秋水仙素相互作用蛋白的筛选与机制解析

Gout is a common form of inflammatory arthritis among the elderly population, and its prevalence is increasing in recent years. Gout has a close relationship with hypertension, hyperlipidemia, diabetes and other rich man's diseases which are an enormous burden to society. Colchicine is a typical microtubule inhibitor and also is an ancient and powerful drug used to treat gout so far. Results from preliminary studies show that colchicine can inhibit the release of chemokines and reduce the recruitment of inflammatory cells. However, the exact mechanism is still unclear if the anti-inflammatory effect of colchicine is mediated by inhibiting microtubule or other pathway. This project aimed to identify the interacting protein with colchicine and to clarify the colchicine mediated signaling pathway, and the possibility of prevention and treatment of gout by targetting the key molecules in the colchicine mediated signaling pathway will also be explored. The implementation of this innovative project will not only help to reveal the molecular mechanisms of colchicine and to deepen our understanding of the basic biological processes of gout, but also provide some new ideas for the development of new treatment strategies and methods of gout.

痛风是在中老年人群中常见的一种炎症性关节炎，近年来其发病率呈逐年上升趋势，并且与高血压、高脂血症、糖尿病等"富贵病"的发生密切相关，给社会带来巨大的负担。秋水仙素是一种典型的微管抑制剂，也是非常经典且至今仍被作为治疗痛风急性发作的药物。前期研究结果显示其可以抑制趋化因子释放，减少炎症细胞募集，从而发挥抗痛风作用。但是秋水仙素是否通过抑制微管来发挥抗炎作用，抑或存在其他的作用通路，具体机制仍不清楚。本项目拟以筛选秋水仙素的相互作用蛋白为核心，阐明秋水仙素治疗痛风所介导的信号通路，并探讨以秋水仙素治疗痛风所介导的信号通路中关键节点分子为靶点进行痛风防治的可行性。本项目的实施有助于揭示秋水仙素分子调控机制，为我们对痛风基本生物学过程的了解提供新视角，并且对发展痛风治疗新策略和新方法提供行之有效的新思路。

秋水仙素可以作为治疗急性痛风的有效药物，但目前对其发挥作用的具体机制仍未研究清楚。在本项目中，我们首先成功完成了秋水仙素的生物素标记，体外和体内检测结果表明标记后的化合物仍能保持秋水仙素的生物学活性；然后利用计算机模拟技术编写基于配体相似性的算法进行了秋水仙素与相互作用蛋白的亲和活性预测，筛选出其潜在的靶点蛋白GLRA3，并通过免疫共沉淀进行了验证；mRNA表达谱芯片结果显示秋水仙素处理后Cytokine-cytokine receptor interaction和Jak-STAT两个通路相关基因变化最为明显。进一步研究发现，GLRA3可与CCR5结合并抑制其磷酸化水平。上述结果表明，GLRA3和CCR5在秋水仙素所调控的信号通路中扮演着重要的角色，秋水仙素可能通过GLRA3/CCR5信号来阻滞中性粒细胞等炎症细胞向痛风炎症部位的浸润。