淫羊藿苷防治阿尔茨海默病的自噬溶酶体途径的作用机制研究

The accumulation of insoluble proteins is a pathological hallmark of several neurodegenerative disorders, including AD. Clearance of the abnormally modified long-lived protein mainly occur by the autophagy-lysosomal pathway. Macroautophagy, a major pathway for organelle and protein turnover, has been implicated in the development of AD. The basis for the profuse accumulation of autophagic vacuoles (AVs) in affected neurons of the AD brain. Recent studies have found that reversal of autophagy dysfunction in AD brain can ameliorate amyloid pathologies and memory deficits. Activation of autophagy may have therapeutic potential in AD, however, there is a big side-effects of gene or drug treatments. Thus, looking for natural products which could modulate autophagy has broad prospects. Icariin, a compound from Epimedium brevicornum Maxim, has been reported to have many neuroprotective effects. Our preliminary study found icariin markedly decreased intraneuronal beta-amyloid in intracerebral infusion beta-amyloid 42 AD model, but the mechanism is unknown. Now, we are in an attempt to explore the protective mechanism of icariin in AD through autophagy-lysosomal pathway, in order to provide theoretical basis for this drug research. In this study, immunohistochemistry, ELISA and western blot will be used to detect accumulations of beta-amyloid especially in autophagic substrates within autolysosomes/lysosomes in vivo and in vitro. To observe the effect of icariin on autophagy-lysosomal pathway, autophagic associated makers, ultrastructural analyse and enzymatic activity assays for cathepsins will be detected. Then AVs fusion with lysosomes and autophagic flux inhibitors will be used to study the targets of icariin in autophagy-lysosomal pathway and explore the protective mechanism of icariin in AD.

阿尔茨海默病(Alzhermer's disease, AD) 是一种常见的神经退行性疾病，是老年痴呆最常见的病因，其特征为毒性蛋白异常聚集。自噬溶酶体途径是清除这些长寿命蛋白最重要的机制，其在AD发病机制中的作用已成为研究热点。近年研究发现改善AD脑内自噬溶酶体功能失调可清除AD脑内蛋白聚集，而针对单一靶点的基因或药物治疗存在较大副作用，寻找调控自噬的小分子天然产物具有广阔的前景。前期研究发现淫羊藿苷可降低AD模型大鼠神经元内淀粉样多肽的聚集，但其作用机制不明。本研究旨在研究淫羊藿苷防治AD的自噬溶酶体途径的作用机制。通过整体和细胞两个层面，结合自噬多种检测指标观察淫羊藿苷对自噬溶酶体途径的影响。以自噬泡与溶酶体融合和初、次级自噬流抑制剂为工具药，通过免疫印迹及免疫荧光研究淫羊藿苷的作用靶点，探讨淫羊藿苷干预AD的分子机制，为淫羊藿苷的临床治疗提供实验理论基础。

AD病变中毒性Aβ异常聚集可造成神经元和突触的损害，进而引起神经功能缺损。细胞自噬能清除具有细胞毒性、长时间存活及具有聚集倾向的蛋白质。由于AD 中轴突逆向转运受损，神经元轴突末梢的AVs无法转运至溶酶体与其融合而降解，导致其大量聚集。研究发现，改善AD脑内自噬溶酶体功能失调可有效减低Aβ从而改善突触功能。但是针对单一靶点的基因及药物治疗可干扰正常生理功能，副作用较大。淫羊藿苷可减少脑内APP和Aβ含量，但其机制尚不完全清楚。自噬溶酶体途径在淫羊藿苷干预AD病变中的作用尚未见报道。本项目通过整体和细胞两个层面，结合自噬多种检测指标观察淫羊藿苷对自噬溶酶体途径的影响。我们发现淫羊藿苷可改善侧脑室注射Aβ1-42多肽模型大鼠学习记忆障碍、突触损伤、减少脑内Aβ聚集及SAPPβ的表达。采用免疫印迹检测自噬流活性标记物，结果显示淫羊藿苷可降低AD模型脑内LC3Ⅱ/ LC3 I、Beclin及Cathepsin D表达，改善自噬溶酶体功能。我们还发现预先给予淫羊藿苷亦可改善AD模型大鼠行为学障碍、自噬溶酶体功能及海马神经元超微结构，与淫羊藿苷治疗效果相似。为体现中医理论“肾虚”是AD病机及中医临床证型，采用皮下注射氢化可的松结合侧脑室定位注射Aβ25-35建立肾虚型老年痴呆大鼠模型，发现淫羊藿苷和补肾复方（含淫羊藿）可改善肾虚型老年痴呆模型大鼠学习记忆能力、脑内基本病变、突触结构及自噬功能。体外结果显示淫羊藿苷可减少Aβ25-35诱导的N2a（mouse neuroblastoma N2a，小鼠神经瘤细胞）细胞内外Aβ的含量。淫羊藿苷可通过提高mTOR信号通路中pT389 P70/P70水平（P70S6K活性形式）抑制自噬过度激活。并以初、次级自噬流抑制剂为工具药，探讨自噬溶酶体途径在淫羊藿苷干预AD的分子机制。本研究为淫羊藿苷的临床应用提供有力理论基础和实验依据。