Department of Physiology, Kyungpook National University School of Medicine, 101 Dongin 2 Ga, Jung Gu, Daegu 700-422, Korea.
J Neuroinflammation. 2013 Feb 4;10:21. doi: 10.1186/1742-2094-10-21.
Hypothermic protection against ischemic stroke has been reported by many studies. Hypothermia is supposed to mitigate the effects of deleterious genes and proteins and promote the activity of protective genes and proteins in the ischemic brain. Metallothionein (MT)-1/2 is thought to be a crucial factor for metal homeostasis, immune function, and apoptosis. This protein was found to exert protective effects in models of brain injury as well. In the present study, we investigated the effect of hypothermia on MT expression and the underlying mechanisms.
Cultured bEnd.3 brain endothelial cells were exposed to oxygen glucose deprivation and reperfusion (OGD+R). Reverse transcription PCR and western blot analyses were performed to measure the expression of MT, transcription factors, and methylation regulating factors. Transcription factor binding assays were also performed. Methylation profiles of the promoter area were obtained with pyrosequencing.
Hypothermia protected bEnd.3 cells from OGD+R. When the cells were exposed to OGD+R, MT expression was induced. Hypothermia augmented MT levels. While OGD+R-induced MT expression was mainly associated with metal regulatory transcription factor 1 (MTF-1), MT expression promoted by hypothermia was primarily mediated by the signal transducer and activator of transcription 3 (STAT3). Significantly increased STAT3 phosphorylation at Ser727 was observed with hypothermia, and JSI-124, a STAT-3 inhibitor, suppressed MT expression. The DNA demethylating drug 5-aza-2'-deoxycytidine (5-Aza) enhanced MT expression. Some of the CpG sites in the promoter MT=> it should be "the CpG sites in the MT promoter" showed different methylation profiles and some methylation regulating factors had different expressional profiles in the presence of OGD+R and hypothermia.
We demonstrated that hypothermia is a potent inducer of MT gene transcription in brain endothelial cells, and enhanced MT expression might contribute to protection against ischemia. MT gene expression is induced by hypothermia mainly through the STAT3 pathway. DNA methylation may contribute to MT gene regulation under ischemic or hypothermic conditions.
许多研究报道了低温对缺血性中风的保护作用。低温被认为可以减轻有害基因和蛋白质的作用，并促进缺血性大脑中保护性基因和蛋白质的活性。金属硫蛋白（MT）-1/2 被认为是金属动态平衡、免疫功能和细胞凋亡的关键因素。这种蛋白质在脑损伤模型中也被发现具有保护作用。在本研究中，我们研究了低温对 MT 表达的影响及其潜在机制。
培养 bEnd.3 脑内皮细胞，使其经历氧葡萄糖剥夺再灌注（OGD+R）。采用逆转录 PCR 和 Western blot 分析来测量 MT、转录因子和甲基化调节因子的表达。还进行了转录因子结合分析。采用焦磷酸测序获得启动子区域的甲基化图谱。
低温可保护 bEnd.3 细胞免受 OGD+R 损伤。当细胞暴露于 OGD+R 时，MT 表达被诱导。低温增强了 MT 水平。虽然 OGD+R 诱导的 MT 表达主要与金属调节转录因子 1（MTF-1）有关，但低温诱导的 MT 表达主要是由信号转导和转录激活因子 3（STAT3）介导的。低温下观察到 STAT3 丝氨酸 727 磷酸化显著增加，STAT3 抑制剂 JSI-124 抑制 MT 表达。DNA 去甲基化药物 5-氮杂-2'-脱氧胞苷（5-Aza）增强了 MT 表达。MT 启动子中的一些 CpG 位点在 OGD+R 和低温存在下显示出不同的甲基化图谱，一些甲基化调节因子在 OGD+R 和低温存在下的表达谱也不同。
我们证明了低温是脑内皮细胞中 MT 基因转录的有效诱导剂，增强 MT 表达可能有助于对抗缺血。MT 基因表达受低温诱导主要通过 STAT3 途径。DNA 甲基化可能有助于在缺血或低温条件下调节 MT 基因。
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