转录因子EB通过激活自噬及溶酶体功能改善缺血性卒中小鼠神经功能  

Transcription factor EB improves neurological function in mice with ischemic stroke by activating autophagy and lysosomal function

作　　者：周大旺 张晨禹 黎博 张强[1,3] 刘聪 詹浩洪[2,3] 胡春林 廖晓星 ZHOU Da-wang;ZHANG Chen-yu;LI Bo;ZHANG Qiang;LIU Cong;ZHAN Hao-hong;HU Chun-lin;LIAO Xiao-xing(The Emergency and Disaster Medical Center,The Seventh Affiliated Hospital,Sun Yat-sen University,Shenzhen 518107,China;Department of Emergency,The First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510080,China;Department of Assisted Circulation Laboratory,The First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510080,China;Research Institute of Sun Yat-sen University in Shenzhen,Shenzhen 518057,China)

机构地区：[1]中山大学附属第七医院急诊与灾难医学中心,广东深圳518107 [2]中山大学附属第一医院急诊科,广东广州510080 [3]中山大学附属第一医院卫生部辅助循环重点实验室,广东广州510080 [4]中山大学深圳研究院,广东深圳518057

出　　处：《中国病理生理杂志》2021年第7期1178-1186,共9页Chinese Journal of Pathophysiology

基　　金：广东省基础与应用基础研究基金项目(No.2020A1515010120);深圳市科技计划项目(No.JCYJ20160608142215491)。

摘　　要：目的:观察外源性导入转录因子EB(TFEB)对缺血性脑卒中小鼠神经功能损伤的影响。方法:(1)15只10周龄雄性C57BL/6小鼠用于建立永久性大脑中动脉阻塞(pMCAO)模型,随机分为假手术组、梗阻2 h组、梗阻4 h组、梗阻8 h组和梗阻24 h组,每组3只,Western blot法检测pMCAO后皮层神经元TFEB、溶酶体相关膜蛋白1(LAMP-1)、微管相关蛋白1轻链3B(LC3B)和cleaved caspase-3蛋白的表达;(2)45只8周龄雄性C57BL/6小鼠分为假手术组、TFEB过表达组、过表达对照组、TFEB敲减组和敲减对照组,每组9只。除假手术组外,其余4组小鼠均经侧脑室注射9型腺相关病毒(AAV9),分别为AAV9-TFEB、AAV9-NC、AAV9-shTFEB和AAV9-shNC,注射2周后建立pMCAO模型,24 h后采用Longa评分法评估小鼠神经功能,磁共振法检测并计算脑梗死体积,TUNEL染色检测皮层神经元细胞凋亡情况,尼氏染色观察皮层神经元细胞形态及数量,Western blot法检测皮层神经元TFEB、LAMP-1、LC3B、cleaved caspase-3蛋白的表达。结果:与假手术组相比,pMCAO后2~24 h,皮层神经元TFEB、LAMP-1及LC3B-II蛋白表达水平均显著下降,而cleaved caspase-3表达升高(P<0.05)。侧脑室注射AAV9-TFEB可改善pMACO后小鼠Longa评分,而注射AAV9-shTFEB则恶化Longa评分(P<0.05)。磁共振结果显示侧脑室注射AAV9-TFEB可减少脑梗死体积,而注射AAV9-shTFEB则增加脑梗死体积(P<0.05)。尼氏染色显示AAV9-TFEB组小鼠皮层神经元细胞数量显著多于AAV9-NC组,AAV9-shTFEB组小鼠皮层神经元细胞数量则显著少于AAV9-shNC组(P<0.05)。Western blot结果显示过表达TFEB可增加皮层脑组织LAMP-1和LC3B-II表达,降低cleaved caspase-3表达(P<0.05);敲减TFEB则降低皮层脑组织LAMP-1和LC3B-II表达,增加cleaved caspase-3表达(P<0.05)。结论:利用AAV9外源性导入TFEB可通过激活自噬及溶酶体功能而减少pMCAO后皮层神经元细胞凋亡,改善小鼠神经功能。AIM:To observe the effect of exogenously introduced transcription factors EB on neurological inju-ry in ischemic stroke mice.METHODS:In the first part,male C57BL/6 mice(10-week-old,n=15)were used to estab-lish a ischemic stroke model by permanent middle cerebral artery occlusion(pMCAO),and animals were randomly divided into sham operation group,2 h of infarction group,4 h of infarction group,8 h of infarction group and 24 h of infarction group.The protein levels of TFEB,lysosome-associated membrane protein 1(LAMP-1),microtubule-associated protein 1 light chain 3B(LC3B)and cleaved caspase-3 in the cortical neurons after pMCAO were determined by Western blot.In the second part,male C57BL/6 mice(8-week-old,n=45)were divided into sham operation group,TFEB over-ex-pression group,over-expression control group,TFEB knock-down group,and knock-down control group.Except for sham group,the mice in other 4 groups were injected with adeno-associated virus serotype 9(AAV9)into the lateral ventricle,namely AAV9-TFEB,AAV9-NC,AAV9-shTFEB and AAV9-shNC.Two weeks later,the pMCAO model was es-tablished.After 24 h,the Longa score method was used to evaluate the neurological function of the mice,the MRI was used to calculate the cerebral infarct volume,the TUNEL staining was used to detect the apoptosis of cortical neurons,and the morphology and number of cortical neurons were observed by Nissl staining.The protein levels of cortical neurons TFEB,LAMP-1,LC3B and cleaved caspase-3 were determined by Western blot.RESULTS:Compared with sham opera-tion group,the protein levels of TFEB,LAMP-1 and LC3B-II were decreased significantly after pMCAO in different infarct time groups,while the protein levels of cleaved caspase-3 were increased(P<0.05).Injection of AAV9-TFEB into the lateral ventricle improved the Longa score of the mice after pMACO,while injection of AAV9-shTFEB worsened the Longa score(P<0.05).MRI results showed that AAV9-TFEB reduced the volume of cerebral infarction,while AAV9-shTFEB increased the volume of cerebral infarction

关 键 词：脑缺血性卒中 转录因子EB 自噬 细胞凋亡 溶酶体 

分 类 号：R743.3[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]