CAJ | 학술논문

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Western Blot技术分别检测各组脑组织中总akt、p-akt1、p-gsk-3β的蛋白表达变化；应用real time RT-PCR方法检测Akt1 mRNA、Akt2 mRNA和Akt3 mRNA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＞0.05)；再灌注24 h和72 h模型组的p-akt1、p-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-akt1、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72 h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2 h Akt1 mRNA、Akt2 mRNA和Akt3 mRNA的表达三组间差异无统计学意义(P＞0.05)；再灌注24 h和72 h模型组的Akt1 mRNA和Akt3 mRNA表达高于sham组和wortmannin组,且差异有统计学意义(P＜0.05),而Akt2 mRNA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PI3K/Akt信号通路,进而调节Akt下游蛋白糖原合成激酶-3β的磷酸化水平而实现的.Akt1 mRNA和Akt3mRNA表达增高可能与p-akt1和p-gsk-3β蛋白表达增高有关.
목적 탐토PI3K/Akt신호통로대심저온저류량소서모형적뇌보호작용급초보분석기발휘작용적궤제.방법 장90지3주령적C57BL/6건강소서수자수궤분위가수술조(sham)、모형조(model)화wortmannin조,매조30지.모형조화wortmannin조건립심저온저류량동물모형,가수술조불조단쌍측경총동맥,기여조작여모형조일양.응용Western Blot기술분별검측각조뇌조직중총akt、p-akt1、p-gsk-3β적단백표체변화；응용real time RT-PCR방법검측Akt1 mRNA、Akt2 mRNA화Akt3 mRNA적변화급대각시간단뇌조직진행2,3,5-록화삼분기사담서(TTC)염색、고정、박조급분석뇌조직손상적정황.결과 각조중총akt단백적표체차이무통계학의의(P＞0.05)；재관주24 h화72 h모형조적p-akt1、p-gsk-3β단백표체증고,차이유통계학의의(P＜0.05)；이응용wortmannin후p-akt1、p-gsk-3β단백표체강저,차이유통계학의의(P＜0.05),여가수술조상비차이무통계학의의(P＞0.05).재관주24h화72 h모형조화wortmannin조상비뇌손상적정도감경,차차이유통계학의의(P＜0.05).재관주2 h Akt1 mRNA、Akt2 mRNA화Akt3 mRNA적표체삼조간차이무통계학의의(P＞0.05)；재관주24 h화72 h모형조적Akt1 mRNA화Akt3 mRNA표체고우sham조화wortmannin조,차차이유통계학의의(P＜0.05),이Akt2 mRNA적표체삼조간차이무통계학의의(P＞0.05).결론 심저온저류량소서모형유뇌보호작용,기발휘작용적궤제가능시통과격활PI3K/Akt신호통로,진이조절Akt하유단백당원합성격매-3β적린산화수평이실현적.Akt1 mRNA화Akt3mRNA표체증고가능여p-akt1화p-gsk-3β단백표체증고유관.
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 ischemia-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-akt1 and p-gsk-3β were determined by Western blot and mRNA expressions of akt1,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-akt1 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-akt1 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 akt1,akt2 and akt3 mRNA expressions at 2 h after reperfusion.The mRNA expressions of akt1 and akt2 were increased at 24 and 72 hours after reperfusion in model group than those in wortmannin 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-akt1 and p-gsk-3β.The findings of real time PCR imply that akt1 mRNA and akt3 mRNA may play a role in the expression of p-akt1 and p-gsk-3β undergoing deep hypothermic low flow in mice.