviral knockout vector pLKO.1 was used to construct the DNMT1 and p16 knockout system.The methylation status of p16 promoter region was determined by the methylation-specific PCR method.Results:Ticagrelor promoted the viability,proliferation and migration ability of human umbilical endothelial cell(HUVEC)treated with ox-LDL,and significantly inhibited ox-LDL-induced apoptosis.Ticagrelor down-regulated the expression of p16 in HUVECs induced by ox-LDL,and overexpressed p16 could reverse the protective effect of ticagrelor on HUVECs induced by ox-LDL.DNMT1 inhibited the expression of p16 by affecting the methylation level of p16 promoter region.Conclusion:Ticagrelor could reduce the injury of HUVECs induced by ox-LDL and improve endothelial function.The mechanism might be related to the regulation of DNMT1/p16 signaling pathway.

关 键 词：替格瑞洛 氧化型低密度脂蛋白 人脐静脉内皮细胞 P16 DNA甲基化转移酶1 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学]