Objective: To observe the effect of electroacupuncture at "Baihui"(GV20) and "Shenshu"(BL23) on the expression of autophagy-related proteins in the hippocampus of rats with Alzheimer's disease (AD)，so as to explore its underlying mechanisms on improvement of AD.
Methods: Forty-eight male SD rats were randomly divided into blank control group, model group, electroacupuncture group and sham electroacupuncture group, with 12 rats in each group. The AD rat model was establish by intraperitoneal injection of D-galactose for 6 weeks. Rats in the electroacupuncture group received electroacupuncture (50 Hz, 1 mA)at GV20 and BL23 for 20 min each time after daily intraperitoneal injection. Rats in the sham electroacupuncture group received acupuncture at the local skin of GV20 and BL23 without electricity. After the intervention, Morris water maze and open field test were used to evaluate the learning and cognitive ability of rats in each group. The transmission electron microscope was used to observe the numerical density of synaptic in hippocampus, and the immunohistochemical staining was used to observe the paired helical filament protein-1 (PHF-1) in the hippocampus. Western blot was used to detected the expression of autophagy-related proteins phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), phosphorylated AKT (p-AKT), mammalian target of rapamycin (mTOR) in the hippocampus.
Results: Compared with the blank control group, the escape latency of the rats in the model group increased from day 2 to day 5 (P<0.01), and the ratio of the time through the quadrant of the original platform reduced （P<0.01）, in the open field test the distance of exercise, the number of uprights and the rate of exercise time in the central area decreased (P<0.01), meanwhile the density of hippocampus synapses decreased (P<0.01), the positive expression of PHF-1 and the relative expression of PI3K, AKT, p-AKT, and mTOR all increased (P<0.01). Compared with the model group, the escape latency of rats in the electroacupuncture group was shortened from day 2 to day 5 (P<0.01), and the ratio of the time through the quadrant of the original platform meanwhile, the distance of the open field test, the number of uprights, and the rate of central area exercise time up-regulated (P<0.01), the numerical density of hippocampus synatic increased (P<0.01), the positive expression of PHF-1 and the relative expression of PI3K, AKT, p-AKT, and mTOR all down-regulated (P<0.01). Compared with the model group, the expression of PI3K in the sham electroacupuncture group decreased (P<0.05).
Conclusion: Electroacupuncture can improve learning and memory and cognitive impairment in AD rats, which may be associated with its effects in regulation of hippocampal autophagy and removal of neurofibrillary tangles by suppressing PI3K/AKT/mTOR signaling pathway.
目的：观察电针对阿尔茨海默病(AD)大鼠海马区自噬相关蛋白表达的影响,从神经元自噬的角度探讨电针“百会”“肾俞”穴治疗AD的机制。方法：SD大鼠随机分为空白对照组、模型组、电针组和假电针组，每组12只。除空白对照组外，其余各组予D-半乳糖连续腹腔注射6周建立AD大鼠模型。电针组大鼠于每日腹腔注射后给予电针“百会”“肾俞”穴，假电针组仅刺入“百会”“肾俞”穴区皮肤且电针仪不通电,每次20 min，1次/d，共6周。干预结束后，采用Morris水迷宫和开放旷场实验评估各组大鼠的学习记忆和认知能力，透射电镜观察海马区突触数密度，用免疫组织化学染色法观察海马区双螺旋细丝蛋白-1(PHF-1)表达水平，用Western blot法检测海马区自噬相关蛋白磷酸肌醇-3-激酶(PI3K)、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)的相对表达量。结果：与空白对照组比较，模型组大鼠第2天至第5天逃避潜伏期增长，经过原平台象限时间比率减小(P<0.01)，旷场实验运动距离、直立次数、中心区域运动时间比率均减少(P<0.01)，海马区突触数密度降低(P<0.01)，PHF-1的阳性表达及PI3K、AKT、p-AKT、mTOR相对表达量均增加(P<0.01)。与模型组和假电针组比较，电针组大鼠第2天至第5天逃避潜伏期缩短，经过原平台象限时间比率升高(P<0.01)，旷场实验运动距离、直立次数、中心区域运动时间比率，以及海马区突触密度增高(P<0.01)，PHF-1的阳性表达及PI3K、AKT、p-AKT、mTOR相对表达量均减少(P<0.01)。与模型组比较，假电针组PI3K表达降低(P<0.05)。结论：电针能改善AD样大鼠的学习记忆和认知障碍，其机制可能是与调控PI3K/AKT/mTOR信号通路，诱导自噬，促进神经原纤维缠结清除有关。.
Keywords: Alzheimer’s disease; Autophagy; Cognitive function; Electroacupuncture; PI3K/AKT/mTOR signaling pathway.