PURPOSE: The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta(4)) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of Tbeta(4) in preventing mitochondrial disruption and in inhibiting caspase-mediated 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 Tbeta(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: Tbeta(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 Tbeta(4) treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. Tbeta(4) treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure.
CONCLUSIONS: Tbeta(4) plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. Tbeta(4) may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases.