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J. Baryla, D. Bhayana, H. Liu, R. L. McKown, G. W. Laurie, C. M. L. Hutnik; The Effect of Lacritin on Human Corneal Epithelial Cells Exposed to Benzalkonium Chloride or Tert-Butyl Hydroperoxide. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4617.
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To determine if lacritin can protect human corneal epithelial (HCE) cells from the ocular bacteriocidal agent benzalkonium chloride (BAK) and oxidative stressor tert-butyl hydroperoxide (t-BOOH). In previous studies, lacritin has demonstrated prosecretory and mitogenic properties, thus it may offer protection to cells exposed to different stressors. BAK is a commonly used preservative in eye drops with well-documented toxicity, whereas t-BOOH causes oxidative damage to various ocular cells.
Recombinant human lacritin and negative control fragment C-25 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 or t-BOOH, then repeated in the presence of lacritin or C-25. Real-time PCR examined lacritin-dependent expression of lacritin, SDC1, HPSE, and MUC16 mRNA’s. Data were collected as the mean +/- SD from at least 3 experiments, and analyzed via the T-test.
BAK (0.001-0.005%) and t-BOOH (0.1-5 mmol) killed HCE cells in a time and dose dependent manner. Even lower concentrations of BAK induced apoptosis. The optimal lacritin protective dose (1 nM) reduced 0.004% BAK-dependent cell death by 10% (p=0.01) compared to BAK alone after only 1 min of treatment. Preincubation with 1 nM lacritin for 24 h followed by 0.004% BAK for 1 min improved survival by 12% (p=0.007) when compared to BAK alone. However, when additional lacritin was added at 1min to the pre-treated cells, cell viability decreased. Similar trends were seen in t-BOOH experiments. Lacritin (1 nM) autostimulated lacritin mRNA expression by HCE’s.
Incubation of HCE cells with lacritin (1 nM) either 24 hrs before or simultaneously with BAK or t-BOOH promotes HCE cell survival. These results are very promising, and continued investigations are warranted to further characterize the protective properties of lacritin on corneal epithelial cells.
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