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  

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

作　　者：张明洋 王静[1] 李敏超[1] ZHANG Mingyang;WANG Jing;LI Minchao(Department of Respiratory Medicine,Second Affiliated Hospital,Chongqing Medical University,Chongqing 400010,China)

机构地区：[1]重庆医科大学附属第二医院呼吸内科,重庆400010

出　　处：《南方医科大学学报》2020年第10期1432-1438,共7页Journal of Southern Medical University

基　　金：国家自然科学面上基金(81270102);重庆市自然科学基金(cstc2012jjA10050);重庆市教委科学技术研究项目(KJ120301)。

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。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

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 

分 类 号：R562.25[医药卫生—呼吸系统]