海马神经元线粒体DNA 6mA在血管性认知障碍中的作用研究  

Role of mitochondrial DNA 6mA in the hippocampal neurons in vascular cognitive impairment

作　　者：陈子怡 杨凌飞 王开昕 李庆生 贾延劼[1] 龚哲[1] Chen Ziyi;Yang Lingfei;Wang Kaixin;Li Qingsheng;Jia Yanjie;Gong Zhe(Department of Neurology,First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]郑州大学第一附属医院神经内科,郑州450000

出　　处：《中华神经医学杂志》2024年第8期757-768,共12页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金青年科学基金(82001290)。

摘　　要：目的探讨海马神经元线粒体DNA N6-脱氧腺苷甲基化(6mA)在血管性认知障碍中的作用及相关机制。方法(1)体内实验:采用随机数字表法将SPF级健康雄性大鼠分为假手术组及慢性脑低灌注(CCH)组,每组12只。CCH组大鼠结扎双侧颈总动脉建立CCH模型;假手术组大鼠仅分离双侧颈总动脉,不结扎。2组大鼠于造模后第50天采用旷场实验检测探索能力,第51~53天采用新物体识别试验进行认知功能评估,第54~59天采用Morris水迷宫法检测学习记忆能力。随后处死大鼠检测海马组织ATP浓度及活性氧(ROS)荧光强度并通过TUNEL染色检测海马CA1区神经元凋亡情况。(2)体外实验:HT-22细胞分为正常对照(NC)组、氧糖剥夺(OGD)组、OGD+siControl组、OGD+siMETTL4组。其中NC组正常培养细胞,OGD组给予低糖低氧处理12 h,OGD+siControl组及OGD+siMETTL4组分别给予NC-siRNA、METTL4-siRNA转染细胞后低糖低氧处理12 h。通过透射电镜观察细胞线粒体形态,采用流式细胞术检测细胞ROS荧光强度,通过JC-1荧光染色检测细胞线粒体膜电位,使用试剂盒检测线粒体复合物Ⅰ、Ⅲ活性。(3)体内及体外实验:采用免疫荧光染色实验及Western blotting实验检测大鼠海马组织神经元线粒体及HT-22细胞线粒体中METTL4及DNA 6mA表达。结果(1)体内实验:与假手术组比较,CCH组大鼠出现认知障碍表现,表现为旷场实验中进入中心区域次数明显增多,探索新物体时间明显减少,水迷宫实验中穿越平台次数明显减少,逃避潜伏期明显延长,差异均有统计学意义(P<0.05)。与假手术组大鼠比较,CCH组大鼠海马ATP浓度明显下降[(18.820±1.177)nmol/Lvs.(10.190±0.519)nmol/L],ROS荧光强度明显增加[(4488.00±255.70)AUvs.(11644.00±530.20)AU],差异均有统计学意义(P<0.05)。TUNEL染色实验结果显示CCH组大鼠海马神经CA1区凋亡神经元数量较假手术组大鼠明显增多。免疫荧光染色结果显示CCH组大鼠海马神�ObjectiveTo investigate the role and mechanism of mitochondrial DNA N6-methyladenine(6mA)in the hippocampal neurons in vascular cognitive impairment.Methods(1)In vivo experiments:SPF male rats were randomly divided into sham-operated group and chronic cerebral hypoperfusion(CCH)group(n=12).CCH models in the CCH group were established by ligating bilateral carotid arteries,while rats in the sham-operated group were only bilaterally dissected without ligation.Exploratory ability was detected by open field test 50 d after modeling,cognitive function was evaluated by novel object recognition test 51-53 d after modeling,and learning and memory abilities were tested by Morris water maze 54-59 d after modeling.And then,rats were sacrificed;ATP concentration and reactive oxygen species(ROS)level in the hippocampal tissues were detected,and neuron apoptosis in the hippocampal CA1 area was detected by TUNEL.(2)In vitro experiments:HT-22 cells were divided into normal control(NC)group,oxygen-glucose deprivation(OGD)group,OGD+siControl group,and OGD+siMETTL4 group.Cells in the NC group were cultured routinely,cells in the OGD group were subjected to low sugar and low oxygen for 12 h,and cells in the OGD+siControl group and OGD+siMETTL4 group were,respectively,transfected with NC-siRNA or METTL4-siRNA after being subjected to low sugar and low oxygen for 12 h.Mitochondria morphology was observed by transmission electron microscopy,ROS was detected by flow cytometry,mitochondria membrane potential was detected by JC-1 fluorescent staining,and mitochondrial complex I and III activity was detected by kit.(3)In vivo and in vitro experiments:METTL4 and DNA 6mA expressions in neuronal mitochondria of rat hippocampal tissues and mitochondria of HT-22 cells were detected by immunofluorescent staining and Western blotting.Results(1)CCH rats had cognitive impairment:compared with the sham-operated group,CCH group had significantly increased frequency of entering the central area and reduced time in exploring new objects in open field e

关 键 词：血管性认知障碍 慢性脑低灌注 线粒体DNA 6mA METTL4 

分 类 号：R749.13[医药卫生—神经病学与精神病学]