基于肠道微生物-肠-脑轴的肉苁蓉多糖和苯乙醇苷协同抗阿尔兹海默病作用及机制研究

Alzheimer’s disease is characterized by cognitive dysfunction and the pathological and therapeutic mechanisms are not fully understood. The incidence rate is increasing year by year, however there is no effective prevention and treatment for it. Cistanche deserticola, also known as Rou Cong Rong in China, which has been considered one of the best Chinese herbs to strengthen kidneys and to tonify Yang for a long time, has neuroprotective activity. The major chemical components of Cistanche deserticola are phenylethanoid glycosides and polysaccharides, and the phenylethanoid glycosides are always identified as neuroactive substances. Previous study has found that Cistanche polysaccharides also have neuroprotection and the ability of regulating gut microbiota. It is possible that different compounds of Cistanche deserticola show pharmacological effect through gut microbe-intestinal-brain axis. Therefore, the scientific hypothesis that Cistanche polysaccharides and phenylethanoid glycosides play a neuroprotective role through the "microbiome-gut-brain" axis is proposed. In order to confirm the hypothesis, in vitro fermentation of polysaccharides and phenylethanoid glycosides with gut microbiota model are used; In SAMP8 mice model, non-targeted and targeted metabolomics method will be used to find the difference of metabolites between individual and combination administration. High-throughput sequencing technology will be used to analyze the changes of gut microbiota, to find biomarkers associated with biological effects. Different pathways, including endogenous compounds changes, drug metabolism, anti-inflammatory, and immune enhancement, will be used to reveal the synergistic effect of polysaccharides and phenylethanoid glycosides. Fecal bacteria will be transplanted from SAMP8 or SAMR1 mice into pseudo germ-free mice to find the role of gut microbiota in treating disease. This research will provide a new idea for the study of polysaccharide-rich Traditional Chinese Medicine.

阿尔兹海默病是一种严重的中枢神经系统退行性疾病，发病率逐年增高且无有效的防治药物。肉苁蓉是临床常用补肾中药，有神经保护作用，化学成分主要为苯乙醇苷和多糖。课题组前期发现多糖有调节肠道微生物的作用，能促进苯乙醇苷体内吸收，由此提出了肉苁蓉多糖经由“肠道微生物-肠-脑”轴与苯乙醇苷协同发挥神经保护作用的科学假说。为证实以上假说，本项目拟利用肠道微生物体外代谢转化方法研究多糖经调节肠道微生物对苯乙醇苷代谢的影响；采用非靶向和靶向代谢组学技术，考察单独给药和联合给药SAMP8小鼠代谢物的差异，采用高通量测序技术检测肠道微生物的变化，寻找与生物效应相关的生物标志物，从改变内源性化合物、调节药物代谢、抗炎、增强免疫多个途径揭示多糖和苯乙醇苷联合使用，经由肠道微生物协同发挥神经保护的机制。最后，采用伪无菌鼠模型，验证肠道微生物是中药发挥作用的始动因素，为富含多糖的中药及其复方作用机制的研究提供新思路。

阿尔兹海默病是一种严重的中枢神经系统退行性疾病，发病率逐年增高且尚无有效的防治药物。肉苁蓉是临床常用补肾中药，苯乙醇苷类成分是抗阿尔兹海默病的主要物质之一，另外，肉苁蓉多糖也具有调节肠道菌群和神经保护的作用，但目前尚缺乏其结构和活性机制的相关报道。本课题首先对肉苁蓉多糖和苯乙醇苷类成分进行分离纯化及详细的结构表征。利用肠道菌群体外发酵模型，研究发现肠道菌群酵解肉苁蓉多糖，发酵液 pH在0-48小时内，随时间的推移而降低，分子量也逐渐降低，而发酵产物短链脂肪酸：乙酸、丙酸的浓度随时间的增加而增加，同时，肠道菌群也影响苯乙醇苷代谢。采用SD大鼠模型研究肉苁蓉多糖对肠道菌群和松果菊苷体内代谢的影响，发现肉苁蓉多糖包括粗多糖、高分子量和低分子量多糖均可促进松果菊苷的体内吸收，调节肠道菌群多样性，增加有益菌群，如普雷沃氏菌的生长，同时促进短链脂肪酸的产生。采用快速老化模型小鼠SAMP8研究肉苁蓉多糖、松果菊苷对其行为学及肠道菌群的影响。结果发现，肉苁蓉多糖单独给药及与松果菊苷联合给药均能延长SAMP8小鼠生存率，改善小鼠生存质量，且对肠道菌群紊乱具有改善和恢复作用，但Morris水迷宫实验结果显示对SAMP8小鼠学习记忆能力无显著影响。采用细胞模型和C57小鼠，研究肉苁蓉多糖的免疫调节作用。在体外，肉苁蓉多糖通过调节巨噬细胞发挥抑制肿瘤细胞的增殖和转移作用，提高巨噬细胞对肿瘤细胞的吞噬能力；在体内，肉苁蓉多糖通过增加CD8 T细胞的浸润和降低M2型巨噬细胞的比例达到抗肿瘤的效果。本研究结果为今后肉苁蓉的开发利用提供科学指导。