Exposure to cigarette smoke extract and lipopolysaccharide modifies cytoskeleton organization in bronchial epithelial cells
Exposure to cigarette smoke extract and lipopolysaccharide modifies cytoskeleton organization in bronchial epithelial cells
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DOI：
10.1080/01902148.2017.1377784
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发表时间：
2017-01-01
影响因子：
1.7
通讯作者：
Pace, Elisabetta
中科院分区：
文献类型：
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作者：
D'Anna, Claudia;Cigna, Diego;Pace, Elisabetta
呼吸道上皮的完整性对于气道稳态至关重要。烟草烟雾暴露和气道反复感染在慢性阻塞性肺病（COPD）呼吸功能的进展和下降中起着至关重要的作用。本研究的目的是通过使用二维电泳 (2DE) 分析结合基质辅助激光解吸/电离飞行时间质谱法，检测单独和/或组合使用香烟烟雾提取物 (CSE) 和脂多糖 (LPS)（革兰氏阴性菌的成分）刺激的支气管上皮细胞系 (16-HBE) 中的差异表达蛋白。应用蛋白质印迹分析来确认显着调节的蛋白质的表达。流式细胞术和免疫荧光用于通过鬼笔环肽法评估F-肌动蛋白聚合。在不同的实验点鉴定出 14 种蛋白质，其强度有显着变化（p < 0.05）：6 种上调，8 种下调。正如预期的那样，生物信息学分析表明，大多数这些蛋白质都参与抗氧化和免疫反应以及细胞骨架稳定性。 Western blot 分析证实：CSE 和 LPS 共同暴露后，蛋白酶体激活剂复合物亚基 2 (PSME2)、Peroxiredoxin-6 (PRDX6)、Annexin A5 (ANXA5) 和热休克蛋白 beta-1 (HSPB1) 减少，辅乳糖蛋白样蛋白 (COTL-1) 增加。此外，LPS 和 CSE 增加了肌动蛋白聚合。总之，虽然需要进一步的验证研究，但我们的研究结果表明，CSE 和 LPS 可能导致肺功能进行性恶化，改变支气管上皮细胞中参与代谢过程和细胞骨架重排的蛋白质的表达。
The integrity of the respiratory epithelium is crucial for airway homeostasis. Tobacco smoke exposure and recurrent infections of the airways play a crucial role in the progression and in the decline of the respiratory function in chronic obstructive pulmonary disease (COPD). The aim of this study was to detect differentially expressed proteins in a bronchial epithelial cell line (16-HBE) stimulated with cigarette smoke extract (CSE) and lipopolysaccharide (LPS), a constituent of gram-negative bacteria, alone and/or in combination, by using two-dimensional electrophoresis (2DE) analysis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot analysis was applied to confirm the expression of significantly modulated proteins. Flow cytometry and immunofluorescence were used to assess F-actin polimerization by phalloidin method. Fourteen proteins, with significant (p < 0.05) changes in intensity, were identified at various experimental points: 6were up-regulated and 8were down-regulated. As expected, bioinformatic analysis revealed that most of these proteins are involved in anti-oxidant and immune responses and in cytoskeleton stability. Western blot analysis confirmed that: Proteasome activator complex subunit 2 (PSME2), Peroxiredoxin-6 (PRDX6), Annexin A5 (ANXA5) and Heat shock protein beta-1 (HSPB1) were reduced and Coactosin-like protein (COTL-1) was increased by co-exposure of CSE and LPS. Furthermore, LPS and CSE increased actin polimerization. In conclusion, although further validation studies are needed, our findings suggest that, CSE and LPS could contribute to the progressive deterioration of lung function, altering the expression of proteins involved in metabolic processes and cytoskeleton rearrangement in bronchial epithelial cells.