Toll样受体4上调miR-181b在特发性肺纤维化中的抗纤维化作用及机制研究

The etiology and pathogenesis of idiopathic pulmonary fibrosis (IPF) are still unclear and thus no proven effective therapies exist. Toll-like receptor 4 (TLR4) was believed to play an important regulatory role in pulmonary fibrosis, but the specific mechanism has not been well defined. Our previous research demonstrated that TLR4 increased the expression of miR-181b in airway epithelial cells (AECs), and miR-181b could target transforming growth factor receptor I (TGFBR1), an important protein in promoting fibrosis. To further investigate whether TLR4 could downregulate TGFBR1 expression on AECs via miR-181b and inhibit the pulmonary fibrosis process in IPF in this way, we intend to carry out a following study in cells and animal models using bioinformatics, molecular biology and immunology technology such as gene transfection. The project is shown below: (1) To explore the specific mechanism that TLR4 activation increased miR-181b expression; (2) To investigate the regulatory effects of miR-181b on pulmonary fibrosis via its targets TGFBR1, as well as the specific mechanism. This study would help to complement to the pathogenesis thesis of IPF, and to provide a new pathway in discovering strategies which could be used to prevent progression of pulmonary fibrosis.

特发性肺纤维化（IPF）病因及发病机制不明，目前尚无有效的治疗药物。本课题组及国内外相关研究表明Toll样受体4(TLR4)对肺部纤维化具有重要的调控作用，但具体机制不明。本课题组前期研究工作发现：TLR4上调呼吸道上皮细胞中miR-181b的表达，且初步证明miR-181b可靶向作用于重要促纤维化蛋白转化生长因子受体I（TGFBR1）。为进一步明确TLR4是否通过上调miR-181b影响呼吸道上皮细胞表面TGFBR1的表达，从而抑制IPF的肺纤维化进程，本项目拟结合生物信息学及基因转染等分子生物学及免疫学技术，在细胞水平和动物模型体内进行如下研究：①TLR4激活后上调miR-181b表达的具体机制；②miR-181b通过靶点TGFBR1对肺纤维化的调控及具体机制。本研究将有助于对现有IPF发病机制的阐述作出补充，并为探索延缓或阻止肺纤维化进展的策略提供新思路。

项目背景： 研究表明Toll样受体4（TLR4）活化是发生肺纤维化所必需的。然而，miRNA是否参与TLR4激活的抗纤维化作用仍没有确定。.项目目标：验证miRNA在抗TLR4激活的抗纤维化中发挥的作用，重点关注miR-181b及其作用靶点TGFBR1。.方法：通过miRNA阵列和qRT-PCR方法，确定TLR4活化诱导的人上皮细胞系A549细胞中的miRNA。使用Western印迹和qRT-PCR证实miR-181b对TGF-β受体1（TGFBR1）表达的调节。使用Western印迹，qRT-PCR和免疫荧光（IF）评估miR-181b对TGF-β1诱导的A549细胞上皮 - 间质转化（EMT）的影响。 在博来霉素诱导的肺纤维化小鼠中，miR-181b通过静脉注射编码miR-181b基因的重组腺相关病毒载体过表达，通过免疫组化，IF，qRT-PCR和形态测定法测定其作用。结果：用LPS处理后，miR-181b在A549细胞中显着上调。 miR-181b可通过靶向3'UTR而抑制TGFBR1基因表达而不影响其在A549细胞中的mRNA水平。过表达miR-181b可以减轻体外TGF-β1诱导的EMT。 miR-181b体内过表达减轻了肺泡间隔增厚，并导致胶原和MMPs表达减少。.结论：我们的结果表明TLR4 / miR-181b / TGFBR1轴在IPF中的关键作用。该作用轴对肺纤维化的治疗提出了新的治疗策略与思路，同时对药物的研究提供了新的方向。