PURPOSE. The purpose of this study was to determine the effect of thymosin beta 4 (T 4 ) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of T 4 in preventing mitochondrial disruption and in inhibiting caspasemediated apoptosis was examined. METHODS. Nontransformed human corneal epithelial cells (HCECs) at passage 4 were untreated or treated with ethanol (20% for 20 seconds) or a combination of ethanol and T 4 . The cells were allowed to recover from ethanol treatment for 24 hours. Mitochondrial membrane integrity and the release of cytochrome c to the cytoplasm were assessed using microscopy, Western blot, and ELISA. Bcl-2 expression and cell proliferation were measured using ELISA. Colorimetric activity assays were completed for caspase-2, -3, -8, and -9. RESULTS. T 4 treatment decreased deleterious mitochondrial alterations, significantly decreased cytochrome c release from mitochondria, and increased Bcl-2 expression in ethanol-exposed human corneal epithelial cells. In ethanol-exposed corneal epithelium T 4 treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. T 4 treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure. CONCLUSIONS. T 4 plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. T 4 may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases. (Invest Ophthalmol Vis Sci
