Authors: Jiang Zhixin, Hao Peng, Tang Xin, Li Xuan
Laser assisted subepithelial keratomileusis (LASEK) is one of surgical procedures for refractive correction. Dilute alcohol that is used for the removal of epithelium during LASEK induces the apoptosis of corneal epithelial cells. Researches showed that thymosin β4 (Tβ4) can arrest apoptosis, but whether Tβ4 plays inhibitory effect on ethanol-induced damage of corneal epithelial cells is still unelucidated.
The aim of this study was to investigate the protective effects of Tβ4 on ethanol-induced rabbit corneal epithelial cell damage in vitro.
The corneal tissue of deendothelium was obtained from 10 New Zealand white rabbits. Corneal epithelial cells were cultured in vitro by using explant culture method. Cultured cells were identified by detecting the expression of keratin 12 and connexin 43 with reverse transcription PCR (RT-PCR). The cells of second generation were collected and divided into 4 groups. The cells were regularly cultured in the normal control group, and Tβ4 was added in the culture medium at the final concentration of 1 μg/ml in the Tβ4 treated group. Ethanol-induced rabbit corneal epithelial cell damage models were established by adding PBS containing 20% alcohol in medium for 20 seconds in the model group, and Tβ4 was added in the medium of model cells at the final concentration of 1 μg/ml in the model+ Tβ4 group. The survival rate of the cells was detected by MTT assay, and the apoptosis rate of the cells was examined by TUNEL method. The relative expression levels of cyclin D1 mRNA and cyclin-depensent protein kanase 4 (CDK4) mRNA in the cells were detected by real-time flurescence quantitative PCR. The content of bcl-2 protein in the cells was detected by ELISA assay. Spectrophotometry was employed to measure the activity of caspase-3. The study complied with the Regulation for the Adminstration of Affairs Concerning Experimental Animals by State Science and Technology Commission.
The mean cell survival rate was (52.1±14.07)% in the model group, which was significantly reduced in comparison with 100% of the normal control group and (77.7±19.60)% of the model+ Tβ4 group (P=0.001, P=0.035). TUNEL staining revealed more positive cells in the model group and the model+ Tβ4 group, and the percentage of TUNEL positive cells was (30.0±6.7)% in the model+ Tβ4 group, showing an evident decline in comparison with (42.4±4.0)% of the model group (P=0.01). The relative expression levels of cyclin D1 mRNA and CDK4 mRNA were 0.93±0.17 and 0.88±0.09 in the model+ Tβ4 group, which were significantly higher than 0.68±0.05 and 0.54±0.07 in the model group (P=0.027, 0.002). ELISA assay exhibited that bcl-2 content in the cells was considerably lower, and caspase-3 activity was significantly higher in the model group than those in the model+ Tβ4 group (P=0.030, 0.021).
Tβ4 plays a protective effect on rabbit corneal epithelial cells from apoptosis by lowing the caspase 3 activity and increasing bcl-2 content in ethanol-damaged rabbit corneal epithelial cells. In addition, Tβ4 promotes the regrowth of corneal epithelial cells by up-regulating the expression of cyclin D1 and CDK4.