电针对阿尔茨海默病模型大鼠GSK3β信号通路相关蛋白的影响

Effects of Electroacupuncture on GSK3β Signaling Pathway Related Proteins in Alzheimer’s Disease Model Rats

目的观察电针"百会""肾俞"穴对阿尔茨海默病(AD)模型大鼠学习记忆能力及海马CA1区GSK3β信号通路相关蛋白的影响及机制研究。方法30只SPF级SD雄性大鼠随机分成假手术组、模型组和电针组,每组10只。除假手术组外,其余各组大鼠双侧海马区各注射5μLβ淀粉蛋白25-35(Aβ25-35)制备AD模型,假手术组给予同等剂量生理盐水注射。假手术组、模型组正常喂养,不做干预处理,电针组选取百会及肾俞穴(两侧肾俞穴交替选择)进行电针治疗,频率2 Hz,电流1 mA,每日1次。每个疗程6天,共2个疗程,两个疗程间隔1天。治疗结束后第2天上午8点开始Morris水迷宫(Morris water maze,MWM)检测大鼠学习记忆能力,行为学实验结束后第2天取材。苏木精-伊红染色(Hematoxylin eosin staining,HE)观察海马CA1区病理形态改变。免疫组化法(Immunohistochemical method,IHC)检测大鼠海马区Tau蛋白磷酸化的表达,及糖原合成酶激酶3β(glycogen synthase kinase3β,GSK3β)信号通路相关蛋白表达。免疫印迹法(Western blot,WB)检测磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)、蛋白激酶B(protein kinase B,AKT)、GSK3β相关蛋白的表达。聚合酶链反应(polymerase chain reaction,PCR)检测AKT、GSK3βmRNA的表达。结果与假手术组相比:模型组大鼠逃避潜伏期明显延长(P<0.01),穿越平台次数明显减少(P<0.01),目标象限停留时间明显缩短(P<0.01);模型组海马区Tau蛋白磷酸化水平明显提高(P<0.01),GSK3β蛋白表达水平明显提高(P<0.01);模型组PI3K(P85)、AKT(Ser473)、GSK3β(Ser9)蛋白相对表达量降低(P<0.05);模型组AKT mRNA表达水平明显降低(P<0.01),GSK3βmRNA表达水平明显提高(P<0.01)。与模型组相比:电针组大鼠逃避潜伏期明显缩短(P<0.01),穿越平台次数明显增加(P<0.01),目标象限停留时间明显延长(P<0.01);电针组海马区Tau蛋白磷酸化水平明显降低(P<0.01),GSK3β蛋白表达水平明显降低(P<0.01);电针组PI3K(P85)、AKT(Ser473)、GSK3β(Ser9)蛋白相对表达量增加(P<0.05);电针组AKT mRNA表达水平明显提高(P<0.01),GSK3βmRNA表达水平明显降低(P<0.01)。结论电针治疗能够有效改善AD大鼠学习记忆能力,其机制可能与影响GSK3β信号通路相关蛋白表达从而抑制Tau蛋白磷酸化有关。

Objective To observe the effects of electroacupuncture at“Baihui”(GV 20)and“Shenshu”(BL 23)on memory and GSK3βprotein in hippocampal CA1 region of Alzheimer’s disease(AD)model rats and its mechanism.Methods Thirty SPF grade male SD rats were randomly divided into sham operation group,model group and electroacupuncture group,with 10 rats in each group.Except for sham operation group,rats in other groups were injected with 5μLβAmyloprotein 25-35(Aβ_(25-35)).The sham operation group was given the same dose of normal saline injection.The sham operation group and model group were fed normally without intervention,while the electroacupuncture group was given electroacupuncture treatment.Baihui(GV 20)and left Shenshu(BL 23)were selected on the first day,then Baihui(GV 20)and right Shenshu(BL 23)were selected on the next day,and bilateral Shenshu(BL 23)were selected alternately.The frequency was 2 Hz,and the current was 1 mA,once a day.Each course of treatment is 6 days,a total of 2 courses,1 day between the two courses.Learning and memory were measured in the Morris Water maze(MWM)at 8 am on the second day after treatment and on the second day after behavioral study.Hematoxylin eosin staining(HE)was used to observe the pathological changes of hippocampal CA1 area.The phosphorylation of tau protein and the expression of glycogen synthase kinase3β(GSK3β)signal pathway related protein in hippocampal region were detected by immunohistochemistry(IHC).Western blot(WB)was used to detect phosphatidylinositol 3 kinase(PI3 K),protein kinase B(Akt)and the expression of GSK3βrelated proteins.Akt and GSK3βmRNA expression were detected by polymerase chain reaction(PCR).Results Compared with the sham operation group,the escape latency of the model group was significantly longer(P<0.01),the number of crossing the platform was significantly reduced(P<0.01),and the residence time in the target quadrant was significantly shorter(P<0.01).And compared with the model group,the escape latency of the EA group was significantly shortened(P<0.01),the frequency of crossing platform was significantly increased(P<0.01),and the residence time in the target quadrant was significantly shortened(P<0.01).Besides,compared with sham operation group,Tau protein phosphorylation level and GSK3βprotein expression level were significantly increased in model group(P<0.01);Compared with model group,Tau protein phosphorylation level and GSK3βprotein expression level were significantly decreased in EA group(P<0.01).In addition,compared with the sham operation group,the relative expressions of PI3 K(p85),AKT(ser473)and GSK3β(ser9)were decreased in the model group(P<0.05);Compared with the model group,the relative expressions of PI3 K(p85),AKT(ser473)and GSK3β(ser9)were increased in the EA group(P<0.05).Compared with sham operation group,AKT mRNA expression level in model group was significantly lower(P<0.01),and GSK3βmRNA expression level was significantly higher(P<0.01);Compared with model group,AKT mRNA expression level in EA group was significantly higher(P<0.01),and GSK3βmRNA expression level was significantly lower(P<0.01).Conclusion Electroacupuncture can improve the learning capacity and memory of AD rats,and it may be related with the expression of GSK3βsignaling pathway related proteins and inhibiting the phosphorylation of Tau protein.