Effects of 17β-estradiol on human corneal wound healing in vitro

To investigate the effect of 17β-estradiol on corneal wound healing, particularly on epithelial mitosis and migration.

Immortalized human corneal epithelial cells (HCECs) were cultured in media with different concentrations of 17β-estradiol (10, 50, 100, and 200 pg/mL), Dulbecco modified Eagle medium: Nutrient Mixture F-12 (negative control), and serum-containing Dulbecco modified Eagle medium: Nutrient Mixture F-12 (positive control). After 6 or 24 hours of hormone treatment, to evaluate the migratory potential of 17β-estradiol, wound healing assays were conducted via the manual scraping of HCECs and western blot analysis of fibronectin and matrix metalloproteinase 9 (MMP9). The proliferative potential of 17β-estradiol was evaluated via a proliferation assay using western blot analysis for proliferating cell nuclear antigen. In addition, epidermal growth factor (EGF) was measured by reverse transcription-polymerase chain reaction, and for the inhibition of epidermal growth factor receptor (EGFR)-mediated signal transduction, a wound healing assay was conducted after HCECs cultured with EGFR small interfering RNA were stimulated with 100 pg/mL 17β-estradiol.

Wound healing assay rates were enhanced as 17β-estradiol increased, with statistically significant changes seen in 50, 100, and 200 pg/mL 17β-estradiol-treated and positive control cells, compared with negative control cells (P < 0.05, in each group). Western blot analysis revealed that the expression of the MMP9 gene was upregulated by 17β-estradiol, and the expression of the fibronectin gene was downregulated by 17β-estradiol. The mitosis assay via western blot analysis showed that the expression cell cycle-associated protein, proliferating cell nuclear antigen, increased gradually as a result of 17β-estradiol treatment. Reverse transcription-polymerase chain reaction showed that EGF was upregulated by 17β-estradiol, and the EGFR small interfering RNA did not totally block the wound healing of the 17β-estradiol-treated cells but statistically significantly reduced the wound healing rate (P = 0.031).

17β-Estradiol facilitated the maintenance of the beneficial effect on corneal epithelial migration and proliferation, and the promoting effect of 17β-estradiol is partially related to increased EGF in vitro.