Minocycline is Cytoprotective in Human Corneal Endothelial Cells and Induces Anti-Apoptotic Bcl-2 and XIAP
Résumé
Introduction: Loss of corneal endothelial cells is one major factor limiting transplant clearness and survival after keratoplasty. Beside others, apoptosis due to cellular stress is responsible for this decrease. This study investigates possible anti-apoptotic and cytoprotective effects of Minocycline on a cultured human corneal endothelial cell line (HCEC-SV40) under oxidative stress and TGF-beta. Methods: Corneal endothelial cells (CEC) were treated with 1µM to 150µM Minocycline. Cell viability and IC50 were evaluated after 48h and after H2O2 treatment (tetrazolium-dye-reduction assay and live-dead-assay). Expression of Bcl-2 and XIAP (X-linked inhibitor of apoptosis) and their mRNA were assessed by RT-PCR and Western-Blot analysis after treatment with Minocycline alone and consecutive incubation with 200µM H2O2 and TGF-beta-2 . A quantitative detection of histone-associated DNA fragmentation by ELISA was performed. Results: Minocycline concentrations from 1µM to 50µM showed no toxic effects on CECs. Pre-treatment with 10-40µM Minocycline led to an increase in viability after H2O2 treatment. In addition, Minocycline pre-treatment attenuated the increase of histone-associated DNA fragmentation after treatment with H2O2 and TGF-beta-2 significantly. When CECs cells were treated with Minocycline and then consecutively with H2O2 or TGF-beta-2, RT-PCR and Western-Blot analysis yielded an overexpression of Bcl-2 and XIAP. Conclusion: In this study Minocycline prevented apoptotic-cell-death in cultured CECs in vitro. Our results suggest, that Minocycline might offer cytoprotective properties that might help to prevent loss of corneal endothelial cells in vivo.
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