缺氧预处理可能通过下调糖原合酶激酶3β和信号转导及转录激活蛋白3保护大鼠局灶性脑缺血

Hypoxia precondilioning may protect focal cerebral ischemia in rats by downregnlation of glycogen synthase kinase 3β, phosphorylated signal transducer and activator of transcriplion 3

目的探讨磷酸化糖原合酶激酶3β（phosphorylated glycogen synthase kinase 3β，pGSK3β）和磷酸化信号转导及转录激活蛋白3（phosphorylated signal transducer and activator of transcription 3, pSTAT3）在缺氧预处理保护大鼠缺血性脑损伤中的作用。 方法将60只SD大鼠随机分为假手术组、脑缺血组和缺氧预处理组，每组20只。采用改良线栓法建立大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）模型，缺氧预处理组大鼠在制备MCAO模型前置于模拟海拔5 000 m的低压氧舱中（压力：0.53×105 Pa；氧分压：81 mmHg，1 mmHg＝0.133 kPa），每天3 h，连续5 d。在MCAO模型制作后24 h对大鼠进行神经行为学评分（n＝6）和脑梗死体积测定（n＝6），应用免疫组化染色法检测缺血皮质神经元核抗原（neuronal nuclei, NeuN）和pGSK3β（Ser9）表达（n＝7），应用蛋白质印迹法检测缺血皮质pGSK3β（Ser9）和pSTAT3（Tyr705）表达（n＝7）。 结果缺氧预处理组神经功能缺损评分[（1.833±0.408）分对（2.667±0.516）分；t＝3.101，P＝0.011]和脑梗死体积[（18.137±0.801）%对（24.125±0.694）%；t＝13.840，P〈0.001]均显著低于和小于脑缺血组。免疫组化染色显示，脑缺血组和缺氧预处理组NeuN阳性细胞数量均显著少于假手术组[（48.000±1.414）个/高倍视野、（124.833±3.061）个/高倍视野和（213.500±2.429）个/高倍视野，F＝7 150.550，P〈0.001]，缺氧预处理组显著多于缺血组（P〈0.001）；脑缺血组和缺氧预处理组pSTAT3阳性细胞数量均显著多于假手术组[（57.667±1.366）个/高倍视野、（29.167±1.941）个/高倍视野和（3.500±1.049）个/高倍视野，F＝1 962.649，P〈0.001]，缺氧预处理组显著少于缺血组（P〈0.001）。蛋白质印迹分析显示，脑缺血组和缺氧预处理组缺血皮质pGSK3β和pSTAT3表达水平显著高于假手术组[pGSK3β：（2.336±0.102）、（0.876±0.196）和（0.440±0.012），F＝1 610.826，P〈 0.001；pSTAT3：（8.368±0.230）、（4.883±0.123）和（0.595±0.138），F＝4 018.051，P〈 0.001]，缺氧预处理组显著低于缺血组（P均〈0.001）。 结论缺氧预处理对缺血性脑损伤大鼠具有神经保护作用，可能与下调pGSK3β和pSTAT3表达有关。

ObjectiveTo investigate the roles of phosphorylated glycogen synthase kinase 3β （pGSK3β） and phosphorylated signal transducer and activator of transcription 3 （pSTAT3） in hypoxic preconditioning-induced neuroprotection against ischemic brain injury in rats. MethodsSixty SD rats were randomly divided into a sham operation group, a cerebral ischemia group, and a hypoxia preconditioning group （n=20 in each group）. A model of middle cerebral artery occlusion （MCAO） was induced by the modified suture method. Before the preparation of MCAO model, the rats in the hypoxia preconditioning group were put into a hypobaric oxygen chamber at a simulated altitude of 5 000 m （pressure： 0.53 × 105 kPa; partial pressure of oxygen： 81 mmHg; 1 mmHg=0.133 kPa）, 3 h a day for 5 days. At 24 h after MCAO modeling, the rats were subjected to neurobehavioral score （n=6） and cerebral infarction volume measurement （n=6）. Immunohistochemical staining was used to detect the expression levels of neuronal nuclei （NeuN） and pGSK3β （Ser9） （n=7）. Western blot was used to detect the expression levels of pGSK3β （Ser9） and pSTAT3 （Tyr705） in the ischemic cortex （n=7）. ResultsThe neurological deficit score （1.833±0.408 vs. 2.667±0.516; t=3.101, P=0.011） and cerebral infarction volume （18.137%±0.801% vs. 24.125%±0.694%; t=13.840, P〈0.001） in the hypoxia preconditioning group were significantly lower or smaller than those in the cerebral ischemia group. Immunohistochemical staining showed that the numbers of NeuN positive cells in the cerebral ischemia group and the hypoxia preconditioning group were significantly less than that in the sham operation group （48.000±1.414/high power field [HPF], 124.833±3.061/HPF, and 213.500±2.429/HPF; F=7 150.550, P〈0.001）, the hypoxia preconditioning group was significantly more than the ischemia group （P〈0.001）; the numbers of pSTAT3 positive cells in the cerebral ischemia group and the hypoxia preconditioning group were significantly higher than that in the sham operation group （57.667±1.366/HPF, 29.167±1.941/HPF and 3.500±1.049/HPF; F=1 962.649, P〈0.001）, and the hypoxia preconditioning group was significantly less than the ischemia group （P〈0.001）. Western blot analysis showed that the expression levels of ischemic cortical pGSK3β and pSTAT3 in the cerebral ischemia group and the hypoxia preconditioning group were significantly higher than those of the sham operation group （pGSK3β： 2.336±0.102, 0.876±0.196 and 0.440±0.012; F=1 610.826, P〈0.001; pSTAT3 ： 8.368±0.230, 4.883±0.123 and 0.595±0.138; F=4 018.051, P〈0.001）, the hypoxia preconditioning group were significantly lower than the ischemia group （all P〈0.001）. ConclusionsHypoxia preconditioning has neuroprotective effect for ischemic brain injury in rats. It may be associated with the down-regulation of the expressions of pGSK3 and pSTAT3.