Abstract
Objective
To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5 (RGC-5) cells, pro-protein convertase-2 (PC2), carboxypeptidase-E (CPE) and preproneuropeptide Y (preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed.
Methods
The RGC-5 cell was differentiated in 0.1 μmol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation (OGD). The acute or chronic OGD-induced cell death rates were obtained by using PI or TUNEL staining. The protein expression levels were determined by using the Western blot method and PC2 activity analysis.
Results
The ischemia caused substantial cell death in an OGD dose-dependent manner. In the cells, proPC2 and preproNPY protein levels gradually increased whereas proCPE gradually decreased. After OGD, PC2 activity was decreased. In the conditioned medium, proPC2 and PC2 proteins gradually decreased whereas proCPE, CPE, and preproNPY proteins gradually increased.
Conclusion
These results demonstrated that OGD inhibited the neuropeptide pro-protein processing system by reducing PC2 activity and the maturation of proPC2. The aggregation of the pro-proteins and the increase of the active CPE excision adversely exacerbated the cell injury. The pro-protein processing system might play a critical role in the ischemic stress of RGC-5 cells.
摘要
目的
通过分析细胞和培养液中的原蛋白转化酶-2, 羧基肽酶-E和前神经肽原-Y蛋白的表达水平, 研究视网膜神经节细胞-5 (retina ganglion cell-5 cells, RGC-5) 在应激损伤后的神经肽原蛋白加工系统的变化。
方法
在培养 液中加入终浓度为0.1 μmol/L 的staurosporine并保温24 h完成RGC-5的分化; 使用氧葡萄糖剥夺(oxygen and glucose deprivation, OGD) 的细胞模型, 诱导分化的RGC-5的应激损伤; 利用PI 和 TUNEL染色方法测定急性和慢性应激模型的细胞死亡率; 使用Western blot 和原蛋白转化酶2的活性测定方法, 检测细胞和培养液中的原蛋白转化酶-2, 羧基肽酶-E和前神经肽原-Y蛋白表达水平。
结果
应激反应引起了以剂量依赖性的细胞死亡。 随OGD应激剂量的增加, 原蛋白转化酶-2 和前神经肽原-Y 蛋白表达水平逐渐增加, 而羧基肽酶原-E 逐渐降低。 细胞应激损伤后, 原蛋白转化酶-2 的活性降低。 在培养液中, 原蛋白转化酶-2 和原蛋白转化酶原-2 逐渐降低, 而羧基肽酶-E, 羧基肽酶原-E和前神经肽原-Y渐渐增加。
结论
应激损伤通过降低原蛋白转化酶-2的活性和原蛋白转化酶原-2的激活, 抑制了神经肽原蛋白加工系统, 而应激后原蛋白的堆积和羧基肽酶-E 细胞外排的增加又反过来加重了细胞的损伤。 原蛋白加工系统在应激损伤中起重要作用。
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The retraction note to this article can be found online at http://dx.doi.org/10.1007/s12264-009-0519-x.
An erratum to this article can be found at http://dx.doi.org/10.1007/s12264-009-0519-x
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Tang, SS., Zhang, JH., Liu, HX. et al. RETRACTED ARTICLE: Pro-protein convertase-2/carboxypeptidase-E mediated neuropeptide processing of RGC-5 cell after in vitro ischemia. Neurosci. Bull. 25, 7–14 (2009). https://doi.org/10.1007/s12264-009-1027-8
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DOI: https://doi.org/10.1007/s12264-009-1027-8
Keywords
- neuropeptide pro-protein processing system
- retina ganglion cell-5
- in vitro
- oxygen and glucose deprivation
- pro-protein convertase-2
- carboxypeptidase-E
- preproneuropeptide-Y