鹿茸蛋白提取物口服保护帕金森病神经损伤的蛋白/多肽活性成分研究

This study was to explore the active ingredient of Pilose antler extractions protected nerve injury on Parkinson's disease after oral administration. Parkinson's disease is the focus and difficulty of the nervous system disease research, current treatments can not slow disease progression and have serious side effects. Pilose antler is a precious Chinese medicine herbs which can periodically regenerate. Pilose antler contains protein component of up to 55.26% which regulate nervous system by local injection or in vitro experiments. The pharmacological effects are related to the physiological characteristics of Pilose antler. But due to the limitations of the experimental method, up to now, there have no clear active ingredients was discovered, which resulting in the value of Pilose antler is difficult to be exploited. Our previous studies set up a protein tracing method solved the thorny issue for medical researchers whether proteins from animal drugs used in Traditional Chinese Medicine could be absorbed into bloodstream after oral administration. We extracted a group of proteins from Pilose antler (PAE) which had a good therapeutic effect on Parkinson's disease and we proved the Pilose antler protein could be absorbed into bloodstream after oral administration, but the protein components in promoting regeneration of nervous system need for further experimental validation. On the basis of the work above, in this project, we will first use protein tracing method, to determine which components could be detected in the rat cerebrospinal fluid after oral administered. With the information provided on the the antler protein library, clinical information and comprehensive analysis of the modern Pharmacology, the absorbed protein from Pilose Antler were selected for the further research of the activity to clarify the following issues:1.Whether the PAE can blood brain barrier? 2. What protein / polypeptide are the similarities and differences be absorbed in physiological and pathological state ? 3.Are the protein/polypeptides same in serum/cerebrospinal fluid and component having pharmacological activity consistent? This studies illustrate the material basis of PAE on protection of nerve injury in Parkinson’s disease after oral administration and provide a new method for developing an oral formulation of PAE, lay a foundation for finding a safe and effective drugs for Parkinson’s disease.

帕金森病是国内外神经系统疾病研究的重点和难点，现有治疗不能延缓病情进展且有严重副作用。研究表明体外实验或局部注射鹿茸提取物可以修复神经系统损伤，但与传统口服给药方式不符。课题组前期从鹿茸中提取了一组蛋白，口服给药对帕金森病动物模型有很好的治疗作用，并证明了其可在生理状态下以蛋白/多肽形式吸收入血。但该组分是否可以通过血脑屏障？在病理状态下被机体吸收的物质与生理状态有何异同？血清和或脑脊液中的蛋白/多肽物质与具有药理活性的成分是否一致？均需要进一步实验证实。本课题首先利用已建立的中药大分子活性示踪法确定口服鹿茸蛋白成分能否进入脑脊液，同时结合蛋白质组学方法对血清和脑脊液中的蛋白/多肽成分进行分析，确定蛋白种类，并用细胞与线虫模型分析其有效成分，最终阐明鹿茸蛋白提取物口服后保护帕金森疾病神经损伤的物质基础，为鹿茸蛋白口服制剂的开发提供新方法，为寻找治疗帕金森疾病的安全有效药物奠定基础。

帕金森病（PD）是国内、外神经学科研究的重点和难点。目前主要治疗手段是药物干预和手术治疗，但难以达到有效根治的目的，因此寻找在疾病早期能够逆转神经元损伤、促进神经元修复的活性成分是各国医药研究者研究PD的主要目标，也是本课题探讨的关键问题。鹿茸是我国传统的珍贵中药材，现代药理学研究表明体外实验或局部注射鹿茸提取物可以修复神经系统损伤，但与传统口服给药方式不符。课题组前期从鹿茸中提取了一组蛋白PAEs，灌胃给药对PD大鼠模型具有神经保护作用，并证明了其可在生理状态下以蛋白/多肽形式吸收入血，但具体发挥神经保护作用的物质基础尚不明确。本研究旨在寻找PAEs中发挥神经系统保护作用的物质基础。.1.PAEs成分的鉴定和分析：（1）建立鹿茸Denovo转录组蛋白库；（2）测定PAEs中蛋白含量为53.16%；（3）通过SDS-PAGE对PAEs进行分离。.2.生理状态与病理状态下口服PAEs吸收入血和脑脊液的蛋白/多肽类物质的差异：（1）PAEs经过人工肠液的分解后仍有部分蛋白能通过肠系膜；（2）LSCM联合FCS检测表明末次给药2h后为最佳采集时间点；（3）病理状态下PAEs在50 kDa附近与在生理状态下吸收较为一致，而其他分子量大小的蛋白条带均存在明显差异，比较不同状态PAEs吸收入脑脊液的情况，结果提示PD模型大鼠吸收PAEs分子量较大；（4）与模型组相比，血清中49种差异蛋白来源于PAEs，其中与神经生长相关的蛋白有11个；脑脊液中47种差异蛋白来源于PAEs，其中与神经生长相关的蛋白有8个。 .3.PAEs中具有神经保护作用的蛋白成分：（1）利用AKTA AVANT快速蛋白分离系统联合阴、阳离子柱及分子筛共筛选出6段分段蛋白；（2）阴、阳离子结合性的大分子量的蛋白促PC12细胞增殖和诱导分化的作用最为显著；（3）阴、阳离子结合性的大分子量蛋白恢复A30P线虫运动最为显著，阳离子柱结合①段恢复A30P线虫树突最为显著；（4）通过免疫荧光与 Western blot检测 NF-H的表达，阴离子柱结合组①段且中浓度组表达最显著；（5）对最有效蛋白段质谱鉴定，其中与神经生长、及其保护机制相关的蛋白有5个。