Protective Effects of Resveratrol Against Antibody-Induced Cell Death in Retinal Cells

Abstract

Purpose:: Autoantibodies against recoverin and enolase have been associated with retinal degeneration in patients with autoimmune and paraneoplastic retinopathies. The purpose of this study was to determine whether resveratrol, an anti-aging polyphenol derived from purple grapes, has protective effects against antibody-induced apoptosis in retinal cells.

Methods:: E1A.NR3 retinal cells (developed by Gail Seigel) were treated with either 0.8 mg/ml of rat normal IgGs, anti-recoverin (Rec-1) antibodies, or anti-α-enolase (Enol-1) antibodies in the presence or absence of resveratrol (10-40 µM). We measured intracellular ATP levels with a luminescent cell viability assay, intracellular calcium and caspase 3 activity levels with fluorescent assays, and the expressions of sirtuin (SIRT1) and apoptosis inducing factor (AIF) proteins by Western blotting.

Results:: When E1A.NR3 cells were exposed to resveratrol before treatment with Enol-1 or Rec-1 antibodies, 30% more of the cells were protected from apoptotic death. A 16 or 24-hour treatment significantly inhibited caspase 3 activity as compared to the control untreated cells (p<0.01). In Rec-1 and Enol-1 treated cells we observed an increase in intracellular calcium, but even a short 10 min pretreatment with resveratrol significantly reduced the intracellular calcium levels (p<0.0001). Also, resveratrol dramatically reduced (p<0.001) the sudden burst in intracellular calcium caused by thapsigargin, a specific inhibitor of the Sarco-Endoplasmic Reticum Ca²⁺-ATPase (SERCA) channel. In Enol-1 treated cells, intracellular ATP dropped by 30% of the untreated cells (p<0.01), but resveratrol abolished such changes. Normal rat IgG (without specificity to enolase and recoverin as tested by Western blot) did not induce apoptosis and did not show any significant changes in intracellular ATP or calcium levels. Resveratrol also triggered the overexpression of anti-apoptotic SIRT1 (~70-fold increase) compared to untreated control cells.

Conclusions:: Resveratrol-induced SIRT1 expression is a newly identified pro-survival mechanism that provides protection against antibody-induced apoptosis in retinal cells. Resveratrol can be useful in the treatment of cancer-associated and autoimmune retinopathies where antibody-induced apoptosis is one of the main causes of disease pathogenicity.

Keywords: autoimmune disease • apoptosis/cell death • protective mechanisms