朱茯苓水提物对小鼠的催眠作用和分子机制研究  

Hypnotic Effect of Zhufuling Water Extract on Mice and Its Molecular Mechanism

作　　者：周扬[1] 王蕴涵 刘竞天[1] 钟其新 朱晔[1] 朱冰影 曾峰 Zhou Yang;Wang Yunhan;Liu Jingtian;Zhong Qixin;Zhu Ye;Zhu Bingying;Zeng Feng(Suzhou Key Laboratory of Medical Biotechnology,Suzhou Vocational Health College,Suzhou 215009;Artemisinin Research Center,Guangzhou University of Chinese Medicine,Guangzhou 510445;Shenzhen Hospital,Guangzhou University of Chinese Medicine,Shenzhen 518034)

机构地区：[1]苏州卫生职业技术学院,苏州检验医学生物技术重点实验室,苏州215009 [2]广州中医药大学青蒿研究中心,广州510445 [3]广州中医药大学深圳医院(福田),深圳518034

出　　处：《中药药理与临床》2021年第3期140-145,共6页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金青年项目(编号:31901002);江苏省卫生健康委医学科研项目(编号:Z2019051);苏州市科技局民生科技项目(编号:SYSD2018072);福田区卫生公益科研项目(编号:FTWS2019024、FTWS2020050);2020年江苏省高等学校自然科学面上项目(编号:20KJB360012);2021年江苏省高校“青蓝工程”优秀青年骨干教师培养项目。

摘　　要：目的:研究朱茯苓对小鼠的催眠作用和对小鼠海马神经元细胞的影响,从分子层面探讨其催眠机制。方法:ICR小鼠随机分为正常对照组、艾司唑仑0.1 mg/kg组、朱茯苓水提物1.1、2.3、4.5 g/kg组,连续灌胃给药7 d后,分别观察小鼠自主活动次数;阈下剂量戊巴比妥钠所致小鼠入睡率;阈剂量戊巴比妥钠致小鼠睡眠时间的测定,取小鼠海马组织,以酶联免疫吸附法测定(ELISA)法测定睡眠相关因子γ-氨基丁酸(GABA)和5-羟色胺(5-HT)的含量。体外培养小鼠海马神经元HT22细胞,采用CCK-8法检测细胞活性,优选合适的朱茯苓水提物和艾司唑仑浓度用于后续实验。将小鼠海马神经元细胞随机分为为空白对照组、艾司唑仑20μg/mL组、朱茯苓水提物80、160、320μg/mL组,给药24 h后采用流式细胞术分析海马神经元细胞早期凋亡情况,采用实时荧光定量PCR(RT-PCR)检测细胞内Gaba、5-Ht受体和钟基因mRNA表达情况,采用蛋白免疫印迹(Western blot)检测细胞内GABA、5-HT受体和钟基因蛋白表达情况。结果:小鼠自主活动结果表明,与正常对照组相比,朱茯苓2.3、4.5 g/kg组小鼠自主活动次数显著减少(P<0.01);朱茯苓水提物4.5 g/kg组小鼠入睡率明显提高(P<0.05);朱茯苓水提物1.1、2.3、4.5 g/kg组小鼠睡眠潜伏期显著缩短(P<0.01),睡眠时间显著延长(P<0.01),朱茯苓水提物2.3、4.5 g/kg组小鼠海马组织GABA和5-HT含量明显提升(P<0.05或P<0.01)。细胞实验结果表明培养第7 d的小鼠海马神经元细胞更为饱满,光晕变得立体,细胞间网络连接更加紧密,突起进一步增多和增长,生长成熟。与正常对照组比较,朱茯苓水提物80、160、320μg/mL组细胞早期凋亡率显著降低(P<0.01),朱茯苓水提物320μg/mL组GABRA1、GABRA2、5-HT1A、5-HT1B、Cry1、Per1、Per2基因蛋白表达和mRNA表达水平显著上调(P<0.01)。结论:朱茯苓对小鼠具有一定的催眠作用,可能通过提高海马组织内GABA�Objective:To study the hypnotic effect of Zhufuling(朱茯苓) on mice and its effect on hippocampal neurons and to explore its hypnotic mechanism from the molecular level. Methods:ICR mice were randomly divided into a blank control group, an estazolam(0.1 mg/kg) group, and low-(1.1 g/kg), medium-(2.3 g/kg), and high-dose(4.5 g/kg) Zhufuling water extract groups. After 7 days of intragastric administration, the frequency of spontaneous activity, the sleeping rate induced by a subthreshold dose of pentobarbital sodium, and the sleep time of mice induced by a threshold dose of pentobarbital sodium were observed. Then the hippocampus was detected for the content of γ-aminobutyric acid(GABA) and 5-hydroxytryptamine(5-HT) by the enzyme-linked immunosorbent assay(ELISA). In cell experiments, H22 cells in hippocampal neurons were cultured in vitro, and the cell activity was detected by Cell Counting Kit 8(CCK-8). The Zhufuling water extract and estazolam with appropriate concentrations were selected for subsequent experiments. The mouse hippocampal neurons were randomly divided into a blank control group, an estazolam(20 μg/mL) group, and low-(80 μg/mL), medium-(160 μg/mL), and high-dose(320 μg/mL) Zhufuling water extract groups. After 24 hours of administration, the early apoptosis of hippocampal neurons was analyzed by flow cytometry. The real-time fluorescent quantitative polymerase chain reaction(PCR) and Western blot were used to detect the mRNA and protein expression of GABA, 5-HT receptor, and clock gene, respectively. Results:Compared with the results in the blank control group, the frequency of spontaneous activities in the medium-and high-dose Zhufuling water extract groups was reduced(P<0.01) and the sleeping rate of mice in the high-dose Zhufuling water extract group was elevated(P<0.05). Additionally, the sleep latency in the Zhufuling water extract groups was shortened(P< 0.01), while the sleep time was prolonged(P<0.01). The content of GABA and 5-HT in the hippocampus of mice in the medium-and high-d

关 键 词：朱砂拌衣 朱茯苓 催眠作用 凋亡 神经递质 

分 类 号：R285.5[医药卫生—中药学]