小柴胡汤加味对慢性束缚抑郁模型大鼠海马谷氨酸转运体EAATs,VGLUTs表达的影响  

Effect of Modified Xiao Chaihutang on Expression of EAATs and VGLUTs in Hippocampus of Chronic Restraibt Stress Depression Model Rat

作　　者：许二平[1,2] 毛梦迪 尚立芝 王国强[1] 刘保光 李耀洋[4] 白明 栗俞程 汪保英[1,2] 陈晓辉 XU Er-ping;MAO Meng-di;SHANG Li-zhi;WANG Guo-qiang;LIU Bao-guang;LI Yao-yang;BAI Ming;LI Yu-cheng;WANG Bao-ying;CHEN Xiao-hui(Academy of Chinese Medical Sciences,Henan University of Chinese medicine,Zhengzhou 450046,China;Henan Key Laboratory for Modern Research of Zhongjing Compound Traditional Chinese Medicine,Zhengzhou 450046,China;School of Basic Medical Science,Henan University of Chinese Medicine,Zhengzhou 450046,China;The First Clinical School of Henan University of Chinese Medicine,Zhengzhou 450006,China)

机构地区：[1]河南中医药大学中医药科学院,郑州450046 [2]河南省仲景方药现代研究重点实验室,郑州450046 [3]河南中医药大学基础医学院,郑州450046 [4]河南中医药大学第一临床医学院,郑州450006

出　　处：《中国实验方剂学杂志》2020年第23期27-36,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81973739);河南省高等学校重点科研项目(20B360011,21A360020);河南省教育科学“十三五”规划一般课题(〔2019〕-JKGHYB-0101,〔2019〕-JKGHYB-0114);河南省中医药科学研究专项(2019JDZX2121,2019ZY3048)。

摘　　要：目的:观察小柴胡汤加味对慢性束缚抑郁模型大鼠海马谷氨酸膜转运体(EAATs)及囊泡转运体(VGLUTs)表达的影响,探讨小柴胡汤加味基于谷氨酸转运的抗抑郁机制。方法:120只SD大鼠随机分为正常组、模型组、小柴胡汤加味组低、中、高剂量组、利鲁唑组,每组20只。除正常组外,其余各组采用束缚应激制备大鼠抑郁模型,小柴胡汤加味低、中、高剂量组分别灌胃(ig)小柴胡汤加味6.5,13,26 g·kg-1;利鲁唑组腹腔注射利鲁唑20 mg·kg-1;正常组和模型组ig等量生理盐水;1次/d,共干预21 d。采用强迫游泳实验(FST)和悬尾实验(TST)评价大鼠的抑郁行为;采用高效液相色谱法(HPLC)检测海马组织中谷氨酸含量;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠海马中EAAT1,EAAT2和EAAT3 mRNA表达;蛋白免疫印迹法(Western blot)检测海马组织EAAT1,EAAT2,EAAT3,VGLUT1,VGLUT2蛋白表达;用尼氏染色法观察大鼠海马神经元形态,免疫组化(IHC)检测海马CA1区EAAT1,EAAT2和NeuN蛋白在神经细胞中的定位表达。结果:与正常组比较,模型组大鼠悬尾和强迫游泳不动时间均显著延长(P<0.01),海马内EAAT1,EAAT2,EAAT3 mRNA和蛋白表达均显著下降(P<0.01),VGLUT1和NeuN蛋白表达均显著降低(P<0.01);而谷氨酸水平和VGLUT2表达均显著增高(P<0.01)。与模型组比较,小柴胡汤加味中、高剂量组大鼠悬尾和强迫游泳不动时间明显缩短(P<0.05,P<0.01),海马内EAAT1,EAAT2和EAAT3mRNA和蛋白表达量均显著增加(P<0.01),VGLUT1和NeuN蛋白表达均显著增强(P<0.01),谷氨酸水平和VGLUT2表达显著回降(P<0.01),海马神经元结构明显复原。结论:小柴胡汤加味有明显的抗抑郁作用,其机制可能与其上调大鼠海马内EAAT1,EAAT2,EAAT3基因和VGLUT1蛋白表达。Objective: To study the effect of modified Xiao Chaihutang on the expressions of excitatory amino acid transporters(EAATs)and vesicle glutamate transporters(VGLUTs)in hippocampus of rats with chronic depression,in order to explore the anti-depressant mechanism of modified Xiao Chaihutang based on glutamate transport. Method: A total of 120 SD rats were randomly divided into normal group,model group,and low,middle and high-dose modified Xiaochaihutang groups(6.5,13,26 g·kg-1)and riluzole group,with 20 rats in each group. Except normal group,the depression model of rats was prepared through Chronic restraint stress(CRS). The normal group and the model group were intragastrically(ig)given normal saline. The modified Xiao Chaihutang groups were intragastrically given corresponding herbal drugs(6.5,13,26 g·kg-1),and the Riluzole group was given Riluzole 20 mg·kg-1 through intraoeritoneal injection for 21 days,once a day. Then the depressive behaviors of rats were observed by forced swimming test(FST)and tail suspension test(TST). The level of glutamic acid(Glu) in rats hippocampus was determined by high performance liquid chromatography(HPLC). The m RNA expressions of EAAT1, EAAT2 and EAAT3 in hippocampus were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)method. Western blot was used to detect the protein expressions of EAAT1,EAAT2,EAAT3,VGLUT1 and VGLUT2 in rat hippocampus tissue. Nissl staining was used to observe the morphology of hippocampal neurons in rats. Immunohistochemical(IHC)S-P method were used to detect the location expressions of EAAT1,EAAT2 and Neu N proteins in rat hippocampal CA1 region tissue. Result: The immobility times in FST and TST were increased significantly(P<0.01),the m RNA and protein expressions of EAAT1,EAAT2,EAAT3 were decreased significantly(P<0.01),and as well as the expressions of VGLUT1 and Neu N were decreased significantly(P<0.01),while the level of Glutamate and the expression of VGLUT2 were increased significantly(P<0.01)in model grou

关 键 词：抑郁症 小柴胡汤 酸枣仁汤 兴奋性氨基酸转运体(EAATs) 谷氨酸囊泡转运体(VGLUTs) 

分 类 号：R2-0[医药卫生—中医学] R22