柴牡安神颗粒抗焦虑作用及其活性机制
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摘要
目的:观察柴牡安神颗粒的抗焦虑作用,并探讨其作用机制。方法:ICR小鼠按体质量随机分为正常组、地西泮(0. 002 g·kg~(-1))组和解郁安神颗粒(0. 001 4 g·kg~(-1))组,柴牡安神颗粒高、中、低剂量(0. 001 98,0. 000 99,0. 000 495 g·kg~(-1))组,每组20只。为了考察药物抗焦虑作用,小鼠每天上午灌胃给药4周后,测试明暗箱、开场活动实验,然后动物取血,其中每组6只小鼠通过心脏灌注10%甲醛后取脑组织固定,其余小鼠取血后去取脑海马组织置于液氮保存。采用酶联免疫吸附方法检测小鼠海马组织和外周血中γ-氨基丁酸(GABA)及谷氨酸(Glu)含量,计算GABA/Glu;采用免疫组化法测定海马组织γ-氨基丁酸受体α1蛋白的表达。为了考察药物催眠作用,小鼠灌胃给药7 d后,进行阈下剂量戊巴比妥钠协同催眠实验。结果:与正常组比较,明暗箱实验证实柴牡安神颗粒低、中剂量组显著延长了小鼠明箱停留持续时间(P <0. 05),中剂量组增加了明箱进入次数(P <0. 05);开场实验证实柴牡安神颗粒高剂量显著增加了小鼠在中心的区域距离(P <0. 01)和中心区域停留时间(P <0. 05)。进一步实验证实,在海马组织中,与正常组比较,柴牡安神颗粒高剂量组提高了GABA含量(P <0. 05),显著降低了Glu含量(P <0. 01),提高了GABA/Glu(P <0. 05);在外周血,与正常组比较柴牡安神颗粒高剂量组显著降低了Glu含量(P <0. 01),同时显著增加了GABA含量(P <0. 01),从而显著提高了GABA/Glu(P <0. 01)。海马组织GABAα1受体蛋白免疫组化结果显示柴牡安神颗粒中剂量组明显提高了GABAα1蛋白表达。阈下剂量戊巴比妥钠协同催眠实验证实柴牡安神颗粒中剂量组明显提高了小鼠的入睡率(P <0. 05)。结论:柴牡安神颗粒具有显著抗焦虑作用,作用机制与提高海马GABA含量,降低Glu的含量,且提高了海马GABAα1蛋白表达密切相关。外周血GABA,Glu含量变化与海马组织变化呈正相关,对临床诊断具有参考意义。柴牡安神颗粒同时具有改善睡眠的作用。
Objective: To assess the anxiolytic effect of Chaimu Anshen granules( CMASG) and investigate its bioactive mechanism. Method: ICR mice were randomly divided into normal group,diazepam group( 0. 002 g·kg~(-1)),Jieyu Anshen granules group( 0. 001 4 g·kg~(-1)),high,medium,and low-dose( 0. 001 98,0. 000 99,0. 000 495 g·kg~(-1)) Chaimu Anshen granule groups,with 20 mice in each group. To detect the anxiolytic effect of CMASG,mice were intragastrically administered for 4 weeks in the morning,and light-dark box transition test and open field test were performed once the other day. After the behavior tests,blood samples were collected. Six mice of each group were perfused with formalin through heart,and then the brains were fixed for immunohistochemistry test. Hippocampus of the other mice in each group were collected and stored in liquid nitrogen. The content of γ-aminobutyric acid( GABA) and glutamic acid( Glu) in hippocampus and blood samples were detected by enzyme-linked immunosorbent assay( ELISA), and the ratio of GABA/Glu was calculated. The expression of GABAα1 receptor was evaluated by the immunohistochemistry method. To test the hypnosis effect of CMASG, mice were administered intragastrically for 7 days. The sub-threshold dose of pentobarbital sodium in the sleep experiment was tested. Result: Compared with normal group,the light-dark box transitions test demonstrated that low-dose and medium-dose CMASG groups significantly prolonged the duration in light box( P < 0. 05),and the middle-dose group significantly increased the frequency of entrance to light box( P < 0. 05). The open field test confirmed that the high-dose CMASG significantly increased the travel distance( P < 0. 01) and duration( P < 0. 05) in central region. Further experiments demonstrated that in hippocampus,compared with the normal group,the high-dose CMASG significantly increased the content of GABA( P < 0. 05),but significantly reduced the Glu content( P < 0. 01). The high-dose group significantly increased the GABA/Glu ratio( P < 0. 01). In the blood samples,high-dose CMASG significantly reduced the Glu content( P < 0. 01),and the high-dose group significantly increased the GABA content( P < 0. 01) and the GABA/Glu ratio( P <0. 01). The immunohistochemical results of GABAα1 receptor protein in hippocampus showed that the medium-dose CMASG significantly increased the expression of GABAα1 protein. The sub-threshold dose of pentobarbital sodium on sleep experiments confirmed that the medium-dose CMASG significantly increased the rate of sleep in mice.Conclusion: CMASG showed an anxiolytic effect,and its bioactive mechanism was related with the increase of GABA content,and the decrease of Glu content in hippocampus. Furthermore,it increased the expression of GABAα1 protein in hippocampus. The changes in content of GABA and Glu in peripheral blood were positively correlated with the changes in hippocampal tissues,which provided reference for clinical diagnosis. CMASG also exhibited an effect in improvement of sleep.
