【作者】 吕红；

【导师】 李安源；

【作者基本信息】 山东大学 ， 中西医结合临床， 2009， 博士

【摘要】 背景抽动-秽语综合征(Tourette’s syndrome,TS)是一种无意识的、无目的的、重复的刻板活动为特征的神经精神性疾病。临床表现为头面、腹部及四肢不自主的抽动,喉部发出奇特叫声或罢骂不避亲疏为特征。TS有不规则的发作间歇期,有时可因转移注意力而减轻,睡眠时症状消失,精神紧张时症状加重。该病常伴随注意缺陷、多动冲动或强迫症状。TS诊断标准为:1.具有多种运动性抽动及一种或多种发声性抽动,有时不一定在同一时间出现。2.抽动每天发作多次,通常为一阵阵发作,病情持续或间断发作已超过一年,其无抽动间歇期连续不超过三个月。3.上述症状引起明显的不安,显著地影响社交、就业和其他重要领域的活动。4.发病于18岁前。5.上述症状不是直接由某些药物(如兴奋剂)或内科疾病(如亨廷顿舞蹈病或病毒感染后脑炎)引起。尽管TS是一种轻症的神经精神疾病,但病程长,反复发作,影响正常的生活和学习。近于一半的病人抽动症状会持续到18岁以后,甚者部分病人抽动症状可以持续一生,大约有5%的TS病人有影响生存的症状,称之为恶性TS。多巴胺(dopamin,DA)系统的过度活跃和受体超敏感均有可能引起抽动的发生。因此抑制DA系统功能是治疗TS的重要途径。TS的发病与社会心理因素、遗传因素、中枢神经递质的代谢异常等因素有关。而DA系统功能异常被广泛认为是导致TS发病的主要病理学基础。应用DA受体激动剂在动物身上能产生突发、反复的定型肌肉运动等一系列的酷似TS的症状和体征。DA神经元主要集中在中脑和基底节,其中黑质、纹状体DA的密度约占脑内DA的80%。位于黑质致密层的DA神经元接受纹状体的投射纤维,终止于纹状体背侧神经元;源于脚间核上中线的DA神经元终止于中脑边缘系统通路。黑质-纹状体通路是调节一切行为反应的基本结构,中脑-中脑边缘的DA通道平衡失调致边缘系统抑制障碍被认为是TS的神经生化基础。脑内DA的前体物质来源于食物中的酪氨酸,在限速酶酪氨酸羟化酶的作用下生成多巴,后者又在多巴脱羧酶的作用下生成DA。DA在脑内主要在单胺氧化酶(monoamine oxidase,MAO)的作用下降解为高香草酸(homovanillic acid,HVA),排出体外。HVA是中枢神经系统中DA的主要代谢产物,通常被作为DA活性的主要指标。研究证明TS患者壳核DA释放量明显高于对照组,血清及脑脊液中HVA较正常对照组明显降低,且降低程度与症状严重程度明显相关。被释放的DA分别与纹状体内神经元上的多巴胺D1受体(DopamineReceptor D1,DRD1)和多巴胺D2受体(Dopamine Receptor D2,DRD2)结合,最终使大脑皮质运动区兴奋或去抑制,而产生抽动现象。有研究证明TS患者DA能神经系统异常,尤其是DA能神经系统过度活跃或突触后DA受体超敏感,导致慢性反复出现不自主的动作及发音上的抽动。DRD2被认为与TS关系更为密切,TS患者纹状体内DRD2数目、密度较同年龄、同性别对照明显升高。综上研究表明,TS患者纹状体内DA含量升高,其血清中代谢产物HVA含量减少,纹状体内DRD2活性增强。氟哌啶醇(Haloperidol,Hal)作为DRD2阻滞剂至今仍为治疗TS的有效药物。但氟哌啶醇的副作用相对比较大,其副作用的发生直接与剂量相关。常见的副作用有嗜睡、镇静、乏力、头昏、椎体外系反应(如肌张力障碍、静坐不能、帕金森氏病样震颤等),服用数月后,可能出现类帕金森氏病的症状,如震颤、肌肉僵直等,长期应用氟哌啶醇可导致迟发性运动障碍的发生,表现为不自主的刻板运动,并具有持续性。有如此诸多副作用,一定程度上限制了该药物的临床应用。宁动颗粒(Ningdong granule,NDG)是临床治疗TS的中药复方制剂,已应用于临床多年,并显示出良好的效果。有研究证明天麻素(Gastrodin,Gas)能抑制动物DA系统活性,有显著的镇静、抗癫痫及神经保护作用。本课题采用阿朴吗啡(Apomorphine,Apo)诱导TS大鼠模型,观察宁动颗粒及天麻素对TS大鼠刻板活动的影响;检测TS大鼠血清内HVA、纹状体内DA含量及DRD2基因及蛋白表达的强弱,初步探讨宁动颗粒及天麻素对TS大鼠DA系统的影响,并阐明宁动颗粒及天麻素治疗TS的作用机制,为宁动颗粒及天麻素治疗TS提供科学理论依据。目的1.观察宁动颗粒及天麻素对TS模型大鼠刻板活动的影响。2.观察宁动颗粒对TS模型大鼠纹状体内DA含量、DRD2 mRNA表达和蛋白表达,以及血清中HVA含量的影响。探讨宁动颗粒治疗TS的作用机制。3.观察天麻素对TS模型大鼠血清中HVA含量及纹状体内DRD2 mRNA表达的影响。探讨天麻素抑制TS的机制。方法1.研究对象:4周龄雄性Wistar大鼠96只,随机分为6组,每组16只:(1)空白对照组;(2)阿朴吗啡组(Apo);(3)氟哌啶醇组(Hal);(4)宁动颗粒加氟哌啶醇组(NG+Hal);(5)宁动颗粒低剂量组(NGL);(6)宁动颗粒高剂量组(NGH);(7)天麻素组(Gas)。空白对照组腹腔注射生理盐水5 ml/(kg·d),其余6组腹腔注射Apo 2.0 mg/(kg·d)。连续注射4周。Hal组给予氟哌啶醇1.0mg/(kg·d)(相当于临床用量的10倍);NG+Hal组给予氟哌啶醇1.0mg/(kg·d)+宁动颗粒250mg/(kg·d)(相当于临床用量的10倍);NGL组给予宁动颗粒250mg/(kg·d)(相当于临床用量的10倍);NGH组给予宁动颗粒370mg/(kg·d)(相当于临床用量的15倍);Gas组给予天麻素20.0mg/(kg·d);空白对照组及Apo组分别给予等量蒸馏水。各组大鼠均灌胃给药,每日10:00 am给药1次,连续给药12周。末次给药后禁食24 h,不禁水。3%戊巴比妥钠(30mg/kg,腹腔注射)麻醉,心脏取血分离血清,-20℃保存备用。多聚甲醛全身灌注,留纹状体固定备用,另取新鲜脑组织,-80℃保存备用。然后进行后续实验。2.研究内容:(1)大鼠行为评分测定大鼠用药后行为;(2)酶联免疫吸附剂测定法(enzyme linked immunosorbent assay,ELISA)检测血清中HVA含量;(3)ELISA法检测纹状体内DA含量;(4)免疫组织化学染色法检测纹状体内DRD2蛋白表达;(5)RT-PCR法检测纹状体内DRD2 mRNA表达。结果1.大鼠行为研究与空白对照组比较,Apo可以显著引起大鼠刻板活动。根据大鼠行为测定曲线图所示,连续注射Apo 4周后大鼠出现持续稳定的刻板活动(P＜0.01)。给药后Hal组、NG+Hal组和NGH组大鼠刻板活动与Apo组比较明显减弱(P均＜0.01),且三组间相比差异无统计学意义(P＞0.05)。Gas组大鼠刻板活动与Apo组比较明显减弱(P＜0.05),且与Hal组相比差异无统计学意义(P＞0.05)。