| Background:Based on the close relationship between the dopaminc(DA) system and MT and the discovery in neuroanatomical and neural biochemistry research in recent years, the dysfunction of DA system was considered to the main pathology foundation of MT. But the pathophysiology at molecular or cellar level of MT was not well understood.Objcctive:To study the DA system's pathophysiology at molecular level of MT and to evaluate whether Jing'an oral liquid could effect on the DA concentration, metabolism and transporting of DA and the expression of postsynaptic receptors and signal transduction molecule or not.Materials and Methods:The Chinese medicine and modern medicine's research progress on MT was summarized. MT rat model was established by DOI[1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane].Forty SD male rats were randomly allocated to:normal control group (n=10),MT model group(n=10),Jing'an group(treated by Jing'an oral liquid,n=10), and Tiapride group(treated by Tiapride, n=10). Sixty MT children patients were randomly assigned to Jing'an group and Tiapride group,30cases in each group. Another30healthy children were recruited as the health group. The DA and homovanillic acid (HVA) concentration in plasma of60children patients (compared with30healthy children) before and after treatment and4groups of rat were all measured using High performance liquid chromatography (HPLC). At the same time, DA and HVA concentration in brain tissue of rats were all tested by HPLC.The mRNA expression of catechol-O-mcthyltransferase (COMT) and the distribution in brain of dopamine transporter (DAT) in the rat's brain tissue were tested by Reverse transcriptase-polymerasc chain creation(RT-PCR) and Radioimmunoassay respectively, which were the important matters of the transporting and degradation processes of DA in the synaptic cleft.The mRNA and protein expression of dopamine receptor D1and D2in the brain tissue were observed using RT-PCR and Laser co focal microscopy(LSCM) simultaneously.The total protein and phosphorylation expression of DARPP-32and Phosphor-Thr-34-DARPP-32in the striatum were detected respectively by Immunohistochemitry, Western blot.Result:By HPLC analysis,the HVA concentration in plasma of patients were higher in the Jing' an group (21.2±11.41ng/ml) and Tiapride group (19.94±9.29ng/ml) than in the health group(16.22±4.95ng/ml) before treatment(P<0.05).And the DA and HVA concentrations in brain tissue were much higher in the MT model group(1108.98±241.18ng/g,96.29±23.88ng/g)than in the normal control group (547.66±207.84ng/g,65.53±17.93ng/g)(p<0.01).Compared with the same group before treatment, the HVA concentrations were significantly decreased in the Jing'an group (13.65±10.81ng/ml) and the DA concentrations were significantly decreased in the Tiapride group (12.33±11.12ng/ml)(P<0.05). Compared with the MT model group, the DA and HVA concentrations were all significantly decreased in the Jing'an group (669.67±167.59ng/g,77.92±8.76ng/g) and Tiapride group (759.17±144.42ng/g,68.99±11.42ng/g)(P<0.05).By Radioimmunoassay analysis, the DAT radiation uptaking ratio in the striatum, cerebral cortex and hippocampus were higher in the MT model group (5.43±1.39,4.61±1.58,4.06±1.34) than in the normal control group (3.03±0.83,2.49±0.72,1.98±0.75)(P<0.05), and all lower in the Jing'an group (3.16±1.28,2.58±1.44,2.42±1.08) and Tiapride group (3.21±0.97,2.40±0.97,2.18±1.30) than in the MT model group (P<0.05)By RT-PCR analysis, the mRNA expressing of COMT in the striatum were lower in the MT model group (0.22±0.17) than in the normal control group (0.62±0.25)(P<0.05).The effect of Jing'an oral liquid on COMTmRNA wasn't observed.By RT-PCR and LSCM analysis, the mRNA expressing and fluorescence intensity of DRD1in the prefrontal cortex were higher in the MT model group (0.22±0.07,132.96±17.85) and Tiapride group (0.05±0.03) than in the normal control group (0.13±0.05,94.49±10.61)(P<0.05),and lower in the Jing'an group (0.11±0.06,93.12±8.61) and Tiapride group (0.05±0.03,91.2±9.58) than in the MT model group (P<0.05)By Immunohistochemitry analysis, the DARPP-32total protein Number objects and Integrated optical density(IOD) in the striatum were higher in the MT model group (490.47±186.77,52.62±25.22) than in the normal control group (325.73±137.57,24.36±10.69)(P<0.05),and IOD was lower in the Jing'an group(374.33±60.79)and Tiapride group(24.76±10.65) than in the MT model group(P<0.05),.Compared with the Jing'an group (374.33±160.79),the Number objects in the Tiapride group (248.00±194.09) were much lower(P<0.05).By Western blot analysis, the DARPP-32total protein expression were higher in the MT model group (1.44±0.24) than in the normal control group (1.00±0.32)(P<0.05), and the Phosphor-Thr-34-DARPP-32expressing weren't significantly change in each group (P>0.05) Analysis of total protein revealed a lower expression of DARPP-32in the Tiapride group (1.06±0.29) than in the MT model group(P<0.05).Conclusion:Excessive activity in DA(including the higher concentration in DA and HVA, more distribution in DAT and higher expression in DRD1)and higher expression in DARPP-32total protein which mediates the neurotransmitters and electrical activity as a signaling molecule could participate in the pathophysiology of MT. The speculative molecular mechanism of MT was embodied specifically as follows:After DA corpuscle releasing from the presynaptic DA vesicle, the high expression in DAT prompted the absorption of DA., so the DA cumulated more and more. The next, ultra sensitive receptors of postsynaptic DRD1combined strongly with DA, which could overact the response of DA effectors cell. In the meanwhile,the higher expression of total DARPP-32protein(caused by the phosphorylation of other sites but Thr34) may improve the DA transmission efficiency of the synaptic,which may activate the DA neuron. Lower expression of COMTmRNA perhaps was involved in the pathophysiology of MT.The function mechanisms of Jing'an oral liquid is following. Jing'an oral liquid could inhibit DA neuronal excitability to reduce the tics of MT model rat by decreasing the concentration and lower the expression of DA, HVA, DRD1mRNA and DARPP-32total protein. In short, Jing'an oral liquid could regulate the mutual influence and mutual adjustment of DA, HVA, DRD1mRNA and DARPP-32total protein, thus maintain the stable state of common DA system for motion regulation and the body's normal movement, which reflects its role in the overall results.Evaluation:Compared with previous study on DA system, the overall research on molecular level from the neurotransmitter concentration, the process of transporting and degradation in presynaptic and synaptic space, and the postsynaptic receptor expression to the protein expression of signal transduction is advanced. The correlation between MT and the COMTmRNA expression, DARPP-32total protein and its phosphorylation expression are reported for the first time in the domestic.All of this is bound to have beneficial enlightenment and guiding role for the basic theory and clinical research of MT,and to be benefit for developing more and better Chinese drugs on treating MT.
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