[Study on the mechanism of scalp-acupoint cluster puncture in improving hippocampal synaptic plasticity by regulating the cAMP/PKA/CREB signaling pathway in APP/PS1 mice].

OBJECTIVES: To observe the effect of scalp-acupoint cluster puncture on hippocampal synaptic plasticity and the activity of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway in APP/PS1 mice, so as to explore its possible mechanism underlying improvement of Alzheimer's disease (AD). METHODS: Male APP/PS1 transgenic mice were randomly divided into model, acupuncture, and sham acupuncture groups, with 12 mice in each group. In addition, 12 male C57BL/6 mice with the same genetic background were used as the control group. The mice of the acupuncture group received cluster-needle stimulation at "Baihui"(GV20) and 2 spots (2 mm to GV20 on the left and right sides), and those of the sham acupuncture group received cluster-needle stimulation at the bilateral hypochondrial non-acupoints. Following insertion, the needles were twisted rapidly for about 1 min, then retained for 30 min in the acupuncture group. The intervention was conducted once a day, for a total of 28 d. The Morris water maze test and Barnes maze test were used to evaluate the mouse's learning-memory and cognitive ability. The immunohistochemical staining was employed to detect amyloid-β (Aβ) deposition in the hippocampus. The Nissl staining was used to observe the changes of the number of neurons in the hippocampal CA1 area. A transmission electron microscopy was employed to observe the ultrastructure of hippocampal synapses. The Golgi staining was used to observe the dendritic spine density of hippocampal neurons. The protein expressions of cAMP, PKA, CREB, phosphorylated (p)-CREB, postsynaptic density protein 95 (PSD-95), and synapsin Ⅰ (SYN1) was detected using Western blot. RESULTS: Compared with the control group, the model group showed a significant increase in the escape latency of Morris water maze test and the latency to enter the target hole of Barnes maze test and the percentage of Aβ-positive area (P<0.01, P<0.001), and a striking reduction in the number of target platform crossings in the Morris water maze test, residence time in the target quadrant of the escape box and the number of correct hole entries in the Barnes maze test, number of neurons, dendritic spine density, and the expression levels of cAMP, PKA, PSD-95, SYN1 and p-CREB/CREB ratio (P<0.01, P<0.001). After the intervention, both the increase and decrease of the indexes mentioned above were reversed in the acupuncture group (P<0.01, P<0.001), but not in the sham acupuncture group. Morphological results showed irregular shapes and disordered arrangement of the hippocampal neurons with shrunken nucleoli, atrophied mitochondria, broken mitochondrial cristae, decreased synapses and vesicles in the model group, which was relatively milder in the injury degree, for example, increase in the number of synapses and vesicles, relatively complete mitochondrial cristae, etc., in the acupuncture group. CONCLUSIONS: Scalp-acupoint cluster puncture can improve the learning-memory ability in APP/PS1 mice, which may be associated with its functions in enhancing the hippocampal synaptic plasticity and up-regulating the cAMP/PKA/CREB signaling. 目的: 观察头穴丛刺对APP/PS1小鼠海马突触可塑性的影响并通过环腺苷酸（cAMP）/蛋白激酶A（PKA）/环腺苷酸应答元件结合蛋白（CREB）信号通路探讨头穴丛刺改善阿尔茨海默病（AD）的可能机制。方法: 将雄性APP/PS1转基因小鼠随机分为模型组、针刺组、假针刺组，每组12只，同背景的C57BL/6小鼠12只作为对照组。针刺组给予头穴丛刺治疗，假针刺组取双侧胁下非穴点进行针刺，每次干预30 min，1次/d，共28 d。采用Morris水迷宫和巴恩斯迷宫检测小鼠学习记忆能力和认知功能，免疫组织化学染色检测海马组织淀粉样蛋白-β（Aβ）沉积，尼氏染色观察海马CA1区神经元数量，透射电子显微镜观察小鼠海马突触超微结构，高尔基染色观察海马神经元树突棘密度，Western blot法检测海马cAMP、PKA、CREB、磷酸化（p）-CREB、突触后密度蛋白95（PSD-95）、突触素Ⅰ（SYN1）的蛋白表达。结果: 与对照组相比，模型组小鼠Morris水迷宫逃避潜伏期延长、穿越原平台次数减少（P<0.01），巴恩斯迷宫进入目标洞口的潜伏期显著延长（P<0.01）、逃生箱目标象限的停留时间缩短、正确进入洞口次数减少（P<0.001），Aβ阳性表达面积百分比增多（P<0.001），海马CA1区神经元和突触结构严重损伤，神经元数量、树突棘密度降低（P<0.01），海马cAMP、PKA、PSD-95、SYN1蛋白表达及p-CREB/CREB显著降低（P<0.01）;与模型组相比，针刺组小鼠Morris水迷宫逃避潜伏期缩短、穿越原平台次数增多（P<0.01），巴恩斯迷宫进入目标洞口的潜伏期显著降低、逃生箱目标象限的停留时间和正确进入洞口次数增加（P<0.001），Aβ阳性表达面积百分比减少（P<0.001），海马CA1区神经元和突触结构均有改善，神经元数量、树突棘密度增加（P<0.01），海马cAMP、PKA、PSD-95、SYN1蛋白表达及p-CREB/CREB显著升高（P<0.01）。假针刺组与模型组比较各指标差异均无统计学意义。结论: 头穴丛刺可能通过调节cAMP/PKA/CREB信号通路增强APP/PS1小鼠海马突触可塑性，进而改善AD小鼠认知功能。.