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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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). CONCLUSIONS: 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.