摘要目的 研究PI3K/Akt/GSK3β信号通路在颈部降温的调节作用,初步探讨颈部降温的脑保护作用是否与PI3K/Akt及GSK3β的磷酸化有关.方法 实验在第二军医大学附属长征医院急救科实验室完成.健康雄性新西兰家兔30只随机(随机数字法)分为5组,采用4 min室颤模型.(1)假手术组:常规手术操作置管,不诱导CA,于术后24 h处死取标本.(2)常温复苏组(normothermia theat,NT组):常规致颤复苏并于24 h时间点处死动物取标本.(3)复苏即刻降温组(intra-arrest therapeutic hypothermia,IATH组):于心肺复苏同时启动颈部快速降温,目标脑温为34 ℃,以后维持目标脑温至ROSC后4 h.于24 h时间点处死动物取标本.(4)复苏中降温+特异性抑制剂LY294002(LY294002组):复苏前20 min脑室注射LY294002,其余同组3.(5)复苏后1 h降温组(post-arrest therapeutic hypothermia,PATH组),于心肺复苏后1 h启动颈部快速降温,目标脑温为34 ℃,其余同组3.LY294002溶于DMSO稀释成10 μmol/L,在动物脑立体定位仪下在ROSC前20 min前给予脑室内注射,其余各组给予溶剂DMSO.动物24 h过量麻醉处死,采集标本.应用Western blot检测Akt、p-Akt、GSK-3β、P-GSK-3β (ser9)的蛋白表达,运用TUNEL等方法观察各组组织凋亡变化.所得数据以均数±标准差(x±s)表示,多组间比较采用单因素方差分析.结果 与Sham组比较,常温心肺复苏组兔脑细胞胞质中的Akt (Thr-308)磷酸化水平(P-AKT)和P-GSK3β在心肺复苏后24 h明显减低(P<0.05);复苏即刻颈部降温组P-AKT和P-GSK3β水平在心肺复苏后24 h均较常温复苏组明显增强(P<0.05);复苏后1 h降温组的蛋白表达水平在复苏后24 h也较常温复苏组有增强(P<0.05),但弱于复苏即刻颈部降温组;脑室内注射LY294002去除了亚低温的这一作用,表明了LY294002抑制Akt的磷酸化.复苏即刻降温组和复苏后1 h降温组的凋亡细胞较常规复苏组和LY294002组明显减少(P<0.05).结论 颈部降温能减轻复苏后兔脑神经细胞的损伤,复苏时即刻降温能有更好的脑保护作用.低温的脑保护作用能够被PI3K/Akt通路的阻断剂所阻断.说明亚低温的脑保护作用是通过PI3K/Akt通路的激活来实现的.颈部降温可通过PI3K/Akt/GSK-3β信号通路,使蛋白激酶B的活化,促进GSK3β的磷酸化,发挥神经保护作用.PI3K/Akt通路阻断剂LY294002可抑制复苏后脑组织Akt的活化,从而抑制了下游GSK3β的磷酸化,抵消降温的神经保护作用.

abstractsObjective To study the changes of PI3K/Akt/GSK3β signaling pathway during resuscitation with neck cooling in order to explore the relationship between the protective effect of neck cooling and the phosphorylation of PI3K/Akt and GSK3β.Methods Thirty rabbits were randomly(random number) divided into five groups, and models of cadiac arrest were induced by ventricular fibrillation(VF, the positive electrode in the right ventricle and negative pole on the apex of heart) for 4 min.In sham group,a electrode was placed into right ventricle without electric current conducted, and CA was not induced.The rabbits were sacrificed and specimens were taken at 24 hours after modeling.In normothermia treat group(NT group),resuscitation was carried out to restoration of spontaneous circulation(ROSC),and the rabbits were sacrificed and specimens were taken at 24 hours after modeling.In intra-arrest therapeutic hypothermia group (IATH group), rapid neck cooling was initiated at the same time with CPR,and the target brain temperature was set at 34 ℃ maintained for 4 hours after ROSC.Rabbits were sacrificed and specimens were taken at 24 hours after modeling.In recovery period cooling + LY294002 group(PATH+LY294002 group), LY294002 was injected intra-ventricularly at 20 minutes before resuscitation.Rapid neck cooling was started at the same time with CPR,and the target brain temperature was set at 34 ℃ maintained for 4 hours after ROSC.The rabbits were sacrificed and specimens were taken at 24 hours after modeling.In post-arrest therapeutic hypothermia group (PATH group), rapid neck cooling was begun after CPR for 1 hour,and the target brain temperature was set at 34 ℃ maintained for 4 hours after ROSC.The rabbits were sacrificed and specimens were taken at 24 hours after modeling.Animals were sacrificed by using overdose anesthetic drug.Western blot was used to detect the level of Akt p-Akt GSK-3β p-GSK-3β (ser9) protein, and TUNEL was used to observe apoptosis of tissues in each group.Multiple comparisons were performed with one-way analysis of variance (ANOVA).Results Compared with Sham group, Akt (Thr-308) phosphorylation (P-AKT) and P-GSK-3β levels in the brain neuron cytoplasm in 24 hours after CPR resuscitation in NT group was significantly reduced, and showed a gradual reduction trend (P<0.05);the P-AKT and P-GSK-3β levels in the brain neuron cytoplasm in 24 hours after CPR resuscitation in IATH group were significantly enhanced compared with NT group (P<0.05);the levels of these two kinds of protein at one hour after resuscitation in PATH group were significantly enhanced compared with NT group (P<0.05), but lower in IATH group.Intra-ventricularly injection of LY294002 made the effect of hypothermia lost, indicating that LY294002 inhibited the phosphorylation of Akt.Apoptosis cells were significantly reduced in IATH group and normothermia theatment group compared with PATH group and LY294002 group(P<0.05).Conclusions Neck cooling can reduce apoptosis in rabbit brain cells after recovery, and the protective effect on brain is best in intra-arrest therapeutic hypothermia group.LY294002 specifically block the PI3K/Akt pathway, and the protective effect of cooling on the brain can be abolished,indicating hypothermia protects the neurological function via activation of PI3K/Akt pathway.Neck cooling protects the neurological function by activating PI3K/Akt/GSK-3β, promoting the Akt activation, and increasing the expression of P-GSK3β.Specific Akt inhibitor LY294002 inhibits Akt phosphorylation of brain tissue recovery and further inhibit the phosphorylation of GSK-3β, thus abolishing protective effect of cooling on neurological function.