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 mTOR/P70S6K/IRS-1 signaling pathway in the brain of Alzheimer’s disease rats induced by streptozotocin

摘要:

目的：观察侧脑室（intracerebroventricular，ICV）注射链脲佐菌素（streptozotocin，STZ）对大鼠脑内mTOR/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，mTOR）、磷酸化哺乳动物雷帕霉素靶蛋白（phosphorylated mammalian target of rapamycin，p-mTORSer2448）、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、mTOR、p-mTORSer2448、IRS-1蛋白的表达。结论：ICV-STZ 能增加大鼠脑内mTOR/P70S6K/IRS-1信号通路的激活，提示异常活化的mTOR/P70S6K/IRS-1信号通路可能参与了 AD 的发生发展。

Abstract:

Objective：To investigate the effects of intracerebroventricular injection of streptozotocin on mTOR/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 experi－ment to induce dementia model. Thirty-six days after STZ in－jection，spatial learning and memory of the rats were deter－mined by Morris water maze test. The expression of mammalian target of rapamycin（mTOR），phosphorylated mTOR（p-mTORS－er2448），p70 ribosomal S6 protein kinase（P70S6K），phosphory－lated P70S6K（p-P70S6KThr389），insulin receptor substract-1（IRS-1） and phosphorylated IRS-1（p-IRS-1Ser636） were mea－sured by Western blot. Morphologic changes of neurons in cere－bral 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 hip－pocampus 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 P70S6K，mTOR，p-mTORSer2448 and IRS-1 were not affected by ICV- STZ． Conclusion：ICV-STZ results in increased activation of mTOR/P70S6K/IRS-1 signaling pathway，and the increased activation of mTOR/P70S6K/IRS-1 may play important roles in AD pathogenesis．