Netrin-1 promotes epithelium repair in corneal injury

To explore netrin-1 functions on corneal epithelium in vitro and in vivo.In vitro the human corneal epithelial (HCE) cells were treated with serum free DMEM-F12 basic media containing 0, 50, 100, 200, 300, 500, 800, and 1000 ng/mL of netrin-1, respectively. The cells viability was detected by cell counting kit-8 (CCK-8). The wound-healing assay was applied to assess the migration proficiency of HCE cells. Flow cytometry was used to analyze the cell-cycle distribution and apoptosis. In vivo, normal c57 (6wk) mice were demarcated with a trephine in the middle of the cornea to produce a 3-mm circular wound. Mice corneas were inflicted no epithelium with a 3-mm wound displayed, but remained the limbal epithelium intact. A blunt scalpel blade was used to remove the corneal epithelian cells, followed by topical netrin-1 application (200 ng/mL), and the group treated by PBS as control. The treated group was injected netrin-1 into the normal c57 mice inferior subconjunctival 4h before trauma. Mouse corneal inflammation and neovascularization were observed under slit lamp microscope. The apoptosis of corneal cells was determined by TUNEL staining.A concentration of 200 ng/mL netrin-1 enhanced 25% of the HCE viability. The relative migration rates were 76.3% and 100% in control and netrin-1 treated group after cultured 72h. Treated with netrin-1 (200 ng/mL) decreased the apoptosis of HCE cells, as well as decreased their percentage from 19.3%±0.57% to 12.7%±0.42% of the total. The remaining wound area was 1.22 mm2 in control group but 0.22 mm2 in the netrin-1 treated group. Exogenous Netrin-1 inhibits apoptosis of corneal epithelial cells of c57 mice. TUNEL-positive cells at the epithelial layer of the corneas of the control and netrin-1 treated c57 mice at 24h after wounding were 43.3% and 16.7% respectively.Netrin-1 can reduce HCE apoptosis as well as promote its proliferation and migration. Topical application of netrin-1 promotes the injuryed cornea epithelial wound repair and inhibits apoptosis of corneal epithelial cells. These findings may offer potential therapies to repair the defects of corneal epithelial based on netrin-1.