Cell viability assay

The cell viability in each experimental group was determined by MTT assay (Sigma-Aldrich, St. Louis, MO, USA). Briefly, corneal epithelial cells (10⁵ cells/ml) were seeded in 96-well plates and incubated to reach approximately 70% confluence. The cells were treated with different concentrations (0, 1, 10, 100, 1000 or 10000 nM) MPAPO for 48 h. Then water-soluble tetrazolium salt reagent solution (10 μL) was added to each well followed by immediate incubation for 4 h at 37°C. After discarding the medium and MTT, 150 μL of DMSO was added to each well followed by incubation for another 10 min. Then, the absorbance was measured at a wavelength of 490 nm. The oxidative injury corneal epithelial cell model was established with H₂O₂. Cells were treated with different concentrations of H₂O₂ (6.25, 25, 100, 400, 1600 or 6400 μM) for 4 h, and the treatment without H₂O₂ was used as a control. The condition of cell viability of approximately 50% was selected for the establishment of a corneal epithelial cell model of H₂O₂ injury. In the experiments to evaluate the ability of MPAPO to promote cell wound repair, there were five experimental groups: normal control, PBS, PACAP27, MPAPO and MPAPO+MAX.D.4 (Sigma Aldrich, Shanghai, China) groups. The cells were preincubated with MAX.D.4 (a PAC1 receptor antagonist) for 30 min at 37°C ³⁵, and then MPAPO was added. The proliferation rate of the cells in each experimental group was determined using the MTT assay.