【作者】 陈玉静；

【导师】 田金洲； 盛树力；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2008， 博士

【摘要】 目的:建立散发性老年性痴呆(SAD)大鼠模型,应用具有益气活血、补肾化痰作用的中药复方提取物金思维进行治疗,观察金思维对该模型行为学、海马神经元超微结构、tau蛋白磷酸化相关酶类以及胰岛素信号转导通路相关蛋白表达的影响;探讨金思维治疗AD的作用机理,为指导临床应用和开发新药提供理论和实验依据。方法:将实验动物随机等分6组:假手术组、模型组、盐酸多奈哌齐组和金思维大、中、小剂量治疗组。采用双侧脑室注射微量链脲佐菌素(ICV-STZ)的方法建立拟AD大鼠模型。模型成功后,治疗组分别予盐酸多奈哌齐和金思维(分为大、中、小三个不同剂量)进行三个月的治疗,同时模型组和假手术组给予等体积的双蒸水。采用Morris水迷宫、免疫组织化学、Western blot、电镜以及图像分析技术等手段,观察模型鼠行为学、海马CA1区形态学的改变,分别检测海马组织tau蛋白磷酸化位点(Ser199/202)和相关酶类(GSK-3β、PP-2A、PP-1),以及胰岛素信号转导通路相关蛋白(IR、IGF-I、IRS-1、IRS-2)的含量和表达水平。结果:1金思维能明显改善ICV-STZ模型大鼠出现的学习记忆障碍:定位航行试验中,模型组大鼠平均逃避潜伏期和游泳距离较假手术组明显延长(P<0.05或/P<0.01),金思维各剂量组和盐酸多奈哌齐组平均逃避潜伏期和游泳距离较模型组明显缩短(P<0.05/P<0.01);空间探索试验中,模型组与假手术组比较,原平台象限活动时间明显缩短(P<0.01);金思维各剂量组与模型组比较,原站台象限活动时间均明显延长(P<0.01),且与盐酸多奈哌齐组相比无显著性差异。2 ICV-STZ模型组大鼠海马组织存在胰岛素信号转导通路障碍:IR、IGF-I、IRS-1、IRS-2的含量和表达水平均下调(P<0.01),金思维各剂量组均可不同程度的上调这些蛋白的含量和表达(P<0.05/P<0.01)。3与假手术组相比,ICV-STZ模型组大鼠在Ser199/202位点磷酸化水平明显升高(P<0.01),非磷酸化水平明显降低(P<0.01);而金思维各剂量组和盐酸多奈哌齐组在此二位点的磷酸化水平较模型组显著降低(P<0.05/P<0.01),非磷酸化水平显著升高(P<0.05/P<0.01),尤以大、中剂量组突出。4 ICV-STZ模型组大鼠海马CA1区微管出现断裂和溶解,金思维可修复断裂或溶解的微管;与假手术组相比,模型组蛋白激酶GSK-3β的表达明显升高(P<0.01),磷酸酶PP-2A、PP-1表达明显下降(P<0.01);金思维各剂量组GSK-3β的含量和表达水平较模型组显著降低(P<0.05/P<0.01),PP-2A和PP-1的含量和表达水平显著升高(P<0.05/P<0.01),与假手术组接近。结论:1 ICV-STZ模型大鼠存在胰岛素/ IGF-I信号转导异常以及学习记忆功能障碍,该模型可成功地模拟SAD的病理特征。2金思维提取物可明显改善tau蛋白Ser199/202位点的磷酸化程度,从而防止了过度磷酸化tau蛋白的产生及其带来的毒性作用;金思维能够保持蛋白激酶和磷酸酶表达平衡,确保海马神经元tau蛋白的正常磷酸化,保护微管结构,防止轴突损伤。3金思维可改善胰岛素/ IGF-I信号转导通路,发挥胰岛素及IGF-I营养和保护神经元的功效,从而恢复和促进学习记忆功能。更多还原

