南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (10): 1432-1438.doi: 10.12122/j.issn.1673-4254.2020.10.08

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薄荷醇增强白介素-13诱导的人支气管上皮细胞粘液蛋白5AC的合成与分泌

张明洋，王 静，李敏超   

  • 出版日期:2020-10-20 发布日期:2020-10-20

Menthol enhances interleukin-13-induced synthesis and secretion of mucin 5AC in human bronchial epithelial cells

  • Online:2020-10-20 Published:2020-10-20

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摘要： 目的 研究白细胞介素-13（IL-13）联合薄荷醇对人支气管上皮细胞（16HBE）黏液蛋白（MUC）5AC合成及分泌的影响，并探索瞬时受体电位通道（TRP）melastatin 8（TRPM8）和抗凋亡因子B淋巴细胞瘤-2（Bcl-2）在此环节中所起的作用。方法 将16HBE细胞分为：对照组、IL-13组（培养基加入终浓度10 ng/mL的IL-13连续刺激）、薄荷醇组（于第6天加入1 mmol/L薄荷醇刺激）、IL-13+薄荷醇组（IL-13连续刺激6 d后加入1 mmol/L薄荷醇刺激），观察各组MUC5AC表达量、胞内Ca2+浓度和Bcl-2表达；予以Bcl-2抑制剂ABT-263、TRPM8离子通道特异性抑制剂BCTC干预，观察两者对各组MUC5AC的影响及BCTC对胞内Ca2+浓度、Bcl-2表达的影响。CCK-8法检测细胞活力，流式细胞术检测各组细胞内Ca2+荧光强度，实时荧光定量PCR检测MUC5AC及Bcl-2mRNA水平，酶联免疫吸附法检测培养液中MUC5AC含量。结果 IL-13组、薄荷醇组、IL-13+薄荷醇组MUC5AC mRNA和蛋白的表达均明显高于对照组（P<0.05），且IL-13+薄荷醇组最高并于24 h达到最高峰（P<0.01）；薄荷醇组、IL-13组、IL-13+薄荷醇组胞内Ca2+荧光强度、Bcl-2mRNA表达均明显高于对照组（P<0.05），且IL-13+薄荷醇组最高（P<0.01）；予以BCTC干预后，薄荷醇组、IL-13+薄荷醇组胞内Ca2+荧光强度、Bcl-2mRNA、MUC5AC mRNA和蛋白表达水平均显著低于相应的未干预组（P<0.05），而IL-13组未发生明显变化（P>0.05）；予以ABT-263干预后，各ABT-263干预组MUC5ACmRNA和蛋白表达水平均显著低于相应的未干预组（P<0.05）。结论 IL-13联合薄荷醇对于16HBE细胞MUC5AC的合成及分泌产生协同效应，其机制可能与TRPM8受体诱导的Bcl-2协同性增强有关。

关键词: 支气管哮喘, 白细胞介素-13, 薄荷醇, 黏液蛋白5AC, 抗凋亡蛋白, 瞬时受体电位melastatin 8

Abstract: Objective To investigate the effect of interleukin (IL) -13 combined with cold stimulation on synthesis and secretion of mucin (MUC) 5AC in human bronchial epithelial cell line 16HBE and explore the role of transient receptor potential 8 (TRPM8) and anti-apoptotic factor B-cell lymphoblast-2 (Bcl-2) in this process. Methods 16HBE cells were stimulated with 10 ng/mL IL-13, 1 mmol/L menthol, or both (1 mmol/L menthol was added after 6 days of IL-13 stimulation), and the changes in the expression of MUC5AC, intracellular Ca2 + concentration and Bcl-2 expression were evaluated. The effects of ABT-263 (a Bcl-2 inhibitor) and BCTC (a TRPM8 ion channel inhibitor), alone or in combination, on MUC5AC expression in the cells were tested, and the changes in intracellular Ca2 + and Bcl-2 expression following BCTC treatment were observed. The cell viability was assessed using CCK-8 assay, the mRNA expressions of MUC5AC and Bcl-2 were detected with real-time quantitative PCR, the level of MUC5AC in the culture medium was measured with ELISA, and the intracellular Ca2 + fluorescence intensity was determined with flow cytometry. Results The mRNA and protein expressions of MUC5AC increased significantly in 16HBE cells following stimulation with IL-13, menthol, and both (P<0.05), and were the highest in the combined treatment group with its peak level occurred at 24 h (P<0.01). The intracellular Ca2 + fluorescence intensity and Bcl-2 mRNA expression were also increased in 16HBE cells after the stimulations (P<0.05), and the increments were the most obvious in the combined treatment group (P<0.01). Treatment with BCTC significantly lowered intracellular Ca2 + fluorescence intensity and the expressions of Bcl-2 and MUC5AC mRNA and protein in the cells stimulated with menthol or with both IL-13 and menthol (P<0.05), but caused no significant changes in IL-13-stimulated cells (P>0.05). Treatment with ABT-263 significantly lowered the mRNA and protein expressions of MUC5AC in the cells stimulated with IL-13 and menthol either alone or in combination (P<0.05). Conclusion Menthol combined with IL-13 produces a synergistic effect to promote the synthesis and secretion of MUC5AC in 16HBE cells possibly by activating TRPM8 receptor to upregulate the expression of Bcl-2.

Key words: bronchialasthma, interleukin-13, menthol, mucin 5AC, anti-apoptotic protein, transient receptor potential melastatin 8