Thymosin Beta 4 Inhibits Benzalkonium Chloride–Mediated Apoptosis of Human Corneal Epithelial Cells in vitro

Abstract

Abstract: : Purpose: Thymosin beta 4 (Tß₄), a 43 amino acid molecule, promotes ocular wound healing, decreases ocular inflammation, and has anti–apoptotic effects on corneal epithelium. In this study, the effect of Tß₄ on the survival of cultured human corneal epithelial cells exposed to benzalkonium chloride (BAK) was measured. Methods: Human corneal epithelial cells at approximately 80% confluence were treated with 0%, 0.001%, 0.01%, or 0.1% BAK for 15 minutes. After 3 and 24 hours of recovery in culture medium, cell proliferation was measured using a colorimetric BrdU incorporation assay. Apoptosis was measured using a colorimetric annexin–based cell death assay. Studies were repeated in the presence of 1 µg/ml Tß₄, an in vitro dosage demonstrated effective in several published studies. To further assess the ability of Tß₄ to prevent apoptosis, corneal epithelial cells were treated with 0.01% BAK + Tß₄ over a 5 day time course. Results: At all BAK concentrations used, corneal epithelial cell proliferation was inhibited, and apoptosis was increased, compared to control at 3 and 24 hours recovery time. At the 3 and 24 hour time points, Tß₄ did not abrogate the deleterious effects of BAK; cell proliferation was not promoted by Tß₄ and apoptosis was not inhibited. However, at longer times in culture (2 to 5 days), Tß₄ treatment significantly inhibited the BAK–initiated epithelial cell apoptosis. In addition, Tß₄–treated cells demonstrated decreased apoptosis compared to those cultured in medium alone for 5 days. Conclusions: BAK, a preservative used in many commercially available ocular solutions, induces corneal epithelial cell apoptosis in culture, suggesting that long–term exposure is deleterious to corneal health. The study reported here suggests that Tß₄ may be able to overcome the deleterious pro–apoptotic effects of BAK. Since many BAK–containing eye drops are typically used for extended periods of time, Tß₄ may be a useful additive to solutions containing this preservative.

Keywords: apoptosis/cell death • cell death/apoptosis • cornea: epithelium