PI3K／Akt对深低温低流量小鼠模型的脑保护作用

Effect and molecular mechanism of P13K/Akt signaling pathway on brain undergoing deep hypothermiclow flow in mice

目的探讨PI3K／Akt信号通路对深低温低流量小鼠模型的脑保护作用及初步分析其发挥作用的机制。方法将90只3周龄的C57BL／6健康小鼠数字随机分为假手术组（sham）、模型组（model）和wortmannin组，每组30只。模型组和wortmannin组建立深低温低流量动物模型，假手术组不阻断双侧颈总动脉，其余操作与模型组一样。应用WesternBlot技术分别检测各组脑组织中总akt、p-aktl、p-gsk3β的蛋白表达变化；应用realtimeRT-PCR方法检测AktlmRNA、Akt2mRNA和Akt3mRNA的变化及对各时间段脑组织进行2，3，5-氯化三苯基四氮唑（TTC）染色、固定、拍照及分析脑组织损伤的情况。结果各组中总akt蛋白的表达差异无统计学意义（P〉0．（15）；再灌注24h和72h模型组的p-aktl、l〉gsk-3β蛋白表达增高，差异有统计学意义（P〈0．05）；而应用wortmannin后p-aktl、p-gsk-3β蛋白表达降低，差异有统计学意义（P〈0．05），与假手术组相比差异无统计学意义（P〉0．05）。再灌注24h和72h模型组和wortmannin组相比脑损伤的程度减轻，且差异有统计学意义（P〈0．05）。再灌注2hAktlmRNA、Akt2mRNA和Akt3mRNA的表达三组问差异无统计学意义（P〉0．05）；再灌注24h和72h模型组的AktlmRNA和Akt3mRNA表达高于sham组和wort—mannin组，且差异有统计学意义（P〈0.05），而Akt2mRNA的表达三组间差异无统计学意义（P〉0．05）。结论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P13K／Akt信号通路，进而调节Akt下游蛋白糖原合成激酶一3J3的磷酸化水平而实现的。AktlmRNA和Akt3mRNA表达增高可能与p-aktl和p-gsk3β蛋白表达增高有关。

Objective To explore the effect and molecular mechanism of P13K/Akt signaling pathway on brain tissue of undergoing deep hypothermic low flow in mice. Methods The C57BL/6 mice （n = 90） were randomly assigned into 3 groups： sham group, model group, and wortmannin group. Model group and wortmannin group were cerebral isehemia-reperfusion（I-R） group underwent deep hypothermic low flow and then reperfused and rewarmed, while the sham operation group excluded the arteries occlusion procedure. The protein expressions of akt, p-aktl and p-gsk-3β were determined by Western blot and rnRNA expressions of aktl, akt2 and akt3 were determined by real time RT-PCR at 2 h,24 h and 72 h after I/R respectively. Brain tissue was taken from five mice of each group at reperfusion 2h, 24 h and 72 h timepoint respectively; then coronal slices were made and stained with 2,3,5-triphenyltetrazolium chlorid （TTC）. The samples were postfixed and pictures were taken and analyzed. Results The deep hypothermic low flow had the characteristic changes of I/R injury. The protein expressions of p-aktl and p-gsk-3β in model group were higher than those in other two subgroups at 24 h and 72 h after reperfusion（P〈0. 05）. The expressions of p-aktl and p-gsk-3β were decreased at 24 and 72 hours after reperfusion in wortamnnin group （P〈0. 05）. The model group showed less pathological injury than wortmannin group at 24 h and 72 h after reperfusion（P〈0. 05）. There was no significant difference between aktJ, akt2 and akt3 mRNA expressions at 2 h afterreperfusion. The mRNA expressions of aktl and akt2 were increased at 24 and 72 hours after reperfusion in model group than those in worlmannin and sham group （P〈0.05）. No significant difference detected for akt2 mRNA expression between three groups. Conclusions These results suggest that that PI3K/Akt signaling pathway has cerebral protective effect in mice model undergoing deep hypothermia low flow by increasing the expression of p aktl and p-gsk-3β. The findings of real time PCR imply that aktl mRNA and akt3 mRNA may play a role in the expression of p aktl and p gsk 3β undergoing deep hypothermic low flow in mice.