基于Aβ转基因线虫模型的菖蒲属抗老年痴呆活性成分的发现及作用机制研究

Aβ transgenic Caenorhabditis elegans has become a new screening platform in development of new AD drug in recent years, due to its some advantages, like short lifepan, low costs, and having more reliable results than those by in vitro screening..In our previous work, we found the EtOAc extract of Acorus tatarinowii and Acorus calamus after extracting its volatile oil exhibited significant delay of Aβ-induced paralysis, and some lignans isolated from this extract by bioactive-guided fractionation are comprised by different number of asarone unit linked with rare C8-C7' bond. A notable delay of paralysis was observed in the transgenic worms fed with isolated lignans, and these lignans also inhibit Aβ oligomerization and turn the toxic Aβ oligomer to the nontoxic Aβ monomers by western blot method. Moreover, polymers of lignans showed higher anti-Aβ activity than asarone unit, it suggested that anti-Aβ activity of this kind of lignan is due to the polymerization of asarone unit. On the basis of above research results, this project will systematically make an isolation and identification of this kind of lignans from this two Acorus plants, and investigate the effect of number and agglomeration form of asarone unit on anti-Aβ activity, and elucidate their structure-activity relationship and action mechanism of their having anti-Aβ activity. Based on above chemical and biology research, it will lay a solid foundation to elucidate clearly the material basis and action mechanism of Acorus tatarinowii and Acorus calamus to treat AD in the clinic. It will provide a basis for developing a new AD drug from traditional Chinese medicine in future.

Aβ转基因线虫因其生长周期短、成本低、且比体外筛选更为可靠等优点，已成为近年来国外发现治疗老年痴呆（AD）药物的新筛选模型。.本课题组采用该模型，发现石菖蒲和水菖蒲的去挥发油后的乙酸乙酯部位具有显著抑制Aβ诱导产生的瘫痪作用，并从石菖蒲的活性部位中发现一类由不同数量细辛醚单元通过稀有的C8-C7'聚合而成的木脂素成分具有明显抑制Aβ毒性作用，western blot结果显示其通过抑制Aβ寡聚化并且使毒性Aβ寡聚体解聚为无毒的单聚体而产生。此外，该类木脂素的抗Aβ活性要高于其细辛醚结构单元，表明聚合后有助于活性的提高。本项目将对菖蒲属这两种植物中的该类成分进行系统研究，深入探讨其聚合数量及形式对抑制Aβ活性的影响，阐明其构效关系，并对该类成分产生抗Aβ活性的作用机理展开研究。通过深入的化学和生物学研究，最终阐明益智中药石菖蒲及水菖蒲临床应用的物质基础，为从中药中寻找新的AD药物提供依据。

鉴于阿尔茨海默症（AD）发病机制复杂，与多因素有关，线虫作为已确定完整神经系统的便捷生物在体模型，越来越多的研究表明其在神经系统相关疾病药物研究中的重要性。本项目在前期建立的Aβ转基因线虫筛选平台基础上，建立了tau转基因线虫和polyQ多聚谷氨酰胺蛋白线虫筛选模型，并利用显微注射方法构建了神经元荧光标记线虫模型。上述各类线虫模型的建立为筛选药物及分子机制研究提供了保障。通过利用上述各类转基因线虫模型，我们对细辛醚及其聚合物对线虫的保护作用研究首次发现石菖蒲中挥发油部位及α-细辛醚不仅能有效抑制Aβ诱导的毒性损伤，而且对异常磷酸化tau蛋白和polyQ多聚谷氨酰胺蛋白毒性也有显著的抑制作用，维持线虫体内蛋白质内稳态平衡。在此基础上对挥发油及α-细辛醚维持蛋白稳态功能的分子机制开展研究，发现了它们能通过提高百草枯诱导的线虫氧化应激耐受能力、降低线虫体内ROS水平，促进压力应激关键转录因子的表达，激活自噬发挥作用。进一步利用RNA-seq对α-细辛醚给药处理后的CL4176线虫差异性表达的基因进行了转录组测序分析，发现α-细辛醚给药处理后有1353个差异表达基因。GO和KEGG分析结果表明，α-细辛醚维持线虫体内蛋白稳态平衡与蛋白质合成、降解以及能量代谢过程有关。此外，对细辛醚二聚体、三聚体、四聚体的研究发现，随着聚合度的增加并未明显增加保护作用，这可能与随着分子量变大影响药物吸收有关。本研究提出通过维持蛋白质内稳态平衡、多靶点开发治疗老年痴呆药物，为AD药物的开发提供了新策略。在基金资助下，已发表SCI论文10篇、已毕业硕士生5人、博士生2人。

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