Objective: To assess the anxiolytic effect of Chaimu Anshen granules( CMASG) and investigate its bioactive mechanism. Method: ICR mice were randomly divided into normal group,diazepam group( 0. 002 g·kg~(-1)),Jieyu Anshen granules group( 0. 001 4 g·kg~(-1)),high,medium,and low-dose( 0. 001 98,0. 000 99,0. 000 495 g·kg~(-1)) Chaimu Anshen granule groups,with 20 mice in each group. To detect the anxiolytic effect of CMASG,mice were intragastrically administered for 4 weeks in the morning,and light-dark box transition test and open field test were performed once the other day. After the behavior tests,blood samples were collected. Six mice of each group were perfused with formalin through heart,and then the brains were fixed for immunohistochemistry test. Hippocampus of the other mice in each group were collected and stored in liquid nitrogen. The content of γ-aminobutyric acid( GABA) and glutamic acid( Glu) in hippocampus and blood samples were detected by enzyme-linked immunosorbent assay( ELISA), and the ratio of GABA/Glu was calculated. The expression of GABAα1 receptor was evaluated by the immunohistochemistry method. To test the hypnosis effect of CMASG, mice were administered intragastrically for 7 days. The sub-threshold dose of pentobarbital sodium in the sleep experiment was tested. Result: Compared with normal group,the light-dark box transitions test demonstrated that low-dose and medium-dose CMASG groups significantly prolonged the duration in light box( P < 0. 05),and the middle-dose group significantly increased the frequency of entrance to light box( P < 0. 05). The open field test confirmed that the high-dose CMASG significantly increased the travel distance( P < 0. 01) and duration( P < 0. 05) in central region. Further experiments demonstrated that in hippocampus,compared with the normal group,the high-dose CMASG significantly increased the content of GABA( P < 0. 05),but significantly reduced the Glu content( P < 0. 01). The high-dose group significantly increased the GABA/Glu ratio( P < 0. 01). In the blood samples,high-dose CMASG significantly reduced the Glu content( P < 0. 01),and the high-dose group significantly increased the GABA content( P < 0. 01) and the GABA/Glu ratio( P <0. 01). The immunohistochemical results of GABAα1 receptor protein in hippocampus showed that the medium-dose CMASG significantly increased the expression of GABAα1 protein. The sub-threshold dose of pentobarbital sodium on sleep experiments confirmed that the medium-dose CMASG significantly increased the rate of sleep in mice.Conclusion: CMASG showed an anxiolytic effect,and its bioactive mechanism was related with the increase of GABA content,and the decrease of Glu content in hippocampus. Furthermore,it increased the expression of GABAα1 protein in hippocampus. The changes in content of GABA and Glu in peripheral blood were positively correlated with the changes in hippocampal tissues,which provided reference for clinical diagnosis. CMASG also exhibited an effect in improvement of sleep.
