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

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组大鼠海马神经元中6mA及METTL4分布以线粒体为主,表达较假手术组大鼠均明显增加。Western blotting实验结果显示CCH组大鼠海马组织线粒体中METTL4表达较假手术组大鼠明显升高(1.729±0.168vs.1.000±0.000),差异有统计学意义(P<0.05)。(2)体外实验:透射电镜下,与NC组比较,OGD组细胞表现为线粒体嵴消失、膜断裂、空泡化明显。与OGD组细胞比较,OGD+siMETTL4组细胞ATP生成明显增加,ROS生成明显减少,线粒体膜电位明显升高,线粒体复合物Ⅰ和Ⅲ活性明显增加,差异均有统计学意义(P<0.05)。细胞免疫荧光染色实验结果显示,OGD组细胞mtDNA 6mA及METTL4表达较NC组明显增加,且两者以在线粒体中表达为著;OGD+siMETTL4组细胞线粒体DNA(mtDNA)6mA及METTL4表达较OGD组细胞均明显降低。Western blotting实验结果显示,OGD+siMETTL4组细胞中METTL4表达较OGD组明显降低(1.578±0.261vs.2.970±0.280),差异有统计学意义(P<0.05)。结论CCH后认知障碍大鼠海马组织神经元线粒体特异性高表达的甲基化酶METTL4促使mtDNA 6mA表达增加,进而导致线粒体能量代谢障碍及ROS升高,可能为血管性认知障碍的机制之一。

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 experiment,and significantly decreased frequency of crossing the platform and prolonged escape latency in water maze experiment(P<0.05).Compared with rats in the sham-operated group,rats in the CCH group had significantly decreased hippocampal ATP content([18.820±1.177]nmol/L vs.[10.190±0.519]nmol/L)and increased ROS content([4488.00±255.70]AU vs.[11644.00±530.20]AU,P<0.05).TUNEL results showed that the number of apoptotic neurons in the hippocampal CA1 area of CCH group was obviously increased than that in sham-operated group.Immunofluorescent staining results showed that 6mA and METTL4 mainly distributed in the mitochondria of hippocampal neurons in CCH group,and the 6mA and METTL4 expressions were obviously increased compared with those in the sham-operated group.Western blotting results showed that METTL4 expression in the hippocampal mitochondria of CCH group was significantly higher than that in the sham-operated group(1.729±0.168 vs.1.000±0.000).(2)In vitro experiment:under transmission electron microscope,compared with the NC group,HT-22 cells in the OGD group showed obvious mitochondrial ridge disappearance,membrane rupture and vacuolation.Compared with the OGD group,the OGD+siMETTL4 group had significantly increased ATP production,decreased mtROS production,increased mitochondrial membrane potential,and increased mitochondrial complex I and III activities(P<0.05).Immunofluorescent staining results showed that the mtDNA 6mA and METTL4 expressions in the OGD group were obviously higher than those in the NC group,and both mainly expressed in the mitochondria;mtDNA 6mA expression in the OGD+siMETTL4 group was obviously lower than that in OGD group.Western blotting results showed that METTL4 expression in the OGD+siMETTL4 group was significantly higher than that in the OGD group(1.578±0.261 vs.2.970±0.280).ConclusionSpecific high expression of methylase METTL4 in hippocampal neurons of rats with cognitive impairment after CCH promotes the increased mtDNA 6mA expression and leads to mitochondrial energy metabolism disorders and increased ROS,which is speculated to be one of the mechanisms causing vascular cognitive impairment.