基于Nrf2/ARE信号通路人参皂苷CK靶向调节Aβ沉积耦联氧化应激防治阿尔茨海默病的分子机制研究

Alzheimer's disease (AD) is a neurodegenerative disorder with memory loss and cognitive impairment as the main symptoms. According to traditional Chinese medicine, toxin damaging brain collaterals is an important pathological mechanism of AD and ginseng is traditionally used to promote intelligence. Our previous work showed that ginsenoside Compound K (CK), a metabolite of ginsenoside, can scavenge free radicals in vitro, to improve memory, inhibit neuronal apoptosis, decrease deposition of Amyloid β (Aβ) and protect damaged neurons in AD animal models. We hypothesise that ginsenoside CK alleviates cognitive dysfunction in AD by inhibiting Aβ deposition in neurons through regulating Nrf2/AREdependent oxidative stress switch. Enzymatic reaction kinetics, a primary hippocampal neuronal damage model and an APP/PS1 transgenic animal model will be designed to reveal the modern scientific connotation of toxin damaging brain collaterals of AD at molecular, cellular and animal levels. The mechanisms will be further clarified underlying ginsenoside CK’s efficacy through inhibiting generation and aggregation of oligomer Aβ, protecting damaged neurons, and improving cognitive dysfunction in AD by targeted regulation of Nrf2/ARE signaling. It will also shed light into the molecular mechanisms of ginseng`s traditional efficacy of promoting intelligence and provide a theoretical basis for clinical use of ginseng in the prevention and treatment of Alzheimer's disease.

阿尔茨海默病（Alzheimer's disease，AD）是一种以记忆和认知功能障碍为主要症状的神经退行性疾病，现代医学认为Aβ异常聚集是AD主要发病机制。基于人参益智传统功效及前期工作发现人参皂苷代谢产物CK能清除体外自由基，靶向与Aβ配体亲和并促进Aβ寡聚体解聚，改善AD模型动物的记忆障碍。故提出假设：人参皂苷CK能否通过靶向调节“氧化应激开关” Nrf2/ARE信号通路减少Aβ生成、防止Aβ寡聚体异常聚集。本项目拟用体外筛选评价体系、原代海马神经元损伤细胞模型以及转基因AD动物模型，以Aβ寡聚体的形成和清除为切入点，应用分子模拟、酶反应动力学实验、ThT荧光等分子生物学技术，观察CK靶向调控Aβ耦联神经元氧化损伤的分子机制。初步明确CK可能通过调节Nrf2/ARE信号通路，抑制寡聚体Aβ生成、聚集及清除，改善认知功能障碍，为人参防治AD的临床治疗和防治提供理论依据。

本研究从整体、细胞、分子水平逐层深入，以Aβ寡聚体的形成和清除为切入点，初步明确CK通过激活Nrf2/ARE信号通路以及促进神经元能量代谢，保护受损神经元，改善认知功能障碍。. 分子水平研究人参皂苷CK靶向抑制Aβ寡聚体的效能。利用分子模拟，结合酶反应动力学OpenSPR技术实验，分析人参皂苷CK对Aβ靶向结合效能；并通过ThT，SDS-PAGE电泳实验，检测人参皂苷CK对Aβ寡聚体靶向解聚的能力，透射电镜进一步验证CK抑制Aβ寡集体的解聚能力。细胞水平分别建立AD神经元损伤模型，通过RCT和MTT法证明人参皂苷CK促进神经元增殖和细胞活力，抑制神经元凋亡；应用WB、IHC等分生技术检测人参皂苷CK抑制神经元细胞内Aβ定位表达和聚集；并通过激活Nrf2/ARE信号通路，证明人参皂苷CK能够激活神经元细胞内的氧化应激信号通路进而抑制Aβ聚集保护受损神经元的分子机制。动物水平应用AD模型小鼠，采用MORRIS水迷宫实验阐明人参皂苷CK改善AD小鼠空间探索能力，并采用WB、IHC和电镜技术检测人参皂苷CK抑制AD小鼠脑内Aβ的表达和聚集；应用基因芯片和微生物菌群联合生信分析，其分子机制与调节Nrf2/ARE信号通路和增加神经元细胞能量代谢，激活PPARγ及下游能量代谢途径相关蛋白IRS2、IDE、GLUT1和GLUT3的表达，增加神经元的ATP含量进而减轻由Aβ引起的神经元损伤，赋予人参“大补元气、益智抗衰”的现代科学内涵，为研究人参皂苷CK防治AD获得了更为科学详实的实验资料。