The mechanism of the neuroprotective action of α2-adrenergic receptor agonists in a model of oxygen-glucose deprivation (OGD) in vitro was investigated. Using fluorescent microscopy, immunostaining, inhibitor analysis, and real-time PCR analysis, we demonstrated that OGD evokes a biphasic [Ca2+]i increase in all cells. Initial Ca2+ impulse in astrocytes and Ca2+ oscillations in neurons were followed by a slower global increase of [Ca2+]i in both cell populations. Accumulation of pro-inflammatory factors, such as IL1b and TNFα, was observed during 40-min OGD. It was established that reoxygenation is followed by hyperexcitation of neurons, caspase-3 activation, and subsequent cell death. We showed that a 24-h pretreatment of cell cultures with selective α2-adrenergic receptor agonists guanfacine and UK-14,304 abolished the global [Ca2+]i increase in astrocytes and neurons but did not suppress the first phase of the OGD-induced Ca2+ impulse in astrocytes. In addition, the number of dead cells after OGD was decreased in cell cultures pretreated with the α2-agonists. Guanfacine inhibited caspase-3 activation and suppressed apoptosis in our experiments. In particular, the expression of antiapoptotic genes Stat3 and Bcl-2 was enhanced after the pretreatment with guanfacine. On the contrary, the expression of proapoptotic genes (Socs-3, p53, fas, and Ikk) was decreased. Moreover, application of guanfacine evoked Ca2+ response in astrocytes and led to Ca2+-mediated ATP release and this way suppressed hyperexcitation of the neurons. Thus, activation of astrocytes and Ca2+-mediated ATP release possibly contribute to the complex neuroprotective effects of the α2-adrenergic receptor agonists.

中文翻译：

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Alpha-2-肾上腺素能受体激动剂在脑缺血-体外再氧合模型中的复杂神经保护作用

研究了 α2-肾上腺素能受体激动剂在体外氧-葡萄糖剥夺 (OGD) 模型中的神经保护作用机制。使用荧光显微镜、免疫染色、抑制剂分析和实时 PCR 分析，我们证明 OGD 在所有细胞中引起双相 [Ca2+]i 增加。星形胶质细胞中的初始 Ca2+ 脉冲和神经元中的 Ca2+ 振荡随后是两种细胞群中 [Ca2+]i 的较慢的全局增加。在 40 分钟的 OGD 期间观察到促炎因子的积累，例如 IL1b 和 TNFα。已经确定，再充氧之后是神经元过度兴奋、caspase-3 激活和随后的细胞死亡。我们表明，用选择性 α2-肾上腺素能受体激动剂胍法辛和 UK-14 对细胞培养物进行 24 小时预处理，304 消除了星形胶质细胞和神经元中 [Ca2+]i 的整体增加，但没有抑制星形胶质细胞中 OGD 诱导的 Ca2+ 冲动的第一阶段。此外，在用α2-激动剂预处理的细胞培养物中，OGD 后死细胞的数量减少。在我们的实验中，胍法辛抑制 caspase-3 激活并抑制细胞凋亡。特别是，在用胍法辛预处理后，抗凋亡基因 Stat3 和 Bcl-2 的表达增强。相反，促凋亡基因（Socs-3、p53、fas 和 Ikk）的表达降低。此外，胍法辛的应用引起星形胶质细胞中的 Ca2+ 反应并导致 Ca2+ 介导的 ATP 释放，这种方式抑制了神经元的过度兴奋。因此，