孔圣枕中丹改善SHR大鼠注意缺陷和冲动的多巴胺机制研究
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摘要
注意缺陷多动障碍(Attention Deficit Hyperactivity Disorder,ADHD),又名儿童多动症。近来Sonuga-Barker结合目前对单胺类神经递质在ADHD发病中的认识,提出了“双通路”模型,被认为是目前较好的ADHD心理—病理—生理模型。该模型强调持续性主动注意力障碍和冲动行为在ADHD发病中的中心作用,执行功能障碍(EDF)和“厌恶延迟”(DEL)共同构成ADHD发病的心理—病理基础。在病理生理上,EDF主要因调控前额叶皮质—背侧纹状体环路的相关DA能分支——中脑—皮质束(MC)和黑质—纹状体束(NS)紊乱所致,主要病理位置在前额叶皮质和背侧纹状体(尾状核);而DEL则是调控前额叶皮质—腹侧纹状体的“犒赏”环路的相关DA能分支——中脑—边缘束(ML)功能改变所致,主要病理位置在前额叶皮质、腹侧纹状体和伏核。
儿童多动症患病率高,危害大。目前,中枢兴奋剂是西医治疗ADHD的首选,其虽可在短中期内使注意缺陷、多动、冲动等症状得到一定程度的控制,但不良反应较多,且有效剂量的个体差异性较大,停药后反跳快、复发率高;作为管制药物,潜在的成瘾性作用也使医患双方有诸多顾虑。
近二十多年来,国内中医界开始应用中医药治疗ADHD。大多数学者认为本病的病机主要是脏腑阴阳失调,阴静不足而阳动有余,与心、肝、脾、肾最为密切。国内多家报道都肯定了中医药治疗ADHD的功效,并发现其疗效与中枢兴奋剂大致相当,虽然起效慢,但长期疗效肯定,且未发现明显毒副反应、反跳少,也无成瘾作用。虽然各家使用的方药有所差异,但我们分析多篇报道后发现:孔圣枕中丹是治疗ADHD的基本方剂。该方为唐代孙思邈所创制,功补心肾,长于潜镇安谧,益智强志。由于技术和伦理学的限制,在人体上开展疗效机理的研究非常困难,动物实验就成为重要的替代途径。国内学者在此方面进行了初步探索,发现中药治疗能减少实验动物的自主活动,改善其学习、记忆障碍,不过这些方法存在明显缺陷:动物模型研究缺乏明确的神经病理定位,虽然对实验动物的中枢DA含量及其代谢产物进行了检测,但由于缺乏合理的神经心理-病理-生理模型指导,实验取材全脑组织,考虑到DA在中枢不同部位的作用并不相同,其结果难以确定中医药治疗ADHD的具体作用机制。显然,此缺陷严重限制了动物实验的研究,相关的药效实验也难以开展,极大地影响了有效药物的开发和在临床上的应用,成为ADHD研究中急待突破和解决的关键问题。
一、研究目的
本课题选择中医药治疗ADHD的代表方剂——孔圣枕中丹,研究其对ADHD动物模型自发性高血压大鼠(SHR)中枢前额叶皮质—基底神经节环路的神经递质DA的信号传导的影响,明确孔圣枕中丹在动物实验中的疗效,深入探索中医药治疗ADHD的具体机制。
二、研究内容和方法
本课题运用目前国际上公认的ADHD动物模型——自发性高血压大鼠(SHR),取SHR大鼠双侧中枢调控前额叶皮质—基底神经节环路的DA神经主要分布区域:前额叶皮质和豆状核,运用免疫组化和分子生物学技术来检测孔圣枕中丹对前额叶皮质和豆状核酪氨酸羟化酶、多巴胺转运体、多巴胺受体(包括DRD_1和DRD_2)的基因表达和蛋白表达的影响。并与中枢兴奋剂盐酸哌甲酯对照:
1、把SPF级,8周龄,体重为240—300g的雄性SHR大鼠,随机分成三组,称重后,分别用孔圣枕中丹、盐酸哌甲酯和生理盐水按照一定的药物/体重比计算每只老鼠的灌胃体积(毫升),灌胃4周后,进行下一步实验。
2、免疫组化法检测孔圣枕中丹作用下SHR大鼠前额叶皮质、豆状核TH、DAT、DRD_1、DRD_2蛋白表达特征。
3、蛋白免疫印迹法检测孔圣枕中丹作用下SHR大鼠前额叶皮质、豆状核TH、DAT、DRD_1、DRD_2蛋白表达特征。
4、原位杂交法检测孔圣枕中丹作用下SHR大鼠前额叶皮质、豆状核TH、DAT、DRD_1、DRD_2基因表达特征。
5、荧光定量PCR法检测孔圣枕中丹作用下SHR大鼠前额叶皮质、豆状核TH、DAT、DRD_1、DRD_2基因表达特征。
三、研究结果:
