多奈哌齐对Aβ25-35诱导神经毒性的保护作用  

Neuroprotective effect of donepezil on β-amyloid25-35-induced neurotoxicity

作　　者：杨红旗[1] 李学[1] 蒋秋焕[1] 马建军[1] 徐军[1] 

机构地区：[1]河南省人民医院神经内科、郑州大学人民医院神经内科,郑州450003

出　　处：《中华神经医学杂志》2012年第12期1209-1213,共5页Chinese Journal of Neuromedicine

基　　金：河南省科技攻关计划项目（112102310684）;河南省医学科技攻关计划项目（201203130）

摘　　要：目的观察胆碱酯酶抑制剂多奈哌齐对Aβ25-35诱导的神经毒性的影响及其可能机制。方法PCI2细胞常规培养，四甲基偶氮唑盐（MTT）法检测0、0．5、1、5、10、20、50μmol／β -淀粉样蛋白（AB）β25-35或多奈哌齐对细胞活力的影响；1、5、10、20、50μmol／L多奈哌齐预处理细胞2h后再加入20μmol／L Aβ25-35同时设正常对照组和单纯Aβ25-35组，MTT法检测各组细胞活力和乳酸脱氢酶（LDH）含量的变化；10μmol／L蛋白激酶C（PKC）的抑制剂GF109203X作用细胞30min后加入10μmol／L多奈哌齐，同时设正常对照组、单纯GF109203X组和多奈哌齐组．Western blotting检测各组磷酸化PKC（P．PKC）、磷酸化PKC底物（P-MARCKSl的表达；免疫荧光染色检测10μmol／L多奈哌齐作用2h和正常对照细胞PKCα、PKC∑亚型的表达。结果5、10、20、50μmol／L的Aβ25-35作用于PCI2细胞24h后可以引起细胞活力下降，差异有统计学意义（P＜0．05）。与正常对照组比较，20μmol／LAβ25-35组细胞活力下降、LDH释放增加；与Aβ25-35组比较，Aβ25-35＋5、10、20、50μmol／L多奈哌齐组细胞活力增加，LDH释放下降，差异有统计学意义（P〈0．05）。Westem blotting检测结果显示．与正常对照组相比，10μmo/L多奈哌齐组P-PKC和P-MARCKS的表达增加；与多奈哌齐组相比较，GFl09203X＋多奈哌齐组P-PKC和P-MARCKS的表达降低，差异有统计学意义（P〈0．05）；免疫荧光染色证实正常对照组PKCα和PKC∑较多地在细胞浆内表达，多奈哌齐组PKCα和PKC∑在膜部分表达增多。结论多奈哌齐可以拮抗Aβ25-35的神经毒性作用，激活PKC表达可能是其发挥保护作用的机制之一。Objective To investigate the neuroprotective effect of cholinesterase inhibitordonepezil on β-amyloid25-35 （Aβ25-35）-induced neurotoxicity and its related mechanism. Methods PC12 cells were conventionally cultured. Serial concentrations of Aβ25-35 and donepezil （0, 0.5, 1, 5, 10, 20 and 50μzmol/L） were added to PC12 cells and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） staining was then employed to detect their effects on PC12 cell viability; pretreatments with 1, 5, 10, 20 and 50 μmol/L donepezil were given to the PC12 cells 2 h before adding 20 μmol/L A1325.35 as pretreatment groups A, B, C, D and E, and normal control group I and 20 txmol/L Aβ25-35 treatment group were chosen; MMT assay was again used to detect the PC12 cell viability and level of lactate dehydrogenase （LDH）. Pretreatment with 10 txmol/L GF109203X （protein kinase C [PKC] antagonist） given to the PC12 cells 30 min before adding 10 μmol/L donepezil was carried out as pretreatment group F, and normal control group II, 10 μmol/L GF109203X treatment group and 10 txmol/L donepezil treatment group were chosen; the expressions of phosphorylation-PKC （P-PKC） and its major substrate phosphorylated myristoylated alanine-rich protein C kinase substrate （P-MARCKS） were measured by Western blotting; the effect of donepezil on PKCα and PKC∑ isoforms subcellular distribution were detected by immunofluorescence staining. Results Aβ25-35（5, 10, 20 and 50 μmol/L） treatment for 24 h could decrease the cell viability in PC12 cells in a dose-dependent manner （P〈0.05）; as compared with PC12 cells in the control group, the 20 μmol/L Aβ25-35 treatment group enjoyed lower PC12 cell viability and higher release of LDH. As compared with 20μmol/L Aβ25-35 treatment group, pretreatment groups B, C, D and E enjoyed increased cell viability and decreased LDH release （P〈0.05）. Western blotting indicated that 10 μmol/L donepezil treatment can promote PKC and MARCKS phosphorylation as compa

关 键 词：多奈哌齐 Β-淀粉样蛋白25-35 阿尔茨海默病 蛋白激酶C 

分 类 号：R749.16[医药卫生—神经病学与精神病学]