The Molecular Biological Mechanism Study Of Ningdong Granule And Gas On The Tourette's Syndrome

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.