引文
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[5]罗荣.浅谈焦虑症的临床表现及诊断[J].求医问药:学术版,2012,10(8):229-230.
[6]韩玲娜,王春雷,张莉.外侧缰核中5-HT_(2C)受体在帕金森病模型大鼠焦虑行为中的调节作用及其机制[J].吉林大学学报:医学版,2016,42(3):473-480.
[7]宋灵云,李苗苗,于海玲.芥酸酰胺对小鼠的抗焦虑样作用[J].沈阳药科大学学报,2017,34(4):333-337.
[8]任莹莹,周良良,吴华丽,等.靶向单胺受体及谷氨酸系统抗抑郁药的研究进展[J].药物资讯,2016,5(2):38-44.
[9]周春,韦少俊.重性抑郁障碍患者相关脑代谢物的水平变化及其作用研究进展[J].山东医药,2016,56(15):98-100.
[10] Andrade T G,Avanzi V,Graeff F G. Effect of estradiol benzoate microinjection into the median raphe nucleus on contextual conditioning[J]. Behav Brain Res,2009,204(1):112-116.
[11]马丽华.发育期遗传性癫痫大鼠海马组织Glu与GABA循环通路重要靶点的研究[D].沈阳:中国医科大学,2013.
[12] LI Q, Bartley A F, Dobrunz L E. Endogenously released neuropeptide Y suppresses hippocampal shortterm facilitation and is impaired by stress-induced anxiety[J]. J Neurosci,2017,37(1):23-37.
[13]余思邈,朱云,王立福,等.焦虑和抑郁状态对原发性肝癌患者免疫功能的影响[J].肝脏,2018,23(6):496-498.
[14] Belleville G,Cousineau H, Levrier K, et al. The impact of cognitive-behavior therapy for anxiety disorders on concomitant sleep disturbances:a Meta-analysis[J]. J Anxiety Disord,2010,24(4):379-386
[15] Mellman T A. Sleep and anxiety disorders[J].Psychiatric Clinics,2006,29(4):1047-1058.
[16]赵文清,宋立升.焦虑障碍患者的共病失眠问题[J].精神医学杂志,2016,29(5):392-396.
[17]舒山,程楠,韩咏竹.广泛性焦虑障碍的药物治疗进展[J].转化医学电子杂志,2016,3(3):51-54.
[18]公晋,王永生.符为民运用黄连温胆汤治疗焦虑症经验[J].世界中西医结合杂志,2018,13(2):187-189,273.
[19]程新慧,刘奇,张承舜,等.焦虑症的针灸临床治疗及选穴特点分析[J].时珍国医国药,2015,26(2):431-433.
[20]于泽鹏,高佳琪,刘聪,等.五味子醋镇静催眠抗焦虑作用及其作用机制[J].中国实验方剂学杂志,2018,24(11):139-143.
[21]陈陆松,江琛,齐小强.柏子养心方合疏肝宁神散治疗广泛性焦虑障碍[J].中国实验方剂学杂志,2014,20(22):200-203.
[22]丰广魁,陈隐漪,李乐军,等.柴龙解郁丹治疗广泛性焦虑症的临床研究[J].南京中医药大学学报,2015,31(3):214-217.
[23]王停,郑燕飞,王济,等.第二十一讲关于治疗焦虑症医案的探讨[J].中医药通报,2015,14(3):7-11.
[24]徐叔云.药理实验方法学.附录[M]. 3版.北京:人民卫生出版社,2002:1861.
[25] Contrera J F,Matthews E J, Kruhlak N L, et al.Estimating the safe starting dose in phase I clinical trials and no observed effect level based on QSAR modeling of the human maximum recommended daily dose[J]. Regul Toxicol Pharmacol,2004,40(3),185-206.
[26]孙世光,李自发,魏盛,等.昆明小鼠状态焦虑动物模型明暗箱实验的结构维度分析[J].中华行为医学与脑科学杂志,2011,20(7):627-629.
