Gu Limin, Xu Hua, Wang Fang, Xu Guoxu, Sinha Debasish, Wang Juan, Xu Jing-Ying, Tian Haibin, Gao Furong, Li Weiye, Lu Lixia, Zhang Jingfa, Xu Guo-Tong
Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China.
Department of Ophthalmology, Second Affiliated Hospital of Soochow University, Suzhou, China.
Invest Ophthalmol Vis Sci. 2014 Oct 21;55(12):8208-22. doi: 10.1167/iovs.14-14435.
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.
视网膜神经元细胞功能障碍甚至细胞死亡与视网膜中兴奋性毒性谷氨酸（Glu）水平升高有关。我们的目的是研究Glu导致视网膜细胞死亡的机制，并探讨促红细胞生成素（EPO）对糖尿病视网膜中Glu神经毒性的神经保护作用。
本研究采用雄性Sprague-Dawley（SD）大鼠和R28细胞系。通过腹腔注射链脲佐菌素（STZ）诱导SD大鼠患糖尿病。糖尿病发病两周后进行玻璃体内注射；4天后，采集视网膜进行检测。R28细胞分别用Glu、Glu+EPO或Glu+EPO+可溶性EPO受体（sEPOR）处理24小时，然后收集细胞进行后续研究。用谷氨酸检测试剂盒测量视网膜中的谷氨酸水平。用TUNEL染色法测定细胞死亡情况。用RT-PCR、蛋白质印迹法和免疫荧光法研究谷氨酰胺合成酶（GS）、谷氨酸-天冬氨酸转运体（GLAST）、离子型谷氨酸受体（iGluRs）、凋亡诱导因子（AIF）和聚（ADP-核糖）（PAR）聚合物的变化。
在糖尿病2周的大鼠视网膜中，Glu浓度约为正常对照组的1.21倍。TUNEL染色显示视网膜细胞死亡增加。视网膜GS和GLAST表达降低，而iGluRs（如KA1和NR1）以及PAR聚合物表达增加。在R28细胞中，Glu（10 mM）处理24小时后，细胞活力下降了52.7%；KA1、NR1、PAR聚合物和细胞核AIF的表达均增加。上述情况在体内和体外均可被EPO在很大程度上逆转。sEPOR消除了EPO的保护作用。
促红细胞生成素在糖尿病大鼠视网膜和Glu处理的R28细胞中均表现出对Glu介导的神经毒性的神经保护作用。神经保护机制主要是通过维持谷氨酸-谷氨酰胺循环相关蛋白的正常表达以及抑制AIF易位和PAR聚合物形成。
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