2-(α-羟基戊基)苯甲酸盐改善痴呆小鼠认知的机制研究

The pathogenesis of Alzheimer's disease (AD) is complicated, at present,there are no effective radically curative drugs. However, the two specific pathological changes in AD are definitive, which are the extracellular deposition of amyloid-βpeptide (Aβ) and intracellular neurofibrillary tangle. 2-(α- Hydroxypentyl)-benzoate (HPB) is a new anti-cerebral ischemia compound synthesized by Chinese scientists. In our previous studies, we found that HPB could improve the learning and memory of a variety of AD animals and reduce the expressions of Aβ and phosphorylated tau protein(p-tau,main component of neurofibrillary tangle). Now, the objective of this study is to verify the improvement of HPB on the cognition of amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice. Meanwhile, the Aβ and p-tau in their brains will be detected with enzyme linked immunosorbent assay (ELISA) and western blot, and their brain morphological changes (e.g. morphous and number of neuron, extracellular senile plaque and intracelluar neurofibrillary tangles) will be observed with pathological method. Then, APP/PS1 transfection cells will be adopted, and the effects of HPB on APP metabolism, cell growth and p-tau expression will be investigated with immunocytochemical method, real-time polymerase chain reaction (PCR) and flow cytometry and so on. Meanwhile, specific inhibitors will be used in order to research the influences of HPB on APP secretases (α-,β-,and γ-), Aβ degradation enzymes （neprilysin and insulin degrading enzyme） and its upstream protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and tyrosine protein kinase (TPK) signal pathways. The influences of HPB on upstream protein kinases (GSK-3 and CDK-5)and downstream phosphatases (PP2A and PP1)of p-tau will be also investigated. Eventually, we shall elucidate its main action mechanisms against AD and provide experimental data for developing it a new anti-AD drug.

阿尔茨海默病（AD）机制复杂，目前仍缺乏有效的根治药物。但其两个特征性病理改变-神经细胞外淀粉样蛋白（Aβ）沉积和神经原纤维缠结是确定的。2-(α-羟基戊基)苯甲酸盐（HPB）是我国自主合成的新型抗脑缺血化合物。我们前期研究发现，HPB能改善多种AD动物的学习记忆，同时减少脑Aβ和磷酸化tau蛋白（p-tau）表达。本课题拟确证HPB对APP/PS1转基因小鼠认知的改善，并用ELISA、免疫印迹法研究其对脑Aβ和p-tau表达的影响，用病理学方法观察其对脑组织形态的影响。再以APP/PS1转染细胞为对象，采用免疫细胞化学法、PCR、流式细胞术等，观察药物对APP代谢和细胞生长以及p-tau的影响，同时使用阻断剂，研究HPB对APP分泌酶、Aβ降解酶和其上游PKC、MAPK、TPK信号通路以及p-tau上游激酶和下游磷酸酶的影响。阐明其抗AD主要机制，为将其开发成新型抗AD药物提供实验依据。

研究内容和结果：本研究观察了HPB对APP/PS1双转基因小鼠学习记忆的改善作用，并初步探讨其作用机制。实验动物随机分六组：正常组（非转基因C57BL/6J小鼠）、模型组、安理申组、HPB15、45和135mg/kg组。灌胃给药或溶剂3个月。用Morris水迷宫检测小鼠的空间学习记忆能力。用病理学方法（HE染色、刚果红染色、改良 Bielschowsky银染法）观察小鼠脑组织神经元形态、老年斑等。用Western blot法检测脑组织p-tau蛋白的表达水平。结果显示，HPB可剂量依赖性地改善痴呆小鼠的空间学习记忆能力，以及海马神经元形态异常。可以减少脑组织老年斑的形成，并改善神经纤维排列异常。但脑组织p-tau蛋白的表达在各组间没有差异。表明，HPB可以改善APP/PS1双转基因小鼠的认知，同时可以减少其老年斑的形成，并改善神经元形态及神经纤维排列。.完成情况：研究内容按照计划进行，绝大部分工作已完成。其余少量指标的测定工作正在进行，3月份可完成。之所以有少量指标测定未完成，是因转基因动物昂贵，使用少，必须以订购方式获得，且动物提供方动物繁殖速度有限，难以短时间满足需求，仅供应动物就花费7个月。加之，给药时间长达3个月，而研究时间仅有一年。.成果：已发表相关署名文章3篇，其中SCI和核心期刊各一篇。研究内容正在整理补充，将尽快成文投稿。

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