NGL组刻板活动虽有所减弱,但与Apo组相比差异无统计学意义(P＞0.05),与Hal组、NG+Hal组和NGH组相比有显著性差异(P＜0.05)。2.血清中HVA含量比较与空白对照组比较,Apo组大鼠血清HVA含量明显降低(4.76±0.95 mg/mlvs 3.49±0.66 mg/ml,P＜0.01)。各治疗组大鼠血清HVA含量均有所升高,与Apo组相比差异具有显著性(P＜0.01),其中NG±Hal组升高最为显著(4.94±0.86 mg/ml vs 3.49±0.66 mg/ml,P＜0.01)。其次为NGH组(4.86±1.48mg/ml vs 3.49±0.66 mg/ml,P＜0.01)。Hal组(4.81±1.60 mg/ml vs 3.49±0.66 mg/ml,P＜0.01)。NGL组(4.77±1.15 mg/ml vs 3.4±0.66 mg/ml,P＜0.01),4治疗组之间相比差异无统计学意义(P＞0.05)。Gas组(4.69±1.30 mg/ml vs 3.49±0.66mg/ml,P＜0.05),与Hal组之间相比差异无统计学意义(P＞0.05)。3.纹状体中DA含量比较与空白对照组比较,Apo组大鼠纹状体内DA含量明显升高(103.42±44.39ng/ml vs 240.23±72.32 ng/ml,P＜0.01)。给药后Hal组、NG+Hal组、NGL组和NGH组大鼠纹状体内DA含量均有所降低。NGH组纹状体内DA含量降低最显著,与Apo组相比差异具有显著性(136.04±50.95 ng/ml vs 240.23±72.32ng/ml,P＜0.01)但Hal组、NG+Hal组和NGL组DA含量与Apo组相比无显著性差异(208.20±104.01 ng/ml vs 240.23±72.32 ng/ml,P＞0.05;202.36±102.22ng/ml vs 240.23±72.32 ng/ml,P＞0.05;214.40±81.07 ng/ml vs 240.23±72.32 ng/ml,P＞0.05),三组间相比差异无统计学意义(P＞0.05),与NGH组比较有显著性差异(P＜0.01)。4.大鼠脑组织切片纹状体DRD2光镜下DAB染色,灰度比较与空白对照组比较,Apo组大鼠纹状体内DRD2灰度明显升高(P＜0.01)。给药后Hal组、NG+Hal组、NGL组和NGH组大鼠纹状体内DRD2灰度均有所降低。NGH组下降最为显著(P＜0.01),其次为Hal组和NG+Hal组,两组与Apo比较均有显著性差异(P＜0.05),NGL组与Apo组相比差异无统计学意义(P＞0.05)。NGL组和NGH组相比差异具有显著性(P＜0.01)。5.大鼠纹状体内DRD2 mRNA表达比较与空白对照组比较,Apo组大鼠纹状体内DRD2 mRNA表达明显升高(P＜0.01)。给药后Hal组、NG+Hal组、NGL组和NGH组大鼠纹状体内DRD2mRNA表达均有所降低。NG+Hal组下降最为显著(P＜0.01),其次为Hal组和NGH组,两组与Apo比较均有显著性差异(P＜0.05),Hal组、NG+Hal组和NGH组与NGL组相比差异具有显著性(P＜0.05)。NGL组与Apo组相比差异无统计学意义(P＞0.05)。Gas对大鼠纹状体内DRD2 mRNA表达的影响无统计学意义(P＞0.05).结论宁动颗粒及天麻素对TS模型大鼠具有明显抑制刻板活动的作用,能抑制DA系统兴奋性,促进DA代谢,增加血清中HVA含量;同时表明宁动颗粒还具有显著降低纹状体内DA含量、抑制纹状体内DRD2蛋白及mRNA表达的作用。更多还原

【Abstract】 BackgroundTourette’s syndrome(TS) is a neuropsychiatric disorder characterized by stereotypic,involuntary,purposeless and repetitive movements.The motor tics include headshakes,violent clonic tics consisting of thrusting head jerks and orofacial tics such as facial grimacing,eye blinking and throat clearing.It dosen’t occur regularity.The symptoms will decrease by divert attention,vanish after sleep,and aggravate after actuation.It often associate with attention-deficit hyperactivity disorder,motor tics and obsessive-compulsive disorder.The diagnostic criteria of TS are 1.The character of TS are motor tics and vocal tics,the two symptoms often occur not at the same time.2.Tics break out several times every day.It can last more than 1 year,and the period with no tics less than 3 months.3.The tics can cause conspicuouse restlessness,which can influence social intercourse,employment and some other social activities.4.It often occur before the age of 18 years.5.The symptoms are not cause by some drugs(such as stimulant),and morbus internus(such as Humtington’s chorea and viral encephalitis).For some individuals,tics can cause lifelong impairment and about 5%of TS patients have life-threatening symptoms, which were defined malignant TS.It is widely believed that abnormalities of dopamin (DA) neurotransmission