基于脑保护的藏药蔓菁抗高原缺氧药效物质基础与作用机制研究

Brain edema is the late period of acute mountain sickness (AMS), and high expression of hypoxia inducing factor (HIF-1) is the first measures of prevention and treatment of AMS. In the prophase study, Brassica rapa can effectively prevent and treat cerebral white matter edema and functional injuries caused by low pressure oxygen. Based on the key link in the process of the project for AMS brain damage, the hypothesis, puts forward that "multiple targets regulated HIF – 1 signaling pathway is an important mechanism of brain protection, and it’s efficacy material base may be related to the compounds of polysaccharide, thioglycoside, flavonoid , etc. " To isolate and purificate of chemical parts of polysaccharide, thioglycoside and flavonoid, and then apply oxygen damage brain cells model and simulated plateau hypoxia rats model, the cell morphology scanning electron microscope (SEM) is used to analysis. RT-PCR, Western bloting techniques detect HIF - 1, VEGF, EPO and other related cytokines. Using GC/MS，the metabonomics of plateau hypoxia rats model is to study. And through the principal component analysis (PCA), and other statistical methods, the effect and chemical information correlation are analyzed. With multidisciplinary methods, the material base and its mechanism of action of brain protection of Brassica rapa are explored and it puts further scientific evidence for the prevention and treatment of AMS.

脑水肿是急性高原病（AMS）的晚期症状，抑制缺氧诱导因子（HIF-1）的高表达是防治AMS的首要措施。前期研究发现藏药蔓菁可以有效预防和治疗低压缺氧导致的脑白质水肿及功能损伤。本项目针对 AMS脑损伤的关键环节，提出“多靶点调控HIF-1信号通路是藏药蔓菁脑保护的的重要机制，其药效物质基础可能与蔓菁多糖、硫苷等多成分有关”的假说，分离纯化蔓菁多糖、硫苷、黄酮等化学部位，应用缺氧损伤脑细胞模型、模拟高原缺氧大鼠模型，结合细胞形态扫描电镜（SEM）分析，RT-PCR、Western bloting等技术检测HIF-1、VEGF、EPO等相关细胞因子，结合GC-MS等现代分析手段进行代谢组学研究，通过主成分分析（PCA）等统计方法分析药效与化学信息相关性，多学科交叉，探索藏药蔓菁脑保护的药效物质基础及其作用机制，进一步为防治AMS提供科学证据。

本项目从资源-质量-药效-机制等4个层次研究了藏药蔓菁，开展了蔓菁药材资源的调查，采用DNA条形码技术对55份蔓菁样本进行了研究，结果表明蔓菁序列聚为一只，表现出良好的单一性，种内存在1个变异位点。证实了ITS2序列作为蔓菁药材DNA 条形码具有很好的稳定性和准确性。建立了蔓菁药材的质量标准，并采用PMP柱前衍生-HPLC法建立了蔓菁药材中葡萄糖的含量测定及其单糖特征图谱分析方法，13批蔓菁药材中葡萄糖的含量为10.32%~21.43%；特征图谱6个共有峰稳定重现。采用急性毒性和亚急性毒性初步评价蔓菁膏的安全性；采用小鼠常压耐缺氧、小鼠急性脑缺血性缺氧、小鼠亚硝酸钠中毒模型研究蔓菁膏的抗缺氧作用，结果表明小鼠经口给予蔓菁膏的LD50为56.73 g·kg-1，Dm为86.80 g ·kg-1·d-1，Dn为35.55 g·kg-1·d-1；蔓菁膏具有提高缺氧耐受力的作用，确定有效剂量为0.388 g·kg-1·d-1；开展了急性高原缺氧脑损伤模型及蔓菁多糖的分离纯化研究，测定了蔓菁多糖的单糖组成，由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖等7种单糖组成，其摩尔比为0.26:0.54:0.05:4.17:0.84:1:1.06。研究了蔓菁多糖抗高原缺氧脑损伤的保护作用，研究了蔓菁多糖抗高原缺氧脑损伤的保护作用，结果表明蔓菁多糖能减轻低压缺氧引起的脑损伤，与低压缺氧模型组相比，其他给药组均能改善小鼠脑记忆功能，蔓菁多糖高剂量组脑组织SOD活性升高，MDA活性及HIF-1α、VEGF、EPO蛋白表达降低，表明蔓菁多糖能通过调控HIF-1α信号通路改善低压缺氧引起的脑损伤。开展了蔓菁多糖对CCl4致急性肝损伤的保护作用，结果表明蔓菁多糖能缓解肝脏病变，与模型组相比，蔓菁多糖中、高剂量组肝组织SOD、GSH-Px活性升高，MDA、TNF-α、IL-1β和IL-6级血清ALT、AST、ALP含量均降低，且降低了肝组织caspase3蛋白的表达。研究成果发表论文5篇，其中4篇为核心期刊；正在申请专利1项，获得中国民族医药学会科学技术奖一等奖。