气道上皮分泌的CLCA作为防治肺内型ARDS干预新靶位的基础研究

The mechanism of the origination of pulmonary inflammation in ARDS is still unclear. Because airway epithelium is the first barrier of pulmonary immune system, the goblet cells` can secret a sort of specific calcium activated chloride channel (CLCA) when being induced by various kinds of pathogens. It is was reported that the chemokine CXCL in the alveolar of pneumonia model with CLCA-/- mouse decreased, in the meantime the number of neutrophils infiltration also dropped significantly. We also found that the content of CLCA in bronchoalveolar fluid (BALF) of intrapulmonary ARDS patients increased and would gradually decrease with the remission. Therefore, we puts forward a hypothesis that pathogen-induced CLCA can promote airway epithelial cells to produce CXCL, which can initiate and deteriorate the inflammation of ARDS patients. Based on this, this project aims to finish the researches as following. Firstly, a mouse model with intrapulmonary ARDS will be established, where the time relationship between the level of CLCA and the characteristics of ARDS will be studied. Secondly, the effects of positive and passive intervention in the expression and function of CLCA will be checked on CXCL content and disease characteristics. Thirdly, the CXCL synthesis and the signal transduction pathways will be investigated with the purifying CLCA interfering the airway epithelial cells of primary culture. Finally, whether the CLCA content of sputum and BALF in ARDS patients can act as biological predictor will be explored. The subject aims to clarify the role of airway epithelial cells and CLCA in the initiative and development of ARDS, in order to provide a new target of prevention and treatment for ARDS.

ARDS失控的炎症风暴的启动机制仍不清楚。气道上皮是肺脏第一道免疫屏障，多种病原体能诱导杯状细胞分泌特异性的钙激活Cl-通道（CLCA），且CLCA-/-肺炎小鼠肺泡趋化因子CXCL含量减少，导致中性粒细胞浸润显著降低。本课题组也发现，肺内型ARDS患者肺泡CLCA含量明显升高，并随着病情缓解而降低。因此提出“病原体诱导分泌的CLCA能促进上皮细胞产生CXCL，从而启动和加重ARDS炎症发生”的假设。基于此，本项目拟：①建立肺内型ARDS小鼠模型，研究CLCA表达水平与ARDS特征的时程关系；②正向和反向干预ARDS小鼠气道CLCA表达与功能，观察对肺内CXCL含量及疾病特征的影响；③提纯CLCA并干预培养气道上皮细胞，探讨CXCL合成及信号转导通路；④探讨ARDS患者痰液及肺泡液CLCA含量，做为疾病生物预测因子的临床价值。旨在阐明上皮细胞及CLCA在ARDS发病中的始动地位及防治靶点。

ARDS的主要科学问题是以中性粒细胞为主的炎症风暴启动机制仍不清楚。气道上皮是具有生物学活性的肺脏第一道防御屏障，其在肺內型ARDS（ARDSp）形成中的角色尚无研究。内毒素能诱导上皮杯状细胞分泌特异性的钙激活Cl-通道（Calcium activated chloride channel,CLCA），分为异种同源的人hCLCA1和小鼠mCLCA3。基于此，本课题组提出“病原体诱导气道上皮分泌的CLCA能启动和（或）加重ARDSp炎症发生”的假设。本研究内容拟通过临床、在体动物及离体细胞模型，探讨CLCA在ARDSp发生中的角色及机制。主要结果：1. hCLCA1与ARDSp患者临床预后相关性的临床研究：ARDSp患者BALF中hCLCA1、TNF-α含量及中性粒细胞百分比均明显高于CAP患者及正常肺叶者。ARDSp患者hCLCA1含量与炎症指标呈正相关性，生存组的hCLCA1含量明显低于死亡组。2. mCLCA3在ARDS小鼠早期炎症及损伤中的作用：LPS致ARDS小鼠肺损伤水平在6h达到高峰，之后逐渐缓解。ARDS小鼠BALF中IL-1β、TNF-α在2h达到高峰，IL-6、CXCL-1、CXCL-2在6h达高峰，之后均呈逐渐下降趋势。ARDS小鼠肺组织mCLCA3表达在2~6h达高峰，之后渐降。Ab-mCLCA3干预后，肺损伤程度及部分炎症因子水平下降。3. mCLCA3对肺泡巨噬细胞的活化作用：mCLCA3对肺泡内巨噬细胞具有活化作用，并提高CXCL-1、CXCL-2等炎性因子表达水平。4.mCLCA3在ARDS小鼠后期炎症及损伤中的作用：mCLCA3在LPS诱导ARDS模型24h时表达水平下降，黏蛋白MUC5AC表达水平仍高，两者表现出反向趋势。CLCA抗体干预未能降低ARDS炎症水平，提示巨噬细胞在炎症风暴后期扮演非重要角色，此时中性粒细胞是主要功能细胞。NFA能降低黏蛋白MUC5AC水平，并加重ARDS损伤程度，推测此时高水平的黏蛋白发挥着新近报道的抑制炎症的保护性作用。此时“CLCA/MUC5AC平衡”的极向向后者偏移，对于ARDS炎症具有一定的抑制效应。综述所述，气道上皮CLCA在ARDSp早期的表达水平升高，并对炎症水平及肺损伤程度具有促进作用，与患者的预后具有正相关性。因此早期特异性抑制气道上皮CLCA表达和分泌是潜在的ARDSp防治新方案。