[27] Kulesskaya N,Voikar V. Assessment of mouse anxietylike behavior in the light-dark box and open-field arena:role of equipment and procedure[J]. Physiol Behav,2014,133:30-38.
[28]黄继忠,王祖承.焦虑动物模型的实验方法和药理学评价[J].上海精神医学,2003,15(2):106-109.
[29]孙文军,曲淼,徐向青,等.焦虑障碍中医临床诊疗指南释义[J].北京中医药,2018,37(2):105-110.
[30]陈国统,何杨伟,汪碧云.柴胡龙骨牡蛎汤加减结合抗焦虑抑郁药物对躯体化焦虑、抑郁状态的疗效及机制分析[J].中华中医药学刊,2017,35(3):758-760.
[31]冯美珍.加味栀子豉汤治疗抑郁症患者43例[J].光明中医,2017,32(5):667-669.
[32]黄继忠,王祖承.焦虑动物模型的实验方法和药理学评价[J].上海精神医学,2003,15(2):106-109.
[33] Nikolaus S,Antke C,Beu M,et al. Cortical GABA,striatal dopamine and midbrain serotonin as the key players in compulsive and anxiety disorders-results from in vivo imaging studies[J]. Rev Neurosci,2010,21(2):119-140.
[34]王承展,黄庚娣,方青,等.创伤性应激障碍样大鼠海马GABA受体表达的变化[J].中国心理卫生杂志,2016,30(12):952-956.
[35] Kita I,Seki Y, Nakatani Y, et al. Corticotropinreleasing factor neurons in the hypothalamic paraventricular nucleus are involved in arousal/yawning response of rats[J]. Behav Brain Res,2006,169(1):48-56.
[36]张雪,黄仕营,张燊,等.安神方的抗焦虑作用[J].南方医科大学学报,2012,32(1):80-84.
[2] Kessler R C,Chiu W T,Demler O,et al. Prevalence,severity,and comorbidity of 12-month DSM-Ⅳdisorders in the National Comorbidity Survey Replication[J].Arch Gen Psych,2005,62(6):617-627.
[3]胡强,万玉美,苏亮,等.中国普通人群焦虑障碍患病率的荟萃分析[J].中华精神科杂志,2013,46(4):204-211.
[4] Pigott T A. Gender differences in the epidemiology and treatment of anxiety disorders[J]. J Clin Psych,1999,18(1):4-15.
[5]罗荣.浅谈焦虑症的临床表现及诊断[J].求医问药:学术版,2012,10(8):229-230.
[6]韩玲娜,王春雷,张莉.外侧缰核中5-HT_(2C)受体在帕金森病模型大鼠焦虑行为中的调节作用及其机制[J].吉林大学学报:医学版,2016,42(3):473-480.
[7]宋灵云,李苗苗,于海玲.芥酸酰胺对小鼠的抗焦虑样作用[J].沈阳药科大学学报,2017,34(4):333-337.
[8]任莹莹,周良良,吴华丽,等.靶向单胺受体及谷氨酸系统抗抑郁药的研究进展[J].药物资讯,2016,5(2):38-44.
[9]周春,韦少俊.重性抑郁障碍患者相关脑代谢物的水平变化及其作用研究进展[J].山东医药,2016,56(15):98-100.
[10] Andrade T G,Avanzi V,Graeff F G. Effect of estradiol benzoate microinjection into the median raphe nucleus on contextual conditioning[J]. Behav Brain Res,2009,204(1):112-116.
[11]马丽华.发育期遗传性癫痫大鼠海马组织Glu与GABA循环通路重要靶点的研究[D].沈阳:中国医科大学,2013.
[12] LI Q, Bartley A F, Dobrunz L E. Endogenously released neuropeptide Y suppresses hippocampal shortterm facilitation and is impaired by stress-induced anxiety[J]. J Neurosci,2017,37(1):23-37.
[13]余思邈,朱云,王立福,等.焦虑和抑郁状态对原发性肝癌患者免疫功能的影响[J].肝脏,2018,23(6):496-498.
