肥大细胞多巴胺D3受体通过负调控TLR4延缓类风湿性关节炎软骨细胞凋亡的作用机制研究

TLR4 activation on mast cells may promote the occurrence and development of synovitis and subsequent cartilage destruction in rheumatoid arthritis (RA). Chondrocytes apoptosis induced by inflammatory cytokines in the synovium is the primary cause of cartilage destruction in RA. Our previous studies showed that dopamine D3 receptor (D3R) on mast cells negatively regulated TLR4 activation and its downstream inflammatory cytokines production. Our preliminary data revealed that D3R on mast cells in synovial fluid was negatively correlated with disease activity in RA patients, and D3R on mast cells was involved in the negative regulation of chondrocytes apoptosis. So we speculate that D3R on mast cells may delay chondrocytes apoptosis via negatively regulating TLR4 activation. In this study, we would first analyze the relationship between D3R on mast cells and chondrocyte apoptosis in RA patients. Secondly, we would establish the animal model of RA using wild-type and D3R-deficient mice to validate the role of D3R on mast cells in chondrocyte apoptosis through negatively regulating TLR4 on mast cells. Finally, we would construct mast cells which are high or low expression of TLR4/D3R using gene delivery system, then mast cells and chondrocyte were cocultured to investigate the molecular mechanism through which D3R and its negative regulation of TLR4 on mast cells could influence chondrocyte apoptosis and the underlying mechanisms. The aim of our study was to investigate the negative regulatory role of D3R on mast cells in chondrocyte apoptosis and its underlying mechanisms. It will provide vital experimental evidence for exploring a novel diagnostic and therapeutic target in RA.

肥大细胞TLR4活化促进RA滑膜炎和软骨破坏，滑膜炎性因子诱导的软骨细胞凋亡是RA软骨破坏的主要原因。我们前期发现肥大细胞多巴胺D3受体(D3R)负调控TLR4活化及下游炎性因子表达。预实验发现RA患者滑液肥大细胞D3R与RA活动度呈负相关，肥大细胞D3R对软骨细胞凋亡发挥负调控作用。因此我们推测肥大细胞D3R通过负调控TLR4进而延缓RA软骨细胞凋亡。然而目前关于肥大细胞D3R对RA软骨细胞凋亡的作用和机制研究尚属空白。本研究拟从病例分析肥大细胞D3R与RA软骨细胞凋亡的相关性；建立WT和D3R-/-小鼠RA模型，验证肥大细胞D3R通过负调控TLR4在RA软骨细胞凋亡中的作用；构建D3R和TLR4高/低表达肥大细胞，将肥大细胞与软骨细胞共培养，探讨肥大细胞D3R对软骨细胞凋亡的作用机制。本研究旨在揭示肥大细胞D3R抑制RA软骨细胞凋亡的作用和机制，为探寻RA诊疗新靶点提供重要的实验依据。

肥大细胞TLR4活化促进类风湿关节炎滑膜炎和软骨破坏，滑膜炎性因子诱导的软骨细胞凋亡是类风湿关节炎软骨破坏的主要原因。我们前期研究发现，肥大细胞多巴胺D3受体（D3R）与类风湿性关节炎患者的疾病活动性高度相关，且D3R具有阻断活化的肥大细胞TLR4-MAPK信号级联的抑炎作用，但D3R如何负调控TLR4信号通路尚需进一步研究。本研究中，我们联合应用DBA/1小鼠和D3R基因敲除小鼠构建胶原诱导的小鼠关节炎（CIA）模型，并应用此模型探究D3R在肥大细胞TLR4信号及其相关炎症因子、关节炎症和软骨破坏过程中的调控作用。另外，我们在细胞水平通过检测肥大细胞泛素和自噬相关分子来阐明TLR4降解的机制。我们的研究结果表明D3R缺陷小鼠比野生型小鼠的类风湿关节炎病理特征更严重，D3R通过TLR4依赖性途径在体内外抑制肥大细胞活化。且更重要的是，本研究发现D3R可促进肥大细胞LC3脂质转化，从而加速泛素标记的TLR4降解。D3R可抑制活化的肥大细胞mTOR和AKT磷酸化，同时增强AMPK磷酸化，随后介导TLR4的自噬依赖性蛋白降解。总之，本研究发现肥大细胞D3R可通过mTOR/AKT/AMPK-LC3-泛素-TLR4信号通路减轻小鼠类风湿性关节炎。本研究明确了D3R对类风湿关节炎发生中肥大细胞过度活化引起的炎症反应的保护作用，提出了对于类风湿关节炎发病机制认识的重要新观点，并为未来探索类风湿关节炎治疗的新靶点提供了客观依据。

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