| Tourette Syndrome(TS)is a developmental neuropsychiatric disorder characterized by multiple,brief,stereotypical but nonrhythmic movements and by vocalizations called tics.The onset of tic symptoms in TS typically occurs during early childhood and adolescence.Till now,the etiology and pathophysiology of TS are not clear.Many epigenetic factors have been implicated in the pathogenesis of TS,including perinatal injuries,exposure to androgens and psychological stress,as well as the imbalance of neurotransmitters.The cortico-striato-thalamo-cortical circuits play a critical role in coordinating motor and cognitive processes,and dysfunction of the basal ganglia has long been suggested to be the major cause of tic symptoms,but it can't elaborate the mechanism of TS and its comorbidities wholly.Based on the close relation between the dopamine and the amino acid neurotransmitters,and the importance of balance between excitatory amino acid and inhibitory amino acid to keep the normal function of the brain,more and more scholars begin to pay more attention to the amino acid and its function in the pathophysiology of TS.Based on the children's physiological character of "excessive of the liver and insufficient of the spleen",my tutor professor wang hypothesized that deficiency of the spleen could cause spleen phlegm,and excessive energy of the liver could cause liver wind.The liver wind agitated and spleen phlegm obstructed the channels.According to this hypothesis,the principle of treatment was strengthening the earth(spleen)and suppressing the wood(liver).Under this guide,we created a recipe named Jian-pi-zhi-dong decoction(JPZDD).In clinic,JPZDD had good curative effect,but the target of the formula was unclear.In order to further explore the neurochemical mechanism of JPZDD,this subject aimed to induce TS model by iminodipropionitrile(IDPN),based on the techniques such as the animal ethology,high performance liquid chromatography(HPLC),immunohistochemistry(IHC),Real-time PCR(RT-PCR),Western Blot(WB)and vivo microdialysis.We studied the influences of the formula on the stereotyped behavior of TS mice(rat)model,and detected the contents of the amino acid transmitters,and the expression of type A receptor of GABA(GABAAR)in the striatum.This study was divided into four parts:The first part,the behavioral change was observed before and after the treatment.At the same time,the contents of Glu and GABA in the striatum of mice were detected by HPLC.The second part,the expression of GABAAR in the striatum of mice was detected by IHC and RT-PCR.The third part,the contents of Glu and GABA in the striatum of rats were detected by microdialysis-HPLC.The fourth part,the expression of GABAAR in the striatum of rats was detected by WB and RT-PCR.Experiment 1 The effect of JPZDD on the stereotyped behavior of TS mice model and the influence on the amnio acid neurotransmitters in the striatumObjective To study the influences of JPZDD on bilateral striatal amino acid neurotransmitters in mice with TS.Methods Thirty mice were injected with IDPN,350mg·kg-1·d-1 for 7 consecutive days except the ten mice of the saline group which were injected with saline.TS mice were randomly divided into three groups:IDPN group?IDPN+JPZDD group(the Chinese herbal group)?IDPN+Tiapride group(the western medicine group).The IDPN group and the saline group were gavaged with saline(0.9%)at 20 ml·kg-1·d-1,the Tiapride group with Tiapride at 50mg·kg-1·-1,and JPZDD group with JPZDD at 20g·kg-1·d-1,respectively for six weeks.Then behavioral recordings were conducted,and the contents of Glu and GABA in the bilateral striatum of the mice were detected by HPLC.Results TS mice model induced by IDPN showed abnormal stereotypy in different degrees.Remarkably,severity of stereotypy in either IDPN+Tia or IDPN+JPZDD group decreased significantly before and after the treatment(P<0.05).There is no statistical significance between the TS group and the saline group(P>0.05).The contents of Glu and GABA decreased from high to low as follows,IDPN group,IDPN+Tia group,IDPN+JPZDD group and the saline group.Compared with the blank contorl group,the contents of Glu and GABA in IDPN group,the IDPN+Tia group and the IDPN+JPZDD group were higher(P<0.05).The content of Glu in IDPN+JPZDD group was lower than the IDPN group(P<0.05).The content of GABA was also lower,but this difference did not reach statistical significance(P>0.05).There was no statistical significance between the IDPN+Tia group and IDPN+JPZDD group.Compared to the saline group,the ratio of Glu and GABA decreased in IDPN group,IDPN+Tia group and IDPN+JPZZD group,but there was no statistical significance between these groups(P>0.05).Conclusion JPZDD can regulate the contents of Glu and GABA in the striatum,and