Introduction Loss of corneal endothelial cells (CECs) is one major factor limiting transplant clarity and survival after keratoplasty. Amongst other factors, apoptosis due to cellular stress is responsible for these problems. This study investigates the possible anti-apoptotic and cytoprotective effects of minocycline on a human corneal endothelial cell line (HCEC-SV40) cultured under oxidative stress and with transforming growth factor beta (TGF-β).
Methods CECs were treated with 1–150 μM minocycline. Cell viability and the median inhibitory concentration (IC50) were evaluated after 48 h and after H2O2 treatment (tetrazolium dye reduction assay and live–dead assay). Expression of B-cell CLL/lymphoma 2 (Bcl-2) and X-linked inhibitor of apoptosis (XIAP) and their mRNA were assessed by reverse transcriptase (RT)-PCR and western blot analysis after treatment with minocycline alone and consecutive incubation with 200 μM H2O2 and TGF-β2. A quantitative detection of histone-associated DNA fragmentation by ELISA was performed.
Results Minocycline concentrations from 1–50 μM showed no toxic effects on CECs. Pre-treatment with 10–40 μM 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-β2 significantly. When CECs were treated with minocycline and then consecutively with H2O2 or TGF-β2, 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.
- corneal endothelium
- organ culture
- pharmacology, transplantation
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Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.