摘要
水通道蛋白5敲除对呼吸道粘液表达谱的影响及其信号转导机制的研究
第一部分
水通道蛋白5在慢性哮喘小鼠模型气道炎症和粘液分泌中的作用
目的气道粘液高分泌是慢性气道疾病的特征,水通道蛋白5对于呼吸道液体分泌的量起着很重要的作用,本研究从动物水平探讨在屋尘螨致敏的慢性哮喘小鼠模型中,水通道蛋白5基因敲除对气道炎症和粘液分泌的影响。
方法应用水通道蛋白5基因敲除小鼠和野生型小鼠,屋尘螨滴鼻致敏制备慢性哮喘小鼠模型,HE检测病理、炎症评分,AB-PAS染色检测小鼠气道杯状细胞的增生、免疫组化和Realtime-PCR检测小鼠气道粘蛋白MUC5AC、MUC5B的表达及定量,ELISA检测支气管肺泡灌洗液(BALF)细胞因子IL-2、IL-4、IL-10、IFN-γ和MUC5AC的表达。
结果屋尘螨慢性滴鼻致敏后,和野生型小鼠相比,水通道蛋白5基因敲除鼠病理评分减轻,BALF中Th2细胞因子减少,Thl细胞因子增加,BALF液中MUC5AC蛋白、肺组织中MUC5AC、MUC5B基因和蛋白表达减少。
结论水通道蛋白5基因敲除慢性哮喘小鼠过敏性炎症和气道粘液分泌较野生型小鼠减轻。
第二部分
水通道蛋白5对PMA诱导的原代小鼠气道上皮细胞MUC5AC合成的影响
目的气道上皮是呼吸道和外环境相互作用的非特异性屏障,原代培养的气道上皮细胞保留在体上皮细胞的特征和功能,是研究呼吸道上皮细胞很好的模型。本研究从细胞水平,探讨AQP5对PMA诱导的原代培养小鼠气道上皮细胞MUC5AC合成的影响及可能机制。
方法原代培养两种小鼠(水通道蛋白5基因敲除鼠和野生鼠)气道上皮细胞,transwell建立气液平面,2周后扫描电镜及角蛋白免疫组化鉴定气道上皮细胞。PKC特异性激动剂PMA刺激原代小鼠气道上皮细胞及PKC通路特异性阻断剂Calphostinc阻断该通路,ELISA检测两组小鼠MUC5AC蛋白水平的变化,用western blotting检测两组小鼠气道上皮细胞PMA刺激后PKC、p-PKC、p-p38、p38、ERK、p-ERK的表达差异。
结果气液平面培养后,扫描电镜显示培养的细胞上皮有微绒毛和纤毛覆盖,细胞角蛋白14免疫组化染色为阳性。PMA 20ng/ml刺激24小时后,两组小鼠气道上皮细胞分泌的MUC5AC明显升高,水通道蛋白5基因敲除鼠增加更显著,两者有显著性差异。PKC特异性阻断剂Calphostinc能阻断PMA引起的两组小鼠MUC5AC分泌。原代小鼠气道上皮细胞经PMA刺激后,western blotting检测显示PKC、p38磷酸化激活,ERK通路无变化。
结论AQP5通过Ca2+-PKC-p38信号通路影响粘蛋白MUC5AC的合成和分泌。
第三部分
LPS诱导的粘液下腺细胞MUC5AC和AQP5的变化和调控机制
目的气道粘液分泌物由水和粘蛋白MUC5AC等组成,水／粘蛋白的比例失调导致痰液粘稠,影响纤毛清除功能。本研究从细胞水平探讨在LPS刺激粘液下腺SPC-A1细胞后,MUC5AC和AQP5的变化和调控机制。
方法用LPS刺激SPC-A1细胞,Realtime-PCR检测AQP5和MUC5AC mRNA水平的变化,western blotting、ELISA法检测AQP5和MUC5AC蛋白的变化。用EGFR抑制剂AG1478及MAPKs抑制剂(ERK抑制剂PD98059、p38 MAPK抑制剂ML3404、JNK抑制剂SP600125)干预,LPS刺激SPC-A1细胞6小时后Realtime-PCR检测AQP5和MUC5ACmRNA的表达。
结果LPS刺激SPC-A1细胞后,MUC5AC基因和蛋白水平呈时间、浓度依赖性升高,同时AQP5基因和蛋白水平呈时间、浓度依赖性降低,两者呈负相关。EGFR抑制剂、p38/JNK抑制剂显著降低LPS诱导的MUC5ACmRNA的升高,p38/JNK抑制剂减轻AQP5mRNA的降低。
结论P38/JNK抑制剂为AQP5和MUC5AC共同的信号通路,应用P38/JNK抑制剂能同时减轻LPS诱导MUC5AC和AQP5的变化,改善水／粘液比例。
PartⅠ
Potential Effects of Aquaporin-5 in Allergic Inflammation and Airway Mucin Secretion in a Murine Model of Asthma
Objective Airway mucus hypersecretion is one of the characteristics of chronic airway diseases. The aquaporin (AQP)-5 has been proposed to contribute to the volume of liquid secreted from the upper airways. The purpose of the present study was to determine whether deletion of AQP5 impacts on mucin hypersecretion in the lower airways with dust mite (HDM)-induced chronic allergic pulmonary inflammation in AQP5+/+and AQP5-/-mice.
