CTCFL/RPS3A通过抑制内质网应激减少神经元细胞凋亡缓解阿尔兹海默症大鼠认知功能障碍实验研究  

CTCFL/RPS3A alleviates cognitive dysfunction in Alzheimer’s disease rats by inhibiting endoplasmic reticulum stress and reducing neuronal apoptosis

作　　者：孟清琳 潘娜 刘养凤 苟英之 吴雅欣 赵锦华 MENG Qinglin;PAN Na;LIU Yangfeng;GOU Yingzhi;WU Yaxin;ZHAO Jinhua(Department of Neurology,Air Force Medical University Air Force 986 Hospital,Xi’an 710054,China)

机构地区：[1]空军军医大学空军第九八六医院神经内科,陕西西安710054 [2]咸阳市第一人民医院神经内科,陕西咸阳712000

出　　处：《陕西医学杂志》2024年第10期1314-1319,1325,共7页Shaanxi Medical Journal

基　　金：陕西省自然科学基础研究计划项目(2020JQ-928)。

摘　　要：目的:探究RPS3A在阿尔兹海默症(AD)中的作用及其分子机制。方法:通过将Aβ注射到大鼠双侧海马区,构建AD模型。采用RPS3A的腺病毒载体(Ad-RPS3A)注射AD大鼠,饲养14 d后通过水迷宫测试评估大鼠的学习与记忆能力,通过Y迷宫测试评估大鼠的空间探索能力。此外,采用RPS3A的过表达载体(RPS3A)、shRNA(sh-RPS3A)、CTCFL的过表达载体(CTCFL)、shRNA(sh-CTCFL)分别转染神经元HT22细胞48 h,然后采用30μmol/L的Aβ处理细胞24 h。使用JASPAR预测CTCFL在RPS3A启动子的结合,并通过Ch-IP分析和荧光素酶报告基因分析进行验证。RT-qPCR检测过表达和沉默效率;Western blot检测RPS3A和CTCFL蛋白,以及内质网应激相关蛋白和凋亡相关蛋白表达水平;CCK-8和流式细胞术分别检测细胞活力和细胞凋亡率。结果:与假手术组相比,AD模型组大鼠海马组织中RPS3A的mRNA和蛋白水平显著下调。Aβ诱导的HT22细胞中RPS3A蛋白表达显著下调;过表达RPS3A促进HT22细胞增殖,抑制Aβ诱导的细胞凋亡,降低Cleaved caspase-3和Bax蛋白表达,促进Bcl-2蛋白表达,抑制内质网应激相关蛋白CRP78、CHOP、ATF6和XBP1表达,降低IRE1和PERK磷酸化水平。机制研究显示CTCFL通过与RPS3A启动子区结合,促进RPS3A转录激活。过表达CTCFL促进RPS3A蛋白表达;而沉默CTCFL作用相反。体内结果显示,过表达RPS3A降低AD大鼠抵达平台的时间、到达平台距离以及第一次进入SW区的时间,增加大鼠自发交替率和进入新异臂次数的比值,改善Aβ诱导的AD大鼠的学习与记忆能力障碍和空间探索能力障碍。结论:CTCFL介导的RPS3A通过内质网应激诱导神经元细胞凋亡,促进阿尔兹海默病小鼠的认知功能障碍。Objective:To explore the role of ribosomal protein S3A(RPS3A)in Alzheimer’s disease(AD)and its underlying molecular mechanism.Methods:The AD rat model was established by injecting Aβinto the bilateral hippocampus of rats.AD rats were injected with adenoviral containing RPS3A overexpression vector(Ad-RPS3A).After feeding for 14 days,the learning and memory abilities and spatial exploration abilities of rats were evaluated by water maze test and Y maze test.Moreover,neuronal HT22 cells were transfected with RPS3A overexpression vector(RPS3A)or shRNA(sh-RPS3A),or transfected with CTCFL overexpression vector(CTCFL)or shRNA(sh-CTCFL)for 48 hours,and then with treated 30μmol/L Aβfor 24 hours.The binding of CTCFL to RPS3A promoter was predicted by using JASPAR database and verified with Ch-IP analysis and the luciferase reporter gene analysis.RT-qPCR was used to detect the overexpression and silencing efficiency.Western blot was used to detect the protein levels of RPS3A and CTCFL,as well as expression levels of Endoplasmic-reticulum(ER)stress-related proteins and apoptosis related proteins.Cell viability and apoptosis were detected with CCK-8 and flow cytometry,respectively.Results:Compared with the sham group,the mRNA and protein levels of RPS3A in the hippocampus of AD model rats were significantly downregulated.Moreover,the protein expression of RPS3A was significantly downregulated in Aβ-induced HT22 cells.Overexpression of RPS3A promoted HT22 cell proliferation and inhibited Aβ-induced cell apoptosis,decreased the expression of Cleaved caspase-3 and Bax proteins,promoted Bcl-2 protein expression,inhibited the expression of ER stress-related proteins CRP78,CHOP,ATF6 and XBP1,and decreased the phosphorylation levels of IRE1 and PERK.Mechanistic studies revealed that CTCFL promotes RPS3A transcriptional activation by binding to RPS3A promoter.Overexpression of CTCFL promoted RPS3A protein expression,while silencing CTCFL has the opposite effect.The in vivo results showed that overexpression of RPS3A reduced th

关 键 词：阿尔兹海默症 认知功能障碍 神经元凋亡 RPS3A CTCFL 内质网应激 

分 类 号：R749.16[医药卫生—神经病学与精神病学]