摘要
背景阿尔茨海默病是老龄化社会人类健康的主要威协之一,目前,阿尔茨海默病(AD)的临床治疗仍然面临没有几种有效药物可以选择的困境,虽然有些药物可以暂时提高阿尔茨海默病患者的认知能力,但还没有一种治疗方法能够完全抑制或逆转阿尔茨海默病的神经退行性变进程。研究AD的发病机制,寻找能有效阻止AD发展进程的新药,及药物靶点是神经退行性变领域的艰巨任务之一。近年来,中医药在治疗老年痴呆方面的研究,取得很大的进步,己经成为一个不可忽视的力量。本文从对中枢神经系统有较好疗效的中药单体中挑选出12种,利用APPswe/PSAE9转基因阿尔茨海默病小鼠模型,筛选得到可改善阿尔茨海默病模型小鼠学习记忆能力的中药单体吴茱萸碱,我们从多个层次研究其药理作用并进行机制的研究。
方法淀粉样前体蛋白基因瑞典突变(APPswe)和早老素基因缺失突变基因(PSAE9)的双转基因(APPswe/PSAE9)小鼠是目前公认的痴呆动物模型。快速老化型小鼠具有均一的遗传背景和稳定的老化病态特征,是一种比较理性的痴呆模型,选择5月龄的APPswe/PSAE9转基因阳性鼠和SAMP8小鼠进行实验,并选择20只5月龄野生鼠和R1小鼠作为对照组,这些小鼠在性别、体重相匹配,将模型小鼠分为4组：AD模型组、安理中治疗组(2 mg/kg/d)、吴茱萸碱治疗组(50 mg/kg/d)、吴茱萸碱治疗组(100 mg/kg/d)。模型组及对照组小鼠给予常规的标准鼠粮,安理中治疗组和吴茱萸碱治疗组在常规的标准鼠粮中加入相应剂量的药物,小鼠每天鼠粮的摄入量按下列计算：每只体重为20g的小鼠,每天摄入鼠粮为3g,给药治疗时间为28天,给药后进行水迷宫实验。PET观察葡萄糖摄取情况,免疫组织化学检测老年斑变化,ELISA检测脑组织中细胞因子IL-1β、IL-6、TNF-α的表达,Western blot检测脑组织cox-2,iNOS, p-tau,Aβ寡聚体的表达。
结果水迷宫实验显示：吴茱萸碱能够显著改善APPswe/PSAE9转基因阿尔茨海默病小鼠和SAMP8小鼠呈现出的学习记忆能力损伤,能够改善APPswe/PSAE9转基因和SAMP8小鼠学习记忆能力最为有效的药物剂量为100mg/kg/d。PET观察脑组织葡萄糖摄取实验结果显示,吴茱萸碱能显著改善APPswe/PSAE9转基因阿尔茨海默病小鼠脑组织能量代谢障碍；吴茱萸碱治疗可以降低APPswc/PSΔE9转基因阿尔茨海默病小鼠脑组织中增强炎症的细胞因子IL-1β、IL-6、TNF-α(?)过度表达；Western blot结果显示,吴茱萸碱治疗也可以明显抑制和炎症相关的环氧化酶-2(COX-2)的表达。
结论在转基因小鼠AD模型和自发突变小鼠AD模型体内,吴茱萸碱能够改善阿尔茨海默病的认知功能,并表现出比现在临床用药安理中更好的效果,吴茱萸碱可以抑制脑组织中炎症因子的表达,由于目前公认炎症反应是AD病理发生的因素之一,所以吴茱萸碱对AD的治疗作用是通过抑制脑组织中炎症因子的表达实现的。我们结果揭示,吴茱萸碱可作为治疗阿尔茨海默病的候选药物。
第二部分APPswe/PSAE9转基因阿尔茨海默病小鼠模型的脑组织炎症和行为改变
目的目的研究阿尔茨海默病APPswe/PSAE9双转基因小鼠自主行为的改变,并研究其与认知功能损伤的相关性,以研究自主行为改变作为阿尔茨海默病行为特征的可行性。并从细胞、分子水平上研究与炎症相关的改变
方法对APPswe/PSAE9双转基因小鼠和野生鼠分别在4月龄、6月龄、8月龄进行旷场自主行为实验,并同Morris水迷宫分析进行比较,利用SPSS16.0软件统计分析。用免疫组织化学方法检测6月龄小鼠脑组织星形胶质细胞活化状态。用Western blotting检测cox-2表达。
结果在4月龄、6月龄转基因小鼠的学习记忆能力、以及自主性探究性行为与野生鼠比较有明显的差异。表现为：模型鼠学习记忆能力减弱,兴奋性增高,自主活动增加。这些表现与临床患者的症状有许多相似性,8月龄时,差异减小。星形胶质细胞活化明显,表现为GFAP明显增多,细胞因子分泌增加。Cox-2表达明显增加。
结论APPswe/PSAE9双转基因小鼠的自主行为学表现为烦躁不安,自主活动增加,与阿尔茨海默病患者的临床表现有许多相似之处,并与认知损伤同时发生,可作为小鼠模型判别的行为标志之一,在分子。细胞水平上,模型小鼠出现明显的炎症反应。因此APPswe/PSAE9双转基因小鼠,在阿尔茨海默病的病因病机研究和药物研发等方面具有一定的应用价值。
Backgroud Alzheimer's disease is one of the main threats to human health in an aging society. Although drugs can temporarily improve cognitive abilities of Alzheimer patients. At present there are no treatments that can totally stop or reverse neurodegenerative process of Alzheimer. The management of Alzheimer's disease (AD) has been a long-standing challenge and area of interest. In recent years, Chinese medicine in the treatment of Alzheimer's research, great progress has been made to become a force to be reckoned. Here, using the APPswe/PSAE9 transgenic mouse model of AD, evodamine was screening out from 12 kinds of compounds from Chinese herbs which showed beneficial effect on central nervous system, as the potential drugs for improvement ability of learning and memory in AD. We further studied the effects and the possible underlying mechanisms of evodamine on learning and memory improvement of AD in APPswe/PSΔE9 transgenic mouse.
