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Experimental eye research

Diquafosol promotes corneal epithelial healing via intracellular calcium-mediated ERK activation.


PMID 26505315

Abstract

Diquafosol is known as a purinergic P2Y2 receptor (P2Y2R) agonist that stimulates water and mucin secretion from conjunctival epithelial cells and goblet cells, leading to tear film stability in dry eye. However, its effect on corneal epithelial healing has not yet been elucidated. The aim of the present study was to evaluate the effect of diquafosol on corneal epithelial healing inxa0vivo and on P2Y2R-related downstream signaling pathways inxa0vitro. We administered 3% diquafosol ophthalmic solution on 3xa0mm-diameter epithelial defects made in rat corneas and assessed the wound closure over time. Corneal epithelial healing was significantly accelerated in diquafosol-treated eyes compared to control eyes at 12 and 24xa0h. During wound healing, P2Y2R staining appeared stronger in the re-epithelized margin near the wound defect. To evaluate whether diquafosol stimulates epidermal growth factor receptor/extracellular-signal-regulated kinase (EGFR/ERK)-related cell proliferation and migration, simian virus 40-transfected human corneal epithelial (THCE) cells were used for inxa0vitro experiments. Cell proliferation was accelerated by diquafosol at concentrations from 20 to 200xa0μM during 48xa0h, but inhibited at concentrations over 2000xa0μM. The intracellular calcium ([Ca(2+)]i) elevation was measured in diquafosol (100xa0μM)-stimulated cells using Fluo-4/AM ([Ca(2+)]i indicator). [Ca(2+)]i elevation was observed in diquafosol-stimulated cells regardless of the presence of calcium in media, and suramin pretreatment inhibited the calcium response. The effect of diquafosol on phosphorylation of EGFR, ERK and Akt, and cell migration was determined by western blotting and inxa0vitro cell migration assay. Diquafosol induced phosphorylation of EGFR at 2xa0min post-stimulation, and phosphorylation of ERK at 5xa0min post-stimulation. Phosphorylation of ERK was attenuated in cells pretreated with suramin or BAPTA/AM ([Ca(2+)]i chelator), and partially with AG1478 (EGFR inhibitor). Likewise, diquafosol-treated cells showed acceleration of gap closure in cell migration assay, which was inhibited by suramin, BAPTA/AM, AG1478, and U0126 (MEK inhibitor). These studies demonstrate that diquafosol is effective in promoting corneal epithelial wound healing and that this effect may result from ERK-stimulated cell proliferation and migration via P2Y2R-mediated [Ca(2+)]i elevation.