随着人口老龄化程度的日益严峻，衰老及其相关的神经退行性疾病已成为医药领域的研究重点。我们前期工作显示，茜草多糖不仅能够抑制阿尔茨海默症相关β-淀粉样肽（Aβ）在体外和细胞内的聚集，还能够缓解Aβ聚集对细胞和秀丽线虫整体动物的神经毒性，并能延长秀丽线虫疾病模型的寿命。因此，本项目拟利用多种转基因和突变秀丽线虫模型，深入阐释茜草多糖对Aβ体内聚集介导的神经毒性的抑制作用机制。研究方法涉及实时定量PCR、免疫印迹和行为学等，主要内容包括：利用Aβ聚集模型分析茜草多糖对聚集过程的抑制作用；利用Aβ体内聚集模型分析茜草多糖对蛋白质降解功能的增强机制；利用信号通路模型分析茜草多糖对胁迫和寿命相关信号途径的调控机制。本项目将为开发中药多糖类神经保护和神经退行性疾病药物提供药效物质理论基础，促进非营养性多糖在中药领域的深入开发利用，并推动秀丽线虫模型在中药研发领域的应用。

Aging and its associated diseases has become a common hotspot in medicinal research. A number of studies have shown that many aging-related neurodegenerative diseases are characterized by abnormal aggregation and accumulation of disease-related proteins. We have recently demonstrated that Rubia cordifolia polysaccharide (RPS) is able to modulate aging and proteotoxic stress pathways in C. elegans models. RPS can not only alleviate the neurotoxicity mediated by amyloid β peptide but also extend the adult lifespan of Aβ C. elegans. Since a number of signaling pathways related to both lifespan regulation and stress resistance contribute to the neuroprotective effec of polysaccharides, we aim to investigate the inhibitory mechanism of RPS on Aβ neurotoxicity using C. elegans models. We will firstly investigate the suppressing effect and manner of RPS on Aβ aggregation process, and then explore the modulating effect of RPS on ubiquitin-proteasome system and autophagy-lysosome pathway. Furthermore, we will examine the effect of certain signaling cascades, including insulin/insulin-like growth factor-1 signaling, target of rapamycin signaling and oxidative stress on the neurotoxicity-suppressing and lifespan-extending activities of RPS. These pathways will be explored in a range of transgenic and mutant C. elegans models using real-time PCR, Western blotting, behavior assay and other molecular techniques. Revelation of the mechanism of action of RPS will provide an important insight into the potential of polysaccharides from traditional Chinese medicine and other glycotherapeutics in the prevention of proteotoxic disorders, and promote more applications of the model animal C. elegans in the studies of traditional Chinese medicine.

本项目选择β-淀粉样肽（Aβ）转基因和相关信号通路秀丽线虫模型，针对Aβ聚集、蛋白质降解体系以及寿命和胁迫相关信号通路，采用行为学和寿命实验、定量PCR、免疫印迹等手段，探讨了茜草多糖抑制Aβ聚集毒性的作用机理，已按计划完成研究任务。主要结果包括：（1）采用硫磺素T染色法，发现茜草多糖能有效抑制Aβ的体外聚集，进一步采用圆二色谱法发现该多糖可阻止β-折叠的形成，进而延缓Aβ聚集进程；（2）通过行为学和点印记实验证实茜草多糖能缓解Aβ在秀丽线虫体内异常聚集导致的瘫痪症状，并能减少Aβ寡聚体；（3）茜草多糖能够提高Aβ转基因秀丽线虫糜蛋白酶样蛋白酶体的活性，蛋白酶体抑制剂可以抑制多糖的神经保护作用，同时该多糖对19S蛋白酶体中一些基因的表达有调控作用，说明茜草多糖可通过增强蛋白酶体功能来抑制Aβ聚集毒性。此外，茜草多糖对自噬相关基因的表达没有影响；（4）采用胰岛素信号通路突变株和转基因模型，发现茜草多糖不能延长daf-16突变株的寿命，其能够促进DAF-16的入核，说明该多糖的延寿作用与调节DAF-16密切相关。茜草多糖能够抑制Aβ秀丽线虫体内p38磷酸化，但对ERK1/2和JNK磷酸化水平没有影响，而且多糖的神经保护作用与p38抑制剂的作用不叠加，说明茜草多糖缓解Aβ聚集毒性的作用与p38通路相关。此外，利用RNA Seq技术检测茜草多糖对Aβ秀丽线虫模型基因表达的影响，通过差异表达基因的功能分析发现多糖对阿尔茨海默症等相关的通路有调控；（5）茜草多糖能够延长秀丽线虫在百草枯胁迫下的存活，降低活性氧自由基和脂质过氧化产物水平，提高抗氧化酶活力，并能维持线粒体膜电位并增加ATP含量，说明该多糖的神经保护作用与缓解氧化胁迫和恢复线粒体功能有关；（6）利用Aβ和多聚谷氨酰胺等神经退行性疾病相关的多种秀丽线虫模型，发现其它中药多糖也具有缓解蛋白聚集毒性的神经保护作用。本项目将促进非营养性多糖在中药领域的深入研发，为开发神经退行性疾病中药多糖类药物提供药效物质理论基础。