Clk1基因对Aβ诱导的阿尔茨海默病自噬水平的影响  

Effect of Clk1 gene on level of Aβ-induced autophagy in Alzheimer′s disease

作　　者：司文英 孟宪勇[2] 孙晓静 侯宇清[2] 董晓华[1] SI Wen-ying;MENG Xian-yong;SUN Xiao-jing;HOU Yu-qing;DONG Xiao-hua(Dept of Pharmacy,Hebei North University,Hebei Key Laboratory of Neuropharmacology,,Zhangjiakou Hebei 075000,China;The First Affiliated Hospital of Hebei North University,Zhangjiakou Hebei 075000,China)

机构地区：[1]河北北方学院药学院,河北省神经药理学重点实验室,河北张家口075000 [2]河北北方学院附属第一医院,河北张家口075000

出　　处：《中国药理学通报》2023年第9期1662-1668,共7页Chinese Pharmacological Bulletin

基　　金：河北省自然科学基金资助项目(No H2019405081);河北省卫生厅资助项目(No 20231553);河北北方学院自然科学研究计划项目(No H2022405027)。

摘　　要：目的探讨siRNA转染沉默Clk1基因对阿尔茨海默病(Alzheimer′s disease,AD)模型细胞自噬水平的影响。方法采用MTT法及siRNA转染沉默Clk1基因技术,观察Aβ25-35、Clk1基因对细胞存活率的影响。细胞免疫荧光观察自噬标志物微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)表达情况。Western blot检测Clk1、HIF-1α、PI3K/AKT/mTOR、自噬相关蛋白Beclin1、LC3-Ⅱ/Ⅰ、p62表达情况。实时荧光定量聚合酶链式反应(RT-qPCR)检测Beclin1、LC3、p62 mRNA水平的变化。结果与NC+NS组相比,NC+Aβ组HT22细胞的活力明显降低(P<0.01),而沉默Clk1后siClk1+Aβ组细胞活力较NC+Aβ组明显升高(P<0.01)。细胞免疫荧光结果显示NC+Aβ组LC3表达较NC+NS组明显升高(P<0.01),siClk1+Aβ组LC3表达较NC+Aβ组明显降低(P<0.01)。Western blot结果显示与NC+NS组相比,NC+Aβ组Beclin1、LC3-Ⅱ/Ⅰ蛋白表达明显增加(P<0.01),p62、PI3K、p-AKT/AKT、p-mTOR/mTOR蛋白表达明显降低(P<0.01);与NC+Aβ组相比,siClk1+Aβ组Beclin1、LC3-Ⅱ/Ⅰ蛋白表达明显降低(P<0.01),p62、PI3K、p-AKT/AKT、p-mTOR/mTOR蛋白表达明显增加(P<0.01);加入LY294002和YC-1可以逆转沉默Clk1对Beclin1、LC3-Ⅱ/Ⅰ、p62、PI3K、p-AKT/AKT、p-mTOR/mTOR蛋白的影响。RT-qPCR结果显示siClk1+Aβ+YC-1组与siClk1+Aβ组相比,Beclin1、LC3、p62 mRNA水平无差异(P>0.05),其余各组间比较Beclin1、LC3、p62 mRNA与蛋白水平变化一致。结论沉默Clk1表达可以缓解Aβ25-35诱导的HT22细胞过度自噬引发的细胞存活率降低,其机制可能与PI3K/AKT/mTOR信号通路及HIF-1α相关。Aim To investigate the effect of siRNA transfection of silencing Clk1 gene on autophagy levels in AD model cells.Methods The Clk1 gene was silted using siRNA transfection techniques.MTT was used to observe the effects of Aβ25-35 and Clk1 genes on cell survival.Cellular immunofluorescence was applied to observe the expression of the autophagy marker microtubule-associated protein 1 light chain 3(LC3).Western blot was employed to detect the expression of Clk1,HIF-1α,PI3K/AKT/mTOR,autophagy-related proteins Beclin1,LC3-II/I,and p62.Quantitative real-time polymerase chain reaction(RT-qPCR)was utilized to detect the expression of Beclin1,LC3,p62 mRNA.Results Compared with the NC+NS group,the viability of HT22 cells in the NC+Aβgroup was significantly reduced(P<0.01),while the viability of cells in the siClk1+Aβgroup was significantly higher than that in the NC+Aβgroup(P<0.01).The results of cellular immunofluorescence showed that the expression of LC3 in the NC+Aβgroup was significantly higher than that in the NC+NS group(P<0.01),and the expression of LC3 in the siClk1+Aβgroup was significantly lower than that in the NC+Aβgroup(P<0.01).Compared with the NC+NS group,the expression of Beclin1 and LC3-II/I proteins in the NC+Aβgroup was significantly raised(P<0.01),and the expression of p62,PI3K,p-AKT/AKT,and p-mTOR/mTOR was significantly reduced(P<0.01).Compared with the NC+Aβgroup,the expression of Beclin1 and LC3-II/I proteins in the siClk1+Aβgroup decreased significantly(P<0.01),and the expression of p62,PI3K,p-AKT/AKT,and p-mTOR/mTOR proteins increased significantly(P<0.01).The addition of LY294002 and YC-1 reversed the effect of silencing Clk1 on Beclin1,LC3-II/I,p62,PI3K,p-AKT/AKT,p-mTOR/mTOR proteins.RT-qPCR results showed that there was no significant difference in Beclin1,LC3 and p62 mRNA levels in the siClk1+Aβ+YC-1 group compared with the siClk1+Aβgroup(P>0.05),and Beclin1,LC3 and p62 mRNA were consistent with the protein levels in the other groups.Conclusions Silencing Clk1 expression can allevi

关 键 词：Clk1 阿尔茨海默病 Β淀粉样蛋白 自噬 PI3K/AKT/MTOR HIF-1Α 

分 类 号：R392[医药卫生—免疫学] R394.2[医药卫生—基础医学] R745.7R977.6