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

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 in vivo and on P2Y2R-related downstream signaling pathways in vitro. We administered 3% diquafosol ophthalmic solution on 3 mm-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 24 h. 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 in vitro experiments. Cell proliferation was accelerated by diquafosol at concentrations from 20 to 200 μM during 48 h, but inhibited at concentrations over 2000 μM. The intracellular calcium ([Ca(2+)]i) elevation was measured in diquafosol (100 μM)-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 in vitro cell migration assay. Diquafosol induced phosphorylation of EGFR at 2 min post-stimulation, and phosphorylation of ERK at 5 min 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.

Ocular Effects of Sulfur Mustard and Therapeutic Approaches

“…They showed that topical administration of RvE1 significantly increased tear production and down regulates cyclooxygenase-2 (COX-2) expression, and increased the density of superficial epithelial cells in the dry-eyed mouse model [Li et al, 2010]. Diquafosol is reported to be beneficial in tear film stabilization and repair of corneal epithelial damages [Yarmola et al, 2007;Bremond-Gignac et al, 2014;Byun et al, 2016]. Diquafosol sodium and rebamipide.…”

“…This drug is a P2Y2 purinergic receptor agonist that stimulates the receptors in ocular tissues and thus increases mucin (conjunctival goblet cells stimulation) and the aqueous portion of tear film (conjunctival epithelial cells stimulation) [Shigeyasu et al, 2015]. Diquafosol is reported to be beneficial in tear film stabilization and repair of corneal epithelial damages [Yarmola et al, 2007;Bremond-Gignac et al, 2014;Byun et al, 2016].…”

Comparison of cytotoxicities and wound healing effects of diquafosol tetrasodium and hyaluronic acid on human corneal epithelial cells

“…Interestingly, we observed this effect at a low concentration (2 mg/ml). Byun et al reported that the effect of diquafosol on corneal epithelial healing was accelerated at concentrations from 20 to 200 µM, but inhibited at concentrations over 2000 µM [23]. This concentration dependence might be related to BAC concentrations in diluted diquafosol, and that whereas diquafosol promotes corneal epithelial wound healing, BAC at higher concentration inhibits this process.…”

Corneal Blindness Caused by Mustard Gas

“…Diquafosol is a P2Y2 purinergic receptor agonist that stimulates the receptors in ocular tissues and thus increases mucin (conjunctival goblet cells stimulation) and the aqueous portion of tear film (conjunctival epithelial cells stimulation) [37]. Three percent diquafosol ophthalmic solution is effective for the treatment of dry eye disease through tear film stabilization and repair of corneal epithelial damages [38,39]. Another P2Y2 receptor agonist is uridine.…”