TREK-1通道活性改变对大鼠局灶性脑缺血后细胞凋亡和凋亡相关蛋白的影响

目的:观察双孔钾通道TREK-1活性改变对大鼠局灶性脑缺血后细胞凋亡和凋亡相关蛋白的影响。方法:45只大鼠随机分为假手术组(10只)、对照组(10只)和干预组(25只)。建立大鼠光化学脑缺血模型,假手术组不注射玫瑰红,干预组侧脑室注射不同浓度(100μmol/L、250μmol/L、500μmol/L、1 mmol/L)亚麻酸(LIN),对照组注射等量生理盐水。应用免疫荧光双标法观察正常生理情况下TREK-1在大脑神经细胞中的表达,TUNEL及DAPI双标法检测缺血边缘区细胞凋亡,Western blot法检测B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷酸化细胞外信号调节激酶(p-erk)表达。结果:正常生理情况下,TREK-1在神经元和星形胶质细胞中均有表达。与假手术组相比,对照组大量细胞凋亡,Bcl-2/Bax值下降,p-erk蛋白增加(P<0.05);与对照组比较,干预组细胞凋亡显著减少,Bcl-2/Bax值上升,p-erk蛋白降低(P<0.05)。结论:TREK-1在神经元和星形胶质细胞中均有表达。TREK-1激动剂LIN可显著抑制脑缺血后细胞凋亡,上调Bcl-2与Bax比值,抑制erk磷酸化。

Effect of TREK-1 Activation on Cell Apoptosis and the Expression of Apoptosis-associated Protein in Rats Following Focal Cerebral Ischemia

Objective：To investigate the effect of TREK-1 channel activity on cell apoptosis and the protein ex- pression of Bcl-2 and Bax after focal cerebral ischemia in rats. Methods： Forty-five rats were randomly assigned into sham-operated （n=10）, vehicle-treatment （n=10）, and intervention groups （n=25）. Photochemical ischemia was performed and sham-operated animals were given saline instead of Rose Bengal. Alpha-lineonic acid was administrated i.c.v （100 ixmol/L, 250 ~mol/L, 500 txmol/L, or 1 mmol/L）. Vehicle treated animals received the same volumes of 0.9% NaC1. The cellular distribution of TREK-1 in rat brain was detected by immunohistochem- ical staining. Double staining of TUNEL and DAPI was performed to determine the percentage of cell death and the protein expression of B-cell lymphoma-2 （Bcl-2）, Bcl-2-associated X protein （Bax） and Phosphorylated Extra- cellular Signal-Regulated Kinase （p-erk） was detected by western blot. Results： TREK-1 immunoreactivety was expressed in the GFAP positive astrocytes and NeuN positive neurons in the cerebral cortex. Compared with sham-operated groups, the percentage of TUNEL positve cells and the phosphorylation of erk increased signifi- cantly, the Bcl-2 to Bax ratio decreased dramatically （P〈0.05）. However, after treatment with L1N, the percentage of TUNEL positive cells and the phosphorylation of erk was reduced significantly compared to that of vehicle group, and the Bcl-2/Bax ratio was increased （P〈0.05）. Conclusion： TREK-1 was expressed in astrocytes and neurons. TREK-1 activation with LIN remarkably inhibited the cell apoptosis and the phosphorylation of erk, and increased the ratio of protein expression of Bcl-2 and Bax following focal cerebral ischemia.