Anti-Oxidative Capacity of Thymosin Beta-4 in Human Cornea Epithelial Cells

Abstract

Purpose: : We previously demonstrated that internalization was essential for exogenous thymosin beta-4 (Tβ₄) to protect human cornea epithelial (HCE-T) cells against ROS-induced apoptosis, and the purpose of this study is to further elucidate its protective mechanism.

Methods: : HCE-T cells were incubated with histidine-tagged Tβ₄ (1 µg/ml) for two hours before being treated with H₂O₂ (200 µM). Activation of caspases-8 and -9 was examined by flow cytometry and the latter was further analyzed by colorimetric assay. Intracellular reactive oxygen species (ROS) level was measured by flowcytometry using 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFH-DA) as probes. Protein levels and gene expression of manganese-superoxide dismutase (Mn-SOD), copper/zinc-superoxide dismutase (Cu/Zn-SOD) and catalase were analyzed by Western blotting and real-time RT-PCR, respectively.

Results: : It was found that Tβ₄ protected HCE-T cells from H₂O₂-induced apoptosis by reducing both caspase-9 activation and intracellular ROS levels. It was further discovered that Tβ₄ increased the expression of Mn-SOD and Cu/Zn-SOD. Moreover, after the addition of H₂O_2, catalase was also up-regulated by Tβ₄ at both transcription and translation levels.

Conclusions: : Tβ₄ was found capable of up-regulating anti-oxidative enzymes in human corneal epithelial cells and increasing the resistance of the human corneal epithelial cells to ROS-induced apoptosis.

Keywords: oxidation/oxidative or free radical damage • cornea: epithelium • antioxidants