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Abstract
Purpose
To evaluate the response and cellular changes of cultured human corneal epithelium according to the concentration of topical cyclosporine.
Methods
Human corneal epithelial cells were exposed to cyclosporine A concentrations of 1 ug/ml (0.0001%), 10 ug/ml (0.001%), 100 ug/ml (0.01%), and 500 ug/ml (0.05%) for 5 and 10 minutes. An MTT-based colorimetric assay was performed to assess the metabolic activity of cellular proliferation and a lactate dehydrogenase (LDH) leakage assay was used to determine cellular toxicity. The modulations of extracellular matrix proteins such as PIP and laminin were evaluated. The levels of proinflammatory cytokine, TNF-α, and IL-1 were evaluated by ELISA kits.
Results
The inhibitory effects of human corneal epithelial cellular proliferation did not show a concentration- or exposure time-dependent response. Activity of LDH did not show a statistically significant difference for the different concentrations and exposure times. The inhibitory effects of extracellular matrix proteins such as PIP or laminin synthesized from human corneal epithelial cells were compared with those in the control group and showed a statistically significant difference at a cyclosporine concentration greater than 0.01%. Released TNF-α and IL-1 from human corneal epithelial cells did not show any difference according to concentration or cyclosporine exposure time.
Conclusions
To modulate human corneal epithelial cellular proliferation and the levels of extracellular matrix proteins such as PIP and laminin, a concentration of cyclosporine A greater than 0.01% for longer than 5 minutes of exposure is needed. There were little effects of cyclosporine A on proinflammatory cytokine secreted from corneal epithelial cells.
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Figure 1.
The absorption rate of the water-insolubale formazan dye in corneal epithelial cell exposed in 1 ug/ml (0.0001%), 10 ug/ml (0.001%), 100 ug/ml (0.01%), 500 ug/ml (0.05%) cyclosporine by a scanning spectrometer (ELISA reader). Cellular viabilities were not significantly different in proportion to concentration increase. As exposure time lengthened, cell viabilities were not significantly different (p > 0.05).
Figure 2.
LDH titers of cultured corneal epithelial cells exposed in cyclosporine. LDH titer significantly increased in all cases when exposed to 1 ug/ml (0.0001%), 10 ug/ml (0.001%), 100 ug/ml (0.01%), 500 ug/ml (0.05%) cyclosporine (p < 0.017) compared with the control group, but there were no significant differences according to exposure time within the concentrations (p > 0.05).
Figure 3.
PIP level of cultured corneal epithelial cells exposed to cyclosporin. In contrast to the control group, there were significant differences between the high concentration (100 ug/ml, 500 ug/ml) group and control group (p < 0.028) (*), but there were no significant differences between the low concentration (1 ug/ml, 10 ug/ml) group and the control group (p > 0.05).
Figure 4.
Laminin levels of cultured corneal epithelial cells exposed to cyclosporin. Laminin levels were suppressed compared to the control group. There were significant differences between the high concentration (100 ug/ml, 500 ug/ml) group and control group (p < 0.028) (*), however, there were no significant differences between the low concentration (1 ug/ml, 10 ug/ml) group and the control group (p > 0.05).
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