Heilongjiang University of Chinese Medicine, Harbin 150040, China.
Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin 150001.
Zhen Ci Yan Jiu. 2024 Jun 25;49(6):594-603. doi: 10.13702/j.1000-0607.20230180.
To observe the effect of electroacupuncture (EA) on behavior and hippocampal protein phosphorylation in rats with chronic fatigue syndrome (CFS), so as to explore its mechanisms underlying improvement of CFS.
Male SD rats were randomly divided into control, model and EA groups (=12 rats in each group). The CFS model was established by chronic multifactor combined with stress stimulation (treadmill training + restraint stress + sleep disturbance + crowded environment). For rats of the EA group, EA (1 mA, frequency of 10 Hz) was applied to "Shenting" (GV24) (with an acupuncture needle penetrated from GV24 to "Baihui" ［GV20］) and "Dazhui" (GV14) for 15 min, once daily for 28 days. After treatment, the body weight, food intake and water intake of rats in each group were observed. The fatigue degree of rats was evaluated by Semi-quantitative score observation table of the general condition of experimental rats.The open field test (OFT) was used to assess the rats'anxiety severity by detecting the total number of grid-crossing and the times of the central area entered in 5 min, and Morris water maze test was employed to assess the rats' learning-memory ability by detecting the escape latency in 1 min, and the times of the original platform quadrant crossing in 1 min. The hippocampaus was taken for phosphorylated Label-free quantitative proteomics analysis by using Maxquant technology based on full scan mode to calculate the integral of each peptide signal of liquid chromatography-mass spectrometry(LC-MS). The differentially-expressed proteins (>1.5 folds for up-regulation or <0.67 folds for down-regulation) were evaluated by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.
Compared with the control group, the body weight, food intake, and the times of original-platform quadrant crossing of spatial exploring of Morris water maze test were significantly decreased (<0.01, <0.05) , and the score of general conditions, times of grid-crossing and center area-entering of OFT, and the escape latency of navigation task were apparently increased (<0.01) in rats of the model group. After EA intervention, the decreased original-platform quadrant crossing, and the increased score of general conditions, times of grid-crossing and the escape latency of navigation task were all reversed (<0.01, <0.05). Outcomes of proteomics analysis indicated that compared with the model group, there were 297 differentially expressed peptide (48 up-regulated and 249 down-regulated) segments in the control group, and there were 245 differentially expressed peptide (185 up-regulated and 60 down-regulated) segments in the EA group, in which, 25 overlapping peptide segments were reversed after EA treatment, corresponding to 24 proteins, mainly involving cytoskeletal structure. GO function annotation analysis showed that the top three differentially expressed phosphorylated proteins involved in the effect of EA intervention were the actin filament polymerization, protein depolymerization and cytoskeletal tissue in the biological process, the actin binding, structural molecular activity and cytoskeletal protein binding in the molecular function, and the cytoskeleton, dendrites and dendritic trees in the cellular component, respectively. The KEGG pathway annotation analysis for differentially expressed phosphorylated proteins showed that theinsulin secretion, axon guidance, phosphatidylinositol signaling system and lysine biosynthesis, etc. were involved in the effect of EA intervention.
EA of GV24-GV20 and GV14 can improve the general state, anxiety and learning-memory ability of CFS model rats, which may be related to its functions in regulating the hippocampal protein phosphorylation level, and repairing the structure and function of synapses in hippocampus.
观察电针对慢性疲劳综合征（CFS）大鼠行为及海马蛋白磷酸化的影响，探讨其改善 CFS 的作用机制。
雄性 SD 大鼠随机分为对照组、模型组和电针组（每组 12 只）。采用多因素复合应激刺激（跑步机训练+束缚应激+睡眠障碍+拥挤环境）建立 CFS 模型。电针组大鼠给予电针（1 mA，频率 10 Hz），针刺“神庭”（GV24）（针尖从 GV24 向“百会”［GV20］方向透刺）和“大椎”（GV14），每次 15 min，每日 1 次，共 28 天。治疗后观察各组大鼠的体重、摄食量和饮水量。采用实验大鼠一般情况半定量评分观察表评价大鼠的疲劳程度。采用旷场实验（OFT）检测大鼠 5 min 内的总穿越格数和中央区进入次数评估大鼠的焦虑严重程度，采用 Morris 水迷宫实验检测大鼠 1 min 内的逃避潜伏期和 1 min 内原平台象限穿越次数评估大鼠的学习记忆能力。采用 Maxquant 技术基于全扫描模式对海马组织进行磷酸化无标记定量蛋白质组学分析，计算液相色谱-质谱（LC-MS）中每个肽信号的积分。采用基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析评估差异表达蛋白（上调超过 1.5 倍或下调超过 0.67 倍）。
与对照组相比，模型组大鼠的体重、摄食量和 Morris 水迷宫空间探索中原平台象限穿越次数明显减少（<0.01，<0.05），一般情况评分、OFT 中穿越格数和中央区进入次数以及导航任务的逃避潜伏期明显增加（<0.01）。电针干预后，大鼠原平台象限穿越次数减少，一般情况评分、穿越格数和导航任务逃避潜伏期增加均得到逆转（<0.01，<0.05）。蛋白质组学分析结果表明，与模型组相比，对照组有 297 个差异表达肽（48 个上调，249 个下调）片段，电针组有 245 个差异表达肽（185 个上调，60 个下调）片段，其中 25 个差异表达肽经电针治疗后发生逆转，对应 24 个蛋白，主要涉及细胞骨架结构。GO 功能注释分析显示，电针干预效应中差异表达磷酸化蛋白涉及的前 3 个生物学过程分别为肌动蛋白丝聚合、蛋白解聚和细胞骨架组织、肌动蛋白结合、结构分子活性和细胞骨架蛋白结合、细胞骨架、树突和树突树；KEGG 通路注释分析显示，胰岛素分泌、轴突导向、磷酸肌醇信号系统和赖氨酸生物合成等通路参与了电针的干预作用。
电针 GV24-GV20 和 GV14 可改善 CFS 模型大鼠的一般状态、焦虑和学习记忆能力，可能与其调节海马蛋白磷酸化水平、修复海马突触结构和功能有关。
