SHH-CCR3信号通路在RA关节骨破坏中的作用及其分子机制研究

Synovial lesion and bone destruction are basic characteristics of rheumatoid arthritis (RA), and osteoclast activation is the critical factor in bone destruction. Sonic hedgehog (SHH) plays an important role in maintaining metabolic balance of osteoblast and osteoclast. CCR3 is a CC chemokine receptor and closely correlates with osteoclast differentiation and synovial lesion. Our previous studies demonstrated that SHH signaling pathway was aberrantly activated in RA patients. SHH signaling pathway directly contributed to proliferation and migration of synoviocytes and showed its anti-apoptotic effect on vascular endothelial cells. Furthermore, it is reported that osteoclast differentiation was promoted by SHH and bone destruction was decreased in a mouse model of RA by inhibiting SHH. Recently, we find that CCR3 expression is significantly up-regulated in the process of osteoclast differentiation, and its expression is regulated by SHH. However, in the pathological lesions of RA, whether SHH signaling pathway regulates osteoclast differentiation via modulating CCR3 and consequently influences bone destruction remains unclear and needs to be further investigated. In the present study, we will perform the following experiments using models of collagen-induced arthritis (CIA) mice and monocyte/macrophage cells to further explore the effect of SHH-CCR3 signaling pathway on bone destruction of RA, ①Effect of CCR3 on pathological lesion of RA, ②Role of CCR3 in SHH-regulated bone destruction of RA, ③Effect of SHH-CCR3 on osteoclast differentiation. Our study will reveal the molecular mechanism of SHH signaling pathway in bone destruction of RA and provide experimental and theoretical evidence for developing new drugs for the treatment of RA.

关节滑膜病变和骨破坏是类风湿关节炎(RA)的基本特征，而破骨细胞活化是骨破坏的关键。SHH在维持成骨和破骨细胞代谢平衡中起重要作用。CCR3是CC类趋化因子受体，与破骨细胞分化和滑膜病变密切相关。我们前期研究发现SHH在RA中异常活化，直接参与滑膜细胞增殖和迁移、抵抗血管内皮细胞凋亡等过程。研究表明SHH促进破骨细胞分化，抑制SHH可减轻RA模型鼠骨破坏。近期我们发现CCR3在破骨细胞分化中表达上调，并受SHH调控。但在RA病变中，SHH是否通过调控CCR3促进破骨细胞分化，进而影响骨破坏？这有待深入研究。为明确SHH-CCR3与RA骨破坏的关系，本项目将采用CIA小鼠和单核/巨噬细胞模型探索：①CCR3对RA病变的影响；②CCR3在SHH调控RA骨破坏中的作用；③SHH-CCR3对破骨细胞分化的影响。本项目将有助于阐明SHH调控RA骨破坏的分子机制，为开发治疗RA的新药提供实验理论依据。

类风湿关节炎（RA）是一种以对称性多关节炎为主要表现的自身免疫性疾病。RA病情如未得到有效控制，受累的关节出现关节骨侵蚀和破坏的风险显著升高，是致残的主要原因。炎症部位破骨细胞的活化是关节破坏的重要原因，因此，调控破骨细胞可能是抑制RA关节骨破坏的有效治疗靶点。本课题组前期研究表明，SHH信号通路促进RA滑膜细胞异常生物学行为，阻断SHH信号通路可减轻关节炎模型滑膜炎症、骨破坏。既往研究表明，巨噬细胞中SHH信号通路活化可显著上调CCR3的表达。本研究进一步探讨CCR3在SHH介导的RA骨破坏中的作用。.研究内容包括：（1）通过建立关节炎小鼠模型，观察CCR3失活在CIA关节病变和骨破坏中的作用；（2）在SHH过表达的情况下沉默CCR3表达，观察CCR3在SHH参与调控的RA关节炎病变和骨破坏中的作用；（3）利用离体的巨噬细胞模型，观察调控SHH-CCR3信号轴对破骨细胞分化的影响，探讨CCR3在SHH诱导的破骨细胞分化中所起的作用。.体内结果表明沉默CCR3表达可减轻关节炎表现，减轻关节滑膜炎和骨破坏。SHH过表达导致CIA小鼠关节炎表现更严重，促进关节滑膜炎症和关节骨破坏。在SHH过表达的基础上沉默CCR3表达，显著降低了关节炎严重程度，减轻滑膜炎症和骨破坏。通过对关节局部免疫组化染色发现SHH过表达组CCR3表达显著升高；进一步分离关节巨噬细胞进行分析，发现SHH过表达组巨噬细胞CCR3表达升高，提示在关节炎中巨噬细胞CCR3的表达可能受到SHH信号通路的调控，介导了SHH诱导的滑膜炎症和关节骨破坏。体外结果表明，在巨噬细胞分化为破骨细胞过程中，CCR3表达升高，且SHH信号通路可调控CCR3表达。活化SHH信号促进破骨细胞分化，阻断SHH信号后显著抑制破骨细胞分化。在活化SHH信号的基础上，抑制CCR3表达明显抑制破骨细胞分化和破骨相关基因的表达。.本项目进一步阐明了SHH可能通过活化CCR3促进RA滑膜病变和关节骨破坏，为开发靶向SHH信号通路用于治疗RA的新药提供基础。