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N. Ashki, J. Baryla, H. Liu, G. Laurie, C. M. Hutnik; Potential Protective Effects of Lacritin on Human Corneal Epithelial Cells Exposed to Benzyalkonium Chloride. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5464.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study is to determine if a naturally occurring tear protein called lacritin, has protective properties on a cell line of human corneal epithelial (HCE) cells exposed to specific stressors, namely Benzalkonium Chloride (BAK).
Recombinant human lacritin were generated on intein vectors in E. coli, purified on chitin beads, and further purified on DEAE. Time and dose dependent kill curves were established after exposure of subconfluent HCE cells (CRL-11515 line) to BAK, than repeated in the presence of lacritin or control. Western blot analysis was conducted in order to determine the mechanism of BAK-induced cell death. Specific staining for LC-3 was completed in order to determine the possible mechanism of cell toxicity mediated by mechanisms of autophagy.
BAK (0.001% and 0.004%) killed HCE cells in a time and dose dependent manner. Lower concentrations of BAK (0.001%) induced autophagy, where as higher concentrations of BAK (0.004%) resulted in necrosis-mediated cell death. The optimal lacritin protective dose (1 nM) reduced 0.004% BAK-dependent cell death by 30% (p=0.01) compared to BAK alone. Pre-incubation with 1 nM lacritin for 24 h followed by 0.004% BAK for 10 min improved survival by 32% (p=0.001) when compared to BAK alone. Co-treatment of lacritin (1 nM) with BAK (0.004%) resulted in decreased LC-3 expression which was detected by Western blot analysis, suggesting that BAK-induced cell death may be mediated through mechanisms of autophagy.
HCE treated with low concentrations (0.001%) of BAK resulted in a mechanism of autophagy-mediated cell death. But a higher BAK concentrations (>0.001%), the mechanism of BAK-induced cell death switched to necrosis. And incubation of HCE cells with 1nM lacritin either 24 hrs before or simultaneously promoted HCE cell survival.
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