目的：

    研究氧糖剥夺（oxygen-glucose deprivation，OGD）预处理和后处理对大鼠PC-12细胞低温氧糖剥夺/再灌注（oxygen-glucose deprivation/reperfusion，OGD/R）后内质网应激未折叠蛋白反应PERK-CHOP通路蛋白表达的影响。

方法：

将大鼠PC-12细胞进行传代、铺板，随后分为4组：对照组（Sham，n=6），低温OGD/R组（n=6），OGD预处理+低温OGD/R 组(IPC组，n=6）和低温OGD/R组+OGD后处理组(IPost组，n=6）。低温OGD/R组使用无糖培养液将PC-12细胞在17°C (5% CO₂ / 95% N₂)条件下培养2h，完成后使用正常含糖培养液，在37℃ (5% CO₂/95% Air)条件下培养24h。IPC组细胞使用无糖培养液在37°C (5% CO₂ / 95% N₂)条件下培养30min进行OGD预处理，完成后使用正常培养液在37℃ (5% CO₂/95% Air)条件下培养24h诱导缺氧缺糖耐受，然后予以2h低温OGD处理（同低温OGD/R组）。IPost组细胞予以低温OGD处理后，立即进行3个周期的15min复氧复糖和15minOGD处理，然后于37℃ (5% CO₂/95% Air)条件下培养22.5h。Sham组仅与其他各组同步更换含糖培养基，不作其他特殊干预。利用Western Blot半定量法，对葡萄糖调节蛋白78（glucose regulated protein 78，GRP-78）、磷酸化蛋白激酶类RNA 内质网激酶（phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase，p-PERK）、磷酸化真核翻译起始因子2α（phosphorylation of eukaryotic translation initiation factor 2α，p-eIF2α）以及C/EBP 同源性蛋白（C/EBP-homologous protein，CHOP）的蛋白质表达水平进行检测，利用CCK8法进行细胞活力测定。实验所得数据予以普通单向方差分析并进行多重比较，以P＜0.05为差异具有统计学意义。

结果：

1.Western Blot：Sham组、低温OGD/R组、IPC组和IPost组之间GRP78蛋白的表达量呈现逐渐增加趋势；与Sham组相比，低温OGD/R组p-PERK（P＜0.05）、p-eIF2α（P＜0.05）以及CHOP（P＜0.0001）蛋白表达量升高；与低温OGD/R组相比，IPC组 p-PERK （P＜0.05）、p-eIF2α （P＜0.001）以及CHOP （P＜0.01）蛋白表达量降低；与低温OGD/R组相比，IPost组p-PERK （P＜0.01）、p-eIF2α （P＜0.001）以及CHOP （P＜0.001）蛋白表达量降低；IPC组和IPost组之间p-PERK、p-eIF2α以及CHOP的表达量无显著差异。

2.CCK8细胞活力测定：与SHAM组相比，低温OGD/R组、IPC组及IPost组PC-12细胞细胞活力均降低；与低温OGD/R组相比，IPC组及IPost组PC-12细胞细胞活力增加。

结论：

OGD预处理和后处理有助于减弱大鼠PC-12细胞低温OGD/R后内质网应激。

关键词：缺血预处理；缺血后处理；深低温停循环；内质网应激；未折叠蛋白反应；氧糖剥夺/再灌注

Objective：

To investigate the effects of oxygen-glucose deprivation (OGD) preconditioning and OGD post-preconditioning on protein expression of PERK-CHOP pathway in endoplasmic reticulum stress unfolded protein responsein rat PC-12 cells after hypothermic oxygen-glucose deprivation / reperfusion (OGD/R).

Methods：

Rat PC-12 cells were subcultured and plated, and then divided into four groups: control group (Sham), hypothermic OGD/R group (n=6), OGD preconditioning group (IPC group，n=6) and OGD post-preconditioning group (IPost group,，n=6). In the hypothermic OGD/R group, PC-12 cells were cultured in glucose-free medium at 17 °C (5% CO₂/95% N₂) for 2 hours, and then cultured in normal medium at 37 ℃ (5% CO₂/95% Air) for 24 hours. Cells in the IPC group were pretreated with 30min cultured in glucose-free medium at 37 °C (5% CO₂/95% N₂) for OGD, and then cultured in normal medium at 37 ℃ (5% CO₂/95% Air) for 24 hours to induce hypoxia and glucose tolerance, and then treated with 2 hours hypothermic OGD/R (same as hypothermic OGD/R group). After low temperature OGD/R treatment, the cells in IPost group were immediately treated with 15min, reoxygenation, glucose and 15minOGD for 3 cycles, and then cultured at 37 ℃ (5% CO₂/95% Air) for 22.5 hours. The Sham group only changed the culture medium synchronously with other groups without other special intervention. The protein expression levels of glucose regulated protein 78 (GRP-78), phosphorylated protein kinase RNA endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic translation initiation factor 2 α (p-eIF2 α) and C/EBP homologous protein (CHOP) were detected by Western Blot semi-quantitative method, and cell viability was determined by CCK8 method. The experimental data were analyzed by ordinary one-way analysis of variance and multiple comparisons were made, and the difference was statistically significant (P < 0.05).

Results：

1.   Western Blot：The expression of GRP78 protein increased gradually in Sham group, hypothermic OGD/R group, IPC group and IPost group, but there was no significant difference among each group. Compared with Sham group, the expression of p-PERK, p-eIF2 α and CHOP in hypothermic OGD/R group increased. Compared with the cold OGD/R group, the protein expression of p-PERK (P < 0.001), p-eIF2 α (P < 0.001) and CHOP (P < 0.001) decreased after IPC, and the protein expressions of p-PERK (P < 0.001), p-eIF2 α (P < 0.001) and CHOP (P < 0.001) decreased after IPost. There was no significant difference in the expression of p-PERK, p-eIF2 α and CHOP between IPC group and IPost group.

2.CCK8 cell viability: Compared with SHAM group, the cell viability of PC-12 cells decreased in hypothermic OGD/R group, IPC group and IPost group, and increased in IPC and IPost group compared with hypothermia OGD/R group.

Conclusion：

Oxygen-glucose deprivation pretreatment and post-treatment can attenuate endoplasmic reticulum stress of rat PC-12 cells after hypothermic oxygen-glucose deprivation / reperfusion.

Key words: ischemic preconditioning; ischemic postconditioning; deep hypothermic circulatory arrest; endoplasmic reticulum stress; unfolded protein response；oxygen-glucose deprivation/reperfusion