从泛素-蛋白酶体途径研究当归芍药散治疗阿尔茨海默病的生物学机制

To investigate the biological foundation of effective ancient formulas is essential for the inheritance and development of traditional Chinese medicine. Dang Gui Shao Yao San is Zhang Zhong Jing's classic formula that was originally designed to treat women diseases. And interestingly since the late 1980s, it has been widely used in the treatment of Alzheimer's disease(AD) with significant therapeutic effects. However, its biological mechanism has been unclear yet. In recent years, the ubiquitin-proteasome pathway(UPP) has been proven to play important roles in the occurrence and development of AD and it has become a new and effective medicine target for AD treatment. Hereby, from the perspective of protein degradation, we propose the hypothesis that Dang Gui Shao Yao San's efficacy of treating AD is associated with its capacity of regulating UPP. In our study, we will conduct the experiment in vitro (Aβ1-40 induced AD cell model) and in vivo (senescence- accelerated mouse-prone 8) and apply such techniques as real time quantified PCR and Western blot to reveal the changes of important UPP indexes including Ub, E3,26S, UCH-L1, UCH-L3 and UCH-L5/UCH37 so as to reveal the rules and features of Dang Gui Shao Yao San's effect on AD in the whole, cellular and molecular levels. The results of this project will provide a scientific approach and evidence to understand the biological mechanism of Dang Gui Shao Yao San's effect on AD.

确有疗效中医古方防治疾病的生物学基础研究是中医传承和发展的关键科学问题。当归芍药散是张仲景的经典有效古方，本为妇人之疾而设，至上个世纪八十年代末以来被广泛用于治疗阿尔茨海默病，临床疗效确切，但其现代生物学基础尚不明。研究发现泛素-蛋白酶体途径在阿尔茨海默病的发生发展进程中扮演了极其重要的角色，药物可通过调控泛素-蛋白酶体途径的功能治疗阿尔茨海默病。基于此，本研究拟从蛋白质降解角度，提出“当归芍药散可通过调节泛素-蛋白酶体途径发挥抗阿尔茨海默病作用”的假说，以动物实验（SAM/P8快速老化小鼠）和细胞实验（Aβ1-40诱导AD细胞模型）相结合，采用RT-PCR、Western blot等技术检测Ub、E3、26S、UCH-L1、UCH-L3和UCH-L5/UCH37等UPP的重要指标，以期在整体、细胞和分子水平揭示当归芍药散的作用规律和特点，提示当归芍药散治疗阿尔茨海默病的生物学机制。

阿尔茨海默病（Alzheimer’s Disease, AD）是一种起病隐匿的进行性发展的神经系统退行性疾病。其特征性病理改变包括神经元凋亡、细胞外β-淀粉样肽（amyloid beta, Aβ）聚集形成老年斑和细胞内异常磷酸化Tau蛋白聚集形成神经原纤维缠结。泛素-蛋白酶体途径（Ubiquitin Proteasome Pathway, UPP）是细胞内蛋白质降解的主要途径，约有85%以上的蛋白质通过此途径进行降解，AD患者UPP功能异常程度与AD脑内高度磷酸化的Tau蛋白和Aβ的积聚浓度呈明显相关性。AD 属于中医“好忘、喜忘、呆、健忘”等病范畴，肝郁脾虚、血瘀痰浊是AD发生发展的重要原因。当归芍药散出自汉·张仲景所著《金匮要略》：“妇人怀妊，腹中㽲痛，当归芍药散主之”、“妇人腹中诸疾痛，当归芍药散主之。”本为疗妇人之疾而设，但至上个世纪八十年代末以来逐渐被广泛用于治疗阿尔茨海默病，肝脾同调、血水同治，疗效确切，然而其发挥疗效的生物学机制却不甚明了。本研究以UPP为靶点，以SAMP8快速老化小鼠和Aβ1-40诱导的AD细胞模型为受试对象，从肝郁脾虚、血瘀痰浊的核心病机入手，结合血清药理学，采用当归芍药散在体内、外干预的方法，探讨当归芍药散治疗AD的生物学机制。体内实验研究结果显示当归芍药散可明显提高SAMP8早老化小鼠学习记忆功能、改善认知障碍，改善海马区CA1神经元病理变化，降低Aβ和Tau蛋白表达水平；不同程度降低Ub的表达水平，升高26S、E3-Parkin、UCHL1、UCHL3和UCHL5表达水平。体外实验结果显示当归芍药散含药血清干预后，与模型组比较，DSS 含药血清低、中、高剂量组细胞活率不同程度上升，Aβ和Tau表达明显下降，Ub表达水平明显下调，E3、26S、UCHL1、UCHL3和UCHL5表达水平不同程度增加。本研究通过体外、体内实验初步证实调控UPP途径是当归芍药散治疗AD的作用机制之一。