摘要
目的:探究清宫汤调节谷氨酸(Glu)/γ-氨基丁酸(GABA)代谢平衡对焦虑大鼠模型的治疗效果及抗焦虑作用机制。方法:将54只大鼠随机分为正常组、模型组、地西泮组(0.225mg·kg^(-1))和清宫汤低、中、高剂量组(5.085、10.17、20.34 g·kg^(-1)),每组9只,除正常组外,其余各组大鼠均采用不确定空瓶应激和慢性束缚应激12 d以制备焦虑模型,应激的第3天起进行10 d的相应药物干预。在药物治疗结束后,通过高架十字迷宫实验(EPM)、明暗箱实验(LDB)评价各组大鼠焦虑水平并初步分析清宫汤对大鼠焦虑行为的影响,用酶联免疫吸附测定法(ELISA)检测大鼠杏仁核组织中Glu和GABA的水平,电镜技术分别观察各组大鼠杏仁核突触超微结构的变化,实时荧光定量聚合酶链式反应(Real-time PCR)检测杏仁核中谷氨酸脱羧酶65(GAD65)、谷氨酸脱羧酶67(GAD67)、谷氨酰胺合成酶(GS)、谷氨酸转运体-1(GLT-1)mRNA的表达,蛋白免疫印迹法(Western blot)检测其蛋白的表达。结果:与正常组比较,模型组大鼠焦虑状态明显,皮毛黯黄无光泽、易躁、易怒,喜蜷缩于角落,EPM进入开臂次数和开臂停留时间显著减少(P<0.01),LDB明箱停留时间和穿箱次数显著减少(P<0.01),杏仁核中Glu含量增加(P<0.01)、GABA含量降低(P<0.01)、Glu/GAB数值升高(P<0.01),杏仁核突触及前膜囊泡数量减少,突触后膜致密物稀疏,突触间隙增大,内部结构稍有破坏,杏仁核GAD65、GAD67、GS、GLT-1 mRNA和蛋白的表达均降低(P<0.01)。与模型组比较,清宫汤中、高剂量组和地西泮组大鼠皮毛光亮、反应灵敏、行为比较活跃,EPM进入开臂次数和开臂停留时间显著增加(P<0.01),LDB明箱停留时间和穿箱次数显著增加(P<0.01),清宫汤各剂量组和地西泮组大鼠杏仁核Glu含量降低(P<0.05,P<0.01)、GABA含量增加(P<0.05,P<0.01)、Glu/GABA的数值降低(P<0.01),清宫汤各组和地西泮组突触内外结构更完整,突触及囊泡数量更多,突触间隙更清晰,以高剂量组和地西泮组疗效最好,清宫汤高剂量组和地西泮组杏仁核GAD65、GAD67、GS、GLT-1 mRNA和蛋白的表达皆上升(P<0.05,P<0.01)。结论:清宫汤可以改善突触可塑性及影响大鼠杏仁核中GAD65、GAD67、GS、GLT-1的表达以调控Glu/GABA代谢平衡从而发挥抗焦虑作用。
Objective:To investigate the therapeutic effect of Qinggongtang in regulating Glu/GABA metabolic balance and the mechanism of its anxiolytic effect on rat models of anxiety.Method:Fifty-four rats were randomly divided into normal,model,diazepam(0.225 mg·kg^(-1)),and low-dose,medium-dose,and highdose groups of Qinggongtang(5.085,10.17,20.34 g·kg^(-1)),with nine rats in each group.Except for the normal group,the other groups were subjected to indeterminate vacutainer stress and chronic restraint stress for 12 days to prepare the anxiety model.On the 3~(rd)day of the stress,10 days of corresponding drug intervention was started.At the end of the drug treatment,the anxiety level of rats in each group was evaluated by the elevated cross maze experiment(EPM)and the light and dark box experiment(LDB),and the effect of Qinggongtang on the anxiety behavior of rats was preliminarily analyzed.The levels of Glu and GABA in the amygdala tissue of the rats were detected by enzyme linked immunosorbent assay(ELISA),and the changes in the synaptic ultrastructure of the amygdala of the rats in each group were observed by electron microscopy.The mRNA expression of glutamic acid decarboxylase(GAD65 and GAD67),glutamine synthetase(GS),and glutamate transporter-1(GLT-1)in the amygdala were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and their protein expression was detected by Western blot.Result:Compared with those in the normal group,rats in the model group showed an obvious anxiety state and dull yellow and lusterless fur.They were irritable,easy to anger,and preferred to curl up in the corner.The number of times the EPM entered the open arm and the residence time in the open arm were significantly reduced(P<0.01),and the residence time in the open box and the number of times the LDB went through the box were significantly reduced(P<0.01).The content of Glu in the amygdala was increased(P<0.01),and the content of GABA was reduced(P<0.01).The value of Glu/GAB was elevated(P<0.01),and the number of synaptic and pre-synaptic membrane vesicles in the amygdala was decreased.Sparse dense material in the post-synaptic membrane,increased synaptic gap,slightly disrupted internal structure,and decreased mRNA and protein expressions of GAD65,GAD67,GS,and GLT-1 in the amygdala were observed(P<0.01).Compared with those in the model group,rats in the medium-dose and highdose groups of Qinggongtang and the diazepam group had bright fur,sensitive reactions,and more active behavior.The number of times EPM entered the open arm and the residence time in the open arm increased significantly(P<0.01),and the residence time in the open box and the number of times the LDB went through the box increased significantly(P<0.01).The content of Glu in all-dose groups of Qinggongtang and the diazepam group decreased(P<0.05,P<0.01),while GABA content increased(P<0.05,P<0.01).The value of Glu/GABA decreased(P<0.01),and the internal and external synaptic structure of each groups of Qinggongtang and the diazepam group was more complete.Synapses and vesicles were numerous,and the synaptic gap was more clearly defined.The efficacy of the high-dose group of Qinggongtang and the diazepam group was the best,and the mRNA and protein expressions of GAD65,GAD67,GS,and GLT-1 in the amygdala were increased in the high-dose group of Qinggongtang and diazepam group(P<0.05,P<0.01).Conclusion:Qinggongtang can improve synaptic plasticity and affect the expression of GAD65,GAD67,GS,and GLT-1 in the amygdala of rats to regulate Glu/GABA metabolic balance and thus exert anxiolytic effects.
作者
黄潇潇
谢忠礼
谢梦跃
李圣臻
HUANG Xiaoxiao;XIE Zhongli;XIE Mengyue;LI Shengzhen(Traditional Chinese Medicine(Zhong Jing)School,Henan University of Chinese Medicine,Zhengzhou 450046,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第24期28-35,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家中医药管理局第五批全国中医临床优秀人才研修项目(基础)(国中医药人教函[2022]239号)。
关键词
清宫汤
广泛性焦虑症
谷氨酸
Γ-氨基丁酸
突触
Qinggongtang
generalized anxiety disorder(GAD)
glutamate
gamma-aminobutyric acid
synapse
