[Effects of transient receptor potential melastatin 8 cation channels on inflammatory reaction induced by cold temperatures in human airway epithelial cells].

Objective: To explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) in cold-induced production of inflammatory factors in airway epithelial cells and related signal transduction mechanism.

Methods: The 16HBE human airway epithelial cells were stimulated with cold temperature (18°C). In intervention experiments, cells were pretreated with TRPM8 channel antagonist BCTC, protein kinase C (PKC) specific inhibitor calphostin C and transfected with TRPM8 shRNA or control shRNA respectively, and thereafter cold stimulation was applied. Cells were divided into 6 groups: a control group (incubated at 37°C), a cold stimulation group, a cold stimulation + BCTC group, a cold stimulation + TRPM8 shRNA group, a cold stimulation + control shRNA group, a cold stimulation + calphostin C group. Western blot was performed to show the extent of knockdown in TRPM8 protein expression in the TRPM8 shRNA transfected cells. Dynamics of relative concentration of intracellular Ca(2+) in the former 5 groups were measured by calcium imaging techniques. Images were taken at one frame per 10 seconds. The levels of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α mRNA and protein were detected by real-time PCR and ELISA respectively.

Results: The highest relative concentration of intracellular calcium in cold stimulation group (2.36 ± 0.24) was higher than that of control group (1.01 ± 0.02) (t = 12.52, P < 0.01). BCTC and TRPM8 shRNA reduced intracellular calcium (1.05 ± 0.09, 1.08 ± 0.09), compared with single cold stimulation group (t = 6.69 and 9.12, all P < 0.01). IL-6, IL-8, TNF-α mRNA and protein in cold stimulation group[0.66 ± 0.16, 0.77 ± 0.15, 0.73 ± 0.09 and (92 ± 13) ng/L, (125 ± 22) ng/L, (88 ± 12) ng/L ] were significantly higher than those in control group [0.37 ± 0.08, 0.32 ± 0.07, 0.48 ± 0.10 and (52 ± 8) ng/L, (50 ± 9) ng/L, (61 ± 8) ng/L] (t = 3.20 - 6.26, all P < 0.05). IL-6 mRNA, IL-8 mRNA, TNF-α mRNA and protein in cold stimulation + BCTC group [0.42 ± 0.09, 0.52 ± 0.13, 0.52 ± 0.12 and (72 ± 8) ng/L, (92 ± 14) ng/L, (68 ± 11) ng/L], cold stimulation + TRPM8 shRNA group [0.41 ± 0.10, 0.49 ± 0.08, 0.50 ± 0.08 and (60 ± 12) ng/L, (89 ± 14) ng/L, (68 ± 11) ng/L] and cold stimulation + calphostin C group [0.40 ± 0.07, 0.44 ± 0.09, 0.47 ± 0.08 and (69 ± 9) ng/L, (86 ± 15) ng/L, (61 ± 10) ng/L] were significantly lower than those in cold stimulation group (t = 2.47 - 4.21, all P < 0.05). IL-6 mRNA, IL-8 mRNA, TNF-α mRNA and protein in cold stimulation + control shRNA group [0.61 ± 0.10, 0.69 ± 0.11, 0.64 ± 0.13 and (89 ± 13) ng/L, (118 ± 20) ng/L, (79 ± 13) ng/L] showed no significant change, compared with cold stimulation group (t = 0.35 - 1.12, all P > 0.05).

Conclusion: Cold temperature may induce Ca(2+) influx and up-regulate IL-6, IL-8, and TNF-α expression in 16HBE cells by activating the TRPM8 ion channels, and this is via a signaling pathway involving PKC.