基于蛋白质组学和超滤质谱技术研究刺五加治疗帕金森病的作用机制及化学物质基础

Parkinson's disease (PD) is a common neurodegenerative disease, and its seriousness is increasing because of the high incidence and irreversibility. Although numerous synthetic medicne have been 
approved for clinical use in the treatment of PD, most of them can lead to serious side effects. Hence, Chinese herbal medicine have attracted considerable interest for the treatment of PD. Recent studies provide evidence that Acanthopanax Senticosus offer beneficial effects on preventing PD, managing symptoms and reducing the progression of diseases. However, the molecular mechanism of action and the active compounds in Acanthopanax Senticosus for the treatment of PD have not been elucidated. In this study, we propose to determine the molecular mechanism underlying its mode of action, and screen active compounds in Acanthopanax Senticosus based on proteomic analysis and ultrafiltration LC-MS. Firstly, we prepare different effective fractions in Acanthopanax Senticosus and evaluate their inhibitory effects on LPS-induced NO production in the immortalized murine BV-2 microglial cells. Secondly, we select the PD models to investigate the effects of Acanthopanax Senticosus on overall protein changes using deuterium isobaric amine reactive tag (DiART) labeling and followed by liquid chromatography-mass spectrometry (LC-MS) analysis. In addition, we predict signal transduction pathways and identify multiple molecular targets affected by Acanthopanax Senticosus using bioinformatic analysis, Finally, we apply ultrafiltration liquid chromatography-mass spectrometry assay to screen active compounds in Acanthopanax Senticosus. Our study offers important information on the understanding the mechanism of action and active compounds in Acanthopanax Senticosus for the treatment of PD.

帕金森病(PD)是一种严重威胁人类健康的神经退行性疾病，近年来，利用传统中药治疗该病引起科学工作者的广泛关注，但对其作用机制和化学物质基础的研究还十分薄弱。本项目以系统生物学的整体研究思路为指导，利用蛋白质组学及超滤质谱技术，结合生物信息学分析方法,以中药刺五加为研究对象，通过柱色谱技术结合体外药效学评价，获得刺五加抗PD的有效组分；应用稳定同位素标记的定量蛋白质组学方法，分析PD造模前后及刺五加有效组分干预PD模型前后表达差异的蛋白，并利用液质联用技术对其鉴定；探讨刺五加对PD模型动物信号转导通路的影响规律，筛选PD相关发病蛋白及刺五加治疗PD的关键靶点蛋白；在此基础上,采用超滤质谱技术,以筛选得到的蛋白为靶点，从刺五加活性组分中筛选治疗PD的活性化合物,并对其结构进行鉴定，通过揭示化合物作用的蛋白靶点，构建刺五加治疗PD的靶点蛋白网络，阐明中药刺五加治疗PD的作用机制及化学物质基础。

本项目旨在应用蛋白质组学和超滤质谱技术研究中药刺五加治疗帕金森病的作用机制和化学物质基础。经过3年的研究，我们完成了计划中的研究内容，达到了预期目标。此外，我们还拓展研究了不同的刺五加组分总黄酮和总皂苷含量及各组分的抗氧化活性、并对刺五加治疗帕金森病的有效组分进行了全面的质谱鉴定研究。在本项目支持下，我们发表相关学术论文4篇，申请专利3项，培养研究生4名，并参加与课题相关的国内、国际会议多次。取得的成果概述如下：我们首先应用柱色谱技术结合体外药效学评价，获得了刺五加治疗帕金森病的有效组分；其次，应用稳定同位素标记的定量蛋白质组学方法筛选并鉴定了20个与帕金森病相关发病蛋白和23个中药刺五加治疗帕金森病的关键靶点蛋白；此外，我们还构建中药刺五加治疗帕金森病的活性单体化合物的超滤质谱筛选方法，成功筛选并鉴定了9种刺五加治疗帕金森病的单体化合物；在此基础上，通过构建刺五加治疗帕金森病的药物-靶点网络，探讨刺五加治疗帕金森病的药物作用机理，阐明了刺五加治疗帕金森病的多靶点、多成分的整体作用机制及化学物质基础。本项目的研究成果对于研发治疗帕金森病的化学组成与作用机制都明确的中药新药具有重要的参考价值。