血管紧张素Ⅱ及血管紧张素（1-7）对肺成纤维细胞NLRP3炎症小体及自噬调控作用：肺纤维化的新机制

The NLRP3 inflammasome and autophagy have been considered as hot area of research in pulmonary fibrosis at present. The NLRP3 inflammasome, activated by redox imbalance, plays a key role in maintaining the activative phenotype of lung fibroblasts and accumulation of collagen, which can be inhibited by mitochondrial autophagy. Angiotensin Ⅱ(Ang Ⅱ) and angiotensin(1-7) (Ang(1-7)) are the new targets of pulmonary fibrosis therapy, however, the exact molecular mechanism of them in regulating the NLRP3 inflammasome and autophagy is still unclear. Our preliminary findings suggested that Ang Ⅱ, suppressed by Ang(1-7), could induce oxidative stress and activate the NLRP3 inflammasome. Therefore, we suppose that Ang Ⅱaccelerates pulmonary fibrosis via oxidative stress activated NLRP3 inflammasome, which could be reversed by Ang(1-7), these effects might be regulated by mitochondrial autophagy. Our research aims to observe the effects of AngⅡ and Ang(1-7) on NOX4-induced oxidative reaction/Nrf2-induced anti-oxidative reaction, mitochondrial dysfunction, NLRP3 inflammasome activity, and collagen synthesis. Additionally, we also observe the negative regulatory role of p62/SQSTM1 induced selective mitochondrial autophagy in NLRP3 inflammasome signal pathways. This research would provide novel strategy for prophylaxis and treatment of pulmonary fibrosis.

NLRP3炎症小体及自噬是肺纤维化的研究热点。氧化/抗氧化反应失衡激活的NLRP3炎症小体对肺成纤维细胞激活和胶原持续分泌有重要意义，自噬可抑制该效应。血管紧张素Ⅱ（AngⅡ)和血管紧张素（1-7）（Ang1-7)是肺纤维化治疗新靶点，然而对炎症小体和自噬的调控机理不明。本前期研究发现，AngⅡ可诱发氧化应激，激活肺成纤维细胞NLRP3炎症小体，Ang(1-7)可抑制该作用。我们设想：AngⅡ通过氧化应激激活肺成纤维细胞NLRP3炎症小体，促进肺纤维化；Ang(1-7)可抑制该效应；p62/SQSTM1介导的线粒体自噬对此有调控作用。拟从体内外观察AngⅡ及Ang(1-7)对肺成纤维细胞NOX4介导氧化/Nrf-2介导抗氧化反应、线粒体功能、NLRP3炎症小体活化及胶原生成的影响；观察p62/SQSTM1介导的选择性自噬对NLRP3炎症小体通路的负调控作用。本研究为肺纤维化防治提供新策略。

NLRP3炎症小体参与肺纤维化，其活化与氧化应激及自噬相关，RAS系统与肺纤维化相关，然而RAS、自噬、氧化应激、NLRP3在肺成纤维细胞及肺纤维化中的关系尚不明确；外泌体及细胞衰老在肺纤维化中的作用受到越来越多关注，其机制研究尚少。因此，本课题探讨了（1）在AngII刺激下，自噬、氧化应激、NLRP3炎症小体在肺成纤维细胞中的关系及其对肺纤维化的影响，及Ang（1-7）/mas轴对以上指标的作用；（2）在AngII刺激下，巨噬细胞释放的外泌体如何参与肺纤维化；（3）吸烟通过诱导II型肺泡上皮细胞（AT2）衰老加重肺纤维化的机制。结果发现（1）在肺纤维化中，自噬、氧化应激、NLRP3炎症小体均升高；促进自噬可抑制氧化应激、炎症小体及肺纤维化；在肺成纤维细胞中，AngII促进NOX4介导的氧化应激，通过激活炎症小体，促进胶原生成；Ang（1-7）/mas轴诱导自噬，通过下调NOX4介导的氧化应激抑制NLRP3炎症小体，从而减少胶原生成；（2）在肺纤维化中，肺组织中巨噬细胞增加，AngII刺激的巨噬细胞来源的外泌体尾静脉输注可引起肺纤维化，伴随AT1R、TGF-β升高；抑制外泌体可减轻BLM诱导的肺纤维化；体外研究发现AngII刺激的巨噬细胞外泌体包含AT1R，其可被成纤维细胞吸收，通过TGF-β/Smad2/3通路促进胶原生成；（3）吸烟3周后小鼠肺部出现衰老AT2，吸烟小鼠在BLM下肺纤维化更严重；体外发现，烟草萃取物通过下调SIRT1活性抑制自噬促进衰老；SIRT1活性下降与DNA损伤诱导的PARP1活性增加进而消耗NAD+有关；自噬下降可损伤线粒体自噬促进线粒体氧化应激，进一步引起DNA损伤。因此得出结论（1）Ang（1-7）/Mas轴诱导自噬，通过抑制AngII诱导的成纤维细胞的氧化应激抑制NLRP3炎症小体活化，进而减少胶原生成及肺纤维化；（2）AngII刺激的巨噬细胞，通过释放包含AT1R的外泌体，激活成纤维细胞内TGF-β/Smad2/3通路，从而促进肺纤维化；（3）吸烟通过诱导DNA损伤激活PARP1，通过消耗NAD+抑制SIRT1，进而抑制自噬促进AT2衰老；自噬抑制引起线粒体氧化应激，通过诱导DNA损伤，在吸烟引起的衰老中形成环路，进而加重肺纤维化。

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