Klf9上调细胞内ROS水平在闭塞性细支气管炎中的作用及机制研究

Bronchiolitis obliterans (BO) is a kind of small airway disorders, which is caused by inflammation and fibrosis of the bronchiolar walls and contiguous tissues, and characterized by small airway narrowing and/or complete obliteration. It is a rare but severe condition affecting the quality of life seriously. However, the pathogenesis of BO has not been elucidated, and there are no effective treatments. According to existing studies, oxidative stress plays an important role in the pathogenesis of BO, and Kruppel-like factor 9 (Klf9) can increase the body oxidative stress through up-regulating the intracellular reactive oxygen species. As shown in our preliminary studies, blocking the expression of Klf9 can decrease oxidative stress induced cell death and slow the progression of bleomycin induced pulmonary fibrosis. Thus, we make an assumption that Klf9 could also involve in BO through changing the bronchiolar oxidative stress, and might become a therapeutic target of BO. To verify this hypothesis, we intend to conduct experiments on Human small airway epithelial cells and murine (wild type and Klf9 knockout) BO models, in order to illuminating the relationship between Klf9 and oxidative stress induced cell death, airway inflammation and fibrosis, aiming to explore the mechanism of Klf9 in the process of BO and provide a different angle to treating BO.

闭塞性细支气管炎（BO）是一种小气道纤维化性疾病，可导致小气道狭窄或完全闭塞，严重影响患者的生活质量及生存率。迄今BO发病机制尚未完全阐明，缺乏有效治疗，预后不良。现有研究表明氧化应激是BO重要发病机制之一，而Kruppel样转录因子9（Klf9）能通过上调细胞内活性氧水平加重机体氧化应激状态。我们前期发现阻断Klf9能有效减轻氧化应激性细胞损伤及肺纤维化的进程。因此推测Klf9也很可能通过干预氧化应激参与BO这一小气道纤维化性疾病的发病，或将成为BO治疗的新靶点。本研究拟应用人小气道上皮细胞和小鼠（正常小鼠/Klf9基因敲除小鼠）BO模型从体外、体内两方面，BO病程的多个时间点，以及在外界不同氧化应激压力下，观察Klf9与氧化应激性细胞死亡、小气道炎症及纤维化的关系，以期阐明Klf9在BO中的作用及机制，为BO的防治提供新思路。

为证实在BO发病过程中，增加气道氧化应激压力能够诱导Klf9表达上调，且Klf9表达上调后可通过进一步上调细胞内ROS水平加重BO病情，而阻断Klf9通路则可减轻气道炎症及纤维化，增强BO患者对氧化应激的耐受力，本研究应用小鼠BO模型以及人小气道上皮细胞（HSAEpiC）为研究对象揭示Klf9与BO的关系。结果显示Klf9与Nrf2参与的H2O2诱导HSAEpiC氧化应激反应呈正相关。Klf9基因过表达后可以明显提高HSAEpiC对H2O2的敏感性，Klf9基因沉默后可以明显降低HSAEpiC对H2O2的敏感性，在细胞水平上证明了Klf9在氧化应激损伤中的作用。并初步探讨分子机制可能与AMPK/Akt/GSK3β信号通路有关。本研究的难点仍然集中于动物实验部分。研究组成功构建了硝酸雾化吸入BO小鼠模型，但在将BO小鼠饲养于高浓度氧中加大氧化压力后急性期死亡率极高，成模后病理改变分布不均匀，难以采集到病变部位，导致动物实验结果不稳定。现有研究结果能够证明Klf9在氧化应激中具有重要调节作用，因此后期进行了Klf9在急性肺损伤中的作用机制研究，以及应用小分子药物对其保护机制的探讨。研究成果发表SCI论文1篇，中文核心期刊1篇，正在投稿SCI论文1篇。