安寐丹调控Orexin信号通路改善睡眠剥夺斑马鱼模型学习记忆的作用及机制研究  

Study on the Mechanism of AMD Regulating Orexin Signaling Pathway to Improve Learning and Memory of SD zebrafish Model

作　　者：叶子靖 徐波[1,2] 夏婧 鲁海菲 陈雨萌 黄梦莹[1,2] 王平 YE Zijing;XU Bo;XIA Jing;LU Haifei;CHEN Yumeng;HUANG Mengying;WANG Ping(Hubei University of Traditional Chinese Medicine Elderly Brain Health Traditional Chinese Medicine Protection Technology and New Product Research and Development Ministry of Education Engineering Research Center,Wuhan 430065,China;Hubei Shizhen Laboratory,Wuhan 430065,China)

机构地区：[1]湖北中医药大学老年脑健康中医药防护技术与新产品研发教育部工程研究中心,武汉430065 [2]湖北时珍实验室,武汉430065

出　　处：《世界科学技术-中医药现代化》2024年第3期731-741,共11页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会青年项目(82104712):培元固本安神法调控Orexin信号通路改善睡眠剥夺模型大鼠学习记忆的分子机制研究,负责人:徐波。

摘　　要：目的观察培元固本安神法代表方安寐丹对睡眠剥夺(SD)斑马鱼模型学习记忆的作用,并基于食欲素(Orexin)信号通路探讨其可能机制及最佳显效剂量。方法120尾4月龄野生型AB系斑马鱼随机分为空白组、模型组、安寐丹低剂量组(0.009 mg·mL^(-1)·d^(-1))、安寐丹中剂量组(0.027 mg·mL^(-1)·d^(-1))、安寐丹高剂量组(0.081 mg·mL^(-1)·d^(-1))、褪黑素组(0.4 mg·mL^(-1)·d^(-1)),采用LED灯光照诱导方法建立斑马鱼SD模型,利用斑马鱼行为追踪系统监测各组斑马鱼24 h行为学变化,T型迷宫检测各组斑马鱼学习记忆能力差异,苏木精-伊红(HE)染色法观察各组斑马鱼脑部神经元的状态与数量,透射电镜观察各组斑马鱼脑部神经元结构的变化,酶联免疫吸附(ELISA)法检测各组斑马鱼食欲素A(OXA)、食欲素B(OXB)含量水平,蛋白质免疫印迹法(Western blot)、实时荧光定量法(RT-PCR)检测脑组织OXA/p38丝裂原活化蛋白激酶(P38 MAPK)/细胞外调节蛋白激酶(ERK1/2)信号通路蛋白和mRNA表达水平。结果与空白组比较,模型组总静止次数、持续时间以及距离减少(P<0.05),进入目标区域(OR)潜伏期延长(P<0.01);神经细胞数量减少,细胞核变性、胞质透明增多、着色浅,细胞核塌陷、核膜破裂溶解、染色质固缩、线粒体肿胀畸形、内有空腔、形成板层状髓鞘样结构;脑组织OXA、OXB蛋白显著升高(P<0.01),OXA mRNA和蛋白显著上调(P<0.01),P38 MAPK和ERK1/2 mRNA和蛋白显著下调(P<0.01)。与模型组比较,药物干预能延长SD斑马鱼静息运动总时间(P<0.05),缩短斑马鱼到达OR潜伏期(P<0.05);保护神经细胞结构与形态,减轻脑组织损伤;降低脑组织OXA、OXB含量(P<0.01),下调OXA mRNA和蛋白表达量(P<0.01),上调P38 MAPK和ERK1/2 mRNA和蛋白的表达量(P<0.01);且量效关系明显,安寐丹高剂量效果最优。结论培元固本安神法代表方安寐丹可以改善斑马鱼SD模型学习记忆能力,其机制可能与Objective To observe the effects of AnMeiDan(AMD),a representative formula of Peiyuan Guben Tranquilisation method,on the learning memory of zebrafish model of sleep deprivation(SD),and to explore the possible mechanism and the optimal apparent dose based on the Orexin signalling pathway.Methods 1204-month-old wildtype AB line zebrafish were randomly divided into blank group,model group,AMD low-dose group(0.009 mg·mL^(-1)·d^(-1)),AMD medium-dose group(0.027 mg·mL^(-1)·d^(-1)),AMD high-dose group(0.081 mg·mL^(-1)·d^(-1)),and melatonin group(0.4 mg·mL^(-1)·d^(-1)).Zebrafish SD model was established by LED light induction method,24 h behavioural changes of zebrafish in each group were monitored by zebrafish behavioural tracking system,differences in learning and memory ability of zebrafish in each group were detected by T-maze,the state and number of neurons in the brain of zebrafish in each group were observed by Hematoxylin-Eosin(HE)staining method,the structural changes of neurons in the brain of zebrafish in each group were observed by transmission electron microscopy,and changes in neuron structure in the brain of zebrafish in each group were detected by ELISA method.The levels of Orexin A(OXA)and Orexin B(OXB)in zebrafish were detected by enzyme-linked immunosorbent assay(ELISA),and the protein and mRNA expression levels of OXA/p38 mitogen-activated protein kinase(P38 MAPK)/extracellular regulated protein kinase(ERK1/2)were detected by protein immunoblotting and real-time fluorescence quantitative assay(RT-PCR)in zebrafish.Results Compared with the blank group,the total number of rests,duration and distance were reduced in the model group(P<0.05),and the latency to enter the target region(OR)was prolonged(P<0.01);the number of neuronal cells was reduced,the nuclei were degenerated(P<0.01),cytoplasmic hyalinosis was increased(P<0.01),and light colouring was observed,the nuclei were collapsed,the nuclear membranes were ruptured and lysed,the chromatin was solidified,and the mitochondria were swollen and

关 键 词：安寐丹 睡眠剥夺 斑马鱼 学习记忆 食欲素 

分 类 号：R285.5[医药卫生—中药学]