亚低温对大鼠自发性蛛网膜下腔出血急性期脑组织线粒体功能的影响  

Effect of mild hypothermia on mitochondrial function in the brain tissues of rats with acute phase of spontaneous subarachnoid hemorrhage

作　　者：薄海[1,2] 彭朋[1] 秦永生[1] 王大宁[1] 刘暌[3] 康伟民 

机构地区：[1]武警后勤学院军事训练医学教研室,天津300121 [2]天津市心血管重塑与靶器官损伤重点实验室 [3]天津市环湖医院神经外科,300060 [4]解放军第二炮兵总医院神经介入科,北京100080

出　　处：《中华神经医学杂志》2014年第3期239-245,共7页Chinese Journal of Neuromedicine

基　　金：天津市科技计划项目（08ZCGYSF01600,12JCQNJC07900）

摘　　要：目的观察亚低温对大鼠自发性蛛网膜下腔出血（SAH）急性期脑组织线粒体活性氧（ROS）生成及能量代谢的影响，并探讨亚低温作用的相关信号通路。方法SD大鼠按随机数字表法分为假手术组、SAH常温组和SAH亚低温组（每组各6只），后两组采用视交叉前池注血法建立SAH模型，SAH亚低温组在脑池注血后即刻给予亚低温干预（32．5～33．5℃1。各组大鼠于SAH后2h处死取材，电镜下观察海马CA1区超微结构，比色法检测线粒体锰超氧化物歧化酶（MnSOD）、谷胱甘肽过氧化物酶（OPx）、过氧化氢酶（CAT）活性和丙二醛（MDA）含量，极谱法检测线粒体呼吸速率。二氯荧光素法检测线粒体ROS生成速率，JC-1荧光标记法检测膜电位，荧光素酶发光法检测线粒体ATP合成活力。实时荧光定量PCR法检测脑组织Sirtuin．3（SIRT3）mRNA表达，Westernblotting法检测线粒体S1RT3蛋白表达。结果与SAH常温组比较，SAH亚低温组SIRT3mRNA和蛋白表达、线粒体ATP合成活力和膜电位，线粒体态3呼吸速率[（55．26±6．30）nmol02／min·mg蛋白]、呼吸控制比（5．15％±0．63％）、磷氧比（2．16％±0．25％），MnSOD、GPx、CAT活性均明显上调，差异均有统计学意义（P〈0．05）；SAH亚低温组线粒体态4呼吸速率[（10．67±1．69）nmool O2/min·mg蛋白]、ROS产生速率及MDA含量明显降低，差异均有统计学意义（P〈0．05）。结论亚低温可提高线粒体能量代谢效率及ROS清除能力，增强脑组织对SAH的耐受力。Objective To observe the effect of mild hypothermia on mitochondrial reactive oxygen species （ROS） generation and energy metabolism in acute phase of spontaneous subarachnoid hemorrhage （SAH） in rats, and to explore the relative signaling pathway in this process. Methods SD rats were randomly divided into three groups： sham-operated group, normothermic SAH group （SAH-N）, and hypothermic SAH group （SAH-H）. The SAH models were induced by injection of antologous arterial blood into the prechiasmatic cistern. After the injection, rats in SAH-H group were subjected to mild hypothermia intervention （32.5-33.5 ~ C）. All animals were sacrificed by decapitation two hours after SAIl. Ultrastructures of CA1 section of the hippocampus were observed under transmission electron microscopy. The enzyme activities of mitochondrial manganese superoxide dismutase （MnSOD）, glutathione peroxidase （GPx） and catalase （CAT）, and the malonaldehyde （MDA） level were measured with colorimetry method. Mitochondrial respiratory function was measured with polarography method. Mitochondrial ROS generation was determined using dichlorofluorescein （DCF） method. Membrane potent was determined using JC-1 fluorescence labeling. Adenodine triphospate （ATP） synthesis capacity was determined using bioluminescence technique. The Sirtuin-3 （SIRT3） mRNA expression in the brain tissues was detected by real-time quantitative PCR. The mitochondrial SIRT3 protein expression was detected by Western blotting, Results As compared with those in the SAH-N group, mitochondrial SIRT3 mRNA and protein expressions, ATP synthetase activity, membrane potent, state 3 respiratory rate , respiratory control ratio, ratio of ADP to oxygen, and activities of MnSOD, GPx and CAT significantly increased in the SAH-H group （P〈0.05）; and state 4 respiratory rate, mitochondrial ROS generation and MDA level significantly decreased in SAH-H group （P〈0.05）. Conclusion Mild hypothermia could efficiently promote mitochondrial

关 键 词：蛛网膜下腔出血 亚低温 线粒体 能量代谢 氧化应激 Sirtuin-3 

分 类 号：R743.35[医药卫生—神经病学与精神病学]