人气道上皮细胞感知冷刺激的受体机制

Cold-induced signal transduction mechanism in airway epithelial cells

目的探讨温度感受器瞬时受体电位蛋白-8(transient receptor potential melastatin 8,TRPM8)在人气道上皮细胞感受冷刺激过程中发挥的作用及其机制。方法用薄荷醇及冷空气(18℃)刺激人气道上皮16HBE细胞:1以TRPM8通道特异性拮抗剂BCTC、TRPM8 shRNA为干预手段,动态观察在薄荷醇、冷刺激作用下细胞内Ca2+荧光强度变化来监测气道上皮细胞上TRPM8的功能;2以磷脂酰肌醇4,5-二磷酸(phosphatidylinositol 4,5-bisphosphate,PIP2)荧光分子探针PLCδ1-PH-GFP转染细胞,通过动态观察各组细胞内PLCδ1-PH-GFP从细胞膜至细胞质的荧光转位变化来检测PIP2的动态变化来研究冷刺激作用下TRPM8的信号通路。结果 1细胞内相对钙离子浓度变化:薄荷醇组处理4 min后、冷刺激组处理6 min后分别为(5.80±0.34)、(5.02±0.36),明显高于对照组(1.04±0.12)、(1.03±0.08)(P<0.05),1 mmol/L薄荷醇组+BCTC组、1 mmol/L薄荷醇组+转染TRPM8 shRNA组最高值分别为(2.68±0.19)、(1.08±0.16),明显低于1 mmol/L薄荷醇组,18℃+BCTC组、18℃+转染TRPM8 shRNA组最高值分别为(1.77±0.22)、(1.02±0.18),明显低于18℃组(均P<0.05);2PLCδ1-PH-GFP从细胞膜至细胞质的转位变化(Fm/Fc值):1 mmol/L薄荷醇组、冷刺激组最高值分别为(0.90±0.01)、(0.90±0.01),明显高于对照组最高值(1.01±0.02)(P<0.05),细胞处理200 s时1 mmol/L薄荷醇+BCTC15μmol/L组、1 mmol/L薄荷醇组+转染TRPM8 shRNA组分别为(0.86±0.03)、(0.89±0.02),明显低于1 mmol/L薄荷醇组(0.36±0.06)(P<0.05),细胞处理220 s时18℃+BCTC15μmol/L组、18℃+转染TRPM8 shRNA组分别为(0.82±0.02)、(0.93±0.01),明显低于18℃组(0.40±0.06)(P<0.05)。结论TRPM8受体是人气道上皮细胞感受冷刺激的主要受体,并通过TRPM8→Ca2+→PLC→PIP2信号通路引起气道黏液高分泌等反应。

Objective To explore the role of transient receptor potential melastatin 8（ TRPM8）receptor and related cold-induced signal transduction mechanism in airway epithelial cells.Methods The 16 HBE human airway epithelial cells were treated with cold temperature（ 18 ℃） and menthol.By means of Ca^2＋imaging,we explored the function of TRPM8 when the cells were pretreated with TRPM8 channel antagonist BCTC and exon-18-specific TRPM8 shRNA.Hydrolysis of phosphatidylinositol 4,5-bisphosphate（ PIP2） were characterized by means of spatiotemporal dynamics of phospholipase C（ PLC） δ1-pleckstrin homology-green fluorescent protein（ δ1-PH-GFP）.Results The concentration of intercellular calcium in the menthol and cold air treatment group（ 5.80 ± 0.34,5.02 ± 0.36） higher than that in the control group（ 0.94 ± 0.12）（ P〈0.05）.The intracellular calcium concentration was in the menthol ＋ BCTC and menthol ＋ TRPM8 shRNA groups（ 2.68 ± 0.19,1.08 ± 0.16） than in the menthol treatment group（ P〈0.05）.The intracellular calcium concentration in the cold air ＋ BCTC and cold air ＋ TRPM8 shRNA groups（ 1.77 ± 0.22,1.02 ±0.18） were lower than that in the cold air treatment group（ P〈0.05）.The translocation of PLC δ1-PH-GFP from the membrane to the cytosol（ Fm /Fc） in the menthol treatment group and cold air treatment group （ 0.90 ±0.01,0.90 ±0.01） were higher than that in the control group（ 1.01 ±0.02）（ P〈0.05）.Fm/Fc was lower in the menthol ＋ BCTC group and TRPM8 shRNA ＋ menthol group（ 0.86 ± 0.03,0.89 ± 0.02） than in the menthol treatment group（ P〈0.05）,and also lower in the cold air ＋ BCTC group and cold air ＋ TRPM8 shRNA group（ 0.82 ± 0.02,0.93 ± 0.01） than in the cold air treatment group（ P〈0.05）.Conclusion TRPM8 receptor is involved in the response to cold temperature in airway epithelial cells,and contributes to mucus hypersecretion through the TRPM8→Ca^2＋→ PLC→PIP2 signaling pathway.