β-arrestin2调控腺苷A2A受体信号通路在甲氨蝶呤抑制关节滑膜炎中的作用

Methotrexate (MTX) is widely used in treating rheumatoid arthritis (RA) principally via up-regulating adenosine and its receptor A2A (A2AAR) as was revealed in recent years. However, the ACR20 response rate to MTX treatment is between 46%-65% which is considered to associate with the polymorphisms of targets and metabolic enzymes of MTX, but there is no firm evidence shows whether signaling mediated by A2AAR can influence the efficacy of MTX. A2AAR belongs to the G protein coupled receptors (GPCRs) family. β-arrestin2 is an important negative regulator for most GPCRs, and plays a key role in controlling cell proliferation and differentiation. This study will detect the changes of serum and joint adenosine, β-arrestin2 expression and A2AAR signaling in synoviocytes in different disease processes of collagen induced arthritis (CIA) rats and in MTX treating CIA rats. In addition, we will illuminate the characteristics of β-arrestin2 in regulating A2AAR signaling and its effect on MTX efficiency in attenuating synoviocytes hyperplasia by using A2AAR selective agonist CGS-21680, small interfering RNA, and applying advanced techniques for instance flow cytometry, laser scanning confocal microscope, etc. This work should improve our understanding of the pathogenesis of RA and promote rational MTX therapy.

甲氨蝶呤（MTX）是治疗类风湿关节炎（RA）的常用药，目前认为上调腺苷及受体A2A（A2AAR）是其发挥抗炎作用的重要机制。MTX疗效只有46%-65%，一直来认为与MTX作用和代谢的基因多态性有关，但A2AAR偶联信号变化对MTX作用的影响未见报道。A2AAR属G蛋白偶联受体（GPCRs），β-arrestin2是大部分GPCRs的负调节因子，在细胞增殖分化调控中十分重要。本课题拟在以往工作基础上，观察胶原性关节炎大鼠病程不同阶段以及MTX治疗有效或无效时体内腺苷水平、滑膜细胞β-arrestin2和A2AAR信号的变化规律，采用流式细胞术、激光共聚焦等技术，利用A2AAR激动剂（CGS-21680）和小干扰RNA等工具，观察β-arrestin2对A2AAR信号的调节特点及在MTX抑制滑膜细胞异常增殖中的作用。为进一步阐明RA发生机制、病程变化特点和药物合理治疗提供理论依据。

甲氨蝶呤（MTX）是治疗类风湿关节炎（RA）的常用药，目前认为上调腺苷及受体A2A（A2AAR）是其发挥抗炎作用的重要机制。MTX疗效只有46%-65%，一直来认为与MTX作用和代谢的基因多态性有关，但A2AAR偶联信号变化对MTX作用的影响未见报道。A2AAR属G蛋白偶联受体，β-arrestin2是大部分GPCRs的负调节因子。本课题建立了大鼠胶原性关节炎（CIA）模型，关节匀浆腺苷水平从造模d14开始持续升高。正常关节滑膜细胞（FLS）细胞膜上A2AAR和Gs蛋白共表达，β-arrestin2胞膜表达较少，d14胞膜A2AAR和Gs表达轻度减少，较多β-arrestin2分布在胞膜，与A2AAR和Gs共定位；d28细胞膜A2AAR表达明显减少，Gs表达轻度减少，β-arrestin2增多；d42细胞膜A2AAR和Gs蛋白表达恢复，β-arrestin2表达减少，接近正常FLS水平。Gs活性在d14轻度降低，d28明显降低，d42恢复至接近正常水平。细胞内cAMP浓度与A2AAR和Gs活性正相关。提示A2AAR受体的膜表达水平与疾病进程呈负相关，提高A2AAR表达、活性或改善其下游信号通路转导可能有助于疾病的恢复。炎症高峰期β-arrestin2表达升高，因此β-arrestin2是RA治疗的潜在靶点。.本课题大鼠CIA的MTX治疗有效率在53.8%左右，与MTX治疗无效组相比，治疗有效的CIA大鼠血清和关节匀浆中腺苷水平高，IL-1β、IL-6和TNF-α水平低，FLS中A2AAR和Gs胞膜表达和mRNA水平较高，β-arrestin2表达较低，磷酸化IκBα较少，NF-κB胞核分布较低，细胞内cAMP浓度较高。提示A2AAR的表达水平和信号通路活化程度与MTX治疗效果有关。SiRNA抑制β-arrestin2表达的FLS，MTX体外给药显著升高A2AAR和Gs的胞膜表达，磷酸化IκBα较少，NF-κB胞核分布较低，MTX给药使FLS细胞内cAMP浓度明显增高。本课题揭示了大鼠CIA病程不同阶段的病理变化特点和β-arrestin2对A2AAR受体信号的调控规律，明确了β-arrestin2调控的A2AAR信号变化是MTX疗效差异的分子机制，为进一步阐明RA发生机制、病程变化特点和药物合理治疗提供了理论依据。