二氮嗪对深低温低流量小鼠脑的保护作用

Effect of diazoxide with different concentrations on brain tissue in mice model of deep hypothermic low flow

目的观察不同浓度二氮嗪对深低温低流量脑缺血再灌注小鼠模型的脑保护作用，探讨二氮嗪发挥脑保护作用的机制。方法将240只3周龄的C57／BL-6健康小鼠随机分为假手术组（sham）、模型组（m）、二氮嗪（2、5、10mg／kg）组、渥曼青霉素＋二氮嗪（2、5、10mg／kg）组共8组，每组30只。每组再平均分为再灌注2、24、72h3个亚组，每个亚组再平分为蛋白质组和脑组织大体标本组，最小组以5只为单位。根据要求建立深低温低流量动物模型，于再灌注不同时间点取小鼠脑组织。应用Western blot技术分别检测各组脑组织中总蛋白激酶B（AKT）、磷酸化蛋白激酶B（p-AKT）、磷酸化糖原合成酶激酶-3β（p-GSK-3β）的表达变化。对各组脑组织进行2，3，5-氯化三苯基四氮唑（TYC）染色，观察脑组织损伤程度。结果再灌注24h二氮嗪组p-AKT、p-GSK-3β与其他组比较表达增高，差异有统计学意义（P〉0．05）；再灌注24h和72h2mg／kg组与其他二氮嗪组比较表达增高，差异有统计学意义（P〈0．05）。TTC染色结果显示再灌注24h二氮嗪组与其他组比较脑损伤减轻，差异有统计学意义（P〈0．05）；再灌注24h和72h2mg／kg组与其他二氮嗪组比较脑损伤减轻，差异有统计学（P〈0．05）。渥曼青霉素十二氮嗪组与模型组差异无统计学意义（P〉0．05）。结论二氮嗪对深低温低流量小鼠模型有脑保护作用，其发挥作用的机制可能是通过激活磷脂酰肌醇3激酶（P13K）／AKT信号通路，进而调节AKT下游蛋白糖原合成激酶-3β（GSK-3β）的磷酸化水平而实现的。

Objective To observe the influence of diazoxide with different concentrations on brain tissue in mice model of deep hypothermic low flow. Methods The C57BL/6 mice （n = 240） were randomly and equally divided into 8 groups： sham group, model group, diazoxide （2, 5, 10 mg/kg） groups and wortmannin ＋ diazoxide （2, 5, 10 mg/kg） groups. Operation group was subjected to cerebral isehemiareperfusion （I/R） ： Bilateral common carotid arteries were occluded for 120 rain at （18.5 ±0. 5 ）℃ and then reperfused and rewarmed afterwards, and the sham operation group experienced the same procedures except occlusion. The protein expression levels of protein kinase B （AKT）, p-protein kinase B （p-AKT） and p-glycogen synthase kinase-β3 （p-GSK-β3） were detected by using Western blotting at 2, 24 and 72 b after I/R, respectively. Five mice taken from each group at 2, 24 and 72 h after reperfusion respectively were randomly decapitated and the coronal brain slices were stained with triphenyltetrazolium chloride （TTC） solution. The samples were posffixed and photoed. Results The diazoxide group had the characteristic changes of I/R injury, p-AKT and p-GSK-3β expression levels were higher in diazoxide group than those subgroups and at 24 h after reperfusion （P 〈0. 05）. The expression of p-AKT and p-GSK-3β was increased at 24 and 72 h after reperfusion in diazoxide （2 mg/kg） group than that in other diazoxide groups （P 〈0. 05 ）. The diazoxide group had less pathological injury than those groups at 24 h after reperfusion （P 〈0. 05）. The pathological injury was alleviated at 24 and 72 h after reperfusion in diazoxide （2 mg/kg） group as compared with that in other diazoxide groups （P 〈 0. 05 ）. Conclusion Diazoxide exerts the neuroprotective effects against cerebral I/R injury during deep hypothermia low flow probably by activating the phosphatidylinositol 3 kinase（PI3K）/protein Kinase B （AKT） signal pathway, further up-regulating the expression of p-GSK-3β.