脑心舒调控γ-氨基丁酸和谷氨酸平衡的镇静催眠机制研究

Sedative and hypnotic mechanism of Naoxinshu on the balance ofγ-aminobutyric acid and glutamate

目的:研究脑心舒口服液镇静催眠作用及潜在机制。方法:雄性ICR小鼠,随机数字表法分为对照组,艾司唑仑组,全天麻胶囊组、脑心舒口服液高、中、低剂量组,利用动物自发活动分析系统研究小鼠自主活动及戊巴比妥钠诱导睡眠,利用液质联用技术分析脑组织γ-氨基丁酸(GABA)和谷氨酸(Glu),以ELISA分析、免疫组化染色和RT-qPCR技术检测小鼠脑组织中GABA-T和GAD的mRNA转录水平和蛋白表达水平。结果:与对照组比较,脑心舒口服液可减少小鼠自主活动次数(P<0.05),提高小鼠入睡率(P<0.05),缩短睡眠潜伏期(P<0.05),并延长睡眠持续时间(P<0.01);GAGAA受体阻断剂氟马西尼和荷包牡丹碱可逆转脑心舒口服液对小鼠自主活动的抑制作用(P<0.01);脑心舒口服液可升高小鼠额叶皮质和海马GABA水平,降低Glu水平(P<0.05),使小鼠脑组织GABA-T酶活性及其表达量下降,而GAD酶活性及其表达量增加(P<0.01)。结论:脑心舒口服液通过调节GABA-T和GAD蛋白表达和酶活性,调控睡眠和觉醒神经递质GABA&Glu动态平衡,实现镇静催眠作用。

OBJECTIVE To explore the sedative and hypnotic effects of Naoxinshu Oral Solution(NOS)and elucidate its potential mechanisms of action.METHODS Male Institute of Cancer Research(ICR)mice were randomized into six groups of control,eszopiclone,Tianma Capsules,high-dose NOS,medium-dose NOS and low-dose NOS.Animal analysis system was utilized for assessing the spontaneous activity.Additionally pentobarbital sodium-induced sleep experiment was conducted.The levels ofγ-aminobutyric acid(GABA)and glutamate(Glu)in brain were measured by liquid chromatography-mass spectrom-etry.The activities of metabolic enzymes GABA transaminase(GABA-T)and glutamic acid decarboxylase(GAD),as well as protein and mRNA levels,were determined by enzyme-linked immunosorbent assay(ELISA),immunohistochemistry and quanti-tative real-time polymerase chain reaction(qRT-PCR).RESULTS NOS significantly lowered the number of spontaneous activi-ties(P<0.05),enhanced the sleeping rate(P<0.05),shortened sleep latency(P<0.05)and prolonged sleep duration(P<0.01).The sedative effects on spontaneous activity induced by NOS were reversed by GABAA receptor blockers of flumazenil and bicuculline(P<0.01).Furthermore,NOS significantly elevated GABA level in frontal cortex and hippocampus and decreased Glu level(P<0.05).Also NOS boosted GABA and depressed Glu in frontal cortex and hippocampus(P<0.01).Concurrently,the activity and protein levels of GABA-T in hippocampus of mice treated with NOS declined while the activity and expression of GAD spiked(P<0.05).CONCLUSION NOS exerts sedative and hypnotic effects through modulating the activ-ity and expression of GABA-T and GAD,as well as the levels of GABA and Glu.Thus it hints at a potential therapeutic role for NOS in the regulation of sleep and sedation.