二肽基肽酶-4抑制剂对阿尔茨海默病样神经退行性变的保护作用  

The protective effects of dipeptidyl peptidase-4 inhibitor on AD-like neurodegenerative changes

作　　者：陈书仪 郭爱[1] 陈沿霖 付荣霞[2] 赵纲[3] 彭鹏[1] 宋奇骏 邓艳秋[1] CHEN Shu-yi;GUO Ai;CHEN Yan-lin;FU Rong-xia;ZHAO Gang;PENG Peng;SONG Qi-jun;DENG Yan-qiu(Department of Pathophysiology, School of Basic Medical Science, Tianjin Medical University, Tianjin 300070, China;Food Science and Biological Engineering Department, Tianjin Agriculture University;Department of Pathology, Tianjin Cancer Hospital)

机构地区：[1]天津医科大学基础医学院生理学与病理生理学教研室,300070 [2]天津农学院食品工程与生物学院 [3]天津市肿瘤医院病理科

出　　处：《天津医药》2017年第4期342-348,共7页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81270422);国家级大学生创新创业训练计划项目(201610062013)

摘　　要：目的探讨二肽基肽酶-4抑制剂(DDP-4I)对阿尔茨海默病(AD)样神经退行性改变的保护作用及其机制。方法取对数生长期的人神经母细胞瘤细胞SH-SY5Y分为6组:空白对照组(CON组),含1‰二甲基亚砜(DMSO)的磷酸盐缓冲液(PBS)处理12 h;wortmannin干预组(W组),0.03μmol/L wortmannin处理12 h;DPP-4I干预组(DPP-4I组),10μmol/L DPP-4I处理12 h;DPP-4I与wortmannin共同干预组(DPP-4I+W组),10μmol/L DPP-4I预处理2 h,再给予0.03μmol/L wortmannin处理12 h;DPP-4I+wortmannin+Ex9-39共同干预组(DPP-4I+W+Ex9-39组),10μmol/L Ex9-39预处理2 h,再加入10μmol/L DPP-4I作用2 h,最后0.03μmol/L wortmannin处理12 h;Ex9-39干预组(Ex9-39组),10μmol/L Ex9-39处理12 h。MTT法检测各组细胞活性的变化,Western blot法检测各组微管相关的tau总蛋白(tau-5)及其不同磷酸化位点(p Sp S199/202、p T231和p S396)、神经丝(NFs)相关蛋白(NF-H、NF-M)以及胰岛素信号通路PI3K/Akt/GSK-3β中关键酶的磷酸化水平。结果 (1)与CON组相比,W组细胞活力下降,p Sp S199/202、p T231、p S396以及NF-H/M的磷酸化水平升高,而DPP-4I组细胞活力上升,上述指标的磷酸化水平下降;与W组相比,W+DPP-4I组的细胞活力上升,上述指标的磷酸化水平下降。(2)与CON组相比,Ex9-39组细胞活力下降,p Sp S199/202、p T231、p S396以及NF-H/M的磷酸化水平升高;与DPP-4I+W组相比,DPP-4I+W+Ex9-39组上述指标发生相同的变化。(3)与CON组相比,W组p-PI3K、p-Akt、p-GSK3β水平下降,DPP-4I组p-PI3K、p-Akt、p-GSK3β水平上升;与W组相比,DPP-4I+W组p-PI3K、p-Akt、p-GSK3β水平上升。结论 DPP-4I可通过提高GLP-1的浓度和激活PI3K/Akt/GSK-3β信号通路来改善wotmannin诱导的AD样的tau蛋白和神经丝的过度磷酸化,保护神经细胞的退行性变。Objective To explore the protective effects of dipeptidyl peptidase-4inhibitor(DPP-4I)on AD-like neurodegenerative changes and its mechanism.Methods The human neuroblastoma cell line SH-SY5Y on the logarithmic phase was divided into six groups:control group(CON group,treated with PBS contained1‰DMSO for12h),wortmannin intervention group(W group,treated with0.03μmol/L wortmannin for12h),DPP-4I intervention group(DPP-4I group,treated with10μmol/L DPP-4I for12h),both DPP-4I and wortmannin intervention group(DPP-4I+W group,pre-treated with10μmol/L DPP-4I for2h,then0.03μmol/L wortmannin for12h),DPP-4I,wortmannin and Ex9-39intervention group(DPP-4I+W+Ex9-39group,pre-treated with10μmol/L Ex9-39for2h,then10μmol/L DPP-4I for2h followed by0.03μmol/L wortmannin for12h),and Ex9-39intervention group(Ex9-39group,treated with10μmol/L Ex9-39for12h).MTT assay was used to detect the cell vitality.Western blot assay was used to detect the level of total tau protein(tau-5)andphosphorylated tau at different sites(pSpS199/202,pT231and pS396),the level of phosphorylated neurofilaments(NF-H,NF-M)and phosphorylation of critical enzyme in PI3K/Akt/GSK-3βsignaling pathway.Results(1)The cell vitalitydecreased,the levels of pSpS199/202,pT231,pS396and NF-H/M increased significantly in W group than those in CONgroup.However,comparing with CON group,the above mentioned parameters reversed in DPP-4I group.Comparing with Wgroup,the cell vitality increased and phosphorylated levels of above mentioned indices were decreased in DPP-4I+W group.(2)The cell vitality showed a decline trend while the levels of phosphorylation tau at three different sites and NF-H/M werehigher in Ex9-39group than those in CON group.Comparing with DPP-4I+W group,the results of the phosphorylated levelsshowed the same changes in DPP-4I+W+Ex9-39group.(3)Comparing with CON group,the expression levels ofphosphorylated PI3K,Akt and GSK3βincreased significantly in DPP-4I group,while those decreased in W group.Additionally,the expression levels of phosphorylated PI3K,

关 键 词：二肽基肽酶Ⅳ抑制剂 阿尔茨海默病 胰高血糖素样肽类 TAU蛋白质类 蛋白激酶类 神经丝 胰岛素信号通路 

分 类 号：R742[医药卫生—神经病学与精神病学]