基于内质网应激介导高糖条件下血管生成素2敲减对大鼠血管内皮细胞凋亡的影响及PI3K/Akt信号通路的作用机制  

Effects of Ang-2 knockdown on apoptosis of rat vascular endothelial cells under endoplasmic reticulum stress and the mechanism of PI3K/Akt signaling pathway

作　　者：何泉[1] 张雯[2] 张锦娟 韩斌[1] 唐新华[4] HE Quan;ZHANG Wen;ZHANG J injuan(Department of Emergency,The First People＇s Hospital of Yunnan Province,Kunming 650032,China)

机构地区：[1]云南省第一人民医院急诊科,昆明650032 [2]云南省第一人民医院基础研究所,昆明650032 [3]中国科学院昆明动物研究所 [4]云南省第一人民医院遗传诊断中心,昆明650032

出　　处：《中国糖尿病杂志》2018年第11期947-954,共8页Chinese Journal of Diabetes

基　　金：云南省科技厅应用基础研究(昆医联合专项)基金(2015FB091)

摘　　要：目的研究血管生成素2(Ang-2)敲减对高糖环境下内质网应激(ERS)介导的大鼠血管内皮细胞凋亡的影响及对PI3K/Akt信号通路的影响和作用机制。方法用shRNA敲减大鼠血管内皮细胞中Ang-2的表达。检测Ang-2敲减对高糖诱导细胞和ERS诱导剂毒胡萝卜素(TG)诱导的内质网损伤细胞的保护作用。采用CCK8法检测细胞活性,Annexin V-FITC/PI法检测细胞凋亡。采用重氮反应法检测细胞上清液一氧化氮(NO)水平;ELISA检测单核细胞趋化蛋白1(MCP-1)分泌的影响。流式细胞仪检测CD144+内皮微粒的比例。Western blot和RT-PCR分别检测ERS相关基因蛋白表达水平和mRNA表达水平的变化。结果 Ang-2敲减下调高糖和TG处理条件下RAOEC的凋亡水平,减弱高糖和TG处理对RAOEC细胞增殖的抑制作用;Ang-2敲减阻滞MCP-1的分泌,同时促进TG处理后NO水平的升高,但敲减Ang-2的表达对CD144+内皮微粒的释放水平没有明显影响。Ang-2敲减下调ERS相关基因ATF6,iNOS,ORP150,Bip和EIF2α的表达。PI3K/Akt通路中PI3K和Akt的表达水平也随着Ang-2的敲减而下调。结论 Ang-2敲减可下调高糖和TG诱导的ERS相关基因的表达,对细胞有保护作用。Objective To study the effect of Ang-2 knockdown on the apoptosis of rat vascular endothelial cells induced by endoplasmic reticulum stress in hyperglycemia environment and its effect on PI3 K/Akt signaling pathway. Methods The expression of Ang-2 in rat vascular endothelial cells was knockdown by shRNA.To detect the protective effect of Ang-2 knockdown on hyperglycemic-induced cells and thapsigargin（TG）-induced endoplasmic reticulum injury cells,cell activity was detected by CCK8 assay and apoptosis was detected by Annexin v-FITC/PI assay.The levels of supernatant NO were detected by diazo reaction.The MCP-1 secretion was detected by ELISA.The percentage of CD144＋endothelial particles was analyzed by flow cytometry.Western blot and RT-PCR were used to detect the changes of protein expression and mRNA expression of endoplasmic reticulum stress-related genes.Results Ang-2 knockdown reduced the apoptosis level of RAOEC under hyperglycemia and TG treatment and decreased the inhibitory effect of hyperglycemia and TG treatment on the proliferation of RAOEC cells.Ang-2 knockdown blocked the secretion of MCP-1.At the same time,the increase of NO level after TG treatment was promoted.However,the knockdown of Ang-2 expression had no significant effect on the release level of CD144＋ endothelial particles.The expression of ATF6,iNOS,ORP150,Bip and EIF2αwere decreased by Ang-2 knockdown.The expression levels of PI3 Kand Akt in the PI3 K/Akt pathway also decreased with the decrease of Ang-2. Conclusion Ang-2 knockdown can down-regulate the expression of high glucose and TG-induced endoplasmic reticulum stress-related genes and protect the cells from apoptosis.

关 键 词：血管生成素2 高糖 内质网应激 凋亡 PI3K 

分 类 号：R587.2[医药卫生—内分泌]