Laboratory of Clinical Visual Sciences, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Invest Ophthalmol Vis Sci. 2010 Jan;51(1):35-46. doi: 10.1167/iovs.09-3544. Epub 2009 Jul 23.
To characterize the neuroprotective mechanisms of erythropoietin (EPO) against the stress of glyoxal-advanced glycation end products (AGEs) in retinal neuronal cells.
Rat retinal organ culture, primary retinal neuron culture, and retinal cell line (R28 cell) culture under glyoxal-AGEs insult were used as in vitro models. Exogenous EPO was applied to these models. Retinal neuronal cell death was assessed by TUNEL, ethidium bromide/acridine orange staining, and cell viability assay. R28 cell proliferation was evaluated by BrdU incorporation and propidium iodide staining. Real-time RT-PCR and Western blot analysis were used to detect Bcl-xL, Bcl-2, Bax, BAD, and products of extracellular signal regulated kinase (ERK) and Akt pathways. Specific inhibitors and plasmids were used to pinpoint the roles of ERK and Akt pathways. Results. EPO protected the retinal cells from glyoxal-AGE-induced injury in a time- and dose-dependent fashion. The protective function of EPO was proved to be antiapoptotic, not pro-cell proliferative. Glyoxal upregulated Bax expression but suppressed Bcl-xL expression and BAD phosphorylation. In contrast, EPO enhanced BAD phosphorylation and Bcl-xL expression but downregulated Bax. The regulation of these apoptosis-related proteins by EPO was through ERK and Akt pathways.
These data demonstrate that exogenous EPO significantly attenuates the retinal neuronal cell death induced by glyoxal-AGEs by promoting antiapoptotic and suppressing apoptotic proteins. EPO/EPO receptor signaling through ERK and Akt pathways is pivotal in EPO neuroprotective mechanisms.
研究促红细胞生成素（EPO）对糖基化终产物（AGEs）诱导的视网膜神经元细胞应激的神经保护机制。
采用大鼠视网膜器官培养、原代视网膜神经元培养和视网膜细胞系（R28 细胞）培养的体外模型，用AGEs 加葡萄糖醛酸处理这些模型。将外源性 EPO 应用于这些模型。通过 TUNEL、溴化乙锭/吖啶橙染色和细胞活力测定评估视网膜神经元细胞死亡。通过 BrdU 掺入和碘化丙啶染色评估 R28 细胞增殖。实时 RT-PCR 和 Western blot 分析用于检测 Bcl-xL、Bcl-2、Bax、BAD 和细胞外信号调节激酶（ERK）和 Akt 途径的产物。使用特定的抑制剂和质粒来确定 ERK 和 Akt 途径的作用。
EPO 以时间和剂量依赖的方式保护视网膜细胞免受糖基化终产物诱导的损伤。EPO 的保护作用被证明是抗凋亡的，而不是促进细胞增殖的。糖基化增加 Bax 的表达，但抑制 Bcl-xL 的表达和 BAD 的磷酸化。相反，EPO 增强 BAD 的磷酸化和 Bcl-xL 的表达，但下调 Bax。EPO 通过 ERK 和 Akt 途径对这些凋亡相关蛋白的调节。
这些数据表明，外源性 EPO 通过促进抗凋亡蛋白和抑制凋亡蛋白显著减轻糖基化终产物诱导的视网膜神经元细胞死亡。EPO/EPO 受体通过 ERK 和 Akt 途径的信号转导在 EPO 的神经保护机制中起着关键作用。
