AMP-活化蛋白激酶对局灶性脑缺血再灌注小鼠皮质胱天蛋白酶-8、-3表达和细胞凋亡的影响

目的 探讨抑制AMP-活化蛋白激酶(AMP-activated protein kinase,AMPK)活性对局灶性脑缺血再灌注小鼠梗死体积、缺血皮质胱天蛋白酶-8、-3表达以及细胞凋亡的影响.方法 72只雄性C57BL/6小鼠,随机数字表法分为假手术组、缺血再灌注组和AMPK抑制剂组.线栓法建立大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型.AMPK抑制剂组在MCAO即刻腹腔注射Compound C(20 mg/kg),假手术组及缺血再灌注组在相同时间点腹腔注射等量生理盐水.应用2,3,5-三苯基氯化四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色法测量脑梗死体积,免疫组化染色法检测胱天蛋白酶-8、-3表达水平,TUNEL染色法检测细胞凋亡.结果 假手术组未见梗死灶,无胱天蛋白酶-8、-3以及凋亡阳性细胞.与缺血再灌注组比较,AMPK抑制剂组梗死体积显著缩小(45.34±7.20)mm3对(22.71±4.93)mm3;t=5.730,P=0.037],大脑皮质胱天蛋白酶-8(17.58±8.62)个/HP对(6.87±4.32)个/HP;t=3.631,P=0.023]、胱天蛋白酶-3(16.21±5.46)个/HP对(8.22±4.64)个/HP;t=2.630,P=0.021]和凋亡(78.44±8.32)个/HP对(55.73±6.71)个/HP;=5.541,P=0.042]阳性细胞数量均显著减少.结论 抑制AMPK活性可缩小局灶性脑缺血再灌注小鼠梗死体积,其机制可能与下调胱天蛋白酶-8、-3表达和减少细胞凋亡有关.

Effects of AMP-activated protein kinase on the expression levels of caspase-8,-3 and apoptosis in the ischemic cortex following focal cerebral ischemia-reperfusion in mice

Objective To investigate the effects of inhibiting AMP-activated protein kinase (AMPK) activity on the infarct vohune,expressions of caspase-8,-3 and apoptosis in the ischemic cortex following focal cerebral ischemia-reperfusion in mice.Methods A total of 72 male C57BL/6 mice were divided into a sham-operation group,an ischemia-reperfusion group,and an AMPK inhibitor group according to the random number table method.A mode of middle cerebral artery occlusion (MCAO) was induced by the suture method.Immediately after MCAO,the compound C (20 mg/kg) was injected intraperitoneally in the AMPK inhibitor group.The same volume of saline was injected intraperitoneally at the same time point in the sham operation group and the ischemia-reperfusion group.The infarct volume was measured by 2,3,5 triphenyltetrazolium chloride (TTC) staining.The expression levels of caspase-8 and-3 were detected by immunohistochemical staining.Apoptosis was detected by TUNEL staining.Results The sham-operation group had no infarction,no caspase-8,-3,and apoptotic positive cells.Compared with ischemia-reperfusion group,the infarct volume was significantly decreased (45.34 ±7.202 mm3 vs.2.71 ±4.93 mm3;t =5.730,P =0.037),the numbers of positive cells of caspase-8 (17.58 ± 8.62/HP vs.6.87 ± 4.32/HP;t =3.631,P=0.023),caspase-3 (16.21 ± 5.46/HP vs.8.22± 4.64/HP;t=2.630,P=0.021),and apoptosis (78.44 ±8.32/HP vs.55.73 ±6.71/HP;t =5.541,P=0.042) in the cerebral cortex in the AMPK inhibitor group was decreased significantly.Conclusions Inhibition of AMPK activity may reduce the infarct volume of cerebellar ischemia-reperfusion in mice.Its mechanism may be associated with the downregulation of expression levels of caspase-8,-3,and the decrease of apoptosis.