利用人源化AD线虫病理模型筛选两种甘松属植物中抗AD活性倍半萜类成分及分子作用机制研究

In recent years, it has been found that the prevention and elimination of Abeta toxic peptide is considered to be one of highly effective methods for the prevention and treatment Alzheimer's diseas (AD). In our previous work, we found that two species of Nardostachys showed anti-AD activity, and twenty-four sesquiterpenoids possessing various skeleton structures have been isolated from them, of which three compounds have significant efficacy for treating humanized AD Caenorhabditis elegans transferred with human Abeta1-42 gene. In this project, we will isolate and identify more sesquiterpene compounds from the two Nardostachys plants, and then use humanized AD worms to screen the sesquiterpene compounds with anti-AD activity, validate their anti-AD activities in PC12 cell line and APP/PS1 double transgenic mice. Based on the theory of drug molecular design and the analysis of structure-activity relationships, novel sesquiterpene derivatives with high anti-AD activity will be synthesized. Finally, the effects of those sesquiterpenoids and their derivatives on the expressions of Abeta and its deposition, and the expressions of AchE and inflammatory like cytokines, will be also investigated. After knocking down the key components of three stress related molecular signaling pathways in worms by RNA interference, the anti-AD effects of the tested compounds are evaluated, further, whether the tested compounds can activate these key components and induce the expression of their downstream responsive genes are investigated. Molecular mechanisms for anti-AD of sesquiterpenoids and their derivatives obtained in present work will be illuminated. The results of this project will provide high anti-AD activity of sesquiterpenoid lead compounds, and provide a theoretical basis for further anti-AD drug development in the future.

近年来研究发现阻止甚至清除Aβ毒性肽被认为是防治AD的一种有效方法。课题组前期研究发现两种甘松属植物具有抗AD活性，从中分离得到多种骨架倍半萜24个，其中3个化合物对转人Aβ42的人源化AD线虫具有显著治疗作用。本项目拟继续对上述植物中倍半萜进行分离鉴定和富集，采用AD线虫筛选其中的抗AD活性倍半萜，以PC12细胞和APP/PS1双转基因小鼠进行抗AD活性验证。利用药物分子设计理论并结合构效关系，合成结构新颖高抗AD活性的倍半萜衍生物。最后，本项目还将探讨它们对Aβ表达和沉积、AchE和类炎症因子表达的影响。采用RNA干扰敲低机体抗胁迫效应的三条相关分子信号通路的关键组分，观察受试化合物抗AD作用，并考察受试化合物是否激活这些关键组分和诱导下游响应基因的表达，阐明所获倍半萜及其衍生物的抗AD分子作用机制。本项目研究结果将提供高抗AD活性的倍半萜先导化合物，并为进一步的药物研发提供理论依据。

阿尔茨海默症(AD)是一种严重影响老年人社交、工作与生活，给家庭和社会带来沉重负担的疾病。如何安全而有效地阻止甚至清除Aβ毒性肽被认为是预防和治疗AD的一种有效的方法。秀丽隐杆线虫成本低，易于饲养，模型稳定，其与人类保守性高，具有AD发病的同源基因，是很好的AD药物体内筛选模型。本项目通过多种色谱学分离手段对甘松(Nardostachys chinensis)和匙叶甘松(Nardostachys jatamansi)中的化学成分进行分离，并利用理化性质、现代波谱学以及化学等手段鉴定了它们的结构，共分离鉴定113个，其中包含28个新化合物，含3个新骨架化合物。利用秀丽隐杆线虫AD病理模型，系统筛选了所分离获得化合物的抗AD活性，结果发现部分化合物能显著延缓AD线虫的麻痹，提示这些化合物在一定程度上具有预防或治疗AD的潜在活性。进一步，对活性好且量大的化合物Ethyl caffeate和Linarin开展了抗AD作用机制研究。结果表明，Ethyl caffeate能够缓解AD线虫Aβ过度表达诱导的麻痹症状；外源性5-羟色胺实验说明Ethyl caffeate能够缓解Aβ过表达诱导的神经毒性。此外，ThS染色实验证明，Ethyl caffeate能够抑制Aβ沉积的形成。通过qRT-PCR实验进一步研究发现，Ethyl caffeate上调了daf-16、skn-1、hsf-1、sod-3、gst-4和hsp-16.2基因的表达，说明Ethyl caffeate所介导的Aβ毒性的降低可能是通过IIS信号通路实现的。另外，Linarin能够显著延迟AD线虫瘫痪的发作，进一步研究发现，其延迟瘫痪症状与其抑制Aβ沉积斑的形成和降低氧化应激水平有关。进一步研究发现Linarin降低Aβ诱导的毒性可能与其激活sod-3和gst-4的表达有关。本项目的研究丰富了甘松属植物中萜类化学成分的结构多样性，同时为新型抗AD药物的研发提供了新结构类型的先导化合物，对推动新型抗AD药物的研发具有重要意义。本研究发表相关SCI研究论文14篇。