Abstract
Purpose: :
To determine whether the Phase 2 antioxidant resveratrol can prevent hydroquinone (HQ)-induced RPE cell death.
Methods: :
ARPE-19 cells were cultured by standard methods. HQ was added to the cells to establish a kill curve so that a range of experimentally convenient concentrations (250-350 µM) could be used in all subsequent studies. These concentrations have been previously studied and shown to be an accurate model of RPE oxidative damage. ARPE-19 cells were then exposed to HQ and co-incubated with various concentrations of the natural product resveratrol. The number of viable cells was quantified, apoptosis/necrosis was assayed by Annexin V (Calbiochem), and cleaved caspase-3 assays were performed via flow cytometry.
Results: :
A dose-dependent reduction of cell viability in response to HQ exposure was observed. Significant ARPE-19 cell death occurred at 200 µM HQ, and more than 60% of the cells were dead at concentrations of 250 µM and above. The data showed a typical cell death curve with increasing concentrations of HQ. In the presence of increasing concentrations of resveratrol (500nM - 250 µM), there was a significant increase in viable cells at 48 hrs. Annexin V staining revealed an increase in apoptotic cells with increasing HQ dosage, and in the presence of 250 µM resveratrol, a significant increase in unstained cells was observed. Cleaved caspase-3 analysis revealed a drastic reduction in downstream caspase-3 activation in the presence of 250 µM resveratrol.
Conclusions: :
Resveratrol can reduce cell death in an in vitro HQ-induced model of oxidative injury to the RPE. These in vitro studies warrant further exploration of the effect of resveratrol in mouse models of ARMD.
Keywords: apoptosis/cell death • protective mechanisms • retina