BACKGROUND Cyanidin-3-glucoside (C3G), a major anthocyanin present in berries, exhibits a strong antioxidant and has been shown to possess a neuroprotection. Prolonged exposure to glutamate will lead to oxidative damage and endoplasmic reticulum stress which could play a key detrimental role in the development of neurodegenerative disorders (NDs). In the present study, we investigated the neuroprotective effect and underlying mechanisms of C3G on the reduction of oxidative/ER stress-induced apoptosis by glutamate in HT22 mouse hippocampal neuronal cells. METHOD Cells were pre-treated with C3G in various concentrations, followed by glutamate. Cell viability and toxicity were examined using MTT and LDH assays. The apoptotic and necrotic cell death were carried out by Annexin V-FITC/propidium iodide co-staining assays. Generation of intracellular reactive oxygen species (ROS) in cells was measured by flow cytometry using DCFH-DA probe. Expression of antioxidant genes was evaluated by Real-time polymerase chain reaction analysis. The possible signaling pathways and proteins involved were subsequently demonstrated by Western blot analysis. RESULT The pretreatment of the HT22 cells with C3G protected cell death from oxidative toxicity induced by glutamate. We demonstrated that treatment cells with glutamate caused several radical forms of ROS formation, and they were abolished by specific ROS inhibitors. Interestingly, C3G directly scavenged radical activity and inhibited intracellular ROS generation in our cell-based system. In addition, C3G pretreatment suppressed the up-regulation of specific ER proteins namely calpain, caspase-12 and C/EBP homologous proteins (CHOP) induced by glutamate-mediated oxidative and ER stress signal by up-regulating the expressions of survival proteins, including extracellular regulated protein kinase (ERK) and nuclear factor E2-related factor 2 (Nrf2). Furthermore, dramatically activated gene expression of endogenous antioxidant enzymes (i.e. superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPx)), and phase II enzymes (glutathione-S-transferases (GSTs)) was found in C3G-treated with cells. CONCLUSIONS Our finding suggest that C3G could be a promising neuroprotectant via inhibition of glutamate-induced oxidative and ER stress signal and activation of ERK/Nrf2 antioxidant mechanism pathways.

中文翻译：

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Cyanidin-3-glucoside激活Nrf2抗氧化反应元件，并保护谷氨酸诱导的HT22海马神经元细胞的氧化和内质网应激。

背景技术氰基丁-3-葡糖苷（C3G），一种存在于浆果中的主要花色苷，显示出强的抗氧化剂，并且已经显示出具有神经保护作用。长时间暴露于谷氨酸会导致氧化损伤和内质网应激，这可能在神经退行性疾病（NDs）的发生中起关键有害作用。在本研究中，我们研究了谷氨酸降低HT22小鼠海马神经元细胞中C3G对氧化/ ER应激诱导的细胞凋亡的神经保护作用及其潜在机制。方法用不同浓度的C3G预处理细胞，然后用谷氨酸预处理。使用MTT和LDH测定法检查细胞活力和毒性。通过膜联蛋白V-FITC /碘化丙啶共染色测定法进行凋亡和坏死细胞死亡。使用DCFH-DA探针通过流式细胞仪测量细胞中细胞内活性氧（ROS）的生成。通过实时聚合酶链反应分析评估抗氧化剂基因的表达。随后通过蛋白质印迹分析证明了可能涉及的信号途径和蛋白质。结果C3G预处理HT22细胞可保护细胞免于谷氨酸诱导的氧化毒性。我们证明了用谷氨酸处理的细胞引起了几种自由基形式的ROS形成，并且它们被特定的ROS抑制剂所消除。有趣的是，在我们基于细胞的系统中，C3G直接清除了自由基活性并抑制了细胞内ROS的产生。此外，C3G预处理抑制了特定ER蛋白（即钙蛋白酶）的上调，谷氨酸介导的氧化应激和内质网应激信号诱导的caspase-12和C / EBP同源蛋白（CHOP）通过上调存活蛋白的表达，包括细胞外调节蛋白激酶（ERK）和核因子E2相关因子2（Nrf2） ）。此外，在经C3G处理的过程中，发现内源性抗氧化酶（即超氧化物歧化酶（SOD），过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx））和II期酶（谷胱甘肽S-转移酶（GST））的活化基因表达显着增加。与细胞。结论我们的发现表明C3G可能通过抑制谷氨酸诱导的氧化和ER应激信号以及激活ERK / Nrf2抗氧化机制途径而成为有前途的神经保护剂。包括细胞外调节蛋白激酶（ERK）和核因子E2相关因子2（Nrf2）。此外，在经C3G处理的过程中，发现内源性抗氧化酶（即超氧化物歧化酶（SOD），过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx））和II期酶（谷胱甘肽S-转移酶（GST））的活化基因表达显着增加。与细胞。结论我们的发现表明C3G可能通过抑制谷氨酸诱导的氧化和ER应激信号以及激活ERK / Nrf2抗氧化机制途径而成为有前途的神经保护剂。包括细胞外调节蛋白激酶（ERK）和核因子E2相关因子2（Nrf2）。此外，在经C3G处理的过程中，发现内源性抗氧化酶（即超氧化物歧化酶（SOD），过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx））和II期酶（谷胱甘肽S-转移酶（GST））的活化基因表达显着增加。与细胞。结论我们的发现表明C3G可能通过抑制谷氨酸诱导的氧化和ER应激信号以及激活ERK / Nrf2抗氧化机制途径而成为有前途的神经保护剂。在用C3G处理的细胞中发现了II期酶（谷胱甘肽S-转移酶（GSTs））。结论我们的发现表明C3G可能通过抑制谷氨酸诱导的氧化和ER应激信号以及激活ERK / Nrf2抗氧化机制途径而成为有前途的神经保护剂。在用C3G处理的细胞中发现了II期酶（谷胱甘肽S-转移酶（GSTs））。结论我们的发现表明C3G可能通过抑制谷氨酸诱导的氧化和ER应激信号以及激活ERK / Nrf2抗氧化机制途径而成为有前途的神经保护剂。