ATF6与IRE1XBP1在氧糖剥夺/复氧损伤HT22细胞中的交互作用  

Crosstalk between activating transcription factor 6 and the inositol-requiring enzyme 1-X-box binding protein 1 pathway in oxygen-glucose deprivation/reoxygenation-injured HT22 cells

作　　者：唐婷婷 廉应涛 陆礼萍 徐松 余追[1] Tang Tingting;Lian Yingtao;Lu Liping;Xu Song;Yu Zhui(Department of Critical Care Medicine,Renmin Hospital of Wuhan University,Wuhan 430060,Hubei,China;Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,Hubei,China)

机构地区：[1]武汉大学人民医院重症医学科,湖北武汉430060 [2]武汉大学人民医院麻醉科,湖北武汉430060

出　　处：《中华危重病急救医学》2023年第3期278-286,共9页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81772039)。

摘　　要：目的:研究转录激活因子6(ATF6)与需肌醇酶1-X-框结合蛋白1(IRE1-XBP1)在氧糖剥夺/复氧(OGD/R)损伤小鼠海马神经元(HT22)细胞中的交互作用。方法:复制OGD/R损伤HT22细胞模型,观察OGD/R后不同时间点(0、3、6、12、24 h)内质网应激(ERS)、细胞活性和凋亡的变化。将对数生长期的HT22细胞分为空白对照组、对照+ATF6激动剂AA147组、对照+IRE1抑制剂4μ8c组、OGD/R模型组、OGD/R+AA147组、OGD/R+4μ8c组(AA147组和4μ8c组于全程添加10 μmol/L AA147或16 μmol/L 4μ8c)。采用蛋白质免疫印迹试验(Western blotting)检测各组HT22细胞ERS相关蛋白〔葡萄糖调节蛋白78(GRP78)、磷酸化需肌醇酶1(p-IRE1)、磷酸化真核细胞翻译起始因子2α(p-eIF2α)〕以及凋亡相关蛋白Bcl-2、Bax、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)、活化caspase-3的表达;采用实时荧光定量聚合酶链反应(RT-qPCR)检测ERS相关基因以及ATF6〔同型半胱氨酸内质网应激泛素样结构域1(Herpud1)、蛋白二硫键异构酶家族成员4(Pdia4)、Lin-12样抑制子/增强子(Sel1L)〕和剪接型X-框结合蛋白1〔XBP1s,包括DnaJ热休克蛋白成员B9(Erdj4)、Sec24相关基因家族成员D(Sec24d)、信号受体序列3(Ssr3)〕介导的转录反应相关基因的mRNA表达;采用细胞增殖与毒性检测试剂盒(CCK-8)检测细胞活性;采用免疫荧光法检测活化caspase-3表达。结果:与空白对照组比较,ERS相关蛋白p-IRE1和p-eIF2α的蛋白表达量分别在OGD/R 12 h、OGD/R 3 h升高(p-IRE1/β-actin:2.09±0.10比1.00±0.00,p-eIF2α/β-actin:1.39±0.11比1.00±0.00,均 P<0.01),ERS相关基因ATF6、XBP1s、非剪接型X-框结合蛋白1(XBP1u)、转录激活因子4(ATF4)、CCAAT/增强子结合蛋白同源蛋白(CHOP)的mRNA表达量在OGD/R后不同时间点也明显升高,表明ERS在OGD/R作用的HT22细胞中被激活。与OGD/R模型组比较,OGD/R+AA147组p-IRE1蛋白表达量无改变,但XBP1s和XBP1u的mRNA表达量明显下降〔XBP1s(2 -ΔΔCt):0.76(Objective To study the crosstalk between the activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1)-X-box binding protein 1(XBP1)pathway in oxygen-glucose deprivation/reoxygenation(OGD/R)-injured mouse hippocampal neuronal cell line HT22.Methods The OGD/R-injured HT22 cell model was used to observe the changes of the indicators of endoplasmic reticulum stress(ERS),cell viability,and apoptosis at different OGD/R time points(0,3,6,12,and 24 hours).HT22 cells in the logarithmic growth phase were randomized into blank control group,control+ATF6 activator(AA147)group,control+IRE1 inhibitor(4μ8c)group,OGD/R model group,OGD/R+AA147 group and OGD/R+4μ8c group(10μmol/L AA147 or 16μmol/L 4μ8c was given during the whole process in the AA147 group and 4μ8c group).Western blotting was used to detect the expression of ERS-related proteins[glucose-regulated protein 78(GRP78),phosphorylated-inositol-requiring enzyme 1(p-IRE1),and phosphorylated-eukaryotic translation initiation factor-2α(p-eIF2α)],and apoptosis-related proteins(Bcl-2,Bax,caspase-3,and cleaved caspase-3).The mRNA of ERS-related genes,and ATF6[homocysteine-inducible,endoplasmic reticulum stress-inducible,ubiquitin-like domain member 1(Herpud1),protein disulfide isomerase associated 4(Pdia4)and Sel-1 suppressor of lin-12-like(Sel1L)]and spliced XBP1[XBP1s,include DnaJ heat shock protein family member B9(Erdj4),Sec24 related gene family,member D(Sec24d)and signal sequence receptor,gamma(Ssr3)]induced transcriptional response-related genes were measured by real-time quantitative polymerase chain reaction(RT-qPCR).A cell counting kit-8(CCK-8)assay was used to detect the viability of HT22 cells.Immunofluorescence was utilized to test the expression of cleaved caspase-3.Results Compared with the blank control group,the expression of ERS-related proteins p-IRE1 and p-eIF2αwere significantly increased at 12 hours and 3 hours following OGD/R,respectively(p-IRE1/β-actin:2.09±0.10 vs.1.00±0.00,p-eIF2α/β-actin:1.39±0.11 vs.1.00±0.00,both

关 键 词：内质网应激 未折叠蛋白反应 转录激活因子6 X-框结合蛋白1 凋亡 

分 类 号：R338[医药卫生—人体生理学]