HDAC6通过α-tubulin乙酰化调整骨架蛋白结构在香烟烟雾诱导支气管上皮EMT现象的机制研究

Chronic obstructive pulmonary disease(COPD) is characterized with airway remodeling and airflow limitation. The current medicines are basically bronchodilators, which cannot resolve the airway limitation essentially. Our previous work has shown that cigarette smoke could induce epithelial mesenchymal transition(EMT), then produce the airway remodeling characterized with airway wall thickness and fibrosis. But the potential mechanism has not been revealed yet. The activity of HDAC6 is increased by the stimulation of cigarette smoke; while HDAC6 is one of the most important regulatory factors of deacetylation of α-tubulin. Deacetylation of α-tubulin could lead to the reorganization of cytoskeleton proteins, then change the morphology and motility of epithelial cells, which participate into EMT process. Thus in this study, we aim to use the wildtype mice, bronchial epithelial specific HDAC6 knockout mice and human bronchial epithelial cells ,being stimulated with cigarette smoke and cigarette smoke extract, to investigate the role and the activity of HDAC6 after the stimulation of cigarette smoke and cigarette smoke extract, its regulation of deacetylation of α-tubulin, the relationship of acetylated α-tubulin and TGF-β1, Smad, and the effect of the cigarette smoke induced EMT, so as to clarify the mechanism of HDAC6 in the pathophysiology of cigarette smoke induced EMT. This study tries to find the potential mechanism of the airflow limitation and airflow remodeling of COPD, and lay basis for the therapeutic target fundamentally.

慢性阻塞性肺疾病(慢阻肺)最重要的病理机制为与气道重塑相关的不可逆性气流受限，目前的治疗仅为扩张支气管药物，不能从根本上解决气流受限。我们前期研究发现香烟烟雾能引起小气道的上皮细胞间充质化（EMT）改变，引起以气道壁的增厚及纤维化为特征的气道重塑，但机制未明。HDAC6在香烟烟雾刺激后活性增高，且是α-tubulin的去乙酰化重要的调控因子。乙酰化α-tubulin的降低可重组骨架蛋白，改变上皮细胞的形态，迁移能力等，参与EMT。本项目拟利用野生型小鼠及支气管上皮特异性HDAC6敲除小鼠，以及原代支气管上皮细胞给予香烟烟雾或其提取物刺激；以HDAC6的活性，对乙酰化α-tubulin的调控，及其与TGF-β1、Smad的关系等为研究重点，阐释HDAC6在香烟烟雾诱导EMT现象中的作用机制。该研究有助于明确慢阻肺患者气流受限及气道重塑的发病机制，为从根本上解决慢阻肺气道重塑提供借鉴。

背景: 慢性阻塞性肺疾病(COPD)是一种以气道和/或肺泡异常导致的进行性气流限制为主要特征的慢性炎症性气道疾病，气道重构和气道屏障功能障碍是其重要特征。上皮间充质转化(EMT)是气道重塑的核心病理生理因素。HDACs是一类能够修饰组蛋白去乙酰化，调节染色质状态，影响基因表达的蛋白酶。.主要研究内容：本项目明确了HDAC6高选择性抑制剂CAY10603能够减弱香烟烟雾暴露诱导的小鼠肺气肿和气道炎症。构建完成髓系细胞特异性敲除HDAC6的小鼠，并构建了香烟烟雾暴露模型，髓系细胞特异性敲除HDAC6后香烟烟雾诱导的肺气肿和骨骼肌功能障碍的改变情况。.重要研究结果：HDAC6抑制剂可减轻CS诱导的COPD小鼠肺气肿和气道炎症，通过调节上皮屏障功能障碍和逆转EMT抑制CS诱导的小气道重塑。髓系细胞特异性敲除HDAC6能够显著减弱香烟烟雾诱导的肺气肿和骨骼肌功能障碍。.关键数据及科学意义： CAY10603治疗能够逆转香烟烟雾暴露诱导的肺泡平均截距增加和平均肺泡数降低，抑制了CS介导的IL-6和TNF-α的表达上调，减少CS诱导的小气道壁增厚和纤维蛋白沉积，增加小气道上皮细胞中ZO-1和Occludin的表达水平，可有效逆转香烟烟雾诱导的气道上皮屏障功能破坏。香烟烟雾暴露可减少小气道上皮E-cadherin表达并增加间质细胞标志物α-SMA的表达水平；CAY10603治疗组小鼠的小气道上皮细胞的EMT变化得到显著的逆转。HBE细胞在TGF-β作用下上皮细胞标记物降低，间皮细胞标记物增高；使用CAY10603后EMT现象得到部分逆转。在TGF-β作用下HBE细胞p-smad2和p-smad3蛋白表达增高；使用HDAC6i-CAY后p-smad2和p-smad3蛋白表达降低，说明HDAC6i对于EMT的调控现象通过TGF-β及smad-2/3的磷酸化途径。髓系细胞特异性敲除HDAC6能够显著减弱香烟烟雾诱导的小鼠肺气肿和骨骼肌功能障碍。进一步分离并培养了髓系细胞特异性敲除HDAC6的小鼠及同窝对照小鼠的骨髓来源巨噬细胞，RNA-seq结果显示：巨噬细胞中HDAC6参与调控了多种免疫相关通路。.上述结果表明HDAC6抑制剂可减轻CS诱导的COPD小鼠肺气肿和气道炎症，并对远隔脏器如肌肉等产生保护性作用，可作为慢阻肺潜在治疗靶点。