Erythropoietin (Epo) Protects Retinal Neurons From Advanced Glycation End Products (AGEs)-Induced Apoptosis via Erk and AKT Pathways

Abstract

Purpose: : Epo has been shown to be able to prevent the early retinopathy in STZ-treated rats in our recent study. The present study is to explore the biochemical and molecular mechanisms of neuron protective function of Epo.

Methods: : Organ culture, primary cell culture and cell line were used in the present study as follows: neurosensory retina from Sprague-Dawley (SD) rats in the Ame’s medium up to 9h, primary retinal neurons from neonatal (postnatal day 1) SD rats cultured for at least 7 days, and the retinal neuronal cell line R28 kept in DMEM (1000mg glucose) with normal serum. Glyoxal was utilized to induce the AGEs formation with or without Epo. The neuronal death was evaluated by Acridine Orange/Ethidium Bromide (AO/EB) Staining, TUNEL staining and cell viability assay. BrdU incorporation was utilized to see the effect of Epo on cell proliferation. The specific inhibitors, PD98059/U0126 to Erk pathway and wortmannin/LY294002 to AKT pathway, were applied to elucidate the pathways of Epo function. Key molecules in these pathways were detected by western blots.

Results: : Glyoxal-induced cell death in all three in vitro models was observed. Such cell death was attenuated by Epo in a dose and time dependent manner, as showed by AO/EB staining, TUNEL staining and viability assay. By using BrdU incorporation assay, the improved R28 cell viability and decreased cell death after administration of Epo was proved to be not due to the increased cell proliferation. The application of both PD98059 and U0126 abolished the Epo protection for R28 cells under AGEs stress. Similar phenomena were also found after application of wortmannin and LY294002.

Conclusions: : The cyto-protective functions of Epo on retinal neuron death induced by AGEs were demonstrated in the three in vitro models. The present data also indicate that both Erk/MAPK and PI3K/AKT pathways are responsible for anti-apoptosis function of Epo.

Keywords: diabetic retinopathy • apoptosis/cell death • cytokines/chemokines