The human corneal epithelial cell line was donated by Professor Zuguo Liu of the Institute of Ophthalmology of Xiamen University in 2015. Cells were cultured in basal medium (DMEM/F12) mixed with 6% fetal bovine serum (Gibco, CA, USA), 1% penicillin (100 U/ml, Gibco), 1% streptomycin (100 μg/ml, Gibco), 0.1% recombinant human epithelial growth factor (7 ng/ml, Sigma) and 0.35% fetal bovine insulin (7 g/ml, Sigma); growth medium was changed at 2-day intervals. Cells were passaged when they reached 80% to 90% confluence; cells from the third to fifth passages that exhibited good morphology were used for experiments. On the day after cells had been seeded, they were washed twice with Dulbecco’s PBS (Gibco). Cells were then starved with fetal bovine serum-free basal medium overnight (16 h); they were then treated with hyperosmotic medium alone, or with hyperosmotic medium combined with CMC, α-MSH, or CMC + α-MSH. Cells were pre-incubated with CMC, α-MSH, and CMC + α-MSH at twofold greater than the desired final concentration for 30 min before hyperosmotic stimulation; an equal volume of hyperosmotic medium with twofold greater than the desired final concentration was then added to reach the desired concentration. Two LPS (Sigma, US) concentrations of 500 ng/ml and 100 ng/ml were used in this study. Pre-incubation with CMC + α-MSH was performed for 30 min, as above; an equal volume of hyperosmotic medium containing LPS and twofold greater than the desired final concentration of CMC + α-MSH was added. Mcc950 used the same incubation method as LPS. Cells in all groups were then incubated for 24 h.

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