星形胶质细胞来源外泌体对氧糖剥夺再复氧后PC12细胞线粒体功能损伤的保护作用  

Protective Effects of Astrocyte-derived Exosomes on Mitochondrial Functional Damage after Oxygen-glucose Deprivation/Reoxygenation

作　　者：高霄 王正薇 蔡娜 唐智 吴昌学 齐晓岚 官志忠[1,2] 肖雁[1,2] GAO Xiao;WANG Zheng-Wei;CAI Na;TANG Zhi;WU Chang-Xue;QI Xiao-Lan;GUAN Zhi-Zhong;XIAO Yan(Key Laboratory of Endemic and Minority Sexual Diseases,Ministry of Education,Guiyang 550004,China;Key Laboratory of Medical Biology,Guizhou Medical University,Guiyang 550004,China;Editorial Board of Journal of Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]地方病与少数民族性疾病教育部重点实验室,贵阳550004 [2]贵州医科大学分子生物学重点实验室,贵阳550004 [3]贵州医科大学学报编辑部,贵阳550004

出　　处：《中国生物化学与分子生物学报》2024年第6期827-837,共11页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.822602458);贵州省科技厅重点项目(No.黔科合基础[2019]1440号);贵州医科大学国基培育项目(No.20NSP069)资助。

摘　　要：外泌体可以改善由缺氧缺血引起的神经细胞损伤,但星形胶质细胞来源的外泌体(astrocyte-derived exosomes,As-exo)与线粒体功能、线粒体相关内质网膜(mitochondrial associated ER membrane,MAM)的功能及线粒体自噬是否相关目前尚未明确。本研究旨在探究星形胶质细胞来源外泌体对氧糖剥夺再复氧(oxygen and glucose deprivation/reoxygenation,OGD/R)后PC12细胞线粒体功能、MAM以及线粒体自噬的调控作用。超速离心法提取星形胶质细胞培养基上清中的外泌体并对其进行鉴定。利用活细胞工作站观察到荧光标记后的外泌体在24 h时即在PC12细胞内出现明显的富集现象,同时在激光共聚焦扫描显微镜下观察到外泌体与线粒体出现共定位现象;采用Seahorse细胞能量代谢分仪检测线粒体压力变化:与Control组相比,OGD/R组的基础呼吸、质子漏、最大呼吸和ATP相关呼吸都有明显降低(P<0.05或P<0.01),OGD/R+exo组与OGD/R组相比4项指标都升高且差异具有统计学意义(P<0.05或P<0.01);线粒体和内质网共定位结果表明,MAM受到氧糖剥夺再复氧伤害时,结构出现距离减小的聚合现象,而As-exo处理后MAM聚合现象减弱;流式结果表明,As-exo,一定程度恢复由氧糖剥夺损伤带来的线粒体膜电位降低和ROS升高;Western印迹结果显示,As-exo能显著抑制由OGD/R引起的线粒体自噬相关蛋白张力蛋白同源物诱导的假定激酶1(PTEN induced kinase 1,PINK1)和Parkin蛋白(parkin RBR E3 ubiquitin protein ligase,Parkin)升高,加入As-exo可降低LC3Ⅱ/LC3Ⅰ蛋白表达量,升高P62蛋白的表达水平,降低OGD/R引起的线粒体自噬水平。由此可见,OGD/R处理能引起PC12细胞的线粒体功能紊乱、MAM结构改变及线粒体自噬增加,As-exo处理后能改善细胞的线粒体功能、减弱MAM的形成和降低线粒体自噬,从而具有预防缺血性脑卒中再灌注损伤的治疗潜力。Exosomes can ameliorate neuronal cell injury induced by hypoxia-ischemia,but the relationship between astrocyte-derived exosomes(As-exo)and mitochondrial function,mitochondrial associated ER membrane(MAM)function and whether mitochondrial autophagy is relevant is currently unclear.The aim of this study was to investigate the role of astrocyte-derived exosomes in the regulation of mitochondrial function,MAM and mitochondrial autophagy in PC12 cells after oxygen and glucose deprivation/reoxygenation(OGD/R).Exosomes were extracted from the supernatant of the astrocyte culture medium by ultracentrifugation.Using the live cell imaging system,we observed that fluorescently labeled exosomes could show obvious enrichment in PC12 cells at 24 h.Meanwhile,co-localization of exosomes with mitochondria could be observed under the laser confocal scanning microscope;mitochondrial pressure changes were detected using the Seahorse cellular energy metabolism fractionation instrument.The result showed that basal respiration in the OGD/R group,compared with that in the control group,proton leakage,maximal respiration and ATP-related respiration were significantly reduced(P<0.05 or P<0.01),and all four indexes were elevated and statistically significant in the OGD/R+exo group compared with the control group(P<0.05 or P<0.01).The results of the co-localization of the mitochondria and ER showed that the structure of the MAM was harmed by oxygen-sugar deprivation and then reoxygenation,and the structure of As-exo and the mitochondria appeared to have a distance-reduced polymerization phenomenon,while the mitochondria and ER co-localized.The co-localization results of mitochondria and ER showed that the structure of MAM was damaged by oxygen deprivation and reoxygenation,and the aggregation phenomenon of MAM was weakened by the treatment of As-exo;the flow-through results showed that As-exo could restore the decrease of the mitochondrial membrane potential and the elevation of the ROS by oxygen deprivation to a certain degree.Western blo

关 键 词：外泌体 星形胶质细胞 氧糖剥夺再灌注 线粒体自噬 线粒体相关内质网膜 

分 类 号：Q26[生物学—细胞生物学]