淀粉样β蛋白片段25～35下调大鼠海马PI3K/Akt/p70S6K信号传导通路

摘要

目的 探讨阿尔茨海默病(AD)的淀粉样β蛋白(Aβ)的沉积是否损害神经细胞存活的信号传导通路.方法 实验分为生理盐水对照组;Aβ25-35组;Aβ25-35+布洛芬组;Aβ25-35+布洛芬+LY294002组;Aβ25-35+LY294002组.大鼠分别灌胃给予布洛芬7.5或15 mg·kg-1,每日1次,连续3 周后,左侧脑室内注射Aβ25-35(10 μL,1 mmol·L-1),之后继续灌胃给予布洛芬1周.PI3K特异性阻断剂LY294002(5 μL,4 mmol·L-1)在注射Aβ25-35前1 h左侧脑室内注射.注射Aβ25-35后1周,取海马CA1区,Western免疫印迹法观察P53,Bax,FasL,Bcl-2,Akt和p70S6K的蛋白表达水平.应用半胱氨酸天冬氨酸蛋白酶(caspase)3活性测定试剂盒分析caspase 3活性变化,RT-PCR方法观察p70s6k mRNA表达水平.结果 脑室内注射Aβ25-35可引起大鼠海马CA1区磷酸化Akt/PKB和磷酸化p70S6K表达明显降低,分别从对照组1.32±0.14和0.769±0.028下降到0.69±0.08和0.479±0.032.同时,海马CA1区促凋亡蛋白P53,Bax和 FasL表达及caspase 3活性明显增加,抗凋亡蛋白Bcl-2表达明显降低.预先注射LY294002可使caspase 3活性较单独注射Aβ25-35组进一步增加.给Aβ25-35前后连续给予布洛芬4周可明显对抗Aβ25-35引起的上述变化.LY294002可明显减弱布洛芬上调磷酸化Akt/PKB和磷酸化p70S6K表达的作用.结论 Aβ25-35引起抗凋亡通路PI3K/Akt/p70S6K下调可能参与AD的神经元损伤.布洛芬具有较好的对抗作用,这可能与上调PI3K/Akt/p70S6K通路中的一些蛋白有关.%AIM To investigate whether Aβ deposit in Alzheimer disease(AD) impairs signal transduction pathway responsible for neuronal survival.METHODSThe rats were randomly divided into six groups:control group and Aβ25-35 group,Aβ25-35+ibuprofen groups (7.5 and 15 mg·kg-1,respectively),Aβ25-35+ibuprofen+LY294002 group,and Aβ25-35+LY294002 group.Rats were given ibuprofen (7.5 and 15 mg·kg-1 daily,ig) for 3 weeks prior to and 1 week after icv single dose of Aβ25-35 (10 μL,1 mmol·L-1).LY294002 was injected icv 1 h before the injection of Aβ25-35.Seven days after Aβ25-35 injection,the hippocampal expressions of P53,Bax,Fas ligand (FasL),Bcl-2 proteins,phospho-Akt/PKB,and phosphorylated 70 ku ribosomal protein S6 kinase (p70S6K) and caspase 3 were determined in the brain tissue preparations from CA1 area with Western blot.The activity of caspase 3 was measured using a caspase 3 colorimetric activity assay kit.RT-PCR was used to show the change of p70s6k mRNA level.RESULTS Aβ25-35 icv injection significantly down-regulated phosphorylated Akt/PKB from 1.32±0.14 to 0.69±0.08 and p70S6K from 0.769±0.028 to 0.479±0.032 in hippocampal CA1 region.These changes were accompanied by increased expressions of the proapoptotic proteins P53,Bax,and FasL and decreased expression of the anti-apoptotic protein Bcl-2 in rat hippocampus.In addition,caspase 3 activity was significantly enhanced in hippocampal CA1 region in Aβ25-35-treated rats compared with control rats.Ibuprofen can reverse these Aβ25-35-induced changes.CONCLUSION Down-regulated anti-apoptotic PI3K/Akt/p70S6K signaling pathway induced by Aβ25-35 in rat hippocampus may contribute to the neuronal damage in AD.Ibuprofen prevents Aβ25-35-induced down-regulation of PI3K/Akt/p70S6K signaling pathway.