[关键词]
[摘要]
【目的】观察摩腹法结合揿针对焦虑性失眠大鼠睡眠内稳态系统的影响。 【方法】将40只大鼠随机分为正常组、模型组、 摩腹组、揿针组和摩腹加揿针组,每组8只。除正常组,其余各组大鼠采用多因素复合刺激法复制焦虑性失眠模型。相应干 预后,Morris水迷宫实验检测大鼠学习记忆水平,旷场实验检测焦虑应激程度,苏木素-伊红 (HE) 染色观察下丘脑腹外侧视 前核 (VLPO) 神经元病理变化,免疫组织化学法、实时定量聚合酶链反应 (qRT-PCR) 法、Western Blot法检测VLPO脑区N-甲 基-D-天冬氨酸 (NMDA) 受体亚基NR1、NR2B和钙调蛋白激酶Ⅱ (CaMKⅡ) 的蛋白及mRNA表达。 【结果】与正常组比较,模 型组大鼠日间焦虑症状加重,睡眠潜伏期延长、持续时间缩短 (P<0.01) ,水迷宫定向航行实验平均游泳总路程和平均逃避 潜伏期增加 (P<0.01) ,空间探索实验穿越隐藏平台的次数和目标象限滞留时间减少 (P<0.01) ,旷场实验运动距离、中央格 跨越格数与中央格滞留时间均显著减少 (P<0.01) ,修饰频次与直立次数无统计学差异 (P>0.05) ,VLPO脑区神经元出现病理损伤,NR1、CaMKⅡ蛋白及mRNA表达水平降低 (P<0.01) ,NR2B蛋白及mRNA表达水平升高 (P<0.01) ;与模型组比 较,摩腹组、揿针组和摩腹加揿针组大鼠水迷宫实验学习记忆水平与旷场实验焦虑应激程度得到明显恢复 (P<0.05或P< 0.01) ,VLPO脑区神经元损伤改善,NR1、CaMKⅡ蛋白及mRNA表达水平升高 (P<0.05或P<0.01) ,NR2B蛋白及mRNA表 达水平降低 (P<0.05或P<0.01) ;摩腹加揿针组上述各指标改善效果均优于摩腹组或揿针组 (P<0.05或P<0.01) 。 【结论】摩 腹法结合揿针可对焦虑性失眠大鼠起到促眠和抗焦虑的疗效,其相关机制可能与调控VLPO脑区NR1/NR2B之间的动态平衡 及上调CaMKⅡ表达水平,改善VLPO脑区神经元功能,进而恢复下丘脑睡眠内稳态系统调节作用有关。
[Key word]
[Abstract]
Objective To observe the effect of abdominal massage combined with thumb-tack needling for subcutaeous embedding on sleep homeostasis system in rats with anxiety insomnia. Methods Forty rats were randomly divided into normal group, model group, abdominal massage group, thumb-tack needling for subcutaeous embedding group and abdominal massage plus thumb-tack needling for subcutaeous embedding group, with 8 rats in each group. Except for the normal group,the rats in the other groups were used to replicate the model of anxiety insomnia by multi-factor compound stimulation. After the corresponding intervention,Morris water maze test was used to detect the level of learning and memory. Open field test was used to detect the degree of anxiety stress. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of hypothalamic ventral lateral preoptic nucleus (VLPO) neurons. Immunohistochemistry,real-time quantitative polymerase chain reaction (qRT-PCR) and Western Blot were used to detect the protein and mRNA expression of N-methyl-D-aspartate (NMDA) receptor subunits NR1,NR2B and calmodulin kinase Ⅱ(CaMKⅡ) in hypothalamic VLPO area, respectively. Results Compared with the normal group,the daytime anxiety symptoms of the rats in the model group were aggravated,the sleep latency was prolonged and the duration was shortened(P<0.01) . The average total swimming distance and average escape latency of the water maze directional navigation experiment were increased (P<0.01) . The number of crossing the hidden platform and the retention time of the target quadrant in the space exploration experiment were decreased (P<0.01) . The movement distance,the number of central grid crossings and the retention time of the central grid in the open field experiment were significantly reduced (P< 0.01) . There was no significant difference in the modification frequency and the number of uprights (P>0.05) . Neurons in the VLPO brain region showed pathological damage. The protein and mRNA expression levels of NR1 and CaMKⅡ were decreased (P<0.01) in VLPO brain region,and the protein and mRNA expression levels of NR2B were increased (P<0.01) . Compared with the model group,the level of learning and memory in the water maze test and the degree of anxiety stress in the open field test were significantly restored in the abdominal massage group,the thumb-tack needling for subcutaeous embedding group and the abdominal massage combined with thumb-tack needling for subcutaeous embedding group (P<0.05 or P<0.01) ,the neuronal damage in the VLPO brain region was improved,the protein and mRNA expression levels of NR1,CaMKⅡ were increased (P<0.05 or P<0.01) ,and the protein and mRNA expression levels of NR2B were decreased (P<0.05 or P<0.01) . The improvement effect of the above indexes in the abdominal massage plus thumb-tack needling for subcutaeous embedding group was superior to that in the abdominal massage group or thumb-tack needling for subcutaeous embedding group (P<0.05 or P<0.01) . Conclusion Abdominal massage combined with thumb-tack needling for subcutaeous embedding can promote sleep and anti-anxiety in rats with anxiety insomnia. The related mechanism may be related to adjusting the dynamic balance between NR1/NR2B in VLPO brain area and up-regulating the expression level of CaMKⅡ,improving the function of neurons in VLPO brain area,and then restoring the regulation of sleep homeostasis system.
[中图分类号]
R245.31
[基金项目]
国家自然科学基金资助项目 (编号:81960906);新疆维吾尔自治区科技创新团队 (天山创新团队) 项目 (编号:2022TSYCTD0008); 新疆维吾尔自治区研究生科研创新项目 (编号:XJ2023G199)