基于自噬-溶酶体通路的土丁桂中潜在抗阿尔茨海默症树脂糖苷类成分的发现及其作用机制研究

The dysfunction of autophagy-lysosome pathway plays a crucial role in the pathological process of Alzheimer’s disease (AD), and has become a research focus of anti-AD drugs. Evolvulus alsinoides, a traditional medicine used for brain tonic, possesses the anti-AD potential. A novel kind of resin glycoside was isolated from Evolvulus alsinoides in our previous research, which could act on the autophagy-lysosome pathway, inhibit Aβ42 production, and thus possess the potential anti-AD effect. Based on these backgrounds, we plan to effectively isolate these novel resin glycosides from Evolvulus alsinoides. The isolates were then evaluated for their effects on autophagy and lysosome using the GFP-LC3 puncta assays and DQ-Red BSA staining assays, respectively. Molecular biology techniques, such as immunofluorescence technique, Western blot test, RT-PCR test and so on, were further applied for mechanism study. Finally, several in vitro and in vivo anti-AD tests were performed to verify the therapeutic effects of isolated compounds. The ultimate results not only provide theoretical guidance for the comprehensive utilization of Evolvulus alsinoides, but also provide novel lead compounds for the discovery of anti-AD drugs and pave the way to develop new drugs with intellectual property rights.

自噬-溶酶体通路功能紊乱在阿尔茨海默症（Alzheimer’s disease，AD）的病理进程中起着至关重要的作用，为抗AD药物研发的热门靶点。土丁桂为益智健脑的传统草药，具有抗AD潜能。申请人前期研究发现，土丁桂中含有一类新颖的树脂糖苷类成分，能够作用于自噬-溶酶体通路，并抑制Aβ42的产生，具有潜在的抗AD作用。因此，本项目拟在前期工作基础上，以土丁桂为研究对象，定向分离鉴定其富含的新颖树脂糖苷类成分；通过自噬小体形成及DQ-Red BSA染色实验，筛选具有自噬-溶酶体通路调节作用的单体化合物；利用荧光显色、Western blot及RT-PCR等方法对活性单体进行作用机制研究；最后，利用多种体内外模型，验证活性单体的抗AD潜能。本项目的研究成果不仅为土丁桂药材资源的综合利用提供理论指导，而且为抗AD药物研发提供了新颖的活性先导化合物，为开发我国具有自主知识产权的新药奠定良好的基础。

阿尔茨海默症（AD）是一种严重的中枢神经系统退行性疾病。自噬-溶酶体功能异常与AD的多种病理现象之间具有密切的联系,是AD治疗药物研发的热门靶点。旋花科植物在世界各地的民间草药中多有应用，尤其用于中枢神经系统疾病，但其物质基础并不明确。本项目采用多种色谱手段，对旋花科药用植物土丁桂及田旋花的树脂糖苷部位进行了系统的化学成分分离，共获得18个树脂糖苷类化合物，均为新化合物。其中，从土丁桂中分离得到3个糖苷酸类化合物Evolvulic acids A~C和3个大环树脂糖苷类化合物Evolvulins I-III，为首次发现的以三羟基烷酸为苷元的树脂糖苷类化合物；从田旋花中分离得到12个糖苷酸类化合物Arvensic acids A~L，包括多个首次发现的以11S-羟基十七烷酸为苷元的树脂糖苷类化合物。活性研究表明，化合物Evolvulins I和II具有潜在的抗AD及抗肿瘤作用，而其活性可能与自噬-溶酶体通路有关。以上研究为旋花科药用植物的开发利用提供了参考，同时为开发抗AD及抗肿瘤药物研发提供了新的模板分子。