Dynamic changes in and time sequence of ultraviolet B-induced apoptosis in rat corneal epithelial cells.

PURPOSE: To systematically examine the dynamic changes and time sequence in corneal epithelial cell apoptosis after excessive ultraviolet B irradiation.

METHODS

Ultraviolet B (144 mJ/cm2) was used to irradiate rat corneal epithelial cells for 2 h. Cell morphology was observed on differential interference contrast microscopy, and the numbers of the different kinds of apoptotic cells were counted using the ImageJ software. Cell viability was measured with the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide method. Cell apoptotic rate and loss of mitochondrial membrane potential were detected using flow cytometric analyses. The expression levels of 3 apoptotic genes were measured with real-time quantitative polymerase chain reaction at different time points within 0-24 h after irradiation.

RESULTS

After 144-mJ/cm2 ultraviolet B irradiation for 2 h, the expression levels of caspase-8 and Bax were highest at 0 h; furthermore, the mitochondrial membrane potential decreased at 0 h and remained constant for 6 h in a subsequent culture. At 6 h, caspase-3 was activated. The decrease in cell viability and increase in apoptotic rate peaked at 6 h. The caspase-3 expression level decreased within 12-24 h, which led to a decline in apoptotic rate and change in apoptotic stage.

CONCLUSIONS

The corneal epithelial cells exhibited rapid apoptosis after ultraviolet B irradiation, which was associated with both extrinsic and intrinsic pathways.