三七皂苷类成分激活雌激素受体调控自噬障碍抗阿尔兹海默病分子机制研究

Dysfunction of autophagy plays a vital role in the pathogenesis of Alzheimer’s disease. Promoting autophagy flux may provide a novel insight for prevention and treatment of AD. Attenuating dysfunction of autophagy via estrogen receptor-dependent regulation of AMPK/mTOR and PI3K/mTOR signaling pathways is a novel strategy for prevention and treatment of AD. Phytoestrogens are regarded as new drugs for AD because of safety. Panax notoginseng has significant effects against AD. In preliminary experiment, we found that Notoginsenoside R1 and gypenoside XVII, Panax notoginseng saponin compounds, had significant protective effects against AD via estrogen receptor α-dependent activation of PI3K signaling pathway. Moreover, gypenoside XVII could inhibit retention of autophagosome. In this respect, 5 papers were published and 1 patent was authorized. Panax notoginseng compounds might confer neuroprotective effects via estrogen receptor α-dependent inhibition of retention of autophagosome. The present study aims to investigate whether the underlying mechanisms of neuroprotective effects of Panax notoginseng compounds is dependent on attenuating dysfunction of autophagy via estrogen receptor-dependent regulation of AMPK/mTOR and PI3K/mTOR signaling pathways using AD in vivo and in vitro models. The present study may provide a novel target and a novel insight for developing new drugs for AD and theoretical basis for clinical application of Panax notoginseng

自噬障碍是阿尔兹海默病（Alzheimer’s disease，AD）发病的重要病理机制，促进自噬流可为防治AD提供一个新思路。激活雌激素受体调控AMPK/mTOR和PI3K/mTOR信号通路抑制自噬障碍是抗阿尔兹海默病的新途径。植物雌激素样药物由于其安全性成为治疗AD的新策略。三七防治AD疗效显著，我们前期研究发现三七皂苷类成分中的三七皂苷R1和七叶胆苷ⅩⅦ能够激活雌激素受体α，活化PI3K信号通路，发挥抗AD作用；并发现七叶胆苷ⅩⅦ对自噬体滞留具有抑制作用，已发表SCI论文5篇，申请专利1项。研究结果提示三七皂苷类成分可能通过激活雌激素受体α调控自噬体滞留，发挥抗AD作用。本项目拟在前期工作基础上，利用AD体内、外模型，研究三七皂苷类成分的雌激素样作用与防治AD作用的相关性及可能的调控信号通路。论证三七皂苷类成分防治AD作用可能通过非基因途径激活雌激素受体，并通过调控AMPK/mTOR和PI3K/mTOR信号通路进而影响自噬流，发挥抗AD作用，探索其防治AD的分子机制，为AD的防治寻找新的靶点及有效药物，并为三七的临床应用提供理论依据。

自噬障碍是阿尔兹海默病发病的重要病理机制，激活雌激素受体和TFEB信号通路抑制自噬障碍是抗阿尔兹海默病的新途径。我们利用AD体内、外模型，发现三七皂苷类成分包括七叶胆苷ⅩⅦ能够激活雌激素受体α和TFEB信号通路，促进自噬流。我们发现七叶胆苷XVII（GP-17）对Aβ25-35诱导PC12细胞凋亡具有显著的抑制作用，并发现其调控细胞自噬的活性。GP-17 激活雌激素受体介导的 PI3K/Akt 信号通路，抑制GSK-3β信号通路，活化Nrf2/ARE/HO-1 信号通路。我们采用阿尔茨海默病的两种经典模型- APP695瑞典型突变（APP695swe）细胞模型和APP/PS1双转基因小鼠，考察了GP-17的神经保护作用。 GP-17可显著增加Atg5蛋白表达，促进 LC3-I 向LC3-II转化，显著降低P62蛋白表达。GP-17也可显著增加pCMV-GFP-LC3质粒稳转株APP695细胞中的自噬点，促进AβPP, Aβ40, 和Aβ42的清除，抑制APP/PS1双转基因小鼠大脑皮层和海马组织Aβ斑生成，改善其学习记忆能力。我们发现GP-17促使APP695瑞典型突变（APP695swe）细胞、APP/PS1双转基因小鼠皮层和海马的TFEB从TFEB/14-3-3复合体上释放并增加TFEB的核转运，TFEB的核转位，增加LAMP-1的表达以及LAMP-1和LC3-II的共定位，增强溶酶体生物合成和功能；促进LC3-I向LC3-II转化，并增加GFP-LC3瞬转APP695细胞的LC3斑点，促进自噬流；减少APP695swe细胞和APP/PS1小鼠海马和皮层的Aβ水平，并改善APP/PS1小鼠学习记忆功能。沉默TFEB基因可显著抑制GP-17的这些神经保护作用。本研究可为三七的临床应用提供理论依据。