柴达木大肥菇抗缺氧多糖的功能评价及其作用机制

Qaidam Agaricus bitorquis (Quél.) Sacc. is a special mushroom that used to to consume as as folk nutrients and diet possessing anti-fatigue and anti-hypoxic activity in high altitude Qinghai-Tibetan Plateau. Previous research showed that Qaidam Agaricus bitorquis (Quél.) Sacc fruit body possessed high content of polysaccharides, and by means of animal test, which revealing high potential of anti-hypoxic activity. The objective of this project is focused on the polysaccharides of Qaidam Agaricus bitorquis (Quél.), using a combination of purification techniques to preparing monomer-polysacchride, and representative components in turnips to improve the anti-hypoxic ability and compensatory ability of the body against acute hypoxia. We investigate its effect on oxygen-sensitive potassium channel of excitable cells, its protective effect on hypoxic PC-12 and its regulatory mechanism on expression of hypoxia-inducible factor 1 gene and other downstream genes. This research intends to systematically investigate the anti-hypoxia functional polysaccharides and its regulatory mechanism at cell level, the gene level and the whole animal level, respectively, and finally provide the theoretical foundation for development of functional polysaccharides food from macromushroom. In conclusion, this research provides the theoretical basis for functional foods development for mushroom fungus in Qinghai-Tibetan Plateau.

柴达木大肥菇是从青藏高原特殊生境中发现的一种具有抗缺氧、抗疲劳等功能的野生食用真菌。课题组前期研究发现，柴达木大肥菇中存在高量的多糖成分，且经动物实验证实其子实体多糖有显著的抗缺氧作用。本项目拟以柴达木大肥菇活性多糖为研究对象，采用组合分离技术制备多糖单体，以原代培养肺动脉平滑肌细胞、肺神经上皮小体细胞和颈动脉体Ⅰ型细胞构建低氧诱导模型，通过检测乳酸脱氢酶（LDH）含量及CCK-8 法分析流式细胞的凋亡；通过检测Ca2+、多巴胺、乙酰胆碱、 5-羟色胺、内皮素-1及NADPH 氧化酶含量，重点研究其对可兴奋细胞的氧敏感钾离子通道的作用；借助 WB分子杂交技术分析 神经元PC-12 细胞中低氧诱导因子（HIF-1α）、下游促红细胞生成素 (EPO)、血管内皮生长因子 (VEGF)及一氧化氮合酶（NOS）等蛋白表达的调控作用；通过大鼠缺氧模型评价其功效成分对机体急性缺氧代偿的影响。

以柴达木大肥菇子实体、发酵液及菌丝体为原料提取、纯化活性多糖，采用抗缺氧、耐疲劳两种动物模型，比较三种源纯化多糖对缺氧损伤细胞的保护差异，结果表明菌丝体纯化多糖抗缺氧活性最优，能明显提高小鼠抗缺氧耐力；制备菌丝体纯化多糖的三种不同分子量单体，通过细胞模型筛选抗缺氧活性最高的单体为（polysaccharides from mycelium，PM-II）；采用紫外光谱、傅里叶变换红外光谱、核磁共振波谱、质谱等联用解析化学结构，结合两次解谱结果表明：此多糖单体由α-D-吡喃甘露糖，吡喃阿拉伯糖，吡喃果糖单糖，通过1,4糖苷键链接，支链通过1,6-糖苷键链接，含有硫酸酯键和糖酸结构。对PM-II多糖单体进行急性缺氧代偿能力和抗缺氧作用机制研究发现：在缺氧条件下，柴达木大肥菇多糖可以有效降低细胞内Ca2+浓度，使Ca2+外流，通过减少细胞内Ca2+浓度来抑制缺氧诱导的细胞凋亡，增加细胞中的钾电流，上调肺动脉平滑肌细胞中KV1.5蛋白的相对表达量，下调KIR6.2蛋白的相对表达量，疏通因缺氧刺激而受抑制的K+通道；提高细胞中的5-羟色胺、多巴胺、乙酰胆碱、内皮素表达量，降低NADPH氧化酶和LDH酶活性，增强肺通气、肺循环和心血管活动，强化机体的代偿反应；还可以降低缺氧条件下PHD-1、FIH蛋白相对表达量，降低HIF-1α蛋白羟基化反应，抑制了HIF-1α蛋白降解，增加了HIF-1α蛋白的稳定性，促进了下游靶基因一氧化氮合酶（NOS）和乳酸脱氢酶（LDH）等合成，增加机体红细胞及血管的生成、增强糖酵解及葡萄糖转运，机体产生低氧耐受。柴达木大肥菇多糖单体的抗缺氧活性与其组成的单糖α-D-吡喃甘露糖、吡喃阿拉伯糖、吡喃果糖等糖代谢途径有关。