益神宁心方对斑马鱼睡眠剥夺模型行为学及神经递质的影响  

Effects of Yishen Ningxin Formula on behavior and neurotransmitter in sleep deprivation induced zebrafish

作　　者：张瑞[1] 张振贤[2] 史佳宁[2] 蒋天君 ZHANG Rui;ZHANG Zhenxian;SHI Jianing;JIANG Tianjun(Department of Internal Medicine,Jiading Hospital of Traditional Chinese Medicine,Shanghai 201800,China;Traditional Medicine and Prevention of Disease Center,Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200437,China)

机构地区：[1]上海市嘉定区中医医院内科,上海201800 [2]上海中医药大学附属岳阳中西医结合医院传统医学与治未病中心,上海200437

出　　处：《上海中医药杂志》2024年第1期89-95,共7页Shanghai Journal of Traditional Chinese Medicine

基　　金：上海市卫健委上海市中医药事业发展三年行动计划项目[ZY(2018-2020)-ZWB-1001];上海市嘉定区自然科学基金项目(JDKW20200017)。

摘　　要：目的观察益神宁心方对斑马鱼睡眠剥夺模型行为的影响,并对其机制进行初步探索。方法将受精后5 d(5 dpf)野生型AB系斑马鱼随机分为正常组、模型组(咖啡因300μmol/L)、褪黑素组(10μmol/L)、益神宁心方低剂量组(100 mg/L)、益神宁心方中剂量组(300 mg/L)和益神宁心方高剂量组(900 mg/L),咖啡因诱导建立斑马鱼睡眠剥夺模型。应用行为追踪系统定量监测自主活动1 h,明暗交替下连续测定斑马鱼的趋触性行为15 min,酶联免疫吸附试验(ELISA)法检测γ-氨基丁酸(GABA)、5-羟色胺(5-HT)和褪黑素(MT)的含量,实时荧光定量逆转录聚合酶链式反应(RT-qPCR)法检测斑马鱼组织GABA A型受体a1亚单位基因(gabra1)、γ2亚单位基因(gabrg2)和5-HT受体1A类亚型基因(htr1aa)、1D类亚型基因(htr1bd)及MT受体1A类亚型基因(mtnr1aa)、1B类亚型基因(mtnr1ba)mRNA相对表达量。结果与正常组比较,模型组斑马鱼运动状态覆盖距离、运动状态持续时间及中运动时间均显著增加(P<0.05),静止状态持续时间显著减少(P<0.05);中心区域时间百分比及中心区域距离百分比均显著减少(P<0.05);GABA、MT含量及gabra1、gabrg2、mtnr1aa、mtnr1ba mRNA相对表达量均显著降低(P<0.05),5-HT含量及htr1aa、htr1bd mRNA相对表达量均显著升高(P<0.05)。与模型组比较,益神宁心方高、中剂量组运动距离、运动时间、中运动时间减少(P<0.05),静息时间增加(P<0.05);益神宁心方高剂量组中心区域时间百分比及中心区域距离百分比增加(P<0.05);益神宁心方高剂量组GABA、MT含量和gabra1、mtnr1aa、mtnr1ba mRNA相对表达量升高(P<0.05),5-HT含量和htr1aa mRNA相对表达量降低(P<0.05);益神宁心方中剂量组gabrg2、mtnr1ba mRNA相对表达量升高,5-HT含量和htr1bd mRNA相对表达量降低(P<0.05)。结论益神宁心方可以改善咖啡因造模斑马鱼的睡眠剥夺状态,改善焦虑行为,其机制可能与调节GABA、5-HT、MT相关紊乱的�Objective To explore the effects and the mechanism of Yishen Ningxin Formula(YSNXF)on the behavioral changes in sleep deprivation induced zebrafish.Methods Five days after fertilization(dpf),Wild-type AB zebrafish were randomly divided into control group,model group(caffeine 300μmol/L),melatonin group(10μmol/L),YSNXF low-dose group(100 mg/L),YSNXF medium-dose group(300 mg/L)and YSNXF high-dose group(900 mg/L).The sleep deprivation model of zebrafish was induced by caffeine.The autonomous activities were monitored for 1 by behavioral tracking system and the thigmotaxis behaviors were continuously detected for 15 min under the light and dark alternation condition.The levels ofγ-aminobutyric acid(GABA),serotonin(5-HT)and melatonin(MT)were detected by enzyme-linked immunosorbent assay(ELISA).The relative mRNA expression levels of gabra1,gabrg2,htr1aa,htr1bd,mtnr1aa,mtnr1ba in zebrafish´s tissues were detected by real-time fluorescence quantitative PCR.Results Compared with the control group,the duration,coverage distance of the motion state and the duration of medium speed motion state in the model group increased(P<0.05),while the duration of the static state decreased(P<0.05).The percentages of time spent in the central area(%)and the distances covered in the central area(%)both showed a significant decrease(P<0.05).The levels of GABA,MT,and the relative expression of gabra1,gabrg2,mtnr1aa and mtnr1ba mRNA were significantly decreased(P<0.05),while the levels of 5-HT and the relative expression of htr1aa,htr1bd mRNA were significantly increased(P<0.05).Compared with the model group,the high and medium dose groups of YSNXF decreased the duration,coverage distance of the motion state and the duration of medium speed motion state(P<0.05),while increase the duration of the static state(P<0.05).The percentages of time spent in the central area(%)and the distances covered in the central area(%)were increased(P<0.05)in YSNXF high dose group.The levels of GABA,MT,and the relative expression of gabra1,gabrg2,mtnr1aa and

关 键 词：失眠 益神宁心方 斑马鱼 Γ-氨基丁酸 5-羟色胺 褪黑素 中药研究 

分 类 号：R285.5[医药卫生—中药学]