慢性束缚应激对小鼠海马N6-甲基腺苷及相关酶表达的影响  

Effects of chronic restraint stress on the expression of N6-methyladenosine and related enzymes in the hippocampus of mice

作　　者：张圆 时萌萌 贺嘉贝 吴琼 钟小林 向宇燕[3] 徐杨 何洁 ZHANG Yuan;SHI Meng-meng;HE Jia-bei;WU Qiong;ZHONG Xiao-lin;XIANG Yu-yan;XU Yang;HE Jie(Department of Pathology,School of Basic Medicine and Life Sciences,Hainan Medical College/Department of Pathology,the First Affiliated Hospital,Hainan Medical College,Haikou 571199,China;Department of Pattology,the First Affiliated Hospital of Hu’nan Normal University/Hu’man Provincial People’s Hospital,Changsha 420021,China;Clinical Anatomy&Reproductive Medicine Application Institute,School of Medicine,University of South China,Hu’nan Hengyang 421001,China;Key Laboratory of Brain Science Research and Transformation in Tropical Environment of Hainan Province,Hainan Medical University,Haikou 571199,China;Institute of Clinical Medicine,the First Affiliated Hospital of Nanhua University,Hu’nan Hengyang 421001,China;Department of Physiology and Institute of Neuroscience,Hengyang Medical College,University of South China,Hu’nan Hengyang 421001,China)

机构地区：[1]海南医学院基础医学与生命科学学院病理学教研室,海南医学院第一附属医院病理科,海口571199 [2]湖南师范大学附属第一医院/湖南省人民医院病理科,长沙420021 [3]南华大学应用解剖学与生殖医学研究所,湖南衡阳421001 [4]海南医学院热带脑科学研究与转化海南省重点实验室,海口571199 [5]南华大学第一附属医院临床医学研究所,湖南衡阳421001 [6]南华大学衡阳医学院生理学教研室和神经科学研究所,湖南衡阳421001

出　　处：《解剖学报》2023年第2期142-148,共7页Acta Anatomica Sinica

基　　金：湖南省自然科学基金(2019JJ40257);湖南省自然科学基金联合项目(2018JJ3460);湖南省卫生健康委科研立项课题项目(202102011871)。

摘　　要：目的探讨慢性束缚应激对小鼠海马N6-甲基腺苷(m6A)及相关酶表达影响。方法将20只C57BL/6J雄鼠随机分成对照(control)组、慢性束缚应激模型(CRS)组;给予CRS组小鼠3周慢性束缚应激建立小鼠焦虑模型,采用旷场实验和高架十字迷宫实验检测各组小鼠焦虑样行为变化;采用免疫组织化学法和m6A RNA甲基化检测试剂盒检测小鼠海马m6A的表达变化;采用Western blotting和Real-time PCR方法分析海马m6A相关酶表达变化。结果1.小鼠焦虑相关行为学检测结果显示,与control组相比,CRS组小鼠在旷场内框停留时间明显下降(P<0.01);高架十字迷宫开放臂探索时间减少(P<0.0001);2.m6A RNA甲基化检测试剂盒检测结果显示,与control组相比,CRS组小鼠海马m6A含量明显减少(P<0.05);免疫组化结果显示,与control组相比,CRS组小鼠海马m6A阳性产物明显减少(P<0.001);3.PCR结果显示,与control组相比,CRS组小鼠海马去甲基化酶间变性淋巴瘤激酯B(AlkB)同源蛋白5(ALKBH5)(P<0.001)、脂肪和肥胖相关蛋白(FTO)表达显著上调(P<0.05);甲基化酶Wilms肿瘤蛋白1相关蛋白(WTAP)(P<0.05)表达显著下调;m6A甲基化结合蛋白YTH结构域家族蛋白3(YTHDF3)(P<0.05)和YTH结构域蛋白2(YTHDC2)(P<0.01)表达显著上调。Western blotting结果显示,与control组相比,CRS组小鼠海马去甲基化酶ALKBH5(P<0.05)、FTO(P<0.05)表达显著上调;甲基化酶WTAP(P<0.01)表达显著下调;m6A甲基化结合蛋白YTHDF3(P<0.01)和YTHDC2(P<0.05)表达显著上调。结论在慢性束缚应激所致小鼠焦虑与海马m6A表达下调有关,其机制可能与m6A甲基化酶WTAP表达下调或去甲基化酶ALKBH5和FTO表达上调相关。Objective To investigate the effect of chronic restraint stress on the expression of N6-methyladenosine(m6A)and related enzymes in the hippocampus of mice.Methods Twenty C57BL/6J male mice were randomly divided into control group and chronic restraint stress(CRS)group,the model group was given for 3 weeks chronic restraint stress to establish a mouse anxiety model.Open field test and elevated plus maze test were used to detect anxiety-like behavior;Immunohistochemistry and m6A RNA methylation assay were used to detect the expression changes of mouse hippocampal m6A;Western blotting and Real-time PCR were used to analyze hippocampal m6A related enzymes expression.Results 1.The behavioral results showed that,compared with the control group,the CRS group showed significantly reduced time spent in the center of the open field(P<0.01),the CRS group showed significantly reduced exploration time in the open arm of elevated plus maze(P<0.0001);2.Immunohistochemical results showed that,compared with the control group,the hippocampal m6A content in the CRS group reduced significantly(P<0.001);The results of the m6A RNA methylation assay showed that,compared with the control group,the CRS group showed significantly reduced amount of hippocampal m6A(P<0.05);3.Real-time PCR results showed that the expression of hippocampal demethylase anaplastic lymphoma kinase B(AlkB)homolog 5(ALKBH5)(P<0.001)and fat mass and obestity associated protein(FTO)(P<0.05)in the CRS group significantly up-regulated,the expression of methylase Wilms’tumour 1-associating protein(WTAP)(P<0.05)was significantly down-regulated compared with the control group;The expression of m6A methylation binding protein YTH domaincontaining family protein 3(YTHDF3)(P<0.05)and YTH domaincontaining protein 2(YTHDC2)(P<0.01)was significantly up-regulated.Western blotting result showed that,compared with the control group,the mouse hippocampal demethylase ALKBH5(P<0.05)and FTO(P<0.05)expression in the CRS group significantly up-regulated,the expression of WTAP(P<0.01)

关 键 词：慢性束缚应激 焦虑 N6-甲基腺苷 间变性淋巴瘤激酯B同源蛋白5 脂肪和肥胖相关蛋白 Wilms肿瘤蛋白1相关蛋白 免疫印迹法 小鼠 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学]