穿山龙总皂苷对痛风性关节炎“TLR2/4-IL1R受体信号通路”调控的分子机制研究

The incidence of gouty arthritis (GA) is increasing recently. However, there is on effective treating medicine at the moment. Most Chinese therapy is in a condition of early stage. Previous studies of our research group show that there are biological effects of anti-inflammation, analgesia and reducing uric acid of Rhizoma Dioscore Nipponicae on animal models of gouty arthritisand found that “TLR2/4-IL-1R receptor signal pathway”plays a key role in the progress of GA. It regulates the production of inflammatory factors such as IL1β directly. This project is aiming at “TLR2/4-IL-1R receptor signal pathway”to study the effect of total saponins of Rhizoma Dioscore Nipponicae. RealtimePCR,WesternBlot,IF,EMSA and Elisa methods are used to detect the mRNA and protein expressions of TLR2,TLR4,NF-κB,IκB and IKKα etc., the nuclear transfer and activity of NF-κB in the synovium of joint, as well as expressions of inflammatory factors such as IL-1β and IL-6 in the blood serum. At last, UPLC-MS method is used to detect the key biomarkers both in the blood serum and urine from the whole point of view. All above are used to explore the intervene mechanism of total saponins of Rhizoma Dioscoreae Nipponicae. The project is the base of development of total saponins of Rhizoma Dioscoreae Nipponicae to effective new drug in treating GA.

痛风性关节炎（GA）近年呈高发趋势，但无理想的治疗药物。中医药研究尚大多处于初期阶段。本课题组前期研究表明穿山龙对痛风性关节炎动物模型具有良好的抗炎、镇痛、降尿酸等生物学效应，并发现“TLR2/4-IL-1R受体信号通路”在GA发病过程中发挥关键作用，直接调控其相关IL-1β等炎症因子的生成。故本课题以“TLR2/4-IL-1R受体信号通路”为研究对象进行穿山龙总皂苷的干预作用。通过Realtime-PCR、WesternBlot 、IF、EMSA和Elisa法检测关节滑膜组织TLR2、TLR4、NF-κB、IκB、IKKα等的mRNA和蛋白表达、NF-κB的核转移和活化以及血清中IL-1β、IL-6等炎症因子的表达。再通过UPLC-MS法从整体角度检测血清和尿液中关键生物标志物的变化，直接揭示穿山龙总皂苷的干预机制，可为穿山龙总皂苷开发为创新型有效的抗GA药物奠定基础。

本课题组按照项目任务书的计划要求，已完成全部工作计划。共发表SCI论文5篇，国家核心期刊文章7篇。获得黑龙江省中医药科学技术奖一等奖和科学技术奖三等奖(自然类)。培养硕士研究生2名。（预期目标为发表核心期刊文章3-5篇，SCI文章1-2篇）。. 主要研究内容包括：（1）明确了穿山龙总皂苷对“TLR2/4-IL1R”信号通路关键蛋白表达的影响。（2）明确了穿山龙总皂苷对NF-κB活化和炎症因子表达的影响。（3）明确了穿山龙总皂苷对痛风性关节炎大鼠血清生物标志物的影响。（4）明确了穿山龙总皂苷对痛风性关节炎大鼠尿液生物标志物的影响。. 本项目研究在前期研究的基础上，探讨了穿山龙总皂苷通过干预“TLR2/4-IL1R”信号通路影响转录因子NF-κB活化，从而影响炎性因子IL-1β等的表达角度，揭示了穿山龙总皂苷的抗炎分子机制。同时，从血清和尿液生物标志物角度，揭示了穿山龙总皂苷的一般作用机制。实验采用Realtime PCR、WB、EMSA、Elisa和HPLC等多种实验方法，从整体与组织水平分别进行了验证。. 本项目的开展，进一步明确了穿山龙总皂苷的有效干预靶点及作用机制，为其开发为安全有效的抗痛风性关节炎新药提供了药理学方面的依据。