keep the dynamic equilibrium,which might be its pharmacodynamic mechanisms for treating TS.Experiment 2 The effect of JPZDD on the expression of GABAAR alpha4 and betal subunits in the striatum of TS mice modelObjective To explore the positive effects of JPZDD on TS by investigating the expression of GABAAR in the striatum of a TS mice model.Methods Model made and the administering medication were the same with experiment 1.Results The results of IHC showed the expression of GABAAR alpha4 subunit in the IDPN group was higher than the saline group(P<0.01).There was statistical difference between the IDPN+JPZDD group and the saline group(P<0.05).The expression of GABAAR betal subunit in the IDPN group was higher than the saline group,the IDPN+Tia group and the IDPN+JPZDD group(P<0.01).The results of RT-PCR showed the expression of GABAAR alpha4 and betal subunits in the striatum was the highest in the IDPN group,but there was no statistical significance between these groups.Conclusion The mechanism by which JPZDD alleviates TS symptoms may be associated with GABAAR expression downregulation in striatum which may regulate GABA metabolism.Experiment 3 The effect of JPZDD on the stereotyped behavior of TS rat model and the influence on the amino acid neurotransmitters in the striatumObjective To observe the effects of JPZDD on the behavioral changes and the amino acid in striatum of TS rat models induced by IDPN.Methods 48 male SD rats were randomly divided into four groups,the saline group,the IDPN group,the IDPN+Tia group and the IDPN+JPZDD group,12 in each group.TS models were injected with IDPN,250mg·kg-1·d-1 for 7 consecutive days.The IDPN group and the saline group were gavaged with saline(0.9%)at 10 ml·kg-1·d-1,the IDPN+Tia group with Tia at 21mg·kg-1·d-1,and JPZDD group with JPZDD at 16g·kg-1·d-1 respectively.Behavioral tests were conducted every 1 week during the whole 4-week administration.At last,microdialysis probes were unilaterally inserted into the left striatum of the rats,the contents of Glu and GABA in the dialysis samples were determined by HPLC.Results The average score of stereotyped behavior and the distance of locomotor activities in IDPN+JPZDD group declined significantly compared to the IDPN group(P<0.05).The content of Glu in IDPN group was the highest at each time point,while the saline group was the lowest.Five time points(15min,105min,135min,165min,195min)in IDPN+Tia group,four in IDPN+JPZDD group(15min,105min,135min,165min)were significantly lowered compared with the IDPN group(P<0.05,P<0.01).The content of GABA in IDPN group was higher than the saline group at the ninth time point(135min)(P<0.05).There was no statistical significance between other groups(P>0.05).When comparing the ratio of Glu/GABA,there was no statistical significance between these groups.Conclusion JPZDD can significantly inhibit the stereotyped behavior and the locomotor activity of TS rats,and the anti-effect of it may be made through keeping the dynamic equilibrium of the Glu and GABA in the striatum.Experiment 4 The effect of JPZDD on the expression of GABAAR alpha4 subunit in the striatum of TS rats modelObjective To explore the positive effects of JPZDD on TS by investigating the expression of GABAAR in the striatum of a TS rat model.Methods Model made and the medication were the same with experiment 3.Results The results of WB showed the expression of GABAAR alpha4 subunit in IDPN group increased significantly compared to the saline group(P<0.01).The expression in IDPN+Tia group and IDPN+JPZDD group decreased after the treatment compared to the IDPN group(P<0.01),but there was no statistical significance between IDPN+Tia group and IDPN+JPZDD group(P>0.05).The results of RT-PCR showed the expression in IDPN+Tia group and IDPN+JPZDD group decreased after the treatment compared to the IDPN group,but there was no statistical significance(P>0.05).Conclusion The mechanism by which JPZDD alleviates TS symptoms may be associated with downregulating GABAAR expression in striatum which may regulate GABA metabolism.Summary:Based on the physiological character of the children,we think that the location of TS is spleen and liver.The key pathogenesis is excessive energy of the liver and deficiency of the spleen,imbalnce of Yin and Yang.The treating pricinple is strengthening the earth(spleen)and suppressing the wood(liver),and keeping the balance of Yin and Yang.JPZDD is an effective formula for treating TS model induced by IDPN.JPZDD might effectively inhibit stereotyped actions and control TS symptoms by downregulating the GABAAR expression,which may directly decrease the excessive generation of GABA in the striatum so as to keep the balance of Glu and GABA at a new high level. |
PDF Full Text Request