Methods APPswe/PSΔE9 transgenic and SAMP8 mice in 5 months were used,and 20 wild type mice and R1 mice matched with sex, weight were selected. We divided AD model mice into 4 groups:model group, aricept treatment group(2 mg/kg/d), evodamine treatment group(50 mg/kg/d) and evodamine treatment group(100 mg/kg/d). While model group and wild group mice were given routine and standard rat food, aricept treatment group and evodamine treatment group were given an appropriate dose of the drug added to the standard rat food. The daily food intake of rats as the following Weight 20g per mouse, daily food intake of rats for the 3g. The drug was administered for 28 days. After administration the water maze test were performed, PET was used to observe glucose uptake, immunohistochemical was used to detect senile plaques, the expression of brain cytokines IL-1β,IL-6, TNF-a was detected by ELISA and brain cox-2, inos, p-tau, Aβoligomers was by western blot.
Results The water maze test results showed that evodamine administration significantly improved learning and memory deficits presenting in APPswe/PSAE9 transgenic mice and senescence-accelerated mice(SAMP8). The most effective dose of the drug ameliorating learning and memory ability was 100mg/kg/d. Glucose uptake detected by PET observed that evodamine may enhance disturbance of brain energy metabolism in APPswe/PSAE9 transgenic mice remarkably. Evodamine administration may reduce overexpression of cytokines IL-1β,IL-6, TNF-a and obviously inhibit production of COX-2 in APPswe/PSAE9 transgenic mice.
Conclusion Our study showed that evodamine could be the potential drugs for AD therapy. These findings revealed that evodamine could improve cognitive functions of AD in APPswe/PSAE9 transgenic mice, partly through regulating cytokines expression and depressing the COX-2 expression, thereby inhibiting inflammatory damage in Alzheimer's disease brain tissue.
Background To study the locomotor activity changes of APPswe/PSΔE9 double-transgenic mice and to investigate the possibility that the locomotor activity test could be used as behaviour evaluation in Alzheimer Disease. And study the changes in inflammation from the cellular, molecular level.
Methods At 4.6,8 months, Morris water maze and open field trials were performed in transgenic mice and wildtype mice. All collected data were analyzed by SPSS software. The astrocyte activation in 6 months old mouse brain was detected by immunohistochemistry. And the expression of cox-2 was Detected by Western blotting.
Results 4-and 6- months old APPswe/PSΔE9 double-transgenic mice showed decrease in ability of learning and memory, but increase in excitement and in locomotor activity. There was statistically significant difference in the behaviour changes compared with wild type mice, and these features were similar to symptoms of AD patients. The GFAP and cytokines secretion were obviously enhanced, which showed the astrocyte was activated significantly. In addition, the expression of Cox-2 was significantly increased.
Conclusions By the locomotor activity test, the behaviors like dysthesia, increased excitement and increased locomotor activity were observed in the Alzheimer Disease transgenic mice, which are the behavior phenotypes of human Alzheimer Disease. The transgenic mice show obvious inflammatory reaction in molecular and cellular level.It suggested that locomotor activity test could be an useful tool for evaluation of the Alzheimer Disease animal model.
引文
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