安寐丹对睡眠剥夺大鼠自发活动昼夜节律的调节及对生物钟蛋白的影响  

Anmeidan Modulates Circadian Rhythm of Spontaneous Activity and Influences Clock Proteins in Sleep-deprived Rats

作　　者：谢光璟 徐波 黄攀攀 王平 XIE Guang-jing;XU Bo;HUANG Pan-pan;WANG Ping(Institute of Geriatrics,Hubei University of Chinese Medicine,Wuhan 430065,China)

机构地区：[1]湖北中医药大学老年医学研究所,武汉430065

出　　处：《中国实验方剂学杂志》2022年第7期33-39,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家重点研发计划“中医药现代化研究”重点专项(2018YFC1705600)。

摘　　要：目的:探讨安寐丹(AMD)对睡眠剥夺大鼠生物节律及相关蛋白表达的影响。方法:80只大鼠随机分为空白组、模型组、安寐丹组和褪黑素组。自制睡眠剥夺箱进行睡眠剥夺4周,正常与模型组给予等容生理盐水,安寐丹组给药9.09 g·kg^(-1)·d^(-1)。褪黑素组给药0.27 g·kg^(-1)·d^(-1)。自发活动视频分析系统评价自发活动昼夜节律,苏木素-伊红(HE)染色观察下丘脑组织形态,尼氏染色观察下丘脑组织神经元及尼氏小体,蛋白免疫印迹法(Western blot)检测下丘脑cAMP反应元件结合蛋白(CREB)/近日钟基因(Per)通路蛋白表达,免疫组化检测生物钟蛋白核心时钟基因1(Bmal1)、时钟基因(Clock)、Per1、隐花色素1基因(Cry1)的表达。结果:模型组昼夜节律相对紊乱,在6个节点活动时间较空白组显著增加,活动速度及活动次数增加(P<0.01);同时神经元数量减少,排列稀疏,胞核皱缩或碎裂;尼氏体数量减少,出现明显丢失,尼氏体着色变淡;p-CREB、Per1蛋白相对表达降低,Bmal1、Clock、Per1、Cry1蛋白阳性率下降(P<0.01)。与模型组比较,安寐丹组大鼠活动时间、活动速度及活动次数均下降(P<0.01);神经元损伤减轻,数量增加,部分核仁清晰,胞浆明显,尼氏体数量增加;p-CREB、Per1蛋白相对表达升高,Bmal1、Clock、Per1、Cry1蛋白阳性率上升(P<0.01)。结论:安寐丹改善睡眠剥夺大鼠自发活动昼夜节律,其作用可能通过调节CREB/Per信号通路,增加生物钟蛋白Bmal1、Clock、Per1、Cry1表达有关。Objective:To investigate the effect of Anmeidan(AMD)on biological rhythm and related protein expression in sleep-deprived rats.Method:A total of 80 SD rats were randomized into control group(Ctrl,equivalent volume of saline),model group(SD,equivalent volume of saline),AMD group(9.09 g·kg^(-1)·d^(-1)),and melatonin group(MT,0.27 g·kg^(-1)·d^(-1)).Insomnia was induced in rats by self-made sleep deprivation box(4 weeks).Circadian rhythm of spontaneous activity was evaluated by spontaneous activity video analysis system.Morphology of hypothalamus was observed based on hematoxylin-eosin(HE)staining,and the histomorphology of hypothalamus neurons and the Nissl's bodies based on Nissl staining.Western blotting was employed to detect the expression of hypothalamic proteins in cAMP-response element binding protein(CREB)/clock gene period(Per)pathway,and immunohistochemistry the expression of brain and muscle ARNT-like protein 1(Bmal1),Clock,Per1,and cryptochrome circadian regulator 1(Cry1).Result:The model group demonstrated circadian rhythm disorder,as manifested by the significant increase in activity time in 6 designated time periods compared with the control group,and the rise in the activity speed and frequency(P<0.01).Moreover,model group showed decrease in number of neurons which were sparsely arranged with shrunken or fragmented nuclei,reduction in number and loss of Nissl's bodies with light color,and drop in the relative expression of p-CREB and Per1,and the positive rate of Bmal1,Clock,Per1,and Cry1(P<0.01).Compared with model group,AMD group demonstrated reduction in time,speed,and frequency of activity(P<0.01).Moreover,the AMD group also showed alleviation of neuronal damage(P<0.01),and increase in the number of neurons with clear nuclei and cytoplasm in some,and the number of Nissl's bodies.AMD raised the expression of p-CREB and Per1 proteins,and the positive rate of Bmal1,Clock,Per1,and Cry1(P<0.01).Conclusion:AMD ameliorated spontaneous circadian rhythm of sleep-deprived rats by regulating CREB/Per si

关 键 词：安寐丹 睡眠剥夺 昼夜节律 生物钟蛋白 视交叉上核(SCN) 

分 类 号：R2-0[医药卫生—中医学] R22