从蛋白质降解途径新视角探讨补肝养髓中药楮实子防治阿尔茨海默病的分子机制

Conformational disease is a new concept of molecular medicine proposed in recent years, which has been hailed by western scholars as another leap forward in the history of the understanding of human diseases. It has opened up a new direction for the prevention of Alzheimer's disease (AD) and other major incurable diseases. The treatment measures based on the molecular mechanisms of protein conformational disease is no longer a new palliative therapy, which may have a radical significance. Alzheimer's disease (AD) is a typical neurodegenerative protein conformational disease with high incidence rate in the global. At present，there is no ideal treatment method of modern medicine for it，however, Chinese medicine has accumulated rich experiences. This project is based on the basis of many years of research on AD, combining with the latest research progress of AD about the pathology and pathogenesis on endoplasmic reticulum stress (ERS) and protein degradation in disorder. In our study, AD cases will treated with Bugan Yangsui therapy method in according of the TCM principle from the liver. The natural Chinese herbal drug Fructus Broussonetae has the efficacy of reinforcing the liver and nursing the brain. So we will use it as our research drug to detect the key molecules in ERS signaling pathway, the key functional proteins in two important cell protein degradation pathways-autophagy lysosome pathway(ALP) and the ubiquitin proteasome system(UPS), as well as the changes in neuronal apoptosis, by applying Real time quantified PCR, Western blot, Flow cytometry(FCM), Confocal laser scanning microscopy(CLSM), Transmission electron microscopy, and other techniques, combining the experiment in vitro (ERS cell model induced by tunicamycin and AD cell model induced by Aβ) with the experiment in vivo (senescence- accelerated mouse-prone 8,SAM/P8). The results of this project will explore the neuronal protective effect of Fructus Broussonetae and the targets, interpret the molecular mechanism in the genetic, molecular and cellular levels, from the angle of ERS and its associated protein degradation pathway. It can expand a new idea for the research of TCM for the prevention and treatment of AD and other major diseases following important academic and social significance.

构象病的概念是近年提出的分子医学新概念，被西方学者誉为人类在疾病认识史上的又一次飞跃，为阿尔茨海默病（AD）等 重大疑难疾病的防治开辟了新方向；基于蛋白质构象病分子机制的治疗措施不再是新的姑息疗法，而可能具有根治性的意义。AD为典型的神经变性构象病，目前现代医学治疗乏术。本项目立足多年研究基础，结合近年关于AD内质网应激（ERS）和蛋白质降解障碍的病理病机研究最新进展，拟采用补肝养髓天然中药楮实子，以离体实验（ERS细胞模型和AD细胞模型）与在体实验(快速老化小鼠)相结合，运用Realtime-PCR、Western blot、激光共聚焦成像等技术, 检测细胞内重要蛋白质降解途径（自噬溶酶体途径和泛素蛋白酶体系统）关键功能蛋白及神经细胞凋亡的变化；旨在从蛋白质降解新视角，在基因、分子和细胞水平探讨楮实子的神经元保护作用、作用靶点及其分子机制，为中医药防治AD等重大疑难疾病的研究拓展新思路。

阿尔茨海默病（AD) 是一种发病机理复杂、慢性进行性的致死性神经退行性疾病，已成为一个重大的健康和社会、经济问题。目前尚无理想治疗方法。本项目立足于中医药整体综合调治，多环节、多靶点发挥作用的特点和优势，优化筛选出有效天然中药—楮实子，以体内和体外实验相结合，运用先进科学技术，检测自噬溶酶体途径（ALP）和泛素蛋白酶体系统（UPP）重要功能蛋白以及神经细胞凋亡的变化；分析楮实子对AD模型大鼠海马组织以及离体细胞基因表达的影响。.体内动物实验结果显示，楮实子处理的小鼠学习能力和记忆能力明显提高。ERS、ALP和UPP的标记基因检测显示，楮实子可诱导海马组织中ERS / ALP / UPP相关基因表达，抑制细胞凋亡并增强其自噬，且各基因表达呈浓度依赖型变化的趋势。RNA序列分析结果显示，FB处理过小鼠的海马组织的192基因表达上调（>2.0 folds)， 1300个基因表达下调（<0.5 folds)。生物信息学分析发现，这些差异表达基因的生物学活性涉及神经活性配体受体相互作用的信号通路，包括钙信号通路、Hippo信号通路、JcAMP信号通路、cGMP-PKG信号通路等等。.体外细胞实验结果显示：楮实子可改变TM处理PC12细胞模型中的ERS / ALP / UPP相关基因表达，抑制细胞凋亡并增强其自噬。基因芯片分析结果显示，楮实子处理的细胞改变了336个基因的表达（> 2.5倍）。这些差异表达基因功能涉及细胞周期和细胞分裂、碳水化合物的运输和代谢，蛋白质周转和伴侣、细胞内转运和膜泡运输等；其涉及的信号通路包括TNF信号途径、JAK-STAT信号通路、钙信号通路和cGMP-PGK信号通路等。 .总之，楮实子可在多个基因靶点，通过ERS / ALP / UPP多条途径调节大鼠海马功能，进而调节动物脑功能状况，进而发挥神经保护的作用；本研究从ERS及其相关蛋白质降解途径的角度，在基因、分子和细胞水平初步探讨了天然中药楮实子的神经元保护作用、作用靶点及其基因分子机制，为中医药防治AD等重大疑难疾病的研究拓展了新思路，提供了一定的科学实验室依据，具有重要的学术和社会意义。