益智宁对注意缺陷多动障碍动物模型SHR大鼠前额叶皮质DRD1-AC-cAMP-PKA-DARPP32信号通路的影响
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摘要
目的:通过观察中药复方益智宁对注意缺陷多动障碍动物模型SHR大鼠前额叶皮质中腺苷酸环化酶(adenylate cyclase,AC)、环磷酸腺苷(cyclic adenosine monophosphate,cAMP)、蛋白激酶A(protein kinase A,PKA)、多巴胺D1受体(dopamine receptor D1,DRD1)、多巴胺和环磷酸腺苷调节的磷酸化蛋白32(dopamine and cAMP regulated phosphoprotein of 32 kDa,DARPP32)的影响,探讨益智宁治疗ADHD的疗效机制。方法:将30只SHR大鼠随机分为益智宁组、生理盐水对照组、哌甲酯对照组,每组10只,益智宁组给予益智宁煎药液(14.4g·kg~(-1)),哌甲酯组给予哌甲酯(0.0015 g·kg~(-1)),生理盐水组给予生理盐水,每组均按12.5 mL/kg等容量灌胃给药。给药4周后,用ELISA法检测各组大鼠前额叶皮质组织中AC、cAMP的浓度,用RT-PCR和Western blot法检测大鼠前额叶皮质组织中PKA、DRD1、DARPP32的表达水平。结果:与生理盐水组比较,哌甲酯组、益智宁组大鼠的前额叶皮质组织中的AC、cAMP含量均有显著升高(P<0.05);与生理盐水组比较,哌甲酯组、益智宁组大鼠的前额叶皮质组织中PKA蛋白表达及mRNA表达显著升高(P<0.01),且益智宁组与哌甲酯组有显著差异(P<0.05);与生理盐水组比较,哌甲酯组、益智宁组大鼠的前额叶皮质组织中DRD1、DARPP32蛋白表达及mRNA表达显著降低(P<0.01),且益智宁组与哌甲酯组有显著差异(P<0.05)。结论:益智宁组可能通过激活SHR大鼠前额叶皮质AC-cAMP-PKA的信号通路,并通过下调DRD1、DARPP32的水平,负反馈调节脑内多巴胺的含量,从而发挥疗效。
Objective:Through the observation of the influence of Yizhining Decoction on adenylate cyclase(AC),cyclic adenosine monophosphate(cAMP),protein kinase A(PKA),dopamine receptor D1(DRD1),dopamine and cAMP regulated phosphoprotein of 32 kDa(DARPP32)of prefrontal cortex of SHR which is the animal model of ADHD(Attention Deficit Hyperactivity Disorder),to discuss the therapeutic mechanism of Yizhining for ADHD.Methods:Thirty SHR were divided into Yizhining group,normal saline group and Methylphenidate Hydrochloride group(MH),with 10 rats in each group.The Yizhining group was applied with Yizhining Decoction(14.4 g·kg~(-1)).The MH group was treated by Ritalin(0.0015 g·kg~(-1)),while the normal saline group was given normal saline.The gastric volume to the stomach was equal(12.5 mL/kg).After 4 weeks,ELISA was applied to detect the concentrations of AC,cAMP of prefrontal cortex,RT-PCR and Western blot was employed to test the expression levels of PKA,DARPP32 on prefrontal cortex.Results:Compared with normal saline group,the AC content of prefrontal cortex of Yizhining group and Methylphenidate Hydrochloride group significantly increased(P<0.05) and so did the PKA protein expression level and mRNA expression level(P<0.01)and the difference was remarkable between Yizhining group and Methylphenidate Hydrochloride group(P<0.05).And the DRD1,DARPP32 protein expression levels and mRNA expression level on prefrontal cortex notably reduced in the normal saline group(P<0.01)and the difference between Yizhining group and Methylphenidate Hydrochloride group was significant as well(P<0.05).Conclusion:The effect of Yizhining on ADHD may be through activating AC-cAMP-PKA signal pathaway of prefrontal cortex of SHR,and decreasing the levels of DRD1 and DARPP32 and triggering negative feedback of cerebrum dopamine.
Objective:Through the observation of the influence of Yizhining Decoction on adenylate cyclase(AC),cyclic adenosine monophosphate(cAMP),protein kinase A(PKA),dopamine receptor D1(DRD1),dopamine and cAMP regulated phosphoprotein of 32 kDa(DARPP32)of prefrontal cortex of SHR which is the animal model of ADHD(Attention Deficit Hyperactivity Disorder),to discuss the therapeutic mechanism of Yizhining for ADHD.Methods:Thirty SHR were divided into Yizhining group,normal saline group and Methylphenidate Hydrochloride group(MH),with 10 rats in each group.The Yizhining group was applied with Yizhining Decoction(14.4 g·kg~(-1)).The MH group was treated by Ritalin(0.0015 g·kg~(-1)),while the normal saline group was given normal saline.The gastric volume to the stomach was equal(12.5 mL/kg).After 4 weeks,ELISA was applied to detect the concentrations of AC,cAMP of prefrontal cortex,RT-PCR and Western blot was employed to test the expression levels of PKA,DARPP32 on prefrontal cortex.Results:Compared with normal saline group,the AC content of prefrontal cortex of Yizhining group and Methylphenidate Hydrochloride group significantly increased(P<0.05) and so did the PKA protein expression level and mRNA expression level(P<0.01)and the difference was remarkable between Yizhining group and Methylphenidate Hydrochloride group(P<0.05).And the DRD1,DARPP32 protein expression levels and mRNA expression level on prefrontal cortex notably reduced in the normal saline group(P<0.01)and the difference between Yizhining group and Methylphenidate Hydrochloride group was significant as well(P<0.05).Conclusion:The effect of Yizhining on ADHD may be through activating AC-cAMP-PKA signal pathaway of prefrontal cortex of SHR,and decreasing the levels of DRD1 and DARPP32 and triggering negative feedback of cerebrum dopamine.