Methods Bronchoalveolar lavage (BAL) levels of IL-2, IL-4, IL-10, interferon (IFN)-gamma and MUC5AC, and number of peribronchial and perivascular cells were measured.
Results We found that HDM induced more airway inflammation, lung Th2 cell accumulation and mucin hypersecretion in the lungs of C57BL/6 mice rather than AQP5-/-mice. Lung tissue expression of MUC5AC and MUC5B protein and gene expression was significantly reduced in AQP5 knockout mice.
Conclusions Our results implicate involvement of AQP5 in mucin secretion in a mouse model of chronic asthmatic inflammation.
PartⅡ
Role of AQP5 in PMA induced MUC5AC mucin production in murine tracheal epithelia cells.
Objective Primary culture of airway epithelia is a good model for studying differentiation process of epithelial cells. In this study we explored the role of AQP5 in PMA induced MUC5AC mucin production in murine tracheal epithelia cells and possible signaling pathways.
Methods Murine tracheal epithelia cells grown at an air-liquid interface (ALI) were evaluated by scanning electron microscopy at 2 weeks of culture. After murine tracheal epithelia cells were stimulated with PMA for 24 hours, MUC5AC was determined by ELISA. Cells also were pretreated with PKC inhibitor Calphostinc after PMA stimulation. Western blotting was used to assess activation of the protein kinase C (PKC), p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2.
Results Scanning electron micrograph demonstrated the cells were covered with numerous microvillus. Expression of cytokeratin 14 in mouse tracheal epithelial cells was positive. MUC5AC increased more in tracheal epithelia cells of AQP5 (-/-) mice than AQP5 (+/+) mice after PMA exposure. This effect was diminished in cells pretreated with Calphostinc. The phosphorylation of PKC/p38 but not (ERK) 1/2 induced by LPS was activated.
Conclusions AQP5 affected MUC5AC production through Ca2+-PKC-p38 signaling pathways.
PartⅢ
Aquaporin 5 Expression Inhibited by LPS via p38/JNK Signaling Pathway in SPC-A1 Cells
Objective While goblet cell hyperplasia and mucus hypersecretion had been recognized as typical features of chronic obstructive pulmonary disease (COPD), recent studies suggested association of mucin secretion by submucosal glands cell with aquaporin 5 expression, or to be precise, decreased AQP5 expression correlated with increased staining of MUC5AC in submucosal glands of COPD patients. LPS is universally used to induce airway mucin secretion in vitro.
Methods After SPC-A1 cells were stimulated with LPS, AQP5 and MUC5AC mRNA were determined by real-time PCR, AQP5 and MUC5AC protein were measured by western blotting and ELISA. SPC-A1 cells were pretreated with EGFR inhibitor AG1478, ERK inhibitor PD98059, p38 inhibitor ML3404, or JNK inhibitor SP600125 before LPS stimulation. AQP5 and MUC5AC mRNA were determined by RT-PCR.
Results AQP5 transcription and protein expression were decreased while MUC5AC expression was increased by LPS exposure in SPC-A1 cells. Further studies revealed that, AQP5 expression was down-regulated via p38/JNK signaling pathway, while MUC5AC was up-regulated through EGFR-p38/JNK pathway.
Conclusions Therefore, p38 and JNK, their common signaling pathways, may become a promising target in our efforts of preserving AQP5 expression and preventing MUC5AC over expression to restore proper H2O/mucin ratio of airway mucus, which may be ultimately beneficial to COPD patients.
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