play a primary role in the pathophysiology of TS.Inhibit the activity of DA system can weaken TS symptoms.TS is related with social psychic factors,genetic factors,metabolism disorder of central neurotransmitter,and so on.It is Widely believed that abnormalities of dopamine(DA) neurotransmission play a primary role in the pathophysiology of TS. The excitomotory of DA can cause flup and repetitive movements,which just like the symptoms of TS.The DA neurons are concentrate at deutocerebrum and basal ganglia. The density of DA in the substantia nigra and striatum is 80%of the brain.The nerve fiber from striatum project to the DA neurons in the substantia nigra compact layer. The substantia nigra-striatum pathway is the elementary structure of all behavior. The barriers inhibiting of limbic system,which caused by DA pathway disequilibrium of deutocerebrum,is regarded as the biochemistry base.Tyrosine come from food is the precursor of DA,it transform into dioxyphenylalanine by the tyrosine hydroxylase. The dioxyphenylalanine transform into DA by dopa decarboxylase.DA will degrade into homovanillic acid(HVA) by the monoamine oxidase.As main metabolite of DA in central neural system,HVA is generally regarded as major indicator of DA activity. The DA content in dorsal caudate putamen of TS patients was obviously higher than the control group.The content of HVA in sera and cerebrospinal fluid of TS patients was lower than the control group,and the degree of decrease correlate with the the severity of symptoms.DA combine with DA recepters in striatum neuron,and ultimately excites or inhibits cerebral motor cortex,so as to cause tics.There are two families of DA receptors,called D1-like receptor(DRD1) and D2-like receptor (DRD2).Over activity of DA system or super sensitivity of receptor will lead to occurrence of tics.DRD2 was regarded had more intimate relationship with TS.The number and the density of DRD2 in striatum of TS were obviously higher than control group.In conclusion that the DA content in striatum increased,the HVA in sera decreased and the DRD2 activity in striatum enhanced.As the inhibitor of DRD2,Haloperidol(Hal) is regarded as the effective medicine in treatment of TS.Although haloperidol is efficacious for the treatment of TS,a very high proportion of patients eventually discontinue the therapy because of the side effects.It’s side effects are concerned with the dosage.The commen side effects include lethargy,sedation,debilitation,dizziness and extrapyramidal symptoms(such as dysmyotonia,akathisia,thrill like Parkinson’s disease,et al). Tardive dyskinesia will occur after take this medicine for a long time.It will appear heteronomous stereotypy behaviors.Such side effects limited the usage of haloperidol.Ningdong granule(NDG),a traditional Chinese Medicine preparation,has been used as an anti-tics agent for years in clinic.NGD has been proved