链脲佐菌素致阿尔茨海默病模型大鼠脑内mTOR/P70S6K/IRS-1信号通路的变化

Change of m TOR/P70S6K/IRS-1 signaling pathway in the brain of Alzheimer's disease rats induced by streptozotocin

目的:观察侧脑室(intracerebroventricular,ICV)注射链脲佐菌素(streptozotocin,STZ)对大鼠脑内m TOR/P70S6K/IRS-1信号通路的影响。方法:将30只雄性SD大鼠随机分为对照组和模型组,每组各15只,对照组于第1天和第3天ICV注射生理盐水,模型组于第1天和第3天侧脑室注射STZ(ICV-STZ,3 mg/kg)建立AD模型。第1次注射后36 d采用Morris水迷宫检测2组大鼠空间学习和记忆能力的变化;Western blot检测2组大鼠大脑皮质和海马组织哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin,p-m TORSer2448)、核糖体40S小亚基S6蛋白激酶(P70 ribosomal S6 protein kinase,P70S6K)、磷酸化核糖体40S小亚基S6蛋白激酶(phosphorylated P70 ribosomal S6 protein kinase,p-P70S6KThr389)、胰岛素受体底物-1(insulin receptor substract-1,IRS-1)和磷酸化胰岛素受体底物-1(phosphorylated insulin receptor substract-1,p-IRS-1Ser636)蛋白的表达;HE染色观察2组大鼠大脑皮质和海马组织形态学的改变;甲硫素S染色观察2组大鼠脑内Aβ的沉积。结果:Morris水迷宫结果显示ICV-STZ能明显降低大鼠目标象限停留时间[对照组:(55.945±9.447)s,模型组:(31.961±5.346)s,t=8.558,P=0.000]和穿越平台次数[对照组:(7.533±2.560)次,模型组:(2.867±1.506)次,t=6.086,P=0.000];HE染色可见:ICV-STZ大鼠大脑皮质和海马的部分细胞出现了明显的固缩和肿胀;甲硫素S染色发现:在ICV-STZ处理后,大鼠脑内出现Aβ的大量沉积。Western blot结果表明:ICV-STZ能同时增加大鼠大脑皮质p-P70S6KThr389[对照组:(0.268±0.066),模型组:(0.654±0.079),t=-8.409,P=0.000]、p-IRS-1Ser636[对照组:(0.710±0.092),模型组:(1.033±0.135),t=-4.417,P=0.002]和海马p-P70S6KThr389[对照组:(0.405±0.064),模型组:(0.732±0.077),t=-7.339,P=0.000]、p-IRS-1Ser636[对照组:(0.498±0.093),模型组:(0.836±0.102),t=-5.496,P=0.001]蛋白的表达,但不影响大鼠大脑皮质和海马P70S6K、m TOR、p-m TORSer2448、IRS-1蛋白的表达。结论:ICV-STZ能增加大鼠脑内m TOR/P70S6K/IRS-1信号通路的激活,提示异常活化的m TOR/P70S6K/IRS-1信号通路可能参与了AD的发生发展。

Objective：To investigate the effects of intracerebroventricular injection of streptozotocin on m TOR/P70S6K/IRS-1 signaling pathway. Methods：The 30 male SD rats were randomly divided into control group and model group,fifteen rats in each group. The rats were treated with saline or streptozotocin（STZ,3 mg/kg）intracerebroventricularly（ICV）at the 1st day and the 3rd day of the experiment to induce dementia model. Thirty-six days after STZ injection,spatial learning and memory of the rats were determined by Morris water maze test. The expression of mammalian target of rapamycin（m TOR）,phosphorylated m TOR（p-m TORSer2448）,p70 ribosomal S6 protein kinase（P70S6K）,phosphorylated P70S6K（p- P70S6KThr389）,insulin receptor substract- 1（IRS-1）and phosphorylated IRS-1（p-IRS-1Ser636）were measured by Western blot. Morphologic changes of neurons in cerebral cortex and hippocampus were observed by HE staining. Deposition of Aβ in hippocampus was detected by Thioflavin S staining.Results：ICV-STZ can significantly reduce the time spent in the target quadrant（t=8.558,P=0.000）and numbers of platform location crosses（t=6.086,P=0.000）. HE staining demonstrated that some cells were clearly shrank and swelled in cerebral cortex and hippocampus of rats treated by STZ. Meanwhile,the deposition of Aβ detected by Thio-flavin S staining was also significantly increased.Compared with that in control group,the expressions of p-P70S6KThr389（t =-8.409,P=0.000）,p-IRS-1Ser636（t=-4.417,P=0.002）in the cerebral cortex and p-P70S6KThr389（t=-7.339,P=0.000）,p-IRS-1Ser636（t=-5.496,P=0.001）in hippocampus were increased markedly,while the protein levels of P70S6 K,m TOR,p-m TORSer2448 and IRS-1 were not affected by ICV- STZ. Conclusion：ICVSTZ results in increased activation of m TOR/P70S6K/IRS-1 signaling pathway,and the increased activation of m TOR/P70S6K/IRS-1may play important roles in AD pathogenesis.