Ahr干扰Wnt信号通路介导RA模型鼠骨破坏的机制探讨

Rheumatoid arthritis (RA) is a prototypical systemic autoimmune disease. Bone formation of osteoblasts impaires at erosion sites associating with the functional disability of patients with RA. Aryl hydrocarbon receptor (Ahr) is a ligand activated transcription factor and it belongs to the Per-Arnt-Sim superfamily protein. Interestingly, recent evidences demonstrated that this ancient protein has been gained more attention in RA..Increasing Ahr in synovial tissue exacerbated synovitis of RA. In contrast, collagen-immunized Ahr KO mice ameliorated the synovitis due to decreasing the production of Th17 cells and serum levels of such pro-inflammatory cytokines as interleukin (IL)-1β and IL-6. Although Ahr highly expressed in the synovial tissues of patients with RA, its contribution to bone loss have not been completely elucidated. Based on these finding, we hypothesize that activated Ahr locating in osteoblasts involves the bone destruction of arthritis. .The activation of Wnt signaling contributes to bone formation, whereas the blockade of Wnt signaling facilitates bone erosion in patients with RA. Beta-catenin is a cytoplasmic and nuclear protein and involves with“canonical Wnt pathway” signaling cascades, which is essential for bone formation. The removal of beta-catenin from the progenitor cells of osteoblasts prevents the differentiation of osteoblasts. Intracellular proteins interact with beta-catenin and modulate Wnt/beta-catenin signaling activity through activating or inhibiting the phosphorylation of beta-catenin. In this project, we put forward another hypothesis that activated Ahr inhibited Wnt signaling pathway to exacerbate bone destruction in RA, such as suppression the binding of Wnt-coreceptor, inducing the degradation of the beta-catenin complex and declining the transcription of targeting genes with bone formation..In our previous study, we analyzed the expression of Ahr in bone tissue of hind paws derived from collagen-induced arthritis (CIA) mice. Further, we assessed the location of Ahr in subcellular level, and then examined the effects of Ahr on bone destruction. These data showed that higher levels of Ahr expressed in osteoblasts of CIA bone tissues and suppressed the expression of osteoblast marker alkaline phosphatase (ALP) in comparison with normal mice. These previous data provide some experimental evidents to finish this project. .A better elucidation of the involvement of the Ahr on Wnt signaling pathway in RA can improve understanding of the mechanisms of bone destruction and prognosis of the disease. In addition, Ahr may be a candidate of molecular targets in the treatment of RA.

类风湿关节炎（Rheumatoid arthritis ，RA)是我国第二位致残性疾病，其骨破坏机制不清，疾病早期即出现成骨异常的骨流失。芳香烃受体蛋白（Aryl hydrocarbon receptor，Ahr）与RA密切相关，本课题前期研究发现RA模型鼠Ahr高表达抑制了成骨细胞分化成熟，并与骨量呈负相关，提示Ahr高表达可降低骨形成导致RA骨损伤。Wnt通路是骨形成的必需信号，干扰Wnt通路可显著抑制成骨。由此我们推测Ahr的内源性配体可能会通过拮抗Wnt蛋白与受体的结合；作为胞内蛋白的Ahr可能会促进β-catenin磷酸化降解；Ahr入核后可能抑制成骨的靶基因转录，最终导致RA骨破坏。本研究拟采用体外原代细胞培养和RA模型鼠相结合的研究方式，探讨Ahr通路本身及其与Wnt信号通路的关系，揭示Ahr抑制骨形成导致RA早期骨破坏的机制。该研究可能有助于早期发现和干预RA骨破坏。

关注成骨细胞对骨破坏的影响是研究RA早期骨破坏机制的关键所在。近年的研究表明AhR与RA疾病严重程度存在密切关系。但Ahr如何影响成骨细胞发育分化的各阶段进而加剧RA骨破坏的具体机制不十分清楚。本项目主要的研究内容是利用类风湿关节炎（RA）模型鼠——胶原诱导关节炎（CIA）小鼠来探讨芳香烃受体（Ahr）促进RA骨破坏的机制。主要从Ahr信号通路本身及其与经典Wnt成骨信号通路的关系两方面探讨了Ahr抑制成骨细胞功能导致CIA小鼠骨破坏的机制。DKK-1是经典Wnt信号通路的天然抑制分子，Wnt5a是非经典Wnt信号通路中的代表分子，因此，我们以DKK-1和Wnt5a分子为代表，探讨了Wnt5a的在CIA骨破坏中的作用；并研究了DKK-1核酸疫苗和Wnt5a截断片段保护CIA的疗效和机制。这延伸了本项目的研究内容。.关于Ahr及其干扰经典Wnt成骨信号通路促进CIA骨破坏的机制，我们的研究结果显示，CIA小鼠的Ahr表达量增加，增加的Ahr主要定位在成骨细胞，Ahr的增加可以抑制成骨细胞的增殖分化功能。为了进一步明确Ahr抑制成骨细胞功能的具体机制，我们采用骨髓间充质干细胞（MSC）作为研究对象，观察Ahr如何抑制MSC向成骨细胞分化。我们的结果显示，与正常对照组小鼠相比，CIA小鼠MSC中Ahr的表达量明显增高，Ahr信号通路活化，活化的Ahr通过抑制β-catenin的表达影响了CIA小鼠MSC的分化和分泌功能。这表明CIA小鼠中Ahr表达量的增加通过活化Ahr信号通路和抑制经典Wnt信号通路来抑制成骨细胞的功能，进而导致了骨破坏。.关于Wnt5a和DKK-1在RA骨破坏中的作用机制和疗效评估，我们的研究结果显示，CIA小鼠关节成骨细胞中Wnt5a表达上调，并有促进IL-6分泌的作用。其次，我们惊讶地发现Wnt5a截断片段却丧失了加重病情的病理学作用，反而能够抑制炎症和骨破坏。DKK-1核酸疫苗也具有保护骨破坏的作用。.RA最大危害是进展性骨破坏导致的残疾。对其骨破坏发生机制的研究有助于干预疾病进展。改造Wnt5a分子有可能为RA治疗提供新的理论思路。