Metformin and rapamycin protect cells from vital dye–induced damage in retinal pigment epithelial cells and in vivo
To evaluate the effect of autophagy inducers on damage caused by vital dye in adult human RPE (ARPE) cells and in a rat model.
ARPE-19 cells were exposed to ICG or BBG (0.05 mg/ml) with rapamycin (200 nM) or metformin (2 mM) for 30 min and treated with or without 20 μM chloroquine (CQ) to identify the protein levels of LC3 and SQSTM1 by immunoblotting. In vivo study was performed by injecting 10 μl 0.05% ICG and 0.25% BBG into the subretinal space of the rat eyes, and/or co-treated them with metformin and rapamycin. The retinas were used to determine autophagy with the LC3-II level and apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay.
In this study, both ICG and BBG inhibited autophagy flux in adult human retinal pigment epithelium cells (ARPE-19), whereas only ICG consistently reduced autophagy in the retina of rats. Moreover, rapamycin and metformin induced autophagic flux in ARPE-19 cells and increased the LC3-II level in retinal tissues exposed to vital dyes. Both ICG and BBG increased apoptosis in the retina of rats. However, both rapamycin and metformin induced autophagy and reduced the apoptosis caused by vital dyes.
Taken together, these results suggest that rapamycin and metformin may diminish vital dye–induced retinal damage in vivo through activation of autophagy.
KeywordsAutophagic flux Retinal pigment epithelium Vital dyes In vivo
This study was funded by the Kaohsiung Veterans General Hospital (VGHKS106-109 and VGHKS107-131).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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