抑制 AMP 活化的蛋白激酶对局灶性脑缺血再灌注小鼠皮质和海马细胞色素 c表达的影响

Effects of inhibition of AMP-activated protein kinase on cytochrome c expression in the cortex and hippocampus after focal cerebral ischemia-reperfusion in mice

目的：研究抑制 AMP 活化的蛋白激酶（AMP-activated protein kinase, AMPK）活性对小鼠脑缺血再灌注后皮质和海马细胞色素 c（cytochrome c, CytC）的影响。方法36只雄性 C57BL/6小鼠,采用随机数字法分为假手术组、缺血再灌注组和 compound C 组,每组12只。 compound C 组在缺血时腹腔注射 AMPK 特异性抑制剂 compound C（20 mg/kg）。采用改良线栓法制作大脑中动脉闭塞模型,再灌注24 h后采用蛋白印迹法测定缺血侧皮质和海马 AMPK、磷酸化 AMPA（phosphorylated-AMPK, p-AMPK）和细胞质 CytC 表达水平。结果假手术组、缺血再灌注组和 compound C 组皮质 p-AMPK/AMPK 分别为0．701±0．197、1．408±0．322和0．930±0．229（F =12．000,P =0．001）,海马分别为0．685±0．228、1．507±0．418和0．964±0．378（ F =8．530,P =0．003）,缺血再灌注组皮质（ P ＜0．001）和海马（P =0．001）均显著高于假手术组,compound C 组皮质（P =0．005）和海马（P =0．017）均显著低于缺血再灌注组。假手术组、缺血再灌注组和 compound C 组皮质 CytC 水平分别为0．496±0．278、1．461±0．321和1．018±0．175（ F =19．915,P ＜0．001）,海马分别为0．511±0．257、1．610±0．441和0．921±0．228（F =17．795,P ＜0．001）,缺血再灌注组皮质（P ＜0．001）和海马（P ＜0．001） CytC 水平均显著高于假手术组,而 compound C 组皮质（P =0．011）和海马（P =0．002）细胞质 CytC水平均显著低于脑缺血再灌注组。结论抑制 AMPK 活性能下调小鼠脑缺血再灌注后缺血侧皮质和海马细胞质 CytC 表达。

Objective To investigate the effects of of inhibition of AMP-activated protein kinase （AMPK） on cytochrome c （CytC） expression in the cortex and hippocampus after focal cerebral ischemia-reperfusion in mice. Methods Thirty-six male C57BL/6 mice were randomly divided into 3 groups： a sham operation group, an ischemia-reperfusion group, and a compound C group （n = 12 in each group）. The mice of the compound C group were intraperitonealy injected an AMPK specific inhibitor compound C （20 mg/kg） at the time of ischemia. A model of middle cerebral artery occlusion was induced by a modified suture method. After 24 h of reperfusion, Western blot was used to detect the expression levels of AMPK, phosphorylated-AMPK （p-AMPK）, and cytoplasm CytC in the cortex and hippocampus in the ischemic side. Results p-AMPK/MPK levels in the cortex in the sham operation group, ischemia-reperfusion group, and compound C group were 0. 701 ± 0. 197, 1. 408 ± 0. 322, and 0. 930 ± 0. 229, respectively （F = 12. 000, P =0. 001）; p-AMPK/MPK levels in the hippocampus were 0. 685 ± 0. 228, 1. 507 ± 0. 418, and 0. 964 ± 0. 378, respectively （ F = 8. 530, P = 0. 003 ）; p-AMPK/AMPK levels both in the cortex （ P 〈 0. 001 ） and hippocampus （P = 0. 001） in the ischemia-reperfusion group were significantly higher than those in the sham operation group, p-AMPK/AMPK levels both in the cortex （P = 0. 005） and hippocampus （P = 0. 017） in the compound C group were significantly lower than those in the ischemia-reperfusion group. CytC levels in the cortex in the sham operation group, ischemia-reperfusion group, and compound C group were 0. 496 ±0. 278, 1. 461 ± 0. 321, and 1. 018 ± 0. 175, respectively （F = 19. 915, P 〈 0. 001）; CytC levels in the hippocampus were 0. 511 ± 0. 257, 1. 610 ± 0. 441, and 0. 921 ± 0. 228 （F = 17. 795, P 〈 0. 001）; CytC levels both in the cortex （P 〈 0. 001） and hippocampus （P 〈 0. 001） in the ischemia-reperfusion group were significantly higher than those in the sham operation group, while CytC levels both in the cortex （P = 0. 011） and hippocampus （P = 0. 002） in the compound C group were significantly lower than those in the ischemia-reperfusion group. Conclusion Inhibition of the AMPK may down-regulate the cytoplasm CytC expression in the cortex and hippocampus after cerebral ischemia-reperfusion in mice.