引文
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[11] 雷爽,苗竞,韩新民,等.安神定志灵对自发性高血压大鼠前额叶纹状体多巴胺和去甲肾上腺素含量的影响[J].辽宁中医杂志,2017,44(2):411-416.
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[14] 唐彦,陈晓刚,赖东兰,等.益智宁对ADHD大鼠前额叶皮质、纹状体多巴胺的影响[J].山东中医杂志,2008,27(5):332-333.
[15] 陈晓刚,赖东兰,沈凌,等.益智宁方煎剂对SHR大鼠前额叶皮质和纹状体中多巴胺及其代谢产物的影响[J].中医药通报,2013,12(3):61-63.
[16] 赖东兰,彭淑平,沈凌,等.益智宁对注意缺陷多动障碍动物模型SHR大鼠学习工作记忆的影响[J].中医药导报.2017,23(1):18-21.
[17] Napolitano F,Bonito-Oliva A,Federici M,et al.Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine[J].J Neurosci,2010,30(33):11043-11056.
[18] 李凡,舒斯云,包新民.多巴胺受体的结构和功能[J].中国神经科学杂志,2003,19(6):405-410.
[19] Vijayraghavan S,Wang M,Birnbaum SG,et al.Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory[J].Nat Neurosci,2007,10(3):376-384.
[20] 吴剑锋,白洁.多巴胺系统在纹状体和前额叶皮质中的作用[J].中国老年学杂志,2015,35(10):5950-5952.
[21] 王学斌,蒋锦昌,李东风.DARPP32、多巴胺系统与神经元整合[J].生物物理学报,2003,19(1):1-5.
[2] 陈晓刚,练文华,廖永州,等.益智宁方治疗224例注意缺陷多动障碍患儿的临床观察[J].中华临床医药杂志,2004,卷?(11):20-22.
[3] 赖东兰,李宜瑞.益智宁治疗儿童注意缺陷多动障碍21例临床观察[J].山东中医药大学学报,2006,30(5):361-362.
[4] Russell VA,Sagvolden T,Johansen EB.Animal model of attention deficit hyperactivity disorder[J].Behav Brain Funct,2005,卷?(1):9.
[5] Sagvolden T,Behavioral validation of the spontaneously hypertensive rat (SHR)as an animal model of attention-deficit/hyperactivity disorder(AD/HD)[J].Neurosci Biobehav Rev,2000,24(1):31-39.
[6] 周荣易,王娇娇,韩新民.ADHD实验动物模型比较研究[J].中国比较医学杂志,2016,26(9):88-92.
[7] Polanczyk G V,Salum G A,Sugaya L S,et al.Annual research review:a meta-analysis of the wordwide prevalence of mental disorders in children and adlelcents[J].Child Psychol Psychol Psychiatry,2015,56(3):345-365.
[8] 任姣姣,岳婕,帖利军.儿童注意缺陷多动障碍发病机制的研究进展[J].中国妇幼健康研究,2016,27(7):894-896.
[9] Antshel KM,Hargrave TM,Simonescu M,et al.Advances in understanding and treating ADHD[J].BMC Med,2011,9:72.
[10] 赖东兰,李宜瑞,陈晓刚,等.益智宁煎剂改善SHR大鼠持续性主动注意力的实验研究[J].广州中医药大学学报,2009,26(2):169-170,175.
[11] 雷爽,苗竞,韩新民,等.安神定志灵对自发性高血压大鼠前额叶纹状体多巴胺和去甲肾上腺素含量的影响[J].辽宁中医杂志,2017,44(2):411-416.
[12] Arnsten AF.Toward a new understanding of attention deficit hyperactivity disorder pathophysiology:an important role for prefrontal cortex dysfunction[J].CNS Drugs,2009,23 (Suppl 1):33-41.
[13] 万朋,金清华.多巴胺及其受体在中枢神经系统的作用研究进展[J].武汉大学学报(医学版),2017,38(1):169-172.
[14] 唐彦,陈晓刚,赖东兰,等.益智宁对ADHD大鼠前额叶皮质、纹状体多巴胺的影响[J].山东中医杂志,2008,27(5):332-333.
[15] 陈晓刚,赖东兰,沈凌,等.益智宁方煎剂对SHR大鼠前额叶皮质和纹状体中多巴胺及其代谢产物的影响[J].中医药通报,2013,12(3):61-63.
[16] 赖东兰,彭淑平,沈凌,等.益智宁对注意缺陷多动障碍动物模型SHR大鼠学习工作记忆的影响[J].中医药导报.2017,23(1):18-21.
[17] Napolitano F,Bonito-Oliva A,Federici M,et al.Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine[J].J Neurosci,2010,30(33):11043-11056.
[18] 李凡,舒斯云,包新民.多巴胺受体的结构和功能[J].中国神经科学杂志,2003,19(6):405-410.
[19] Vijayraghavan S,Wang M,Birnbaum SG,et al.Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory[J].Nat Neurosci,2007,10(3):376-384.
[20] 吴剑锋,白洁.多巴胺系统在纹状体和前额叶皮质中的作用[J].中国老年学杂志,2015,35(10):5950-5952.
[21] 王学斌,蒋锦昌,李东风.DARPP32、多巴胺系统与神经元整合[J].生物物理学报,2003,19(1):1-5.