蛋白质O-GlcNAc修饰在氧糖剥夺/复氧复糖小鼠神经细胞氧化应激损伤中的作用  

Role of protein O-GlcNAc modification in oxidative stress injury in nerve cells of mice subjected to oxygen-glucose deprivation and restoration

作　　者：吴科帆 张爱宁 邱珍[1] 江梦[1] 夏中元[1] Wu Kefan;Zhang Aining;Qiu Zhen;Jiang Meng;Xia Zhongyuan(Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院麻醉科,430060

出　　处：《中华麻醉学杂志》2021年第11期1396-1399,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(81801309,81671891)。

摘　　要：目的评价蛋白质糖基化(O-GlcNAc)修饰在氧糖剥夺/复氧复糖小鼠神经细胞氧化应激损伤中的作用。方法标准小鼠海马神经细胞系,以5×10^(4)个/ml的密度接种于培养板或培养皿,采用随机数字表法分为4组(n=20):正常组(N组)、O-(连接)N-乙酰葡糖胺水解酶(OGA)抑制剂Thiamet G组(T组)、氧糖剥夺/复氧复糖组(D/R组)以及Thiamet G+氧糖剥夺/复氧复糖组(T-D/R组)。采用低糖培养基在94%N_(2)-5%CO_(2)-1%O_(2)混合气体培养6 h进行糖氧剥夺,随后更换正常培养基进行复氧复糖12 h。T-D/R组在氧糖剥夺/复氧复糖前4 h时细胞培养液中加入终浓度1 mmol/L的Thiamet G,T组于提取细胞蛋白前4 h换成含终浓度1 mmol/L Thiamet G的培养基。复氧复糖完成后,采用DCFH-DA染色法检测细胞ROS累积量,Jc-1染色法检测线粒体膜电位,免疫荧光法检测O-GlcNAc修饰水平,Western blot法检测核因子E2相关因子2(Nrf2)和c-Jun氨基末端激酶(JNK)、磷酸化JNK(p-JNK)、p53抑癌基因(p53)表达水平。结果与N组比较,T组神经细胞O-GlcNAc表达上调,D/R组神经细胞ROS累积量增多,JC-1单体升高,JC-1聚合物降低,Nrf2表达下调,p-JNK、p53表达上调,T-D/R组神经细胞O-GlcNAc表达上调,ROS累积量增多,JC-1单体与JC-1聚合升高,Nrf2表达下调,p-JNK、p53表达上调(P<0.05)。与D/R组比较,T-D/R组神经细胞O-GlcNAc表达上调,ROS累积量减少,JC-1单体降低,JC-1聚合物升高,Nrf2表达上调,p-JNK、p53表达下调(P<0.05)。结论小鼠神经细胞氧糖剥夺/复氧复糖时,蛋白质O-GlcNAc修饰作为内源性保护机制水平增强,可减轻氧化应激损伤,机制可能与调控Nrf2介导的JNK通路有关。Objective To evaluate the role of protein O-linked beta-N-acetylglucosaminylation(O-GlcNAcylation)modification in oxidative stress injury in nerve cells of mice subjected to oxygen-glucose deprivation and restoration(OGD/R).Methods The standard mouse hippocampal neuron cell line was inoculated on a culture plate or dish at a density of 5×10^(4) cells/ml and divided into 4 groups(n=20 each)using a random number table method:normal group(N group),O-(connection)N-acetylglucosamine hydrolase(OGA)inhibitor Thiamet G group(T group),OGD/R group(D/R group)and Thiamet G+OGD/R complex sugar group(T-D/R group).The cells were exposed to a mixed gas of 94%N_(2)-5%CO_(2)-1%O_(2) for 6 h in a low-glucose medium,then medium was replaced with a common medium for restoring oxygen and glucose,and the cells were cultured for 12 h.Thiamet G at a final concentration of 1 mmol/L was added to the culture medium at 4 h before OGD/R in T-D/R group,and the medium was replaced with a medium containing Thiamet G at a final concentration of 1 mmol/L at 4 h before extraction of cellular proteins.After oxygen and glucose restoration was completed,the accumulation of cellular ROS was measured using DCFH-DA staining,mitochondrial membrane potential was measured using Jc-1 staining,O-GlcNAc modification was determined by immunofluorescence,and the expression of nuclear factor E2-related factor 2(Nrf2),c-Jun N-terminal kinase(JNK),phosphorylated JNK(p-JNK),and p53 tumor suppressor gene(p53)was detected using Western blot.Results Compared with group N,the expression of O-GlcNAc in nerve cells was significantly up-regulated in group T,and the accumulation of ROS in nerve cells was significantly increased,JC-1 monomer was increased,JC-1 polymer was decreased,Nrf2 expression was down-regulated,and the expression of p-JNK and p53 was up-regulated in group D/R,and the expression of O-GlcNAc in nerve cells was up-regulated,the accumulation of ROS was increased,the polymerization of JC-1 monomer and JC-1 was increased,Nrf2 expression was down-regulated,an

关 键 词：糖基化 再灌注损伤 脑 氧化性应激 

分 类 号：R743.3[医药卫生—神经病学与精神病学]