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Thymosin-ß4 Inhibits Corneal Epithelial Cell Apoptosis after Ethanol Exposure In Vitro

¹From the Departments of Anatomy and Cell Biology and ²Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan.

PURPOSE. The purpose of this study was to determine the effect of thymosin beta 4 (Tß₄) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of Tß₄ 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 Tß₄. 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. Tß₄ 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 Tß₄ treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. Tß₄ treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure.

CONCLUSIONS. Tß₄ plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. Tß₄ may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases.

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