Klotho对AD神经血管单元的调控机制及川芎苯酞类化合物的干预作用

The neurovascular hypothesis of Alzheimer's disease (AD) suggests that the abnormality of cerebral neurovascular unit (NVU) induces the AD pathogenic cascades, however, the underlying mechanisms remain unclear. Clinical epidemiological studies confirm that aging is the major risk for the development of AD. Aging-suppressor gene Klotho deficiency in mice displays several aging-like phenotypes, such as dementia and atherosclerosis, which is probably associated with the dysfunction of Klotho-regulating signaling transduction pathways. Recent study shows that Klotho levels are lower in cerebrospinal fluid of AD patients compared with controls without AD. In addition, applicant finds that there is a significant inverse correlation between Klotho expression and AD phenotype in mice, and phthalide ligustilide from Rhizoma chuanxiong significantly ameliorates AD-like impairment in mice through regulation of Klotho expression and IGF-1 signaling pathway. This project will prepare series of phthalides from Rhizoma chuanxiong, screen their activity targeting Klotho gene expression. The regulation and underlying molecular mechiansims of Klotho on NVU during the pathogenic process of AD will be investigated by phthalide probe molecules with high activity and cell transfection of Klotho gene. This study will make a contribution to elucidation of molecular basis of high correlation between aging and AD, exploration of potential drug targets against AD and discovery of novel candidates or leading compounds for AD threatment and prevention in future.

阿尔兹海默病（AD）的神经血管假说表明脑内神经血管单元（NVU）异常可启动AD发病级联但致病机制尚未阐明。临床流行病学研究证实衰老是AD的最大危险因素。Klotho（KL）衰老抑制基因缺陷小鼠出现痴呆、动脉粥样硬化等表型改变，其机制可能与衰老过程KL调控的信号转导通路功能障碍有关。新近报道AD患者脑脊液中KL明显减少；申请人发现AD小鼠KL表达水平与AD病变程度呈显著负相关，而川芎苯酞类化合物藁本内酯通过调节KL表达及KL调控的IGF-1通路，明显减轻小鼠的AD病变。本项目拟制备系列川芎苯酞类化合物，靶向KL基因表达的活性筛选，以高活性的苯酞化合物为药物探针分子，结合KL基因的细胞转染技术，研究KL对AD病程中NVU的调控作用与分子机制。本研究工作将有助于深入解析衰老与AD高度关联性的分子基础、探寻防治AD的潜在药物靶点、发现防治AD的新型候选药物或先导化合物，具有重要的科学意义。

阿尔茨海默病（AD）是老年性痴呆最常见的类型。本项目研究Klotho衰老抑制基因在小鼠AD病程中的调节作用及机制、并探讨Klotho增强剂对AD小鼠的防治效应。我们首先检测Klotho基因在不同AD小鼠模型中的表达变化规律；通过分离纯化制备川芎苯酞类化合物及体外活性筛选，发现新型苯酞类Klotho增强剂；再结合基因过表达与敲减的生物技术，研究Klotho基因与其增强剂藁本内酯（LIG）对AD病程的干预作用及其机制。本研究发现：(1) 在散发性AD小鼠（SAMP8小鼠）和家族性AD小鼠（APP/PS1小鼠），Klotho基因的表达与AD病变呈显著负相关；(2) LIG等苯酞类化合物可在体外与体内水平显著上调Klotho表达水平；(3) Klotho过表达慢病毒侧脑室注射或LIG干预可明显减轻APP/PS1小鼠和SAMP8小鼠的认知功能障碍及神经血管病变。Klotho基因及其增强剂LIG对AD的神经保护作用机制主要包括：①增强APP的非淀粉样降解途径，同时调节脑内自噬与转运体介导的Aβ清除功能，降低AD小鼠脑内Aβ的异常累积；②抑制IGF-1/Akt通路，介导FoxO1转录活性及其下游Mn-SOD等靶基因的表达，减轻生物大分子（DNA、蛋白及脂质）的氧化损伤；③抑制RIG-1的表达及多聚化，抑制NF-κB转录活性及其下游IL-6等靶基因的表达，发挥抗神经炎症效应。. 综上所述，本研究发现Klotho基因表达进行性减少参与了慢性AD发生发展过程，Klotho基因本身或其小分子增强剂可通过改善神经血管单元结构与功能发挥神经保护作用，为AD防治提供了新的思路与策略。在该自然基金资助下，本项目已发表SCI论文6篇，授权发明专利1项，培养毕业博士生2名、硕士生1名。