基于TLR/MyD88/NF-kB受体信号通路和NALP3炎性体研究萆薢总皂苷治疗痛风性关节炎的机制

Confirmed Toll-like receptor / myeloid differentiation primary response protein 88 / nuclear factor –κB （TLR/MyD88/NF-κB） pathway synergy NALP3 inflammasome activation IL-1β, which play a major role in sodium urate -induced acute gouty arthritis (GA). The systematic analysis of different literature on gout drugs showed that the Dioscorea hypoglauca is among the top ten in the commonly used prescription. Previous studies show that the total saponin of Dioscorea (TSD) could significantly inhibited the IL-1β production in acute gouty arthritis rat and THP-1 cell stimulated by MSU. We hypothesized that TSD inhibit IL-1β production by adjusting TLRs / MyD88 / NF-κB receptor signaling pathway key protein expression or / and NALP3 inflammasome. This project first study the regulation of TSD on TLR / MyD88 / NF-κB signaling pathway key protein and NALP3 inflammasome in THP-1 macrophages and rat gouty arthritis model, with colchicine as a positive control. On this basis, using NF-κB agonists LPS, caspase-1 activator IPAF, ROS inducers pyocyanin, or plasmid transfection techniques, respectively, to increased signal pathway key protein expression, further investigate the mechanism and target of TSD against gouty arthritis. In-depth study of this subject, providing new ideas and experimental evidence for the drug prevention and treatment of gout, and enrich the theory of Chinese medicine treatment of gout.

已经证实Toll样受体/髓样分化因子/核转录因子κB(TLRs/MyD88/NF-κB)通路与NALP3炎性体协同激活IL-1β在尿酸钠引起的痛风性关节炎（GA）中发挥主要作用。在常用抗痛风中药处方中萆薢使用频率排在前10位。萆薢总皂苷（TSD）显著抑制大鼠GA及尿酸钠刺激THP-1细胞引起的IL-1β的产生。TSD可能是通过调节TLRs/MyD88/NF-κB信号通路关键蛋白表达或/和NALP3炎性体而抑制IL-1β的释放。本课题以THP-1细胞和大鼠GA模型为对象，以秋水仙碱为对照，研究TSD对TLR/MyD88/NF-κB信号通路关键蛋白和NALP3炎性体的调控作用，并分别用NF-κB激动剂LPS、caspase-1激活剂IPAF、ROS诱导剂pyocyanin，或质粒转染技术上调关键蛋白的表达，研究TSD抗痛风的机制和效应靶标。本课题的深入研究为该药防治痛风提供新的思路和实验依据。

TLRs/MyD88/NF-κB信号通路与NALP3炎性体协同激活白细胞介素1β（IL-1β）在尿酸钠引起的痛风性关节炎(GA)中发挥主要作用。本课题以THP-1单核巨噬细胞株和大鼠GA模型为对象，研究萆薢总皂苷(TSD)对TLR/MyD88/NF-κB信号通路和NALP3炎性体的调控作用，并分别用TLR4受体激动剂LPS、ROS激活剂鱼藤酮、TLR4受体特异性抑制剂TAK-242或NF-κB抑制剂PDTC，或质粒转染技术调控通路关键基因和蛋白表达，进一步探讨TSD抗痛风性关节炎的机制。. 结果表明，TSD可明显改善大鼠步态，减轻关节肿胀度，降低炎症细胞因子水平，改善关节病变，降低关节滑膜组织pro-IL-1β、NALP3、ASC、caspase-1前体和活性蛋白表达，降低滑膜组织TLR4、MyD88、NF-κB和pro-IL-1β的mRNA和蛋白表达，抑制NF-κB p65入核。TSD显著降低MSU刺激THP-1巨噬细胞NALP3、caspase-1和凋亡相关斑点样蛋白的含量，降低通路关键蛋白和基因的表达，抑制炎症细胞因子分泌及MSU刺激NF-κBp65的核转位。.进一步研究表明，TSD抑制鱼藤酮诱导的NALP3炎症小体和caspase-1的活化，减轻MSU晶体诱导的炎症反应，抑制LPS诱导的TNF-α、IL-1β分泌及通路关键蛋白和基因的表达。预先给予TAK-242或PDTC，与抑制剂组比较，TSD+抑制剂可进一步下调MSU刺激THP-1源性巨噬细胞关键蛋白的表达，减少TNF-α和IL-1β分泌。成功构建pGPU6-TLR4、pGPU6-NF-κB、pEX-3-TLR4及pEX-3-NF-κB质粒。TLR4和NF-κB共干扰后转入pEX-3-NF-κB质粒，NF-κB mRNA表达水平较共干扰组显著升高，TSD可降低pEX-3-NF-κB组NF-κB mRNA和蛋白的表达；TLR4和NF-κB共干扰后转入pEX-3-TLR4质粒，TLR4 mRNA和蛋白的表达水平较共干扰组显著升高，TSD可降低pEX-3-TLR4组TLR4 mRNA和蛋白表达。结论：TSD对急性痛风性关节炎具有防治作用，其机制可能与抑制TLRs/MyD88/NF-κB信号通路的活化及NALP3炎性体装配和激活，减少下游炎性细胞因子的分泌有关，TLR4受体和NF-κB蛋白可能是其作用靶点。