1.免疫组化法和蛋白免疫印迹法显示,SHR大鼠前额叶皮质和豆状核中酪氨酸羟化酶蛋白的表达,孔圣枕中丹组比盐酸哌甲酯组高,盐酸哌甲酯组又比生理盐水对照组高;多巴胺转运体的表达盐酸哌甲酯组比孔圣枕中丹组高,且两者均低于生理盐水对照组。
2.免疫组化法和蛋白免疫印迹法显示,SHR大鼠前额叶皮质多巴胺受体(包括D1和D2)和豆状核受体D1的蛋白表达,孔圣枕中丹组比盐酸哌甲酯组要高,且两者均比对照组低;豆状核的多巴胺受体D2的蛋白表达,盐酸哌甲酯组要比孔圣枕中丹组高,且均低于对照组。
3.原位杂交和荧光定量PCR表明,SHR大鼠前额叶皮质和豆状核中酪氨酸羟化酶基因的表达,孔圣枕中丹组比盐酸哌甲酯组高,盐酸哌甲酯组又比生理盐水对照组高;多巴胺转运体的基因表达盐酸哌甲酯组比孔圣枕中丹组高,且两者均低于生理盐水对照组。
4.原位杂交和荧光定量PCR表明,SHR大鼠前额叶皮质的多巴胺受体(包括D1和D2)和豆状核受体D1的基因表达,孔圣枕中丹组比盐酸哌甲酯组要高,且两者均低于对照组;豆状核多巴胺受体D2的基因表达,盐酸哌甲酯组要比孔圣枕中丹组高,且均低于对照组。
四、研究结论:
1.孔圣枕中丹能改善ADHD动物模型的核心症状是通过影响调控中枢前额叶皮质-基底神经节环路的DA神经信号传导来实现。
2.孔圣枕中丹促进DA的合成来改善ADHD动物模型的核心症状是通过多靶点、多作用途径来实现的:
(1)增加DA合成限速酶——酪氨酸羟化酶(TH)的含量,促进DA的生物合成及突触前末梢DA的释放;
(2)影响多巴胺转运体的基因表达和蛋白表达,从而减少DA的重摄取;
(3)影响DA受体(包括D1和D2)的基因表达和蛋白表达,上调中枢DA的功能。
ADHD(Attention Deficit Hyperactivity Disorder,ADHD),is also known as ADHD children.By integrating the awareness of monoamine neurotransmitters in the pathogenesis of ADHD,Sonuga-Barker recent put forward a model of the "dual pathway",which is considered better psychological - pathology - physiological model.The model emphasized the Central role of the obstacles to persistent of the initiative to the attention and impulsive behavior in ADHD.the Executive dysfunction(EDF) and the "delay aversion"(DEL) together constitute the psychological - pathologic basis of ADHD.In the pathophysiology, EDF is due to disorder of the cortex beam(MC) and the substantia nigra - Striatum beam (NS) which are related branches of DA,controlling the prefrontal cortex - the dorsal striatum DA loop in the brain.The location of the main pathological is Prefrontal cortex and dorsal striatum;and DEL is due to changes in function of Mesolimbic beam which is related branch of DA,regulating and controlling prefrontal cortex - the ventral striatum of the "reward" loop,the main pathological location are the prefrontal cortex,ventral striatum and nucleus accumbens.