【Abstract】 Objective:To establish rat model of Sporadic Alzheimer’s disease(SAD),and treat with GETO (herbal extract), which have the role function of supplementing Qi, activating blood flow, invigorating the kidney and dissipating phlegm. In this paper, we observe the learning and memorizing ability of the rats,ultrastructure of hippocampal neurons, as well as the expression of enzymes related to phosphorylation of tau and insulin signal transduction pathway in hippocampal neurons of AD rats. So as to investigate the neuroprotective role of GETO on rats with AD, provide further theoretical and experimental evidence for new medicine in clinical applicationMethods:Experimental animals were randomly divided into six groups: sham-operated group, model group, Donepezil group and GETO large, middle and small dosage treated groups. To establish the Sporadic Alzheimer’s disease model by intracerebroventricular injection of STZ (ICV-STZ) bilaterally, GETO groups were treated with gastric perfusion of GETO in three different dosage. The model group and sham group were given double distilled water respectively. 3 months later, Learning and memorizing ability of the rats were inspected through Morris water maze test. Immunohistochemistry(IHC), Western bolt, electron microscope and system of image pro-plus were used to determine ultramicrostructure of hippocampus CA1 region, and the expression of enzymes related to phosphorylation of tau(GSK-3β、PP-2A、PP-1), as well as protein expression of insulin signal transduction pathway(IR、IGF-I、IRS-1、IRS-2)in hippocampal neurons of AD rats.Results:1 GETO can ameliorate the ability of learning and memory of ICV-STZ rats. In place navigation test, Morris water maze test showed that the escape latency and swimming distance were significantly longer in the model group than that in the sham group (P<0.05 or P<0.01), but shorter in GETO groups and Donepezil group(P<0.05 or P<0.01).In spatial probe test, compared with the sham group, swimming time in the platform of previous quadrant was significantly shorter in model group(P<0.01), but significantly longer in GETO groups(P<0.01), and there was no difference between Donepezil group and GETO groups. 2 Compared with the sham group, the levels of phosphorylated tau protein at Ser199/202 epitope were elevated in ICV-STZ rat brain hippocampus, and the levels of non- phosphorylated tau protein at Ser199/202 epitope were decreased (P<0.01); but in GETO groups and donepezil group, the levels of phosphorylated tau at the same epitope were obviously decreased, and non-phosphorylated proteins were obviously increased (P<0.05/ P<0.01), especially in Large and Middle dosage treated groups.3 The microtubules of hippocampal neurons of ICV-STZ model showed prominent changes of fragmentation and dissolution, while the treatment of GETO can significantly ameliorate these changes. The hippocampal neurons of AD rats showed significant intracellular increase in the tau phosphokinase, GSK-3β(P<0.01)and decrease in the tau dephosphry- lating enzymes, PP-1 and PP-2A(P<0.01). Compared with model group, GETO can down- regulate the expression of GSK-3βand up-regulate the expression of PP-2A and PP-1 (P<0.05/P<0.01).4 GETO can regulate brain insulin/IGF signal transduction pathway, there was an insignificant decrease in IR, IGF-I, IRS-1 and IRS-2 in the GETO treated groups as compared to the Model(P<0.01), and GETO groups can up-regulate the expression of them (P<0.05/P<0.01).Conclusion:1 There was dysfunction in learning and memory ability, as well as insulin/IGF-I signal transduction pathway in ICV-STZ rats; this model can simulate the pathological features of AD.2 GETO can ameliorate phosphorylation extent at Ser199/202 sites, and thus prevent the formation and toxicity of hyperphosphorylation of tau protein; GETO can keep balance of protein kinase and phosphatase expression, to ensure normal level of tau protein phosphorylation in hippocampal neurons, protect structure of microtubule, and prevent axonal injury.3 GETO can regulate insulin/IGF-I signaling pathway, help insulin and IGF-I act on protecting neurons from damage and remarkably improving learning and memory function.更多还原