benefit in our clinical study,but the mechanisms and relative effects compared to haloperidol is still unknow.Gastrodin(Gas),extracted from Chinese herb Gastrodia data Blume,has been shown to promote DA transform and have obviously sedative,anticonvulsive and antiepileptic properties.In this study,we induce the TS rats modol by Apomorphine(Apo).We measured the HVA content in the rats sera,the DA content in striatum,the expression of DRD2 protein and mRNA of TS rats modol.We aimed at exploring the possible of NDG and Gas on the DA system of TS rats.ObjectiveTo investigate the possiblem echanism of NDG and Gas in inhibit TS symptoms through observing the changes of content of HVA in sera,the DA content in striatum and detecting the variation of DRD2 mRNA and protein expression.Methods96 male(4 weeks old) wistar rats were randmy divided into 6 groups(n=16):the control group,the model(Apo) group,the(Hal) group,the Ningdong granule+ Haloperidol(NG+Hal) group,the low dosage NDG(NGL) group,the high dosage (NGH) group and the Gas group.TS rat models were induced by intraperitoneal injection(i.p.) with Apo(2mg/kg) in the experimental groups last 4 weeks.Rats in the control group were injected with normal saline(0.9%)(5ml/kg,i.p.).After the injection of Apo(i.p.),rats were treated by intragastric administration(i.g.) with Hal at 1.0 mg/kg(Hal group,equal to 10-folds of clinic dosage);with Hal 1.0mg/(kg·d) +NDG 250mg/(kg·d)(NG+Hal group,equal to 10-folds of clinic dosage);with NDG 250mg/(kg·d)(NGL group,equal to 10-folds of clinic dosage);with NDG 370mg/ (kg·d)(NGH group,equal to 15-folds of clinic dosage);with Gas 20 mg/kg(Gas group);and with normal saline(0.9%) at 10 ml/kg(Control group and Apo group), respectively,once a day at 10am for 12 successive weeks.After the last administration,all rats were fasted with free access to water for 24 h, and anesthetized with intraperitoneal injection of 3%pentobarbital(30 mg/kg) which was followed by other experiments.The detection conten(1) Score the stereotyped behavior after treatmeant of each group;(2) Determination of the content of HVA in sera and DA in striatum by Enzyme linked immunosorbent assay(ELISA);(3) Analysis the DRD2 protein expression by Immunohistochemical method;(4) Analysis the DRD2 mRNA expression by RT-PCR.1.Comparison of stereotyped behaviorApo could induce stereotyped behavior significantly without intervention. According to the polygram of behavior,the rats appeared persistent stereotyped behavior after Apo i.p.4 successive weeks(P＜0.01).After treatment the stereotyped behavior were lower in the Hal group、NG+Hal group and NGH group compared with the Apo group(all P＜0.01).No significant difference was shown in comparison among these three groups(P＞0.05).The scores of Gas group was lower than Apo group conspicuously(P＜0.05).While Gas had the same effect on control stereotyped behavior with Hal(P＞0.05).The