Lu Chen, Ha Tuanzhu, Wang Xiaohui, Liu Li, Zhang Xia, Kimbrough Erinmarie Olson, Sha Zhanxin, Guan Meijian, Schweitzer John, Kalbfleisch John, Williams David, Li Chuanfu
Department of Surgery, East Tennessee State University, Johnson City, 37614, TN.
J Am Heart Assoc. 2014 Apr 10;3(2):e000629. doi: 10.1161/JAHA.113.000629.
Toll-like receptors (TLRs) have been shown to be involved in cerebral ischemia/reperfusion (I/R) injury. TLR9 is located in intracellular compartments and recognizes CpG-DNA. This study examined the effect of CpG-ODN on cerebral I/R injury.
C57BL/6 mice were treated with CpG-ODN by i.p. injection 1 hour before the mice were subjected to cerebral ischemia (60 minutes) followed by reperfusion (24 hours). Scrambled-ODN served as control-ODN. Untreated mice, subjected to cerebral I/R, served as I/R control. The effect of inhibitory CpG-ODN (iCpG-ODN) on cerebral I/R injury was also examined. In addition, we examined the therapeutic effect of CpG-ODN on cerebral I/R injury by administration of CpG-ODN 15 minutes after cerebral ischemia. CpG-ODN administration significantly decreased cerebral I/R-induced infarct volume by 69.7% (6.4±1.80% vs 21.0±2.85%, P<0.05), improved neurological scores, and increased survival rate, when compared with the untreated I/R group. Therapeutic administration of CpG-ODN also significantly reduced infarct volume by 44.7% (12.6±2.03% vs 22.8±2.54%, P<0.05) compared with untreated I/R mice. Neither control-ODN, nor iCpG-ODN altered I/R-induced cerebral injury or neurological deficits. Nissl staining showed that CpG-ODN treatment preserved neuronal morphology in the ischemic hippocampus. Immunoblot showed that CpG-ODN administration increased Bcl-2 levels by 41% and attenuated I/R-increased levels of Bax and caspase-3 activity in ischemic brain tissues. Importantly, CpG-ODN treatment induced Akt and GSK-3β phosphorylation in brain tissue and cultured microglial cells. PI3K inhibition with LY294002 abolished CpG-ODN-induced protection.
CpG-ODN significantly reduces cerebral I/R injury via a PI3K/Akt-dependent mechanism. Our data also indicate that CpG-ODN may be useful in the therapy of cerebral I/R injury.
Toll样受体（TLRs）已被证明参与脑缺血/再灌注（I/R）损伤。TLR9位于细胞内区室，识别CpG-DNA。本研究检测了CpG-ODN对脑I/R损伤的影响。
在C57BL/6小鼠进行脑缺血（60分钟）随后再灌注（24小时）前1小时，通过腹腔注射给予CpG-ODN。乱序ODN用作对照ODN。未处理的进行脑I/R的小鼠用作I/R对照。还检测了抑制性CpG-ODN（iCpG-ODN）对脑I/R损伤的影响。此外，我们在脑缺血15分钟后给予CpG-ODN，检测其对脑I/R损伤的治疗效果。与未处理的I/R组相比，给予CpG-ODN显著降低了脑I/R诱导的梗死体积69.7%（6.4±1.80%对21.0±2.85%，P<0.05），改善了神经功能评分，并提高了存活率。与未处理的I/R小鼠相比，治疗性给予CpG-ODN也显著降低了梗死体积44.7%（12.6±2.03%对22.8±2.54%，P<0.05）。对照ODN和iCpG-ODN均未改变I/R诱导的脑损伤或神经功能缺损。尼氏染色显示，CpG-ODN处理可保留缺血海马区的神经元形态。免疫印迹显示，给予CpG-ODN可使缺血脑组织中Bcl-2水平升高41%，并减弱I/R诱导的Bax水平升高和caspase-3活性。重要的是，CpG-ODN处理可诱导脑组织和培养的小胶质细胞中Akt和GSK-3β磷酸化。用LY294002抑制PI3K可消除CpG-ODN诱导的保护作用。
CpG-ODN通过PI3K/Akt依赖性机制显著减轻脑I/R损伤。我们的数据还表明，CpG-ODN可能对脑I/R损伤的治疗有用。