Prevalence rate of ADHD is High,against is large.At present,central stimulants is the preferred treatment of ADHD in Western medicine,although it may control attention deficit. hyperactivity,impulsive symptoms on certain degree in the short or medium term,but more adverse reactions appear,and the effective dose have greater individual differences,after stopping taking drug,rebound quickly,Relapse soon;as controlled drugs,the potential addiction to the drug make both doctors and patients have many concerns.
Over the past two decades,Chinese medicine is applied to treat ADHD in Chinese medicine practitioners in Chinese.The majority of scholars believe that the pathogenesis of this disease is an imbalance of yin and yang organs,deficiency of yin causes the movement of yang,whose relationship with the heart,liver,spleen,kidney is the most closely.Several reports have affirmed the effectiveness of the traditional Chinese medicine in the treatment of ADHD and found that its effectiveness is similar to Central stimulants,although its effectiveness is slow onset,but effect is positive long-term,and did not find obvious Side effects,less rebound nor addiction.Although each used different prescriptions,we analysis lots of reports and found that Kongsheng Zhen zhong pill is the the basic Prescription in treatment of ADHD.The Prescription is created by Simiao Sun in the Tang Dynasty.effect is to tonify kidney and heart,calm the mind.nourish spirit..Due to restrictions on technology and ethics,it is very difficult to research the mechanism of the efficacy of the human body.the experiment on animals has become an important alternative.Chinese scholars have a preliminary exploration in this regard,founding that traditional Chinese medicine treatment can reduce the autonomy of activities of laboratory animals,improving their learning and memory impairment,but these methods have an obvious flaw:animal model lack of neuropathic clear positioning,although content of the central DA and its metabolites of the experimental animals were detected,the lack of guidance of reasonable neuropsychological - pathology - physical model,materials of the experiment came from the whole brain tissue,the role of DA is not the same in different central parts,the result of Chinese medicine is difficult to determine its specific mechanism of ADHD.Obviously, this defect severely limits the study of animal experiments,the effect related to it will be also difficult to carry out experiments,greatly influenced the development of effective drugs and the clinical application,and research in ADHD need to be a breakthrough on key issues and solutions.
Objective:
This project chooses the classic Chinese medicine prescription on treating ADHD——Kongsheng Zhenzhong Pill and studies it's impact on the signal trasmission of the neurotransmitter DA of the prefrontal cortex-basal ganglial circuit loop in the center of the ADHD animal model——SHR.The project tries to make clear Kongsheng Zhenzhong pill's curative effect in animal experiment and explore in depth the mechanism of treating ADHD with Chinese medicine.
Content and Method:
The project uses the internationally accepted ADHD animal model
nowadays——spontaneous hypertensive rat.In the prefrontal cortex and lentiform nucleus, which are the mainly distributed regions of DA nerves of a rat that control the prefrontal cortex-basal ganglial circuit loop,we use immunohistochemistry and molecular biology technology to detect the influence of the gene expression and protein expression of tyrosine hydroxylase,dopamine transporter,dopamine receptor and compare them with the central stimulant methylphenidate hydrochloride.
1.Use male SHR of SPF grade,which are 8-week old and weigh 240～300g,and randomly divide them into three groups.Then use Kongsheng Zhenzhong Pill, methylphenidate hydrochloride and physiological saline to caculate each rat's gavage volume(ml) in accordance with certain proportion of drug/ weight.4 weeks after gavaging, for the next experimental.
2.Detect with immunohistochemistry the characteristics of the protein expression of SHR's frontal cortex,lentiform nucleusTH,DAT.DRD1,DRD2 under the effect of Kongsheng Zhenzhong Pill.
3.Detect with Western blot the characteristics of the protein expression of SHR's frontal cortex,lentiform nucleusTH,DAT,DRD1,DRD2 under the effect of Kongsheng Zhenzhong Pill.
4.Use in situ hvbridization to detect the characteristics of the gene expression of SHR's lentiform nucleusTH,DAT,DRD1,DRD2 under the effect of Kongsheng Zhenzhong Pill.
5.Detect with fluorescent quantitative PCR the characteristics of the gene expression of SHR's lentiform nucleusTH,DAT,DRD1,DRD2 under the effect of Kongsheng Zhenzhong Pill.
