薄荷醇通过激活瞬时受体电位M8(TRPM8)促进BEAS-2B人支气管上皮细胞的细胞因子分泌  

Menthol promotes cytokine secretion in human bronchial epithelial BEAS-2B cells by activating transient receptor potential melastatin 8(TRPM8)

作　　者：胡艺馨 李敏超[1] HU Yixin;LI Minchao(Department of Respiratory and Critical Care Medicine,Second Affiliated Hospital of 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与危重症医学科,重庆400010

出　　处：《细胞与分子免疫学杂志》2021年第7期577-584,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(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：目的探讨瞬时受体电位M8(TRPM8)在薄荷醇诱导BEAS-2B人支气管上皮细胞表达气道上皮源性细胞因子白细胞介素25(IL-25)、IL-33和胸腺基质淋巴细胞生成素(TSLP)中的作用及其相关信号转导机制。方法用2 mmol/L薄荷醇处理BEAS-2B细胞1、2、3、4 h,选择IL-25、IL-33、TSLP或Ca^(2+)表达较高的时间组,通过小干涉RNA(siRNA)特异性敲低TRPM8(si-TRPM8),采用细胞内钙离子螯合剂1,2-二(2-氨基苯氧基)乙烷-N,N,N′,N′-四乙酸四乙酰氧甲基酯(BAPTA-AM)及核因子κB(NF-κB)抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)处理。CCK-8法检测细胞活力;实时荧光定量PCR检测IL-25、IL-33和TSLP mRNA表达;ELISA检测培养上清液IL-25、IL-33和TSLP蛋白水平;Fluo-4 AM负载结合流式细胞术检测胞内Ca^(2+)荧光强度;Western blot法检测si-TRPM8对BEAS-2B细胞合成TRPM8蛋白的干扰效率及NF-κB p65蛋白表达的影响。结果与空白对照组比,薄荷醇组IL-25、IL-33、TSLP mRNA及蛋白、胞内Ca^(2+)水平和NF-κB p65蛋白表达均增加;敲低TRPM8后,抑制薄荷醇诱导的Ca^(2+)、IL-25、IL-33、TSLP和NF-κB p65表达增加,BAPTA-AM和PDTC均可抑制薄荷醇诱导的IL-25、IL-33和TSLP的高表达。结论薄荷醇通过激活TRPM8引起Ca^(2+)浓度升高、激活NF-κB通路诱导BEAS-2B支气管上皮细胞IL-25、IL-33和TSLP的分泌。Objective To explore the role of transient receptor potential melastatin 8(TRPM8)in the expression of airway epithelial-derived cytokines interleukin 25(IL-25),IL-33 and thymic stromal lymphopoietin(TSLP)in human bronchial epithelial BEAS-2B cells induced by menthol and its related signal transduction mechanism.Methods BEAS-2B cells were treated with 2 mmol/L menthol for 1,2,3 and 4 hours.The groups with the higher expression of IL-25,IL-33,TSLP or Ca^(2+)were chosen to carry out the following experiments.The cells were transfected with siRNA of TRPM8(si-TRPM8)or negative control siRNA(si-NC)and pretreated with intracellular calcium chelator BAPTA-AM,nuclear factorκB(NF-κB)inhibitor pyrrolidine dithiocarbamate(PDTC),and then intervened with menthol.Cell survival rate was measured by CCK-8 assay.The mRNA levels of IL-25,IL-33 and TSLP were detected by real-time quantitative PCR.The protein levels of IL-25,IL-33 and TSLP were detected by ELISA.The intracellular Ca^(2+)fluorescence intensity was detected by flow cytometry with Fluo-4 AM loading.Western blotting was used to detect the interference efficiency of si-TRPM8 on BEAS-2B cells and its effect on the protein expression of NF-κB p65.Results Compared with the blank control group,the mRNA and protein expression of IL-25,IL-33 and TSLP,the level of Ca^(2+),and the protein expression of NF-κB p65 were significantly up-regulated in the menthol group.After knock-down of TRPM8,the menthol-induced increases of Ca^(2+),IL-25,IL-33,TSLP and NF-κB p65 expression were inhibited.Both BAPTA-AM and PDTC could inhibit the high expression of IL-25,IL-33 and TSLP induced bymenthol.Conclusion Menthol can induce the secretion of IL-25,IL-33 and TSLP in human BEAS-2B cells by activating TRPM8 which leads to increased Ca^(2+)concentration and activation of the NF-κB pathway.

关 键 词：瞬时受体电位M8(TRPM8) BEAS-2B细胞 钙离子 人支气管上皮细胞 白细胞介素25(IL-25) IL-33 胸腺基质淋巴细胞生成素(TSLP) 

分 类 号：R965[医药卫生—药理学] R392.11[医药卫生—药学]