Erythropoietin exerts a neuroprotective function against glutamate neurotoxicity in experimental diabetic retina.

Investigative ophthalmology & visual science (2014-10-23)
Limin Gu, Hua Xu, Fang Wang, Guoxu Xu, Debasish Sinha, Juan Wang, Jing-Ying Xu, Haibin Tian, Furong Gao, Weiye Li, Lixia Lu, Jingfa Zhang, Guo-Tong Xu
RESUMEN

Retinal neuronal cell dysfunction and even cell death are associated with increased excitotoxic glutamate (Glu) level in the retina. Our aim was to study a causative mechanism of Glu on retinal cell death and explore the neuroprotective role of erythropoietin (EPO) against Glu neurotoxicity in the diabetic retina. Male Sprague-Dawley (SD) rats and R28 cell line were employed in this study. Diabetes was induced with intraperitoneal injection of streptozotocin (STZ) in SD rats. Two weeks after diabetes onset, the intravitreal injection was performed; 4 days later, the retinas were harvested for testing. R28 cells were treated with Glu, Glu+EPO, or Glu+EPO+soluble EPO receptor (sEPOR), respectively, for 24 hours, and then the cells were collected for the following studies. Glutamate level in the retina was measured with a glutamate assay kit. Cell death was determined with TUNEL staining. The changes in glutamine synthetase (GS), glutamate-aspartate transporter (GLAST), ionotropic glutamate receptors (iGluRs), apoptosis-inducing factor (AIF), and poly(ADP-ribose) (PAR) polymer were studied with RT-PCR, Western blot, and immunofluorescence. In 2-week diabetic rat retinas, Glu concentration was approximately 1.21-fold that in normal control. TUNEL staining demonstrated that retinal cell death was increased. Retinal GS and GLAST expressions were decreased, while the iGluRs, for example, KA1 and NR1, and PAR polymer expression was increased. In R28 cells, 24 hours after Glu (10 mM) treatment, the cell viability was decreased by 52.7%; KA1, NR1, PAR polymer, and nuclear AIF all increased in expression. The above conditions could be largely reversed by EPO both in vivo and in vitro. The protective effect of EPO was abolished by sEPOR. Erythropoietin showed a neuroprotective function against Glu-mediated neurotoxicity both in diabetic rat retina and in Glu-treated R28 cells. The neuroprotective mechanisms were largely through maintaining the normal expression of glutamate-glutamine cycle-related proteins and inhibiting AIF translocation and PAR polymer formation.

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