Result:
1.With the use of Immunohistochemistry and Western blot.the protein expression of tyrosine hydroxylase in the prefrontal cortex and lentiform nucleus of SHR from the Chinese medicine group is higher than that in the western medicine group.Meanwhile,the protein expression from the western medicine group is higher than the control group.The expression of dopamine transporter from the western medicine group is higher than that in the Chinese medicine group while both groups are lower than that in the control group.
2.With the use of Immunohistochemistry and Western blot.the protein expression of dopamine receptors(including D1 and D2) in the prefrontal cortex and DRD1 in lentiform nucleus of SHR from the Kongsheng Zhenzhong Pill group is higher than that from the methylphenidate hydrochloride group while both groups are lower than the control group; the protein expression of dopamine receptors D2 in lentiform nucleus from the methylphenidate hydrochloride group is higher than that in the Kongsheng Zhenzhong Pill group and both groups are lower than that in the control group.
3.The results of in situ hybridization and fluorescence quantitative PCR showed that the gene expression oftyrosine hydroxylase in the prefrontal cortex and lentiform nucleus of SHR from the Chinese medicine group is higher than that in the western medicine group. Meanwhile,the gene expression from the western medicine group is higher than the control group.The expression of dopamine transporter from the western medicine group is higher than that in the Chinese medicine group while both groups are lower than that in the control group.
4.The results of in situ hybridization and fluorescence quantitative PCR showed that the gene expression of dopamine receptors(including D1 and D2) in the prefrontal cortex and DRD1 in lentiform nucleus of SHR from the Kongsheng Zhenzhong Pill group is higher than that in the methylphenidate hydrochloride group and both groups are lower than that in the control group:the gene expression of dopamine receptors D2 in the lentiform nucleus from the methylphenidate hydrochloride group is higher than that in the Kongsheng Zhenzhong Pill group while both groups are lower than that from the control group.
Conclusion:
1.Kongsheng Zhenzhong Pill improves the main symptoms of ADHD animal model bv influencing the signal transmission of the DA nerves in the the prefrontal cortex-basal ganglial circuit loop of the control center.
2.That Kongsheng Zhenzhong Pill can promote DA synthesis to improve the core symptoms of an animal model of ADHD is through a multi-target,multi-role approach to achieve:
(1) To promote the biosynthesis of DA and peripheral presynaptic release of DA by increasing DA synthesis rate-limiting enzymes - tyrosine hydroxylase(TH) content,;
(2) The impact of dopamine transporter gene expression and protein expression, thereby reducing the re-uptake of DA;;
(3) The impact of DA receptors(including D1 and D2) of gene expression and protein expression,increase central DA function.
儿童多动症患病率高,危害大。目前,中枢兴奋剂是西医治疗ADHD的首选,其虽可在短中期内使注意缺陷、多动、冲动等症状得到一定程度的控制,但不良反应较多,且有效剂量的个体差异性较大,停药后反跳快、复发率高;作为管制药物,潜在的成瘾性作用也使医患双方有诸多顾虑。
