Objective: To investigate the role of mammalian target of rapamycin (mTOR) activation in menthol-induced expression of airway inflammation- related factors in human bronchial epithelial cells and explore its mechanism.
Methods: Cultured human bronchial epithelial cells (BEAS-2B) were divided into normal control group, menthol group, rapamycin group, and menthol+rapamycin group with corresponding treatments. The cell viability was measured with CCK-8 method. The mRNA levels of transient receptor potential melastatin 8 (TRPM8), tumor necrosis factor (TNF)-α and interleukin (IL)-1β were detected by RT-PCR, and the protein expressions of phosphorylated mTOR (p-mTOR), TRPM8, TNF-α and IL-1β were determined using Western blotting. The intracellular Ca2+ fluorescence intensity was measured by flow cytometry.
Results: Compared with the normal control cells, menthol- treated cells showed significantly increased TNF-α, IL-1β, and p-mTOR expression and elevated intracellular Ca2+ concentration (P < 0.05), and the rapamycin-treated cells exhibited significantly decreased p-mTOR expression (P < 0.05). No significant difference was found in TNF-α, IL-1β or intracellular Ca2+ concentration between the normal control and rapamycin-treated cells (P>0.05). Compared with the menthol-treated cells, the cells treated with both menthol and rapamycin showed significantly decreased TNF- α, IL-1β, and p-mTOR expression and obviously lowered intracellular Ca2+ concentration (P < 0.05).
Conclusions: Menthol promotes the expressions of airway inflammationrelated factors IL-1β and TNF-α possibly by activating mTOR to cause the increase of intracellular Ca2+ concentration.
目的: 探讨哺乳动物雷帕霉素靶蛋白(mTOR)活化对薄荷醇诱导的人支气管上皮细胞(BEAS-2B)气道炎症相关因子表达的影响及其机制。
方法: 将细胞分为4组:正常对照组、薄荷醇组、雷帕霉素组、薄荷醇+雷帕霉素组。用CCK-8法检测细胞存活率,实时荧光定量PCR检测瞬时受体电位蛋白-8(TRPM8)、肿瘤坏死因子(TNF)-α和白细胞介素(IL)-1β mRNA的表达,Western blot检测BEAS-2B中磷酸化的mTOR(p-mTOR)、TRPM8、TNF-α和IL-1β的蛋白表达。用流式细胞术检测胞内Ca2+荧光强度。
结果: 与正常对照组相比,薄荷醇组中细胞TNF-α和IL-1β mRNA及蛋白表达升高,p-mTOR蛋白表达升高,胞内Ca2+浓度升高(P < 0.05)。与正常对照组相比,雷帕霉素组中细胞TNF-α和IL-1β mRNA及蛋白表达均无差异(P>0.05),p-mTOR蛋白表达降低(P < 0.05),胞内Ca2+浓度无差异(P>0.05)。与薄荷醇组相比,薄荷醇+雷帕霉素组中细胞TNF-α和IL-1β mRNA及蛋白表达降低,p-mTOR蛋白表达降低,胞内Ca2+浓度降低(P < 0.05)。
结论: 薄荷醇可能通过活化mTOR诱导胞内Ca2+浓度升高,进而促进气道炎症相关因子IL-1β和TNF-α的表达。
Keywords: airway inflammation; human bronchial epithelial cells; mammalian target of rapamycin; menthol; transient receptor potential melastatin 8.