eEF2K调控NF-κB对类风湿关节炎滑膜炎症和增殖的调控及其机制研究

Persisting inflammation and abnormal proliferaton of synovial tissue play a key role in the initiation and progress in rheumatoid arthritis (RA), however, the mechanisms contributing to those abnormal changes of biology are still poorly understood. In recent years, it has been shown that eukaryotic elongation factor 2 kinase(eEF2K) participate in modulation of immune responses and growth of tumor cells. Our previous studies demonstrated that NF-κB have an important role in regulating RA synovial inflammation and proliferaton. The expression of eEF2K in RA synovium tissue and RA FLS is significantly elevated compared to osteoarthritis patients, inhibition of eEF2K suppressed proinflammatory cytokines secretion and proliferaton of fibroblast like synoviocytes (FLS) from RA patients.Therefore, we propose that eEF2K modulates synovial inflammation and proliferaton of RA by mediating NF-κB. Our research will be aimed at identifying the inhibitory role of eEF2K on synovial inflammation and abnormal of RA by in vitro and in vivo, CIA model and eEF2K knock-out mice. We will also clarify the molecular mechanisms by co-immunoprecipitation and Western Blot. We propose to clarify the mechanisms underlying joint destruction of RA in the term of protein synthesis, providing the scientific basis for establishment of a novel therapeutic target for RA.

关节滑膜持续炎症和异常增殖在类风湿关节炎（RA）的发病和病情进展中起关键作用，但其机制尚不清楚。近年发现调控蛋白质合成的真核延长因子Ⅱ激酶(eukaryotic elongation factor 2 kinase，eEF2K)可能在免疫反应以及肿瘤的生长调控中发挥重要作用。我们前期发现eEF2K在RA滑膜组织和细胞中表达增高，抑制eEF2K明显抑制RA患者滑膜成纤维细胞分泌炎症因子和增殖，NF-κB通路参与调控RA滑膜炎症和增殖。因此我们推测：eEF2K可能通过介导NF-κB调控RA滑膜炎症和增殖。本研究拟进一步通过体内、外功能实验、CIA模型以及eEF2K基因敲除小鼠明确eEF2K对RA滑膜炎症和增殖的调控，并通过免疫共沉淀和免疫印迹阐明调控机制，旨在从蛋白质合成角度阐明RA发病机制，为RA治疗提供新思路。

滑膜成纤维细胞（FLS）过度活化、增生和具有侵袭性是类风湿关节炎（RA）关节炎症和破坏持续进展的关键环节。近年发现调控蛋白质合成的真核延长因子Ⅱ激酶(eukaryotic elongation factor 2 kinase，eEF2K)在肿瘤的生长和侵袭调控中发挥重要作用。本课题利用现代分子生物学和免疫学技术，通过体外细胞培养和动物模型，证实eEF2K对RA滑膜迁移、侵袭的调控作用，明确eEF2K参与调控RA关节滑膜炎症具体机制。我们研究发现，RA 患者关节滑膜组织和 FLS eEF2K表达显著高于骨关节炎患者和正常对照组，同时 TNF-α 及LPS能刺激 RA FLS eEF2K，而使用MTX及地塞米松可抑制eEF2K表达。利用干扰技术及抑制剂抑制 eEF2K表达能显著抑制 RA FLS炎症因子分泌，同时可抑制RA FLS细胞增殖及迁移侵袭功能，我们同时发现抑制 eEF2K可影响RA FLS细胞骨架形成，此实验表明抑制 eEF2K不仅可抑制RA FLS炎症反应，同时对于增殖及迁移同样有抑制作用。同时我们发现抑制RA FLS eEF2K可以抑制细胞葡萄糖摄取及乳酸生成，继而影响滑膜细胞炎症因子分泌。进一步的机制研究发现，抑制 eEF2K能显著抑制 NF-κB活性，同时可影响p-AKT表达，从而抑制 RA FLS 炎症因子分泌及细胞迁移功能。同时体内使用抑制剂治疗CIA小鼠能够显著改善小鼠的关节炎症及骨质破坏。通过该项目我们揭示 eEF2K在RA滑膜炎症和关节破坏中的关键作用及其分子机制，为确立eEF2K为RA的治疗新靶标提供科学依据。