七氟烷后处理减轻失血性休克复苏小鼠认知功能障碍时METTL3介导的m6A甲基化修饰与SIRT1的关系  

Relationship between METTL3-mediated m6A methylation modification and SIRT1 during sevoflurane post-conditioning-induced mitigation of cognitive impairments in a mouse model of hemorrhagic shock and resuscitation

作　　者：吴雨洁 张丽 陶辉[1] 胡溯 牛志伦 万晓静 胡宪文[1] Wu Yujie;Zhang Li;Tao Hui;Hu Su;Niu Zhilun;Wan Xiaojing;Hu Xianwen(Department of Anesthesiology and Perioperative Medicine,Second Affiliated Hospital of Anhui Medical University,Hefei 230601,China)

机构地区：[1]安徽医科大学第二附属医院麻醉与围术期医学科,合肥230601

出　　处：《中华麻醉学杂志》2023年第11期1386-1391,共6页Chinese Journal of Anesthesiology

基　　金：安徽省教育厅重大研究项目(KJ2021ZD0030);安徽省重点研发项目(2022e07020045);安医大二附院临床培育计划重点项目(2021LCZD02)。

摘　　要：目的评价七氟烷后处理减轻失血性休克复苏(HSR)小鼠认知功能障碍时甲基转移酶样3(METTL3)介导的N6-甲基腺苷(m6A)甲基化修饰与沉默信息调节因子1(SIRT1)的关系。方法清洁级健康雄性C57BL/6小鼠40只, 8~10周龄, 体质量22~26 g, 采用随机数字表法分为5组(n=8):假手术组(Sham组)、HSR组、七氟烷后处理+HSR组(SP+HSR组)、过表达METTL3基因rAAV+七氟烷后处理+HSR组(METTL3+SP+HSR组)和过表达METTL3基因rAAV阴性对照+七氟烷后处理+HSR组(NC+SP+HSR组)。经右颈总动脉在30 min内将小鼠体内总血容量40%的血液匀速抽出, 1 h后在30 min内将抽出的血液原路回输, 建立HSR模型。SP+HSR组先HSR模型, 在输血即刻吸入七氟烷(呼气末浓度2.4%)30 min。Sham组和HSR组于相应时点吸入70%O2-30%CO_(2)混合气体30 min。METTL3+SP+HSR组和NC+SP+HSR组于造模前4周时向小鼠双侧海马注射相应病毒450 nl。于造模后72 h时, 采用Morris水迷宫实验和新物体识别实验检测小鼠学习记忆能力。行为学实验结束后, 采用Western blot法检测海马组织METTL3和SIRT1表达, 采用qRT-PCR法检测海马组织SIRT1 mRNA表达, 采用Dot blot法检测海马组织RNA m6A甲基化水平。结果与Sham组相比, HSR组第1~6天时逃避潜伏期延长, 原平台象限停留时间缩短, 穿越原平台位置次数减少, 新物体识别指数降低, 海马组织METTL3表达上调, RNA m6A甲基化水平升高, SIRT1及其mRNA表达下调(P<0.05);与HSR组相比, SP+HSR组第1~6天时逃避潜伏期缩短, 原平台象限停留时间延长, 穿越原平台位置次数增加, 新物体识别指数升高, 海马组织METTL3表达下调, RNA m6A甲基化水平下降, SIRT1及其mRNA表达上调(P<0.05);与SP+HSR组相比, METTL3+SP+HSR组第2~6天时逃避潜伏期延长, 原平台象限停留时间缩短, 穿越原平台位置次数减少, 新物体识别指数降低, 海马组织METTL3表达上调, RNA m6A甲基化水平升高, SIRT1及其mRNA表达下调(P<0.05), NCObjective To evaluate the relationship between methyltransferase-like 3(METTL3)-mediated RNA N6-Methyladenosine(m6A)methylation modification and silent information regulator factor 1(SIRT1)during sevoflurane post-conditioning-induced mitigation of cognitive impairments in a mouse model of hemorrhagic shock and resuscitation(HSR).Methods Forty clean-grade healthy male C57BL/6 mice,aged 8-10 weeks,with a body weight ranging from 22-26 g,were assigned into 5 groups(n=8 each)using a random number table method:sham operation group,HSR group,sevoflurane post-conditioning+HSR group(SP+HSR group),over-expression of METTL3 gene rAAV+sevoflurane post-conditioning+HSR group(METTL3+SP+HSR group),and over-expression of METTL3 gene rAAV negative control+sevoflurane post-conditioning+HSR group(NC+SP+HSR group).The HSR model was established by withdrawing 40%of the total blood volume from mice through the right carotid artery within 30 min,followed by reinfusion of the withdrawn blood over 30 min 1 h later.The SP+HSR group underwent HSR modeling first and then inhaled sevoflurane(end-tidal concentration 2.4%)for 30 min starting from the time point immediately after blood transfusion.The Sham group and HSR group inhaled a mixture of 70%O2 and 30%CO2 for 30 min at the corresponding time points.In METTL3+SP+HSR group and NC+SP+HSR group,the corresponding virus 450 nl was injected into bilateral hippocampus at 4 weeks before establishing the model.Morris water maze and novel object recognition tests were conducted at 72 h after developing the model to assess the learning and memory abilities.After the end of behavioral tests,the expression of METTL3 and SIRT1 in hippocampal tissues was detected using Western blot,the expression of SIRT1 mRNA was measured using qRT-PCR,and the methylation of RNA m6A was detected using Dot blot.Results Compared to Sham group,the escape latency was significantly prolonged at 1-6 days,the time spent in the target quadrant was shortened,the number of crossing the original platform was decreased,the novel

关 键 词：七氟醚 休克 出血性 复苏术 认知功能障碍 甲基转移酶类 RNA 甲基化 抗衰老酶1 

分 类 号：R614[医药卫生—麻醉学]