PMP对RA-FLS侵袭及软骨侵蚀的影响机制及Gen的干预

We have previously found that PMP concentration was increased in the blood and synovial fluids of the patients with RA in the active stage,and PMP could promote the fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) (RA-FLS) adhesion onto the extracellular matrix (ECM), migration and invasion, regulate the stress fibers reorganization and the formation of filopodia and lamellipodia, up-regulate the expression of p-FAK(Y397) and matrix metalloproteinases (MMPs) in RA-FLS after treatment with PMP, suggesting that PMP regulates RA-FLS adhesion, migration and invasion presumablly through FAK-mediated signal pathway.In this item, we will firstly examine whether PMP led to the erosion and destruction of articular cartilage through regulating RA-FLS adhesion onto ECM, migration and invasion at the cellular level and in vivo model,and then we will further clarify whether the PMP induced activation was mediated by FAK to regulate the PI3K or MARK signal pathway to modulate the expression and activity of MMPs, and the expression and distribution of epithelial-mesenchymal transformation (EMT) markers, and the Rho GTPases expression and activity in RA-FLS after treatment with PMP.Finally, we will verify whether Gen decreased the erosion and destruction of articular cartilage in in vivo models by regulating FAK-mediated signaling pathways to affect RA-FLS adhesion onto ECM, migration and invasion induced by PMP. This study will be helpful to improve our understanding the complex pathological process of RA, and provide a new target for the treatment of RA.

前期工作表明， RA活动期患者血液和关节液中血小板微颗粒（PMP）增高。预实验显示，PMP可促进RA-FLS迁移和侵袭及与Collagen I的粘附，调控RA-FLS中张力纤维装配、丝状和片状伪足的形成，上调RA-FLS中p-FAK（Y397）和MMPs，提示PMP可能通过FAK介导的信号通路调控RA-FLS 的粘附、迁移和侵袭过程。本课题将①明确PMP是否影响RA-FLS与细胞外基质（ECM）（Collagen I、Fibronectin和Matrigel等）的粘附、迁移和侵袭；②明确PMP对RA-FLS的粘附、迁移和侵袭过程是否通过NF-κB介导的信号通路调控MMPs的表达及活性和Rho GTP酶（RhoA、Rac1 和Cdc42等）的表达及活性而实现的；③明确Gen是否影响PMP诱导的RA-FLS与ECM粘附、迁移和侵袭过程。本研究将有助于揭示RA患者体内活化血小板产生的PMP在RA发病中的作用及机制，为继续研究相关信号通路在PMP诱导和Gen抑制RA-FLS与ECM粘附、迁移和侵袭中的作用及在关节软骨侵蚀和骨破坏中的作用及机制的研究打下基础，也可能为RA的治疗提供新靶点。

关节软骨/骨破坏是导致RA致残的主要原因，而关节滑膜细胞在炎性环境下向软骨表面迁移和侵袭是导致关节破坏的重要因素之一。我们的前期工作和相关文献证实血小板及其分泌物参与了RA的病理过程，但其作用的机制尚不完善。我们研究了血小板分泌的微颗粒（PMPs）对RA成纤维样滑膜细胞（RA-FLS）迁移和侵袭的影响，并联合使用金雀异黄素（Gen）进行了干预研究。首先，细胞水平的研究显示，25和50μg/ml的PMPs显著促进RA-FLS的迁移、侵袭以及与细胞外基质的黏附，引起RA-FLS肌动蛋白细胞骨架的重塑。同时，研究结果证实Gen能够抑制PMPs诱导的RA-FLS与ECM的黏附、迁移和侵袭，并影响RA-FLS的肌动蛋白细胞骨架的重构。其次，为了探寻PMPs中促进RA-FLS黏附、迁移和侵袭的主要活性成份，我们采用质谱分析发现PMPs中富含多种趋化因子，为此我们应用这些趋化因子的受体拮抗剂开展了进一步研究，发现BX471、AMG487和STI571均不能阻断PMPs对RA-FLS的促进作用，而CXCR2拮抗剂SB225002则可阻断PMPs促进RA-FLS的迁移。基因芯片检测结合RT-qPCR和Western Blot验证结果显示PMPs可以上调RA-FLS中CXCL5的表达。考虑到CXCL5的受体就是CXCR2，结合受体阻断实验，提示PMPs可能通过CXCL5/CXCR2通路促进RA-FLS迁移和侵袭。最后，我们研究了PMPs促进RA-FLS迁移和侵袭的可能机制，基因芯片分析结合RT-qPCR以及Western Blot验证结果显示PMPs可上调RA-FLS中的MMP1而对其他MMPs的表达无明显影响。进一步研究发现，PMPs能上调RA-FLS中p-Erk、p-NF-κB和p-IκB，提示PMPs可能通过激活Erk活化NF-κB通路上调MMP1而促进RA-FLS迁移和侵袭。另外，我们还发现，PMPs可以上调RA-FLSs中的RANKL并下调OPG，提高了RANKL/OPG的比值，提示PMPs也可能参与了RA患者的软骨/骨破坏。本研究揭示了RA患者体内活化血小板产生的PMPs在RA发病中的作用及机制，为继续研究PMPs在关节软骨侵蚀和骨破坏中的作用及机制的研究打下基础，也可能为RA的治疗提供新靶点。