behavior in the NGL group was higher than that in the Hal group、NG+Hal group and NGH group(P＜0.01).It was no significant difference of the NGL group compared with the Apo group(P＞0.05).2.Comparison of level of HVA in seraThe content of HVA in sera in Apo group was significant lower than that in the control group(P＜0.01).After treatment the HVA content in the Hal group,NG+Hal group, NGL group and NGH group were increased in different degree compared with the Apo group(all P＜0.01),but no difference was shown in comparison among these four groups(P＞0.05).The HVA content was significantly higher in the Gas group than in the Apo group(P＜0.05).There was no significant difference between the Hal group and Gas group(P＞0.05).3.Comparison of level of DA in striatumThe content of DA in striatum in Apo group was significant higher than that in the control group(P＜0.01).After treatment the DA content in striatum in the Hal group,NG+Hal group,NGL group and NGH group were decreased.The DA content in the NGH group was decreased significantly compared with that in the Apo group(P＜0.01).But there was no significant difference in the Hal group,NG+Hal group and NGL group with the Apo group(P＞0.05),and no difference was shown in comparison among these three groups(P＞0.05).4.Observation of sections of striatum stained with DAB,and comparison of the gray scaleThe level of the gray scale of DRD2 proten in striatum in Apo group was significant higher than that in the control group(P＜0.01).After treatment the gray scale in the Hal group,NG+Hal group,NGL group and NGH group were decreased. The gray scale in the NGH group,and in the Hal group and NG+Hal group was decreased significantly compared with that in the Apo group(P＜0.01,P＜0.05). The gray scale was significantly higher in the NGL group than in the NGH group(P＜0.01),and no difference was shown in comparison among the NGL group and the Apo group(P＞0.05).5.Comparison of the expression of DRD2 mRNA in striatumThe level of the expression of DRD2 mRNA in striatum in Apo group was significant higher than that in the control group(P＜0.01).After treatment the expression of DRD2 mRNA in the Hal group,NG+Hal group,NGL group and NGH group were decreased.The expression in the NG+Hal group,and in the Hal group and NGH group was decreased significantly compared with that in the Apo group(P＜0.01,P＜0.05).The expression was significantly higher in the NGL group than in the other three groups(P＜0.05),and no difference was shown in comparison among the NGL group and the Apo group(P＞0.05).Gas had no effect on DRD2 mRNA expression(p＞0.05).ConclusionIn conclusion,NDG and Gas could improve stereotyped behavior.The mechanism was associated with the accommodation of the activity of DA system by increasing the content of HVA in sera.While NDG could decreasing the content of DA and repressing the expression of DRD2 mRNA and proten in striatum.更多还原