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

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 compared with control group, and the expressions of P-PKC and P-MARCKS in the pretreatment group F were significantly lower than those in the donepezil group （P〈0.05）. Immunofluorescence staining indicated that PKCot and PKCe isoforms located mainly in the cytoplasm of PC12 control cells, while donepezil could increase the expressions of PKCα and PKC∑ isoforms in the membrane fraction. Conclusion Donepezil can antagonize Aβ25-35induced neurotoxicity in PC12 cells and PKC activation may underline the donepezil＇ s neuroprotective effect.