基于Nrf2/PPARγ信号通路间串话调节巨噬细胞胞葬的补益肺肾方治疗COPD分子机制

Macrophage efferocytosis plays an important role in resolving inflammatory reaction. COPD has macrophage efferocytosis dysfunction. Crosstalk of PPAR gamma and Nrf2 pathway regulate macrophage efferocytosis and inflammatory response, and participate in COPD development. The prescription of Bufeiyishen can reduce the inflammatory and oxidative stress of COPD, while it can activate the PPAR gamma pathway and inhibit the secretion of inflammatory factors by macrophages. Thus, we hypothesis that PPAR gamma /Nrf2 signal pathway disorder is an important mechanism of COPD macrophage efferocytosis dysfunction, bufeiyishen prescription treat COPD by regulating crosstalk of PPAR gamma /Nrf2 signaling pathway mediated macrophage efferocytosis. On the hypothesis, the rat lung macrophage and THP-1 cell efferocytosis dysfunction model will be established, Bufei Yishen prescription will be applied，combining with the stimulants and inhibitors of pathway and RNAi, transfection and other molecular biology technology to study in vitro and in vivo. The inflammation, oxidative stress, macrophage efferocytosis，efferocytosis and pathway related molecules will be compared in the experimental groups.The research shall be interpretate regulation of macrophage efferocytosis by Nrf2/PPAR gamma pathway crosstalk and the effect on COPD pathology, and explore molecular mechanism of bufeiyishen prescription therapy for COPD ,which provide ideas and reference for the treatment of COPD

巨噬细胞胞葬对结束炎症反应起重要作用，COPD存在巨噬细胞胞葬障碍，PPARγ/Nrf2通路调控巨噬细胞胞葬和炎性反应，参与COPD发生发展，两者相互作用。补肺益肾方可减轻COPD炎症和氧化应激，激活PPARγ通路，抑制巨噬细胞分泌炎症因子。由此，提出假说：PPARγ/Nrf2信号通路失常是COPD巨噬细胞胞葬障碍重要机理，补肺益肾方药可通过调节PPARγ/Nrf2信号通路串话调控巨噬细胞胞葬起到治疗COPD作用。围绕假说，建立大鼠肺巨噬细胞、人THP-1细胞胞葬功能障碍模型，用补肺益肾方，结合通路刺激剂、抑制剂及RNAi、转染和其他细胞分子生物学技术开展体内外研究，比较实验各组炎症反应、氧化应激、巨噬细胞胞葬、胞葬及通路相关分子等变化，阐释Nrf2/PPARγ通路串话对巨噬细胞胞葬调控作用和COPD病理变化的影响，探索补肺益肾方药防治COPD作用分子机制，为治疗COPD提供思路和借鉴

巨噬细胞胞葬对抑制炎症反应起重要作用，COPD存在巨噬细胞胞葬功能障碍，Nrf2/PPARγ通路调控巨噬细胞胞葬和炎症反应，参与COPD发生发展，两者相互作用。本项目确立了巨噬细胞胞葬功能检测技术，通过体内、外实验，利用细胞共培养、流式细胞术和其他细胞分子生物学技术展开研究，从巨噬细胞胞葬功能障碍角度，研究补肺益肾方介导Nrf2/PPARγ通路串话调控巨噬细胞胞葬功能和干预炎症反应治疗COPD的机制。取得了以下成效：①通过对香烟烟雾提取物（CSE）刺激下肺泡巨噬细胞的活力、胞葬率进行时效筛选，最终应用10%CSE构建稳定的体外巨噬细胞胞葬功能障碍模型。②体内实验：基于巨噬细胞胞葬功能障碍的COPD大鼠模型和COPD小鼠模型，证实激活Nrf2、PPARγ通路及其串话可促进肺泡巨噬细胞对凋亡细胞的吞噬，减少肺部凋亡细胞积累，从而缓解局部持续炎症；确证了补肺益肾方可促进Nrf2/PPARγ通路串话，肺功能指标TV、MV、PEF、EF50均有改善，肺实质损伤减轻，炎症因子TNFa、IL-4表达下降，肺组织凋亡细胞积累减少，肺泡巨噬细胞表型因子CD68、iNOS、CD163的表达显著下降，巨噬细胞胞葬功能改善，进而干预COPD进程；③体外实验：基于CSE刺激下的体外巨噬细胞胞葬功能障碍模型，证实激活Nrf2和PPARγ通路可减轻炎症反应，巨噬细胞胞葬率升高，促进巨噬细胞对凋亡细胞的吞噬。补肺益肾方激活Nrf2/PPARγ通路串话可减少炎症因子TNFα分泌，提高抗氧化因子HO-1、吞噬蛋白Rac1和胞葬辅助因子MFGE8、GAS6表达，降低Keap1蛋白表达，改善肺泡巨噬细胞胞葬功能障碍。本课题从巨噬细胞胞葬功能角度研究了补肺益肾方治疗COPD的机制，观察了Nrf2/PPARγ通路串话对巨噬细胞胞葬的调控作用和对COPD病理变化的影响，揭示了补肺益肾方药防治COPD的部分机制，为补肺益肾方的基础与临床研究提供实证依据，为COPD的治疗提供思路和借鉴。