Fig 1.
(A) rhGM-CSF promotes healing of scratch wounds in HCECs. Scratch wounds were made on plates of confluent HCECs and allowed to heal in medium containing 0.1, 1.0, and 10.0 μg/ml rhGM-CSF. The wounded layers were incubated for 48 h. Wound closure was photographed at 0, 24, and 48 h after wounding. Three independent experiments were performed. Scale bar, 500㎛. (B) F-actin staining with phalloidin (red) demonstrates the morphology of migrating HCECs. Scale bar, 200㎛. (C) The migration rate of HCECs was increased by rhGM-CSF treatment in a dose-dependent manner. Plotted data are the mean migration rates of HCECs at 0, 24, and 48 h. The migration rate was defined as the percentage of resurfaced area divided by the initial wound area. *P < 0.05 vs. the migration rate of the control.
Fig 2.
In vivo corneal epithelial wound healing.
(A) rhGM-CSF promoted accelerated corneal wound healing. Slit lamp photographs showed fluorescein staining of corneal epithelial wounds in the control and rhGM-CSF (10, 20, and 50 μg/ml) treatment groups at 0, 12, 24, 36, and 48 h after debridement. (B) Plotted data are the relative percentages of the corneal wound area at 12, 24, 36, and 48 h. The area of the initial corneal epithelial wound was normalized to 100%. The wound-healing rate of rhGM-CSF-treated eyes was much faster than that of untreated eyes. *P < 0.01 vs. control; †P = 0.02 vs. control.
Fig 3.
Flow cytometry analysis of rhGM-CSF effect on HCECs.
HCEC proliferation was quantified by S-phase fraction measurement by flow cytometry. HCECs were treated with 0.1, 1.0, and 10.0 μg/ml rhGM-CSF, and flow cytometric measurements of the S-phase fraction were then performed. The data show the percentages of the cells in the S phase of the cell cycle.
Fig 4.
Evaluation of p38 MAPK signaling pathway by western blotting.
(A) rhGM-CSF induced activation of the p38 MAPK signaling pathway. HCECs were treated with different concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml) for 60 min. The expression of phosphorylated and total p38 MAPK was then analyzed by western blotting. (B) The expression levels of phosphorylated p38 MAPK were normalized to those of total p38 MAPK, which increased with rhGM-CSF treatment. Three independent experiments were performed. *P < 0.05 vs. control.