电针对APP/PS1小鼠学习记忆及海马SIRT1、FOXO3A蛋白表达的影响

Effects of electroacupuncture on learning memory and hippocampal SIRT1 and FOXO3A protein expression in APP/PS1 mice

目的 观察电针对阿尔茨海默病(Alzheimer’s disease,AD)模型小鼠学习记忆能力、海马区神经元形态及沉默信息调节因子1(silent information regulator 1,SIRT1)、叉头框转录因子O3A(forkhead box transcription factor O3A,FOXO3A)表达的影响,探讨电针治疗AD的作用机制。方法30只6月龄雄性淀粉样前体蛋白/早老蛋白1(amyloid precursor protein/presenilin-1,APP/PS1)双转基因小鼠随机分成模型组、电针组、电针+抑制剂组,每组10只。以同源背景C57BL/6野生鼠10只作为空白对照组。除电针+抑制剂组,其余三组在干预前30分钟腹腔注射磷酸盐缓冲液。空白对照组与模型组仅予以束缚,电针组在针刺“百会”“印堂”“水沟”穴的基础上加用脉冲电流,电针+抑制剂组在电针组基础上于干预前腹腔注射3-甲基腺嘌呤溶液。各组干预20分钟/次,每日一次,共15天。15天治疗结束后通过Morris水迷宫实验观察四组小鼠行为学改变;采用苏木素—伊红染色观察各组海马区细胞形态;分别采用免疫组化和蛋白免疫印迹法观察各组小鼠海马组织SIRT1、FOXO3A的表达情况。结果 (1) Morris水迷宫结果显示:电针组较模型组逃避潜伏期明显缩短、穿越站台次数及Ⅰ象限运动距离百分比明显增多(P <0.05,P <0.01);(2)苏木素—伊红染色结果显示:较之模型组,电针组、电针+抑制剂组可不同程度改善海马区神经元病理特征;(3)免疫组化结果显示:较之模型组,电针组SIRT1表达明显上调,总FOXO3A表达下降(P <0.01),电针+抑制剂组SIRT1表达增高(P <0.01);(4)蛋白免疫印迹结果显示:较之模型组,电针组SIRT1、胞核FOXO3A蛋白表达明显上调,胞质FOXO3A蛋白表达明显下调(P <0.01),电针+抑制剂组SIRT1、胞核FOXO3A蛋白表达稍增多(P <0.05),胞质FOXO3A蛋白表达减少(P <0.01)。结论“通督启神”法电针可提高AD小鼠空间学习记忆能力和改善海马区神经元形态结构,其机制可能与增强AD小鼠海马区SIRT1蛋白表达,提高FOXO3A的转录活性有关。

Objective To observe the effects of electroacupuncture on the learning and memory abilities,neuronal morphology and expression of silent information regulator 1(SIRT1) and forkhead box transcription factor 03 A(FOXO3 A) in the hippocampus of mice with Alzheimer’s disease(AD).To investigate the mechanism of action of electroacupuncture in the treatment of AD.Methods Thirty 6-monthold male APP/PS1 mice were randomly divided into the model group,the electroacupuncture group,and the electroacupuncture+inhibitor group,with 10 mice in each group.Ten C57 BL/6 wild mice with homologous background were used as the blank control group.Except the electroacupuncture+inhibitor group,the other three groups were injected with PBS solution intraperitoneally 30 min before intervention.The blank control group and the model group were only restrained,and the electroacupuncture group used pulsed current in addition to acupuncture points "Baihui(GV20)", "Yin Tang(GV29)" and "Shuigou(GV26)".In the electroacupuncture+inhibitor group,3 MA solution was injected intraperitoneally before intervention.After 15 days of treatment,the behavioral changes of mice in the four groups were observed by Morris water maze experiment;the cell morphology of hippocampal area in each group was observed by HE staining;the expression of SIRT1 and FOXO3 A in hippocampal tissue and the expression levels in each group were observed by immunohistochemistry and western blot,respectively.Results(1) Morris water maze results showed that the number of escape latencies was significantly decreased,the number of crossing stations and the percentage of movement distance in quadrant I were significantly increased in the electroacupuncture group compared with the model group(P <0.05,P <0.01).(2) The HE staining results showed that compared with the model group,the electroacupuncture group and electroacupuncture+inhibitor group could improve the neuronal pathological features in the hippocampal region.(3) Immunohistochemical results showed that compared with the model group,the expression of SIRT1 in the hippocampus was significantly upregulated and the expression of total FOXO3 A was decreased in the electroacupuncture group(both P <0.01),while the expression of SIRT1 was increased in the electroacupuncture+inhibitor group(P <0.01).(4) Western blot results showed that compared with the model group,SIRT1 and cytosolic FOXO3 A protein expression was significantly upregulated and cytoplasmic FOXO3 A protein expression was significantly downregulated in the electroacupuncture group(both P <0.01),while SIRT1 and cytosolic FOXO3 A protein expression were slightly increased in the electroacupuncture+inhibitor group(both P <0.05) and cytoplasmic FOXO3 A protein expression was decreased(P <0.01).Conclusion The electroacupuncture method of ’Tongdu Qishen’ can improve the spatial learning and memory ability and the morphological structure of neurons in the hippocampus of AD mice,and the mechanism may be related to the enhancement of SIRT1 protein expression and FOXO3 A transcriptional activity in the hippocampus of AD mice.