安寐丹干预斑马鱼睡眠剥夺模型昼夜节律及能量代谢作用机制研究

Study on the Mechanism of Anmeidan Intervention on Circadian Rhythm and Energy Metabolism in Zebrafish Sleep Deprivation Model

目的探讨安寐丹对斑马鱼睡眠剥夺模型昼夜节律及能量代谢的影响及作用机制。方法75尾4月龄野生型AB系斑马鱼随机分为空白组、模型组、褪黑素组(0.464 mg·L^(-1))、安寐丹低剂量组(0.129 g·L^(-1))、安寐丹高剂量组(0.514 g·L^(-1)),采用咖啡因(25 mg·L^(-1))诱导4天建立成年斑马鱼睡眠剥夺模型。以行为追踪系统监测24 h昼夜节律,T型迷宫检测斑马鱼学习记忆能力,尼氏染色法观察神经细胞损害程度,实时荧光定量PCR检测脑组织clocka、bmal1、cry1a、per1a、per2的mRNA表达水平,定磷法检测肝脏Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶及总ATP酶活力。结果与空白组相比,模型组静止状态总计数、持续时长、覆盖距离减少,大运动状态总计数、持续时长、覆盖距离增加,24 h昼夜节律变化幅度特征降低;第1次进入EC区的时间增加(P<0.05),在EC区累计停留时间减少;中脑神经元尼氏小体减少,着色变浅;bmal1、cry1a mRNA表达水平升高(P<0.01);Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶及总ATP酶活性降低。与模型组相比,安寐丹增加静止状态计数(P<0.05)、减少大运动状态计数(P<0.05),恢复昼夜节律特征;缩短第1次进入EC区的时间,增加EC区的停留时间;保护神经元胞体结构,增加尼氏小体数目;降低bmal1、cry1a mRNA相对表达量(P<0.01);升高Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶及总ATP酶活力(P<0.05,P<0.01),并呈现出剂量相关性。结论安寐丹可以改善斑马鱼睡眠剥夺模型昼夜节律,其机制可能与干预bmal1、cry1a等生物钟基因,提高Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶以及总ATP酶等能量代谢酶活性相关。

Objectivee To investigate the effect and mechanism of Anmeidan on circadian rhythm and energy metabolism in zebrafish sleep deprivation model.Methods 754-month-old wild-type AB zebrafish were randomly divided into blank group,model group,melatonin group(0.464 mg·L^(-1)),Anmeidan low-dose group(0.129 g·L^(-1))and Anmeidan highdose group(0.514 g·L^(-1)).The sleep deprivation model of adult zebrafish was induced by caffeine(25 mg·L^(-1))for 4 days.The 24-hour circadian rhythm was monitored by behavior tracking system,the learning and memory ability of zebrafish was detected by T-maze,the degree of nerve cell damage was observed by Nissl staining,the mRNA expression levels of clocka,bmall,cryla,perla and per2 in brain tissue were detected by real-time fluorescence quantitative PCR,and the activities of Na^(+)-K^(+)-ATPase,Ca^(2+)-Mg^(2+)-ATPase and total ATPase in liver were detected by phosphorus determination.Results Compared with the blank group,the total count,duration and coverage distance of static state in the model group decreased,the total count,duration and coverage distance of large motion state increased,and the amplitude characteristics of 24 h circadian rhythm decreased.The time of entering EC area for the first time increased(P<0.05),and the cumulative residence time in EC area decreased.Nissl bodies of midbrain neurons decreased and the color became lighter.The expression levels of bmall and cryla mRNA increased(P<0.01).The activities of Na^(+)-K^(+)-ATPase,Ca^(2+)-Mg^(2+)-ATPase and total ATPase decreased.Compared with the model group,Anmeidan increased the count of static state(P<0.05),decreased the count of large motor state(P<0.05),and restored the characteristics of circadian rhythm.The time of entering the EC area for the first time was shorten and the residence time in the EC area was prolonged.The cell body structure of neurons was protected and the number of Nissl bodies was increased.The relative expressions of bmall and cryla mRNA were decreased(P<0.01).The activities of Na^(+)-K^(+)-ATPase,Ca^(2+)-Mg^(2+)-ATPase and total ATPase were increased(P<0.05,P<0.01).ConclusionAAnmeidan can improve the circadian rhythm of zebrafish sleep deprivation model,and its mechanism may be related to interfering with biological clock genes such as bmall and cryla,and increasing the activities of energy metabolism enzymes such as Na^(+)-K^(+)-ATPase,Ca^(2+)-Mg^(2+)-ATPase and total ATPase.