抗肺纤维化新药- - 氟非尼酮抑制NALP3炎性体及IL-1 β/IL-1R1/MyD88通路活化的机制研究

Idiopathic pulmonary fibrosis (IPF) is a refractory diffuse lung disease. The efficacy of the currently available treatment is disappointing and new anti-fibrotic medicine with multiple targets and pathways is urgently needed. Although its pathogenetic mechanisms are complex, inflammation plays an important role in the development of IPF. Fluorofenidone(AKF-PD) is a new compound drug designed by Central South University. Its pre-clinical study was already completed according to the requriments of the typeⅠstate new drug of China. The application for the phase Ⅰclinical trials is ongoing. It was recently reported that the activation of inflammasome and the innate immunity pathway of NALP3 inflammasome and IL-1β /IL-1R1/MyD88 play a pivotal role in the development of IPF.Furthermore, in our previous study, we found that the antifibrotic effect of AKF-PD was based on its inhibition of inflammation. In our present experiments, we will use cells, animals and various molecular biology techniques to test if AKF-PD can attenuate bleomycin-induced pulmonary inflammation and fibrosis through suppressing activation of inflammasome and IL-1R1/MyD88 pathway. This project will help us to understand the pharmacological mechanisms of AKF-PD , improve its use in clinic, and establish the theoretical foundation for the develepment of other antifibrotic medicine.

特发性肺纤维化（IPF）是一种难治性弥漫性肺疾病，临床上缺乏有效治疗药物，开发新型多通道多靶点抗肺纤维化药物迫在眉睫。IPF发病机制复杂，而炎症在其发生发展中的作用举足轻重。氟非尼酮（AKF-PD）是中南大学自主设计的新化合物，已按国家Ⅰ类新药要求完成了临床前研究，正在申报Ⅰ期临床试验。本项目基于最近文献报道NALP3炎性体和IL-1β/IL-1R1/MyD88天然免疫反应通路激活在肺纤维化发生发展中起关键作用，以及前期研究发现AKF-PD可通过抗炎机制发挥抗肺纤维化作用的基础上，设计细胞和动物实验，综合应用多种分子生物学技术，明确AKF-PD是否可分别通过抑制炎性体和IL-1R1/MyD88通路的活化，从而多靶点抑制博莱霉素所致肺纤维化早期的炎症反应，发挥抗肺纤维化作用。本项目将有助于加深对AKF-PD药理作用机制的认识，为AKF-PD在临床上的应用及其他抗纤维化新药的研发奠定理论基础

特发性肺纤维（IPF）是一种难治性弥漫性肺疾病，临床上缺乏有效的治疗药物，IPF发病机制复杂，而炎症在其发生发展中的作用举足轻重，氟非尼酮是本课题组自主发明的新型抗纤维化药物，已经申请进入Ⅰ期临床试验阶段。前期研究发现氟非尼酮能够有效地抗炎和抗肺纤维化，然而机制不明。研究表明，NALP3炎性体及IL-1β/IL-1R1/MyD88/NF-KB天然免疫反应激活在肺纤维化发生发展中起关键作用，本项目主要研究氟非尼酮抗纤维化机制是否与抑制NALP3炎性体及IL-1β/NF-KB炎性信号通路有关。C57BL/6J小鼠气管内注射博莱霉素（BLM），并给予氟非尼酮治疗，分别在实验第2天和第14天处死小鼠，肺组织HE和Masson染色，ELISA法检测IL-1β、 IL-6、 MCP-1、MPO，Western blot 和/或RT-PCR检测α-SMA 、fibronectin 、 collagenⅠ、caspase-1、IL-1R1 和 MyD88表达；在尿酸（MSU）刺激的人单核巨噬细胞（THP1）和共转染NALP3炎性体蛋白和pro-IL-1β的293T细胞，Western blot检测caspase-1和IL-1β，免疫共沉淀检测NALP3、ASC和pro-caspase1的相互作用；大鼠肺泡II型上皮细胞（RLE-6TN），IL-1β刺激，Western Blot检测 IL-1R1、MyD88、P-KK、P-IKB、P65表达，Real-time PCR和ELISA的方法检测IL-6、MCP-1水平。动物实验结果表明，氟非尼酮能抑制肺组织炎症和纤维化反应，抑制炎性因子IL-1β, IL-6, MCP-1, MPO表达，抑制Caspase-1, IL-1R1, MyD88 蛋白表达，抑制α-SMA，Fibronectin 和Collagen Ⅰ表达；细胞实验结果表明，氟非尼酮可以抑制炎症细胞NALP3炎性体组成蛋白间的相互结合，抑制Pro-caspase1剪切和IL-1β成熟，抑制肺固有细胞P65入核和NF-KB活化，抑制IL-6和MCP-1产生。本项目研究提示：氟非尼酮通过抑制NALP3炎性体活化，进而抑制炎症因子产生，抑制P65入核和抑制IL-1β/NF-KB信号通路活化来抑制炎性因子表达，发挥抗纤维化作用。