近二十多年来,国内中医界开始应用中医药治疗ADHD。大多数学者认为本病的病机主要是脏腑阴阳失调,阴静不足而阳动有余,与心、肝、脾、肾最为密切。国内多家报道都肯定了中医药治疗ADHD的功效,并发现其疗效与中枢兴奋剂大致相当,虽然起效慢,但长期疗效肯定,且未发现明显毒副反应、反跳少,也无成瘾作用。虽然各家使用的方药有所差异,但我们分析多篇报道后发现:孔圣枕中丹是治疗ADHD的基本方剂。该方为唐代孙思邈所创制,功补心肾,长于潜镇安谧,益智强志。由于技术和伦理学的限制,在人体上开展疗效机理的研究非常困难,动物实验就成为重要的替代途径。国内学者在此方面进行了初步探索,发现中药治疗能减少实验动物的自主活动,改善其学习、记忆障碍,不过这些方法存在明显缺陷:动物模型研究缺乏明确的神经病理定位,虽然对实验动物的中枢DA含量及其代谢产物进行了检测,但由于缺乏合理的神经心理-病理-生理模型指导,实验取材全脑组织,考虑到DA在中枢不同部位的作用并不相同,其结果难以确定中医药治疗ADHD的具体作用机制。显然,此缺陷严重限制了动物实验的研究,相关的药效实验也难以开展,极大地影响了有效药物的开发和在临床上的应用,成为ADHD研究中急待突破和解决的关键问题。
一、研究目的
本课题选择中医药治疗ADHD的代表方剂——孔圣枕中丹,研究其对ADHD动物模型自发性高血压大鼠(SHR)中枢前额叶皮质—基底神经节环路的神经递质DA的信号传导的影响,明确孔圣枕中丹在动物实验中的疗效,深入探索中医药治疗ADHD的具体机制。
二、研究内容和方法
本课题运用目前国际上公认的ADHD动物模型——自发性高血压大鼠(SHR),取SHR大鼠双侧中枢调控前额叶皮质—基底神经节环路的DA神经主要分布区域:前额叶皮质和豆状核,运用免疫组化和分子生物学技术来检测孔圣枕中丹对前额叶皮质和豆状核酪氨酸羟化酶、多巴胺转运体、多巴胺受体(包括DRD_1和DRD_2)的基因表达和蛋白表达的影响。并与中枢兴奋剂盐酸哌甲酯对照:
1、把SPF级,8周龄,体重为240—300g的雄性SHR大鼠,随机分成三组,称重后,分别用孔圣枕中丹、盐酸哌甲酯和生理盐水按照一定的药物/体重比计算每只老鼠的灌胃体积(毫升),灌胃4周后,进行下一步实验。
2、免疫组化法检测孔圣枕中丹作用下SHR大鼠前额叶皮质、豆状核TH、DAT、DRD_1、DRD_2蛋白表达特征。
3、蛋白免疫印迹法检测孔圣枕中丹作用下SHR大鼠前额叶皮质、豆状核TH、DAT、DRD_1、DRD_2蛋白表达特征。
4、原位杂交法检测孔圣枕中丹作用下SHR大鼠前额叶皮质、豆状核TH、DAT、DRD_1、DRD_2基因表达特征。
5、荧光定量PCR法检测孔圣枕中丹作用下SHR大鼠前额叶皮质、豆状核TH、DAT、DRD_1、DRD_2基因表达特征。
三、研究结果:
1.免疫组化法和蛋白免疫印迹法显示,SHR大鼠前额叶皮质和豆状核中酪氨酸羟化酶蛋白的表达,孔圣枕中丹组比盐酸哌甲酯组高,盐酸哌甲酯组又比生理盐水对照组高;多巴胺转运体的表达盐酸哌甲酯组比孔圣枕中丹组高,且两者均低于生理盐水对照组。
2.免疫组化法和蛋白免疫印迹法显示,SHR大鼠前额叶皮质多巴胺受体(包括D1和D2)和豆状核受体D1的蛋白表达,孔圣枕中丹组比盐酸哌甲酯组要高,且两者均比对照组低;豆状核的多巴胺受体D2的蛋白表达,盐酸哌甲酯组要比孔圣枕中丹组高,且均低于对照组。
3.原位杂交和荧光定量PCR表明,SHR大鼠前额叶皮质和豆状核中酪氨酸羟化酶基因的表达,孔圣枕中丹组比盐酸哌甲酯组高,盐酸哌甲酯组又比生理盐水对照组高;多巴胺转运体的基因表达盐酸哌甲酯组比孔圣枕中丹组高,且两者均低于生理盐水对照组。
4.原位杂交和荧光定量PCR表明,SHR大鼠前额叶皮质的多巴胺受体(包括D1和D2)和豆状核受体D1的基因表达,孔圣枕中丹组比盐酸哌甲酯组要高,且两者均低于对照组;豆状核多巴胺受体D2的基因表达,盐酸哌甲酯组要比孔圣枕中丹组高,且均低于对照组。
四、研究结论:
1.孔圣枕中丹能改善ADHD动物模型的核心症状是通过影响调控中枢前额叶皮质-基底神经节环路的DA神经信号传导来实现。
2.孔圣枕中丹促进DA的合成来改善ADHD动物模型的核心症状是通过多靶点、多作用途径来实现的:
(1)增加DA合成限速酶——酪氨酸羟化酶(TH)的含量,促进DA的生物合成及突触前末梢DA的释放;
(2)影响多巴胺转运体的基因表达和蛋白表达,从而减少DA的重摄取;
(3)影响DA受体(包括D1和D2)的基因表达和蛋白表达,上调中枢DA的功能。
ADHD(Attention Deficit Hyperactivity Disorder,ADHD),is also known as ADHD children.By integrating the awareness of monoamine neurotransmitters in the pathogenesis of ADHD,Sonuga-Barker recent put forward a model of the "dual pathway",which is considered better psychological - pathology - physiological model.The model emphasized the Central role of the obstacles to persistent of the initiative to the attention and impulsive behavior in ADHD.the Executive dysfunction(EDF) and the "delay aversion"(DEL) together constitute the psychological - pathologic basis of ADHD.In the pathophysiology, EDF is due to disorder of the cortex beam(MC) and the substantia nigra - Striatum beam (NS) which are related branches of DA,controlling the prefrontal cortex - the dorsal striatum DA loop in the brain.The location of the main pathological is Prefrontal cortex and dorsal striatum;and DEL is due to changes in function of Mesolimbic beam which is related branch of DA,regulating and controlling prefrontal cortex - the ventral striatum of the "reward" loop,the main pathological location are the prefrontal cortex,ventral striatum and nucleus accumbens.
