[Protective effect of thymosin β4 against oxidative damage in cultured rabbit corneal epithelial cells]

The aim of this study was to investigate the effects of thymosin β4 (Tβ4) against oxidative damage in rabbit corneal epithelial cells in vitro.

Experimental study. Primary cultures of rabbit corneal epithelial cells were isolated and cultured from cornea tissue explants. Cell morphology was observed by phase-contrast microscope. The expression of keratin 12 and connexin 43 in cultured cells were examined with RT-PCR. The cultures were divided into 4 groups: control group, Tβ4 group, hydrogen peroxide (H2O2) group, and H2O2+Tβ4 group. The cell viability of cultured corneal epithelial cells was examined by MTT assay. TUNEL staining was used to detect the apoptotic cells. ROS levels were estimated by DCFH-DA using fluorescent microscopy. Cell migration was measured using the scratch wound technique. Data were analyzed using one-way analysis of variance (ANOVA) and secondary analysis for significance with post Hoc tests.

The morphology of cultured cells was round, ovoid, polygonal or paving stone appearance. The results of RT-PCR showed that cultured corneal epithelial cells expressed both keratin 12 and connexin 43, the characteristic genes of corneal epithelial cells. The cell viability in H2O2 group was significantly reduced than that in control group (67.20% ± 5.87% vs. 100.00% ± 9.99%, P = 0.000); the cell viability was significantly improved in Tβ4+ H2O2 group than that in H2O2 group (83.42% ± 7.23% vs. 67.20% ± 5.87%, P = 0.023) . Cell migration was significantly increased in Tβ4 group compared with the controls (117.6% ± 2.22% vs. 100.00% ± 4.06%, P = 0.005) ; Compared with H2O2 group, cell migration was significant increase in H2O2 + Tβ4 group (96.57% ± 8.22% vs. 64.38% ± 11.08%, P = 0.000) . Compared with the control group, the intracellular ROS level of the H2O2 group was significantly increased (234.42% ± 22.15% vs. 100.00% ± 5.28%, P = 0.000) . Intracellular ROS level of H2O2+Tβ4 group was significantly decreased as compared with the H2O2 group (163.26% ± 10.53% vs. 234.42% ± 22.15%, P = 0.000). TUNEL staining indicated that Tβ4 treatment markedly inhibited H2O2-induced apoptosis in cultured corneal epithelial cells.

Tβ4 has a strong protection effect against oxidative damage induced by H2O2 in corneal epithelial cells through promoting cell growth and cell migration, and the underlying mechanisms may due to the antioxidation and anti-apoptotic effects of Tβ4.