基于抑制小胶质细胞活化的石菖蒲抗阿尔茨海默病的物质基础及作用机制研究

Alzheimer's disease (AD) is a common neurodegenerative disease, which threatens the health of the elderly seriously. At present, there are no effective treatments that can totally stop or reverse neurodegenerative process of AD. The inhibition of microglia-mediated excessive activation of neuritis is an important strategy for the prevention and treatment of AD. Traditional Chinese medicine (TCM) Acorus tatarinowii is the most frequently used medicinal plant in the treatment of AD. The mainly active ingredient of Acorus tatarinowii is essential oil. However, Acorus tatarinowii essential oil has obvious toxicity. Our preliminary studies have found that the polysaccharides of Acorus tatarinowii (ATPs) have the ability of improving learning and memory AD mice model, and the inhibition of microglial activation and the activities of anti-neuroinflammation, suggesting that ATPs has anti-neuroinflammation effects and thus treatment of AD. Basis on our preliminary studies, this project intends to establish an in vitro and in vivo model to explore the active ATPs against AD. At the same time, metabolomics was used to analyze the change of metabolite caused by different polysaccharides from Acorus tatarinowii. Then, the overall efficacy of crud ATPs against AD is evaluated and the active ATPs are confirmed. The purified polysaccharides anti-Alzheimer's disease are systematic isolated by the guided of their activity. A variety of chemical reactions and spectroscopy are used to identify the structure of these purified purypolysaccharides, illuminating the material foundation of Acorus tatarinowii in treating AD. Integration of transcriptomics and metabolomics is used to further explore the inhibit mechanism of purified ATPs against excessive activation of microglia. By identifying differential metabolites and genes, the candidate pathways and targets are found. Finally, these targets and pathways are verified by molecular biology methods, thus clarified the mechanisms of Acorus tatarinowii polysaccharides in treating AD, laying a foundation for their development as a new anti-Alzheimer drug with high efficiency and low toxicity.

阿尔茨海默病（AD）是一种最常见的神经退行性疾病，目前尚无有效治疗方法，抑制小胶质细胞过度活化所介导的神经炎是AD防治的重要策略。石菖蒲是中医治疗AD使用频率最高的中药，而人们前期关注的石菖蒲挥发油类成分存在明显毒性，长期服用副作用大。我们前期研究发现，石菖蒲多糖具有改善小鼠学习记忆障碍、抑制小胶质细胞过度活化和抗神经炎的作用，提示石菖蒲多糖可能通过抑制小胶质细胞过度活化而发挥治疗AD的作用。本项目拟在此基础上，利用体内、体外双模型筛选石菖蒲多糖抗AD的活性部位；同时借助代谢组学分析石菖蒲多糖对代谢水平的影响，整体评价药效，确证活性部位；采用活性导向分离追踪石菖蒲抗AD的活性多糖，利用多种化学、谱学手段鉴定其结构，以阐明石菖蒲抗AD的药效物质；进一步以抑制小胶质细胞过度活化为切入点，创新性地整合转录组学与代谢组学聚焦候选通路和靶点，并以分子生物学方法进行验证，以阐明石菖蒲抗AD的作用机制。

石菖蒲 (Acorus tatarinowii Schott)是治疗阿尔兹海默症 (Alzheimer disease, AD) 的方剂中使用频率最高的一味中药。本项目通过行为学实验及代谢组学手段考察石菖蒲粗多糖部位对东莨菪碱诱导记忆获得障碍AD模型小鼠的学习记忆能力、炎症反应以及代谢紊乱的影响，明确活性部位。结果表明ATP50的治疗效果最好。对ATP50进行脱蛋白、透析和冻干，再运用DEAE-Cellulose FF和Sephadex G-75柱层析对其进行分离纯化，得到2个精多糖ATP50a和ATP50b。HPGPC 法检测结果显示ATP50a和ATP50b均为均一糖聚物，相对分子量分别为1.97 kDa和1.25 kDa。通过PMP 柱前衍生化-HPLC 法测定其单糖组成，完全甲基化-GC-MS，结合核磁数据测定其糖残基种类和比例分析各糖残基的连接位点，对ATP50a和ATP50b的结构进行鉴定。结果表明 ATP50a为阿拉伯半乳聚糖，ATP50b为葡聚糖，由t-D-Glcp、→6)-D-Glcp-(1→、→3,6)-D-Glcp-(1→组成，其对应的相对摩尔比为：1.05：1.00：1.38。对ATP50b体外抗神经炎活性进行研究，发现ATP50b能够抑制 LPS诱导的BV2 细胞中的NO、IL-6、TNF-α过度释放。基于核磁代谢组学研究石菖蒲精多糖ATP50b对AD模型小鼠的治疗作用，结果表明ATP50b可通过缓解SCO诱导所引起的AD小鼠能量代谢、氨基酸代谢和脂肪酸氧化等代谢紊乱状态来发挥保护作用。基于16S rRNA测序研究石菖蒲精多糖ATP50b对AD模型小鼠肠道菌群的影响，结果表明AD模型组小鼠的肠道微生物多样性及丰富度明显降低，给予ATP50b干预后，AD小鼠菌群结构明显改善，趋于恢复正常。以上结果表明ATP50b可显著改善AD模型小鼠的空间记忆与学习能力，减轻神经炎症反应，调节小分子代谢物和粪便菌群紊乱。本项目为阐明石菖蒲多糖的药效物质基础提供科学依据，为抗阿尔茨海默病新药研发提供先导化合物。