Prevalence rate of ADHD is High,against is large.At present,central stimulants is the preferred treatment of ADHD in Western medicine,although it may control attention deficit. hyperactivity,impulsive symptoms on certain degree in the short or medium term,but more adverse reactions appear,and the effective dose have greater individual differences,after stopping taking drug,rebound quickly,Relapse soon;as controlled drugs,the potential addiction to the drug make both doctors and patients have many concerns.
Over the past two decades,Chinese medicine is applied to treat ADHD in Chinese medicine practitioners in Chinese.The majority of scholars believe that the pathogenesis of this disease is an imbalance of yin and yang organs,deficiency of yin causes the movement of yang,whose relationship with the heart,liver,spleen,kidney is the most closely.Several reports have affirmed the effectiveness of the traditional Chinese medicine in the treatment of ADHD and found that its effectiveness is similar to Central stimulants,although its effectiveness is slow onset,but effect is positive long-term,and did not find obvious Side effects,less rebound nor addiction.Although each used different prescriptions,we analysis lots of reports and found that Kongsheng Zhen zhong pill is the the basic Prescription in treatment of ADHD.The Prescription is created by Simiao Sun in the Tang Dynasty.effect is to tonify kidney and heart,calm the mind.nourish spirit..Due to restrictions on technology and ethics,it is very difficult to research the mechanism of the efficacy of the human body.the experiment on animals has become an important alternative.Chinese scholars have a preliminary exploration in this regard,founding that traditional Chinese medicine treatment can reduce the autonomy of activities of laboratory animals,improving their learning and memory impairment,but these methods have an obvious flaw:animal model lack of neuropathic clear positioning,although content of the central DA and its metabolites of the experimental animals were detected,the lack of guidance of reasonable neuropsychological - pathology - physical model,materials of the experiment came from the whole brain tissue,the role of DA is not the same in different central parts,the result of Chinese medicine is difficult to determine its specific mechanism of ADHD.Obviously, this defect severely limits the study of animal experiments,the effect related to it will be also difficult to carry out experiments,greatly influenced the development of effective drugs and the clinical application,and research in ADHD need to be a breakthrough on key issues and solutions.
Objective:
This project chooses the classic Chinese medicine prescription on treating ADHD——Kongsheng Zhenzhong Pill and studies it's impact on the signal trasmission of the neurotransmitter DA of the prefrontal cortex-basal ganglial circuit loop in the center of the ADHD animal model——SHR.The project tries to make clear Kongsheng Zhenzhong pill's curative effect in animal experiment and explore in depth the mechanism of treating ADHD with Chinese medicine.
