Objectives: To investigate the effect of "Tongdu Qishen" acupuncture［dredging the Govern Vessel and normalizing mental activities, electroacupuncture (EA) of "Baihui" (GV20), "Yintang" (EX-HN3) and "Shuigou" (GV26) needling］on the learning-memory ability and the mechanism of ferroptosis in Alzheimer's disease (AD) mice.
Methods: Twenty-four male APPswe/PS1dE9 mice were randomly and equally divided into model group and EA group, and 12 normal C57BL/6 mice were used as the control group. In the EA group, EA (2 Hz/100 Hz, 20 min) was applied to GV20 and EX-HN3 in combination with manual acupuncture stimulation of GV26. The treatment was performed once a day, for a total of 28 days. The mice in the three groups were given the same fixation and grasping operation. Morris water maze swimming tests were used to assess the mice's learning-memory ability. Nissl staining and transmission electron microscopy were used to observe the morphological changes of neurons in the hippocampus. The activity of superoxide dismutase (SOD) in the hippocampus tissue was detected by superoxide anionic colorimetric assay kit (WST-1), and malondialdehyde (MDA) contents were detected by thiobarbituric acid (TBA) method. The expression levels of prostaglandin endoperoxide synthase 2 (ptgs2) and glutathione peroxidase 4 (GPX4) mRNA in the hippocampus were detected by fluorescent quantitative real-time PCR.
Results: Behavioral results showed that compared with the control group, the escape latencies at the 2nd, 3rd, 4th and 5th day of Morris water maze swimming test were significantly increased (P<0.05), and the swimming time in the original platform quadrant and the times of cros-sing the original platform were considerably decreased (P<0.05) in the model group. In comparison with the model group, the escape latencies at the 4th and 5th day were strikingly decreased (P<0.05), and the swimming time in the original platform quadrant and times of crossing the original platform significantly increased (P<0.05) in the EA group. Following modeling, the SOD activity and the expression of GPX4 mRNA were obviously down-regulated (P<0.05), and the content of MDA and the expression of ptgs2 mRNA significantly up-regulated (P<0.05) in the model group rele-vant to the control group. The SOD activity and the expression of GPX4 mRNA were apparently increased (P<0.05), and the content of MDA and the expression of ptgs2 mRNA remarkably down-regulated (P<0.05) in the EA group rele-vant to the model group. Histopathological and ultrastructural results showed scattered arrangement of cells, widened space among cells, reduction in the number of cells, and many shrunk of dissolved nucleoli, shrunking and incomplete mitochondria, and high membrane electron density in the hippocampus of the model group, which was relatively milder in the EA group.
Conclusions: "Tongdu Qishen" acupuncture can improve the learning-memory ability of AD mice, which may be related to its functions in up-regulating SOD activity and GPX4 mRNA expression, and down-regulating MDA content and ptgs2 mRNA expression to reduce the lipid peroxidation in the process of ferroptosis.
目的: 观察“通督启神”针法对阿尔茨海默病小鼠学习记忆能力及铁死亡相关因子的影响，探讨其提高阿尔茨海默病小鼠学习记忆能力的机制。方法: APPswe/PS1dE9小鼠随机分为模型组和针刺组，以C57BL/6小鼠作为对照组，每组各12只。针刺组予电针“百会”“印堂”并点刺“水沟”，每日1次，每次20 min，共28 d。采用Morris水迷宫实验检测小鼠学习记忆能力；尼氏染色和透射电镜观察小鼠海马组织神经细胞的形态学及超微结构变化；分别采用WST-1法和TBA法检测小鼠海马组织中超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量；实时荧光定量PCR法检测小鼠海马组织中前列腺素内过氧化物合酶2（ptgs2）、谷胱甘肽过氧化物酶4（GPX4）mRNA的表达水平。结果: 与对照组相比，模型组小鼠第2—5天逃避潜伏期显著延长（P<0.05），原平台象限游泳时间和穿越原平台次数显著减少（P<0.05），SOD活性降低（P<0.05），MDA含量升高（P<0.05），ptgs2 mRNA表达水平升高（P<0.05），GPX4 mRNA表达水平降低（P<0.05）。与模型组相比，针刺组第4—5天逃避潜伏期显著缩短（P<0.05），原平台象限游泳时间和穿越原平台次数显著增加（P<0.05），SOD活性升高（P<0.05），MDA含量降低（P<0.05），ptgs2 mRNA表达水平降低（P<0.05），GPX4 mRNA表达水平升高（P<0.05）。组织病理学和超微结构结果显示，与对照组相比，模型组海马组织神经细胞数量减少，排列结构散乱，线粒体皱缩，嵴不完整，而针刺组上述病理变化在一定程度上得到改善。结论: “通督启神”针法可以提高APPswe/PS1dE9小鼠空间学习和记忆能力，发挥神经保护作用，其机制可能与提高机体抗氧化水平，抑制细胞铁死亡有关。.
Keywords: Acupuncture; Alzheimer’s disease; Ferroptosis; Glutathione peroxidase 4; Prostaglandin endoperoxide synthase 2.