Ischemia causes oxidative stress in the endoplasmic reticulum (ER), accelerates the accumulation of unfolded and misfolded proteins, and may ultimately lead to neuronal cell apoptosis. In the present study, we investigated the effects of protein disulfide-isomerase A3 (PDIA3), an ER-resident chaperone that catalyzes disulfide-bond formation in a subset of glycoproteins, against oxidative damage in the hypoxic HT22 cell line and against ischemic damage in the gerbil hippocampus. We also confirmed the neuroprotective effects of PDIA3 by using PDIA3-knockout HAP1 cells. The HT22 and HAP1 cell lines showed effective (dose-dependent and time-dependent) penetration and stable expression of the Tat-PDIA3 fusion protein 24 h after Tat-PDIA3 treatment compared to that in the control-PDIA3-treated group. We observed that the fluorescence for both 2',7'-dichlorofluorescein diacetate (DCF-DA) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), which are markers for the formation of hydrogen peroxide (H2O2)-induced reactive oxygen species and apoptosis, respectively, was higher in HAP1 cells than in HT22 cells. The administration of Tat-PDIA3 significantly reduced the (1) DCF-DA and TUNEL fluorescence in HT22 and HAP1 cells, (2) ischemia-induced hyperactivity that was observed 1 day after ischemia/reperfusion, (3) ischemia-induced neuronal damage and glial (astrocytes and microglia) activation that was observed in the hippocampal CA1 region 4 days after ischemia/reperfusion, and (4) lipid peroxidation and nitric oxide generation in the hippocampal homogenates 3-12 h after ischemia/reperfusion. Transient forebrain ischemia significantly elevated the immunoglobulin-binding protein (BiP) and C/EBP-homologous protein (CHOP) mRNA levels in the hippocampus at 12 h and 4 days after ischemia, relative to those in the time-matched sham-operated group. Administration of Tat-PDIA3 ameliorated the ischemia-induced upregulation of BiP mRNA levels versus the Tat peptide- or control-PDIA3-treated groups, and significantly reduced the induction of CHOP mRNA levels, at 12 h or 4 days after ischemia. Collectively, these results suggest that Tat-PDIA3 acts as a neuroprotective agent against ischemia by attenuating oxidative damage and blocking the apoptotic pathway that is related to the unfolded protein response in the ER.

中文翻译：

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蛋白质二硫键异构酶A3通过减少氧化和内质网应激显着减少缺血诱导的损伤。

缺血在内质网（ER）中引起氧化应激，加速未折叠和错误折叠的蛋白质的积累，并最终导致神经元细胞凋亡。在本研究中，我们研究了蛋白质二硫键异构酶A3（PDIA3）（一种ER分子伴侣，可催化糖蛋白的一个子集中的二硫键形成）对缺氧HT22细胞系中的氧化损伤和对缺血性损伤的影响。沙鼠海马。我们还证实了通过使用PDIA3-敲除HAP1细胞对PDIA3的神经保护作用。与对照组-PDIA3处理组相比，在Tat-PDIA3处理后24小时，HT22和HAP1细胞系显示出有效的（剂量依赖性和时间依赖性）穿透力和稳定表达的Tat-PDIA3融合蛋白。我们观察到2'的荧光 HAP1细胞中7'-二氯荧光素二乙酸盐（DCF-DA）和末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）分别是过氧化氢（H2O2）诱导的活性氧形成和凋亡的标志物。而不是HT22细胞。Tat-PDIA3的使用显着降低了（1）HT22和HAP1细胞中的DCF-DA和TUNEL荧光；（2）缺血/再灌注1天后观察到的缺血诱导的机能亢进；（3）缺血诱导的神经元损伤和缺血/再灌注后4天在海马CA1区观察到的神经胶质（星形胶质细胞和小胶质细胞）活化，以及（4）缺血/再灌注后3-12小时海马匀浆中脂质过氧化和一氧化氮的产生。与时间匹配的假手术组相比，短暂性前脑缺血在缺血后12 h和4天使海马中的免疫球蛋白结合蛋白（BiP）和C / EBP同源蛋白（CHOP）mRNA水平显着升高。与Tat肽或对照PDIA3处理组相比，Tat-PDIA3的给药改善了缺血诱导的BiP mRNA水平上调，并在缺血后12 h或4天显着降低了CHOP mRNA水平的诱导。总体而言，这些结果表明，Tat-PDIA3通过减轻氧化损伤并阻断与ER中未反应的蛋白质反应有关的凋亡途径，作为抗缺血的神经保护剂。相对于时间匹配的假手术组中的那些。与Tat肽或对照PDIA3处理组相比，Tat-PDIA3的给药改善了缺血诱导的BiP mRNA水平上调，并在缺血后12 h或4天显着降低了CHOP mRNA水平的诱导。总体而言，这些结果表明，Tat-PDIA3通过减轻氧化损伤并阻断与ER中未反应的蛋白质反应有关的凋亡途径，作为抗缺血的神经保护剂。相对于时间匹配的假手术组中的那些。与Tat肽或对照PDIA3处理组相比，Tat-PDIA3的给药改善了缺血诱导的BiP mRNA水平上调，并在缺血后12 h或4天显着降低了CHOP mRNA水平的诱导。总体而言，这些结果表明，Tat-PDIA3通过减轻氧化损伤并阻断与ER中未反应的蛋白质反应有关的凋亡途径，作为抗缺血的神经保护剂。