Thymosin β4 inhibits benzalkonium chloride-mediated apoptosis in corneal and conjunctival epithelial cells in vitro

Thymosin beta-4 (Tβ4) is known to promote ocular wound healing, to decrease ocular inflammation, and to have anti-apoptotic effects on corneal epithelium. In this study, the effect of Tβ4 on the survival of human ocular surface epithelial cells exposed to benzalkonium chloride (BAK) was measured. Human conjunctival epithelial cells (HC0597) or human corneal epithelial cells (HCET) were treated with 0%, 0.001%, 0.01%, or 0.1% BAK for 15 min. After 3 or 24 h of recovery in culture medium containing 1 μg/ml Tβ4, a dosage that has been demonstrated effective in several published studies, DNA synthesis was measured using a colorimetric BrdU incorporation assay. Both conjunctival and corneal epithelial DNA synthesis was inhibited by BAK in a dose-dependent manner. Tβ4 did not protect the epithelial cells from BAK-induced inhibition of proliferation. To assess the ability of Tβ4 to prevent apoptosis, epithelial cells were treated with 0.01% BAK + Tβ4 and cell death was measured using a colorimetric assay. BAK-induced apoptosis increased throughout the duration of the assay, which was carried out to 5 days in culture. Treatment of HC0597 cells with Tβ4 significantly inhibited the apoptosis shown to be initiated by BAK. Treatment of non-transformed human corneal epithelial cells (HCEC) with Tβ4 also significantly inhibited the apoptosis shown to be initiated by BAK at later times in culture. Ocular solutions containing BAK as a preservative are typically used for extended periods of time. This study suggests that Tβ4 may be able to overcome the apoptotic side effect of BAK, and may be a useful additive to solutions containing this preservative.