The Neuroprotective Effects Of Glutathione Combined With Tetrandrine In PD Rats

1.The Effects of Glutathione Combined with Tetrandrine on the biological behavior in Rats of Parkinson's DiseaseObjective:To establish the rat models of Parkinson's Disease(PD) and observe the effects of Glutathione combined with Tetrandrine on the biological behavior in those rats. Methods : The PD rat model was established by stereotactic microinjection of 6-hydroxydopamine.Four weeks later, a rotational test induced by apomorphine was performed to determine the successful rate of models.Then ,the successful models were divided into different groups randomly by peritoneal injection with different candidates .The groups were as following: no treatment group(saline,10 mg·kg-1·d-1),GSH group(GSH300 mg·kg-1·d-1),L-dopa group(L-dopa 25mg·kg-1·d-1), GSH+L-dopa+Tet group(GSH300 mg·kg-1·d-1, L-dopa 25mg·kg-1·d-1 ,Tet 15 mg·kg-1·d-1), GSH+Tet group(GSH300 mg·kg-1·d-1,Tet 15 mg·kg-1·d-1),and the non-PD control group(saline10 mg·kg-1·d-1),each group contains 14 rats. The biological behavior was observed in each rat again after 50 days of treatment.Results:Four weeks later, 73.6% of the rats were successfully established PD induced by apomorphine .After 50 days's treatment, there were no difference on the rotation times in the rats from no treatment, GSH and non-PD groups, compared with those before the treatments. However, the rotation times were significantly decreased in the groups treated with L-dopa (P<0.05), GSH+Tet (P<0.01) and GSH+L-dopa+Tet (P<0.01), especially the GSH+Tet group and GSH+L-dopa+Tet group,because the time of starting rotate of the two groups was longer than other groups and some rats of GSH+L-dopa+Tet group didn't rotate at all.2.The Effects of Glutathione Combined with Tetrandrine on the Antioxidation in PD RatsObjective:To observe the effects of Glutathione combined with Tetrandrine on the antioxidation in PD rats.Methods: Six rats of every group were randomly selected out from the above mentioned each group. Then after anesthetized ,the nigra and striatum of left side from each rat were separate rapidly in iced bath and homogenized and centrifuged. Supernatant liquid were obtained and the the activity of GSH in nigra and the contents of MDA, ROS,MAO-B in striatum were assayed with kits.Results: (1)The levels of MDA, ROS and MAO-B in striatum from GSH+Tet group were significantly decreased compared with other groups except for the non-PD control group. The levels of MDA, ROS and MAO-B in striatum of L-dopa group were significantly increased compared with the no treatment group.(2)The GSH level in nigra of GSH+Tet group was significantly increased compared with other treatment groups except for the non-PD control group.3. The Effects of Glutathione Combined with Tetrandrine on the THmRNA and the dopaminergic neuron in PD RatsObjective:To observe the effects of Glutathione combined with Tetrandrine on the THmRNA and the dopaminergic neuron in PD ratsMethods: (1)Six rats of every group were randomly selected out.After anesthetize, the nigra of left side of each rat was separate rapidly in iced bath and the THmRNA of nigra was detected by RT-PCR. (2) Two rats from each group were randomly selected out and were anesthetized .After perfuse fixation via left ventricle,the rats were decapitated and the brains were taken out.The mesocephalic coronal frozen sections were made for immunohistochemical stain of tyrosine hydroxylase in dopaminergic neuron . The morphology and the number of the tyrosine hydroxylase positive neurons were examined. Results: (1)THmRNA expression in GSH+Tet group and GSH+L-dopa+Tet group were higher than other groups (P<0.05) , and the expression in GSH+Tet group was the highest. ( 2 ) The number of tyrosine hydroxylase positive neurone in GSH group, GSH+L-dopa+Tet group, GSH+Tet group was higher than no treatment group and L-dopa group, and augmented extent of GSH+Tet group was most obviously.4 The Effects of Glutathione Combined with Tetrandrine on Excitatory Amino- acids in PD RatsObjective:To observe the Effects of Glutathione combined with Tetrandrine on glumatic acid,aspartate in PD rats.Methods: Six rats from each group were randomly selected out and were anesthetized.Then the striatum of left side of each group were separate rapidly in iced bath and were homogenized with saline .After 1.5% perchloric acid were added in, the homogenate were centrifuged at 18000r/min for 10 minutes,then the contents of glumatic acid and aspartate were detected by high performance liquid chromatogram- fluorescence (HPLC-FS).Results: Compare with no treatment group, aspartate from GSH group was obviously decreased and glutamic acid was also decreaded with no statistical difference. Compare with GSH group, glutamic acid in GSH+Tet group was considerably decreased, but aspartate was again decreaded with no statistical difference.. In contrast, glutamic acid and aspartate in L-dopa group were significantly increased.Conclusion1,The rat model of unilateral Parkinson's disease was established by stereotactic microinjection of 6-hydroxydopamine , 6-hydroxydopamine was injected into substantia nigra pars compacta and ventral tegmental area. It was a well-established model with high successful rate. The peritoneal injection with GSH +L-dopa+Tet,GSH+Tet and L-dopa could improve the biological behavior in PD model rats. The most significant result was seen in GSH +L-dopa+Tet group.2, Both GSH+Tet and GSH +L-dopa+Tet could significantly enhance the antioxidation in PD model rats but the former treatment showed better enhancement. 3,The expression of THmRNA obviously was increased in GSH+Tet group and GSH +L-dopa+Tet groups, In addition, THmRNA level in GSH+Tet group was higher than that in GSH +L-dopa+Tet group. The treatment of GSH +L-dopa+Tet,GSH+Tet or L-dopa could relieve the damage of dopaminergic neuron. Among them , GSH+Tet showed the best result.4, GSH combined with Tet could lower glumatic acid level in PD rats , and consequently decrease the toxic effect of excitatory amino acids.