ACE-AngⅡ-AT1受体轴/ACE2-Ang（1-7）-Mas轴对吸烟诱发氧化/抗氧化反应失衡导致的肺纤维化的调控作用

Smoking-related interstitial lung disease, such as pulmonary fibrosis, is associated with imbalance between oxidation and antioxidation induced by cigarette smoking. Recent reports have shown that upregulation of ACE-AngⅡ-AT1R axis (ACE axis) promoted pulmonary fibrosis and alveolar epithelial cell apoptosis induced by cigarette smoking. Nevertheless, as our previous studies shown, this effect could be inhibited by ACE2-Ang-(1-7)-Mas axis (ACE2 axis). However, the mechanism about how ACE/ACE2 axis regulates pulmonary fibrosis and cell apoptosis remains unclear. Thus we supposed that ACE/ACE2 axis had positive/negative regulation effects on pulmonary fibrosis and alveolar epithelial cell apoptosis associated with imbalance between oxidation and antioxidation induced by cigarette smoking. To determine the roles of ACE/ACE2 axis and miR-21 in regulating pulmonary fibrosis and alveolar epithelial cell apoptosis induced by cigarette smoking extract (CSE), NOX4-ROS or Nrf2-ARE mediated pathway induced by AngII or CSE will be studied. Besides, whether the above effects induced by AngII or CSE can be inhibited by Ang-(1-7) or lenti-ACE2 remains further studies. Consequently, this project, which is an extension of our original research supported by National Nature and Science Fund, will provide novel strategy for prophylaxis and treatment of smoking-related pulmonary fibrosis.

吸烟相关性肺间质病如肺纤维化与吸烟诱发的氧化/抗氧化反应失衡有关。新近报道，血管紧张素转换酶(ACE)- 血管紧张素II（Ang II）-AT1受体轴（简称ACE轴）可促进吸烟诱发的肺纤维化及肺泡上皮细胞凋亡。本前期工作显示，ACE2-Ang（1-7）-Mas轴（简称ACE2轴）则抑制吸烟诱发的上述作用。然而，ACE轴/ACE2轴在此过程中的调节机制尚不明确。我们设想：ACE轴可促进烟草诱发的氧化/抗氧化反应失衡，导致肺纤维化和肺泡上皮细胞凋亡；ACE2轴可抑制上述效应。拟观察Ang II能否促进NOX4-ROS介导的氧化通路或抑制Nrf2-ARE介导的抗氧化通路，促进吸烟诱发的肺纤维化和肺泡上皮细胞凋亡，观察mir-21在其中的作用；观察Ang II诱导的上述作用能否被ACE2及Ang（1-7）抑制。本研究是原有的国家自然科学基金项目向吸烟诱发的肺纤维化的延伸，为肺纤维化防治提供新策略。

本课题从体内、体外（1）探讨CSE对肺成纤维细胞激活、胶原合成的影响及其机制。深入研究了CSE通过上调ROS影响NLRP3炎症小体和自噬进而促进胶原合成的分子机理。（2）探讨mir-21在AngII或BLM诱导的肺纤维化中作用及机制。深入研究了AngII诱导的mir-21通过靶向降解Spry1激活ERK/NF-kB/NLRP3炎症小体通路促进胶原合成的分子机理。体内研究发现烟熏可诱导大鼠发生肺纤维化，同时伴随着氧化还原失衡，NLRP3炎症小体及自噬的激活，ACE2/Ang(1-7)作用后氧化应激水平降低、NLRP3炎症小体活化减少、自噬改善，肺纤维化得以缓解；在AngII或BLM诱导的肺纤维化肺组织mir-21水平明显升高、ERK/NF-kB/NLRP3炎症小体通路激活，持续性外源性泵入Ang（1-7）或经鼻滴注ACE2过表达慢病毒后可明显抑制AngII或BLM诱导的上述异常。体外研究发现CSE刺激可上调ROS、激活NLRP3炎症小体和不完全自噬进而促进肺纤维化，抗氧化剂NAC、NADPH氧化酶抑制剂DPI或ACE2/Ang(1-7)作用后可降低氧化应激、减轻炎症、改善不完全自噬而抑制成纤维细胞胶原合成。接着我们进一步详细探讨了不完全自噬的机制：即CSE诱到的活性氧通过影响溶酶体主要组织蛋白酶的表达和活性而影响溶酶体的功能，导致不完全自噬的发生。AngII刺激肺成纤维细胞可通过上调mir-21激活ERK/NF-kB/NLRP3炎症小体通路而促进胶原合成、抑制细胞凋亡，U0126（ERK抑制剂）、BAY（NF-kB核转位抑制剂）、慢病毒介导的mir-21干扰可抑制AngII诱导的胶原合成和凋亡抑制。Ang（1-7）或过表达ACE2的肺成纤维细胞可明显抑制AngII诱导的mir-21水平升高及mir-21下游通路的激活，而对mir-21过表达诱导的胶原合成及细胞凋亡抑制无作用。综合体内外结果我们得出：（1）CSE刺激上调ROS激活NLRP3炎症小体和不完全自噬导致炎症组分累积促进成纤维细胞胶原合成，最终加剧肺纤维化的发生。（2）ACE2/Ang（1-7）通过下调AngII诱导的mir-21升高而抑制ERK/NF-kB/NLRP3炎症小体通路激活而抑制胶原合成，进而减轻肺纤维化。