【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Ca²⁺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Ca²⁺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Ca²⁺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Ca²⁺、 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Ca²⁺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。更多还原

【Abstract】 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 Ca²⁺ 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 factor κB（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 Ca²⁺ 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 Ca²⁺, 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 Ca²⁺, 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 Ca²⁺ concentration and activation of the NF-κB pathway.更多还原