[14] Belleville G,Cousineau H, Levrier K, et al. The impact of cognitive-behavior therapy for anxiety disorders on concomitant sleep disturbances:a Meta-analysis[J]. J Anxiety Disord,2010,24(4):379-386
[15] Mellman T A. Sleep and anxiety disorders[J].Psychiatric Clinics,2006,29(4):1047-1058.
[16]赵文清,宋立升.焦虑障碍患者的共病失眠问题[J].精神医学杂志,2016,29(5):392-396.
[17]舒山,程楠,韩咏竹.广泛性焦虑障碍的药物治疗进展[J].转化医学电子杂志,2016,3(3):51-54.
[18]公晋,王永生.符为民运用黄连温胆汤治疗焦虑症经验[J].世界中西医结合杂志,2018,13(2):187-189,273.
[19]程新慧,刘奇,张承舜,等.焦虑症的针灸临床治疗及选穴特点分析[J].时珍国医国药,2015,26(2):431-433.
[20]于泽鹏,高佳琪,刘聪,等.五味子醋镇静催眠抗焦虑作用及其作用机制[J].中国实验方剂学杂志,2018,24(11):139-143.
[21]陈陆松,江琛,齐小强.柏子养心方合疏肝宁神散治疗广泛性焦虑障碍[J].中国实验方剂学杂志,2014,20(22):200-203.
[22]丰广魁,陈隐漪,李乐军,等.柴龙解郁丹治疗广泛性焦虑症的临床研究[J].南京中医药大学学报,2015,31(3):214-217.
[23]王停,郑燕飞,王济,等.第二十一讲关于治疗焦虑症医案的探讨[J].中医药通报,2015,14(3):7-11.
[24]徐叔云.药理实验方法学.附录[M]. 3版.北京:人民卫生出版社,2002:1861.
[25] Contrera J F,Matthews E J, Kruhlak N L, et al.Estimating the safe starting dose in phase I clinical trials and no observed effect level based on QSAR modeling of the human maximum recommended daily dose[J]. Regul Toxicol Pharmacol,2004,40(3),185-206.
[26]孙世光,李自发,魏盛,等.昆明小鼠状态焦虑动物模型明暗箱实验的结构维度分析[J].中华行为医学与脑科学杂志,2011,20(7):627-629.
[27] Kulesskaya N,Voikar V. Assessment of mouse anxietylike behavior in the light-dark box and open-field arena:role of equipment and procedure[J]. Physiol Behav,2014,133:30-38.
[28]黄继忠,王祖承.焦虑动物模型的实验方法和药理学评价[J].上海精神医学,2003,15(2):106-109.
[29]孙文军,曲淼,徐向青,等.焦虑障碍中医临床诊疗指南释义[J].北京中医药,2018,37(2):105-110.
[30]陈国统,何杨伟,汪碧云.柴胡龙骨牡蛎汤加减结合抗焦虑抑郁药物对躯体化焦虑、抑郁状态的疗效及机制分析[J].中华中医药学刊,2017,35(3):758-760.
[31]冯美珍.加味栀子豉汤治疗抑郁症患者43例[J].光明中医,2017,32(5):667-669.
[32]黄继忠,王祖承.焦虑动物模型的实验方法和药理学评价[J].上海精神医学,2003,15(2):106-109.
[33] Nikolaus S,Antke C,Beu M,et al. Cortical GABA,striatal dopamine and midbrain serotonin as the key players in compulsive and anxiety disorders-results from in vivo imaging studies[J]. Rev Neurosci,2010,21(2):119-140.
[34]王承展,黄庚娣,方青,等.创伤性应激障碍样大鼠海马GABA受体表达的变化[J].中国心理卫生杂志,2016,30(12):952-956.
[35] Kita I,Seki Y, Nakatani Y, et al. Corticotropinreleasing factor neurons in the hypothalamic paraventricular nucleus are involved in arousal/yawning response of rats[J]. Behav Brain Res,2006,169(1):48-56.
[36]张雪,黄仕营,张燊,等.安神方的抗焦虑作用[J].南方医科大学学报,2012,32(1):80-84.