Objectives: 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.
Methods: Male SD rats were randomly divided into control, model and EA groups (n=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.
Results: 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 (P<0.01, P<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 (P<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 (P<0.01, P<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.
Conclusions: 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.
目的: 观察电针对慢性疲劳综合征（CFS）大鼠行为学和海马组织蛋白磷酸化的影响，探讨电针治疗CFS的作用机制。方法: 将雄性SD大鼠随机分为空白组、模型组和电针组，每组12只。采用多因素慢性复合应激法制备CFS模型。电针组大鼠给予电针“神庭”（透刺“百会”）和“大椎”治疗，每日1次，每次15 min，连续28 d。治疗结束后观察各组大鼠体质量、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，采用旷场实验及Morris水迷宫实验评价大鼠焦虑程度和学习记忆能力。取各组大鼠海马组织进行磷酸化Label-free定量蛋白质组学检测。结果: 治疗结束后，与空白组比较，模型组大鼠体质量降低（P<0.01），摄食量减少（P<0.05），一般情况半定量评分升高（P<0.01），旷场实验总穿格次数和进入中央区次数均增多（P<0.01），Morris水迷宫实验逃避潜伏期延长（P<0.01）、穿越原平台次数减少（P<0.01）。与模型组比较，电针组大鼠一般情况半定量评分降低（P<0.01），旷场实验总穿格次数减少（P<0.05），Morris水迷宫实验逃避潜伏期缩短（P<0.01）、穿越原平台次数增加（P<0.01）。与空白组比较，模型组有297个差异表达肽段，对应255个蛋白，与模型组比较，电针组有245个差异表达肽段，对应198个蛋白，其中共有24个重合蛋白在电针治疗后表达回调。GO分析表明，电针干预的作用在生物学过程方面主要为对蛋白质聚合和细胞骨架组织的调节，在分子功能聚类上表现为对蛋白质聚合的调节，在细胞组分聚类方面表现为对突触功能的影响。KEGG分析结果显示，电针干预对胰岛素分泌、轴突导引、磷脂酰肌醇信号系统及赖氨酸生物合成等对中枢神经系统组织功能有重要作用的信号通路产生了影响。结论: 电针可以改善CFS模型大鼠的疲劳状态，缓解焦虑情绪，提高学习记忆能力，其机制可能与调节海马组织蛋白磷酸化水平有关。.
Keywords: Chronic fatigue syndrome; Electroacupuncture; Hippocampus; Liquid chromatography mass spectrometry; Phosphorylated proteomics.