Content and Method:
The project uses the internationally accepted ADHD animal model
nowadays——spontaneous hypertensive rat.In the prefrontal cortex and lentiform nucleus, which are the mainly distributed regions of DA nerves of a rat that control the prefrontal cortex-basal ganglial circuit loop,we use immunohistochemistry and molecular biology technology to detect the influence of the gene expression and protein expression of tyrosine hydroxylase,dopamine transporter,dopamine receptor and compare them with the central stimulant methylphenidate hydrochloride.
1.Use male SHR of SPF grade,which are 8-week old and weigh 240～300g,and randomly divide them into three groups.Then use Kongsheng Zhenzhong Pill, methylphenidate hydrochloride and physiological saline to caculate each rat's gavage volume(ml) in accordance with certain proportion of drug/ weight.4 weeks after gavaging, for the next experimental.
2.Detect with immunohistochemistry the characteristics of the protein expression of SHR's frontal cortex,lentiform nucleusTH,DAT.DRD1,DRD2 under the effect of Kongsheng Zhenzhong Pill.
3.Detect with Western blot the characteristics of the protein expression of SHR's frontal cortex,lentiform nucleusTH,DAT,DRD1,DRD2 under the effect of Kongsheng Zhenzhong Pill.
4.Use in situ hvbridization to detect the characteristics of the gene expression of SHR's lentiform nucleusTH,DAT,DRD1,DRD2 under the effect of Kongsheng Zhenzhong Pill.
5.Detect with fluorescent quantitative PCR the characteristics of the gene expression of SHR's lentiform nucleusTH,DAT,DRD1,DRD2 under the effect of Kongsheng Zhenzhong Pill.
Result:
1.With the use of Immunohistochemistry and Western blot.the protein expression of tyrosine hydroxylase in the prefrontal cortex and lentiform nucleus of SHR from the Chinese medicine group is higher than that in the western medicine group.Meanwhile,the protein expression from the western medicine group is higher than the control group.The expression of dopamine transporter from the western medicine group is higher than that in the Chinese medicine group while both groups are lower than that in the control group.
2.With the use of Immunohistochemistry and Western blot.the protein expression of dopamine receptors(including D1 and D2) in the prefrontal cortex and DRD1 in lentiform nucleus of SHR from the Kongsheng Zhenzhong Pill group is higher than that from the methylphenidate hydrochloride group while both groups are lower than the control group; the protein expression of dopamine receptors D2 in lentiform nucleus from the methylphenidate hydrochloride group is higher than that in the Kongsheng Zhenzhong Pill group and both groups are lower than that in the control group.
3.The results of in situ hybridization and fluorescence quantitative PCR showed that the gene expression oftyrosine hydroxylase in the prefrontal cortex and lentiform nucleus of SHR from the Chinese medicine group is higher than that in the western medicine group. Meanwhile,the gene expression from the western medicine group is higher than the control group.The expression of dopamine transporter from the western medicine group is higher than that in the Chinese medicine group while both groups are lower than that in the control group.
4.The results of in situ hybridization and fluorescence quantitative PCR showed that the gene expression of dopamine receptors(including D1 and D2) in the prefrontal cortex and DRD1 in lentiform nucleus of SHR from the Kongsheng Zhenzhong Pill group is higher than that in the methylphenidate hydrochloride group and both groups are lower than that in the control group:the gene expression of dopamine receptors D2 in the lentiform nucleus from the methylphenidate hydrochloride group is higher than that in the Kongsheng Zhenzhong Pill group while both groups are lower than that from the control group.
Conclusion:
1.Kongsheng Zhenzhong Pill improves the main symptoms of ADHD animal model bv influencing the signal transmission of the DA nerves in the the prefrontal cortex-basal ganglial circuit loop of the control center.
2.That Kongsheng Zhenzhong Pill can promote DA synthesis to improve the core symptoms of an animal model of ADHD is through a multi-target,multi-role approach to achieve:
(1) To promote the biosynthesis of DA and peripheral presynaptic release of DA by increasing DA synthesis rate-limiting enzymes - tyrosine hydroxylase(TH) content,;
(2) The impact of dopamine transporter gene expression and protein expression, thereby reducing the re-uptake of DA;;
(3) The impact of DA receptors(including D1 and D2) of gene expression and protein expression,increase central DA function.
引文
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