抑制水通道蛋白4表达对脑缺血再灌注模型小鼠自噬和凋亡的影响  

Impact of inhibiting aquaporin 4 expression on autophagy and apoptosis in a mouse model of cerebral ischemia-reperfusion

作　　者：莫圣龙 朱海燕 陆志成 莫嘉祺 彭晓靖 唐丽娜 杨成敏 简崇东[1,3] 商敬伟 MO Shenglong;ZHU Haiyan;LU Zhicheng;MO Jiaqi;PENG Xiaojing;TANG Lina;YANG Chengmin;JIAN Chongdong;SHANG Jingwei(Department of Neurology,Affiliated Hospital of Youjiang Medical College for Nationalities,Baise 533000,China;Graduate School of Youjiang Medical College for Nationalities,Baise 533000,China;Key Laboratory of Clinical Molecular Diagnosis Research for Diseases with High Incidence of Western Guangxi in Guangxi Higher Education Institutes,Baise 533000,China)

机构地区：[1]右江民族医学院附属医院神经内科,广西百色533000 [2]右江民族医学院研究生学院,广西百色533000 [3]广西高校桂西高发病临床分子诊断研究重点实验室,广西百色533000

出　　处：《中国病理生理杂志》2024年第8期1446-1454,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81860244,No.82160254);右江民族医学院附属医院高层次人才科研项目(No.R20213001);百色市科学研究与技术开发计划课题(百科20224117号);广西研究生教育创新计划项目(No.YCSW2023494)。

摘　　要：目的:探讨抑制水通道蛋白4(AQP4)表达对脑缺血再灌注模型小鼠自噬和凋亡的影响及其机制。方法:采用暂时性大脑中动脉闭塞(tMCAO)建立小鼠脑缺血再灌注损伤模型。将60只小鼠按随机数字表法分成假手术(sham)组、I/R组、AQP4抑制组和自噬抑制剂3-甲基腺嘌呤(3-MA)组,每组15只,sham组和I/R组腹腔注射生理盐水,AQP4抑制组和3-MA组分别腹腔注射AER-271(2 mg·kg^(-1)·d^(-1))和AER-271+3-MA(2 mg·kg_(-1)·d^(-1)),给药3 d,每天1次。观察各组小鼠神经功能Longa评分,记录体重变化;采用苏木精-伊红(HE)染色法观察脑梗死体积及组织病理变化;采用Western blot检测AQP4、LC3-Ⅱ、P62和cleaved caspase 3含量变化,采用免疫荧光法检测LC3-Ⅱ、P62、cleaved caspase-3与NeuN(神经元标志物)的共定位及表达情况。结果:I/R组与AQP4抑制组检测到大面积的脑梗塞和脑水肿,以及较高的Longa评分;与I/R组比较,AQP4抑制组的脑梗塞体积、脑水肿体积和Longa评分均显著降低(P<0.05);与sham组比较,I/R组的AQP4、LC3-Ⅱ和cleaved caspase-3的表达显著增加(P<0.01),而小鼠体重和P62表达则显著降低(P<0.05,P<0.01)。与I/R组比较,AQP4抑制组和3-MA组的AQP4、LC3-Ⅱ和cleaved caspase-3表达显著降低(P<0.05,P<0.01),而小鼠体重和P62表达则显著增加(P<0.05,P<0.01)。然而,与AQP4抑制组比较,3-MA组小鼠Longa评分、脑梗死体积、小鼠体重和AQP4、LC3-Ⅱ、cleaved caspase-3及P62的表达无显著差异。结论:抑制AQP4的表达能够显著减小tMCAO模型小鼠的脑梗死面积和减轻神经损伤程度。同时,抑制AQP4的表达对脑梗死后自噬和凋亡具有减缓作用,而加用自噬抑制剂后对AQP4抑制剂的保护作用无显著影响。AIM:To investigate the impact of aquaporin 4(AQP4)expression inhibition on autophagy and apoptosis in a mouse model of cerebral ischemia-reperfusion(I/R)injury,and to elucidate its underlying mechanism.METHODS:Cerebral I/R injury was induced in mice via transient middle cerebral artery occlusion(tMCAO).Totally 60 mice were randomly divided into sham group,I/R group,AQP4 inhibition group,and 3-methyladenine(3-MA)group,with 15 mice in each group.Among them,the mice in sham and I/R groups received intraperitoneal injections of normal saline,while those in AQP4 inhibition group and 3-MA group received intraperitoneal injections of AER-271(2 mg·kg^(-1)·d^(-1))and AER-271+3-MA(2 mg·kg^(-1)·d^(-1))for 3 d,respectively,once per day.Longa score was adopted to assess the neurological function,and to record changes in body weight.Cerebral infarction volume and histopathological alterations were evaluated using hematoxylin-eosin staining.Western blot analysis was performed to determine the levels of AQP4,LC3-Ⅱ,P62 and cleaved caspase-3,while the LC3-II,P62,cleaved caspase-3 and NeuN(neuronal marker)colocalization and expression assessment were conducted with immunofluorescence.RESULTS:The mice in I/R and AQP4 inhibition groups exhibited extensive cerebral infarction,cerebral edema,and elevated Longa scores.However,in comparision to I/R group,the mice in AQP4 inhibition group showed significantly reduced cerebral infarct volume,cerebral edema volume,and Longa score(P<0.05).Additionally,in contrast to sham group,the mice in I/R group displayed increased expression of AQP4,LC3-Ⅱ and cleaved caspase-3(P<0.01),accompanied by decreased body weight and P62 expression(P<0.05 or P<0.01).Furthermore,compared with I/R group,the mice in both AQP4 inhibition group and 3-MA group demonstrated a decrease in the expression levels of AQP4,LC3-Ⅱ and cleaved caspase-3(P<0.05 or P<0.01),along with increased body weight and P62 expression(P<0.05 or P<0.01).Nonetheless,no significant differences were observed between AQP4 inhibition gro

关 键 词：急性缺血性脑卒中 细胞凋亡 水通道蛋白4 细胞自噬 脑缺血再灌注损伤 

分 类 号：R743[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学] Q255[生物学—细胞生物学]