抑制磷脂酶D1活性可促进缺血性脑卒中模型小鼠神经功能恢复  

Inhibition of Phospholipase D1 Activity Improves the Recovery of Neurological Function in Mice with Ischemic Stroke

作　　者：朱彦兵[1,2] 白帆[3,4] 陶少鑫 潘雨花蕾 王欢 赵羽商[1] 王崧 于艳 ZHU Yanbing;BAI Fan;TAO Shaoxin;PAN Yuhualei;WANG Huan;ZHAO Yushang;WANG Song;YU Yan(Beijing Clinical Research Institute,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;Department of Neurology,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;China Rehabilitation Science Institute,China Rehabilitation Research Center,Beijing 100068,China;Beijing Key Laboratory of Neural Injury and Neural Rehabilitation,Beijing 100068,China)

机构地区：[1]北京市临床医学研究所,首都医科大学附属北京友谊医院,北京100050 [2]首都医科大学附属北京友谊医院神经内科,北京100050 [3]中国康复研究中心中国康复科学所,北京100068 [4]神经损伤与康复北京市重点实验室,北京100068

出　　处：《实验动物与比较医学》2022年第4期322-332,共11页Laboratory Animal and Comparative Medicine

基　　金：国家自然科学基金面上项目(81771235)“磷脂酶D在脑卒中后反应性星形胶质细胞增殖和分泌中的作用及其机制研究”;中央级公益性科研院所基本科研业务费专项资金项目(2019CZ-14)“磷脂酶D在脑卒中后神经元过度自噬中的作用以及机制研究”。

摘　　要：目的探讨小鼠缺血性脑卒中发生后不同时期病灶脑组织自噬的动态变化,以及抑制磷脂酶D(phospholipase D,PLD)1活性与自噬及神经功能的关系。方法先用21只8周龄成年雄性C57BL/6小鼠构建缺血性脑卒中模型,随机分为假手术组和缺血性脑卒中后4 h、6 h、8 h、12 h、24 h、72 h组,每组各3只,采用蛋白质印迹法和免疫荧光染色法检测小鼠病灶脑组织中自噬相关蛋白LC3的表达及分布情况。另将36只8周龄成年雄性C57BL/6小鼠随机分为假手术组、缺血性脑卒中后腹腔注射PBS组和4个浓度(0.3、0.9、1.8和3.6 mg/kg)的PLD活性抑制剂FIPI组,每组各6只;缺血性脑卒中发生后的第1、第3和第7天,分别通过贴纸去除实验、触须诱发前肢放置实验和TTC染色法评估抑制剂最佳浓度及其对神经功能和脑梗死面积的影响。再将9只8周龄成年雄性C57BL/6小鼠随机分为假手术组、缺血性脑卒中后立即腹腔注射PBS和0.9 mg/kg FIPI组,每组3只,通过蛋白质印迹法检测FIPI对自噬相关蛋白表达的影响。最后将36只8周龄成年雄性C57BL/6小鼠随机分为假手术组、缺血性脑卒中后立即腹腔注射PBS组以及不同时间窗(术后立即和术后4 h、8 h、12 h)腹腔注射0.9 mg/kg FIPI组,每组各6只,通过行为学实验和TTC染色法评估缺血性脑卒中小鼠的神经功能和脑梗死面积。结果小鼠缺血性脑卒中发生后,病灶组织中LC3-Ⅱ相对表达水平开始升高,24 h达到峰值(P<0.05),然后逐渐下降。与PBS组相比,给予0.9 mg/kg的PLD1活性抑制剂FIPI组能明显抑制小鼠缺血性脑卒中后病灶组织中LC3-Ⅱ相对表达水平(P<0.05),缩小梗死面积(P<0.05),促进损伤后神经功能的恢复(P<0.05)。与PBS组相比,除了缺血性脑卒中后立即给予FIPI组外,缺血性脑卒中后4 h、8 h和12 h腹腔给予FIPI也都可以明显缩小梗死面积(P<0.05),使小鼠神经功能得到明显恢复(P<0.05)。结论缺血性脑卒中发生后24 h病Objective To investigate the dynamic changes of autophagy in infarcted cortex at different stages after ischemic stroke,and the relationship between phospholipase D1(PLD1)activity,autophagy,and the neurological function in mice.Methods Firstly,twenty-one male C57BL/6 mice at age of 8 weeks were used to establish ischemic stroke models,and they were randomly divided into sham surgery and ischemic stroke groups.The groups were furtherly divided into 4-,6-,8-,12-,24-,and 72-hour groups according to the onset of ischemic strok with three mice in each group.Dynamic expression and change of autophagy-related protein LC3 in the infarcted cortex were observed using Western blotting method and immunofluorescence staining.Secondly,thirty-six male C57BL/6 mice at age of 8 weeks were randomly divided into the sham surgery group,the PBS treatment group,and different concentrations of phospholipase D activity inhibitor 5-fluoro-2-indolyl des-chlorohalopemide(FIPI)treatment groups containing 0.3,0.9,1.8,and 3.6 mg/kg,respectively.Each group comprised six mice.The optimal FIPI concentration and its effects on neurological function and infarcted volume were assessed by adhesive removal and whisker tests and TTC staining.Furthermore,nine male C57BL/6 mice at age of 8 weeks were divided into sham surgery group,intraperitoneal administration of PBS group,and 0.9 mg/kg FIPI group.Each group comprised three mice.The effects of FIPI on changes in autophagy-related protein LC3 were examined by Western blotting.Finally,thirty-six adult male C57BL/6 mice at age of 8 weeks were divided into sham surgery group,intraperitoneal administration of PBS group,and 0.9 mg/kg FIPI treatment groups at four time points(0,4,8,and 12 hours)after ischemic stroke.Furthermore,the neurological functions and infarcted volume were assessed by the adhesive removal and whisker tests as well as TTC staining with six mice in each group.Results The ratio of LC3-Ⅱ/β-actin in the infarcted cortex increased,peaked at 24 hours(P<0.05),and then gradually declined af

关 键 词：缺血性脑卒中 自噬 磷脂酶D1 神经功能 C57BL/6小鼠 

分 类 号：R-332[医药卫生] Q95-33[生物学—动物学]