Low Concentration Of H₂O₂ Promoted-Cell Adhesion And Migration In Cultured Rabbit Corneal Epithelial Cells

Purpose: Fibronectin-integrin system plays an essential role in cornea epithelial cell adhesion and migration. Intracellular ROS have been reported to associate with integrin signaling in promoting cell adhesion in some cell types. The purpose of this study is to investigate the effect of exogenous low dose of H₂O₂ on rabbit corneal epithelial cell adhesion and migration.Methods: Primary rabbit corneal epithelial cells were grown to confluence in SHEM medium with 5% FBS. The cells were gradually serum starved by first culturing in 0.5% FBS overnight and then serum&EGF-free medium for 30 min before use. Serum&EGF-free medium was used in all bolow test.MTT assay was used to test the viability of cells treated with various concentrations of H₂O₂ (0, 1,5, 10, 20, 50, 60, and 70 μM) for 2 and 24 hours. The favored dosages of H₂O₂ were tested for cell adhesion to fibronectin-coated plates. After centrifugation, the adhered cells were counted and photographed at 30 and 60 min. Then N-acetyl cysteine (NAC), a free radical scavenger, was used in the cell suspension before plating as antagonism of H₂O₂. The vinculin-containing focal contacts and F-actin of cells treated with low-dose-H₂O₂ were determined by immunofluorescence. Wound closure assay was used to idtify the effect of low-dose-H₂O₂ on cells migration and wound healing.Results: H₂O₂ atl⁵0μM did not reduce the viability of rabbit corneal epithelial cells compared with non-treated cells (p>0.05) , but concentrations higher than 50 μM were harmful. However, H₂O₂ at the range of 5-50 μM enhanced cell adhesion (P<0.05) and increased cell growth in a dose-dependent manner in comparison with the control (noH2O2). The enhanced adhesion were suppressed in the presence of NAC (/?<0.05) . Immunofluorescence assay confirmed that H2O2 at these low concentrations facilitated the formation of vinculin-containing focal adhesion complexes and F-actin, which were not present in the non-treated cells. In wound closure assay, H2O2 at low concentration (10uM⁵0uM) also exhibited promotion of migration and wound closure (p<0.01) .Conclusion: These findings suggest that exogenous H2O2 at low level can promote cultured rabbit corneal cell adhesion and migration to fibronectin. This is the first report of oxidation-associated cell adhesion and migration in corneal epithelial cells.cornea;epithelium;cell adhesions;fibronectin, cell migration...