羰基化蛋白质组学研究规律运动调控大鼠脑纹状体细胞凋亡的作用与适应性机制  

The Adaptational Regulation Mechanism of the Apoptosis in Aged Rats’Striatum in Response to Regular Aerobic Exercise Based on Carbonylation Proteomics

作　　者：袁顺灵 彭美 向逸 杨启明 雷勇 刘文锋[1,2] 刘霞[1] 汤长发[1] 印大中[2,3] YUAN Shun-Ling;PENG Mei;XIANG Yi;YANG Qi-Ming;LEI Yong;LIU Wen-Feng;LIU Xia;TANG Chang-Fa;YIN Da-Zhong(Hunan Provincial Key Laboratory of Physical Fitness and Sports Rehabilitation,Hunan Normal University,Department of Human Movement Science,College of Physical Education,Hunan Normal University,Changsha 410012,China;The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education,Department of Aging,College of Life Science,Hunan NormalUniversity,Changsha 410081,China;Aging Experimental Center,Qingyuan People's Hospital Affiliated to Guangzhou Medical University,Qingyuan 511500,Guangdong,China)

机构地区：[1]湖南师范大学体适能与运动康复湖南省重点实验室,湖南师范大学体育学院运动人体科学教研室,长沙410012 [2]湖南师范大学蛋白质化学与发育生物学教育部重点实验室,湖南师范大学生命科学学院衰老生化研究室,长沙410081 [3]广州医科大学附属清远人民医院衰老实验中心,广东清远511500

出　　处：《中国生物化学与分子生物学报》2021年第11期1489-1501,共13页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.81702236);湖南省教育厅重点项目(No.20A333);湖南省自然科学基金项目(No.2018JJ3363);湖南省高校青年骨干教师项目(No.[2020]43);湖南师范大学青年优秀人才培养计划(No.ET1507);湖南师范大学大学生创新创业训练计划项目(No.2019154)共同资助。

摘　　要：为研究探讨规律运动对老年大鼠纹状体蛋白质氧化应激与细胞凋亡的影响,通过差异羰基化蛋白质组学特征分析,筛选靶标蛋白质并阐明其作用机制。将24只健康雄性23月龄老年大鼠随机分为静息组(Con-SED)和规律运动组(Aero-EXE)。采用运动强度相当于最大摄氧量(VO_(2max))60%~65%逐渐递增到70%~75%的10周中等强度规律运动。HE染色结果显示,规律运动改善了老年大鼠纹状体基质间区和纹状质间区的结构。TUNEL检测出所有老年大鼠纹状体均出现细胞凋亡核。与Con-SED组相比较,Aero-EXE组纹状体基质间区细胞凋亡指数增加了68.24%(P<0.01),而纹状质间区凋亡指数下降了43.14%(P<0.05)。亲和素珠富集和电喷雾四级杆飞行时间质谱分离鉴定羰基化蛋白质。进一步使用MASCOT服务器的数据库进行生物信息学搜索,结果显示,羰基化蛋白质涵盖28种氧化修饰位点。其中鉴定出Con-SED组羰基化蛋白质有14-3-3蛋白(e)和核内不均一性核糖核蛋白(HNRNPA2B1)等69个;而Aero-EXE组羰基化蛋白质有电压依赖型阴离子选择性通道1(VDAC1)和VDAC2等71个。生物信息学分析羰基化蛋白质的氧化修饰位点及蛋白质相互作用关系,筛选出14-3-3蛋白(e)和HNRNPA2B1羰基化蛋白质与细胞凋亡有着重要关系。免疫组织化学检测结果显示,与Con-SED组相比较,Aero-EXE组的大鼠纹状体SOD和BDNF表达水平显著提高(P<0.01,P<0.05);qRT-PCR检测结果显示,14-3-3蛋白(e)和HNRNPA2B1表达水平显著上调(P<0.05,P<0.01);免疫印迹检测结果显示,PI3K/AKT1/mTOR和CAMKⅡα信号通路相关分子的表达水平显著上调(P<0.05或P<0.01)。综上所述,规律运动刺激了抗氧化系统的适应,改善了与细胞凋亡密切相关的14-3-3蛋白(e)和HNRNPA2B1等蛋白质羰基化,维护了纹状体基质间区和纹状质间区细胞凋亡的稳态,其机制是通过上调14-3-3蛋白(e)和HNRNPA2B1表达水平,促进BDNF的表达,进一步激活并上调下This paper investigated the effects of regular aerobic exercise on protein oxidative stress and apoptosis in aging rat striatum,and further analyzed its target proteins and mechanism based on differential carbonylation proteomics.Totally 24 specific pathogen-free(SPF)23-month-old male Sprague-Dawley(SD)rats were randomly divided into aged sedentary control group(Con-SED,n=12)and aged regular aerobic exercise runner group(Aero-EXE,n=12).The medium intensity of regular aerobic exercise model:The intensity of maximum oxygen consumption(VO_(2max))increased gradually from 50%~55%to 65%~70%of 10 weeks.The results of haematoxylin and eosin(HE)staining showed that regular aerobic exercise improved the structure of interstromal and interstriatal striatum in aged rats.The TdT-mediated dUTP nick end labeling(TUNEL)results showed that apoptotic nuclei can be found in all aged rat striatum.Compared with the Con-SED group,the apoptotic index of the interstromal zone increased by 68.24%(P<0.01),while the striatum the apoptotic index of the interstriatal zone decreased by 43.14%(P<0.05)in the Aero-EXE group.Avidin Bead Enrichment and Electrospray Ionization-Quadrupole-Time of Flight Mass Spectrometer(ESI-Q-TOF-MS)were used to separate and identify carbonylated proteins.The database of MASCOT server was further used for bioinformatics search(Searching and analysis of protein subcellular localization and biological function).The results showed that carbonylated protein cover 28 oxidative modification sites,among which 69 carbonylated proteins have been identified in the Con-SED group,such as heterogeneous nuclear ribonucleoproteins A2/B1(HNRNPA2B1)and 14-3-3 protein(e),etc.;While 71 carbonylation protein in the Aero-EXE group,such as voltage-dependent anion-selective channel 1(VDAC1)and VDAC2,etc.;Bioinformatics and oxidative modification sites analysis indicated that 14-3-3 protein(e)and HNRNPA2B1 carbonylated protein have an important relationship with apoptosis.The immunohistochemical(IHC)test results showed that compared with

关 键 词：规律运动 电喷雾四级杆飞行时间质谱 14-3-3蛋白(e) 核内不均一性核糖核蛋白A2/B1 

分 类 号：R338.2[医药卫生—人体生理学]