基于Notch信号通路调控的益气养阴通痹方干预类风湿关节炎肺间质病变机制研究

Rheumatoid arthritis-Interstitial lung disease (RA-ILD) is the main complications of rheumatoid arthritis. The prognosis of the disease is very poor and it easily leads to a serious threat to the health of patients. So far, no much studies about the disease have been conducted. Notch signaling pathways induce excessive deposition of extra cellular matrix (ECM) constitued of collagen in the intersititial parts of lung and eventually leads to pulmonary fibrosis and play an important role in the development of ILD. However,the relation between Notch signaling pathways and RA-ILD was reported. We had made significant effect for the treatment of the RA-ILD disease by yiqiyangyintongbi formula in the past years. However, the mechanisms of yiqiyangyintongbi formula on RA-ILD is unclear. Our research will investigate the effects of Notch signaling pathways on the expression of α-SMA, collagen Ⅰand collagen Ⅲ expression in lung fibroblasts during the development of RA-ILD by some experimental methods, such as cell culture, gene transfection, real time PCR, western Blot, and so on. In addition, we will investigate the effects of yiqiyangyintongbi formula on the lung function and the lung tissue of RA-ILD rat model.By the research, we attempt to reveal the effects of Notch signaling pathways on the development of RA-ILD disease. Meanwhile, we also attempt to reveal the effects of yiqiyangyintongbi formula on the treatment for the RA-ILD disease.

类风湿关节炎肺间质病变（RA-ILD）是RA肺部受累主要表现形式，预后差，严重威胁着患者健康，目前对该病的研究较少。研究证实Notch信号通路能诱导成纤维细胞增殖分化，促进细胞外基质（ECM）在肺间质的过度沉积，在肺纤维化形成过程中发挥着关键作用，但目前国内外还没有Notch信号与RA-ILD相关的报道。我们前期曾运用自拟益气养阴通痹方治疗本病取得较为确切的临床疗效，但其作用机制还不清楚。本课题拟运用细胞培养、基因转染、RT-PCR、Western Blot等实验方法，观察在RA-ILD模型大鼠肺成纤维细胞增殖过程中Notch信号通路的作用以及益气养阴通痹方的影响，围绕Notch信号通路探讨该方对RA-ILD模型大鼠肺功能、肺间质病变的干预效应。进一步阐释RA-ILD过程中Notch信号通路的作用，探讨该病发生、发展机制；阐释益气养阴通痹方干预RA-ILD分子机制，为临床应用提供依据。

类风湿关节炎肺间质病变（RA-ILD）是RA肺部受累主要表现形式，预后差，严重威胁患者健康。研究证实Notch和Wnt/β-catenin信号通路能诱导成纤维细胞增殖分化，促进细胞外基质（ECM）在肺间质的过度沉积，在肺纤维化形成过程中发挥关键作用。本研究首先利用益气养阴通痹方（防纤方）干预RA-ILD模型大鼠，发现防纤方能够通过抑制Notch1、Jagged1、Jagged2、GSK-3β等蛋白水平表达，进而下调Notch及β-catenin信号通路的过度活化，抑制β-catenin 与NICD的蛋白结合增加，从而能够抑制Snail及ZEB-1 mRNA表达水平并上调E-Cadherin的mRNA表达水平，抑制EMT表型，并最终抑制RA-ILD疾病的发生发展。在使用复方的基础上选取防纤方君药的主要成分黄芪甲苷作为研究对象，黄芪甲苷可通过下调RA-ILD模型大鼠Snail及ZEB-1的m MRA表达水平，并上调E-Cadherin的mRNA表达水平，而两者的上调与下调均依赖于Jagged1的下调作用，抑制Notch通路关键蛋白NICD的表达及β-catenin的表达水平。在体内试验的基础上发现IL-6、TNF-α和CXCL3等炎症因子可能通过血液循环促进肺组织中成纤维细胞的增殖及EMT表型，其中Notch通路和β-catenin信号通路在炎症因子CXCL3的刺激下相互促进共同调控了EMT关键蛋白Snail及ZEB1的表达上调即E-Cadherin的表达下降，从而诱发间质性肺疾病。而黄芪甲苷和雷公藤多苷能够通过抑制Jagged1进而下调Notch及β-catenin信号通路的过度活化，抑制β-catenin 与NICD、β-catenin与LEF、NICD与CSL的蛋白结合增加，能够剂量依赖性的抑制Snail及ZEB-1并上调E-Cadherin的启动子活性、mRNA和蛋白表达水平，抑制成纤维细胞的过度增殖及EMT表型，并最终抑制RA-ILD疾病的发生发展。本研究围绕Notch和Wnt/β-catenin信号通路探讨了益气养阴通痹方（防纤方）对RA-ILD模型大鼠肺间质病变的干预效应，进一步阐释RA-ILD过程中Notch和Wnt/β-catenin信号通路的作用，探讨该病发生、发展机制；阐释益气养阴通痹方（防纤方）及黄芪甲苷干预RA-ILD分子机制，为临床应用提供依