| 1.The Effects of Glutathione Combined with Tetrandrine on the biological behavior and DA,HVA,HVA/DA and MAO-B of PD RatsObjective:To establish the rat models of Parkinson's Disease(PD)and observe the effects of Glutathione combined with Tetrandrine on the biological behavior and DA,HVA,HVA/DA and MAO-B of PD model ratsMethods: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 drugs.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 normal 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.Six rats of every group were randomly selected out.After anesthetize,the striatum of left side of each rat was separate rapidly in iced bath and the contents of MAO-B in striatum were assayed with kits,DA and HVA in striatum were tested with high performance liquid chromatography-electrochemical detection(HPLC-ECD)and DA turnover rate(HVA/DA)was calculated.Results:(1)Four weeks later,73.6%of the rats were successfully established PD models induced by apomorphine.After 50 days' treatment,there was no difference on the rotation times in the rats from no treatment,normal group and GSH group compared with those before treatment.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 levels of DA,HVA in GSH group,GSH+Tet group,GSH+L-dopa+Tet group and L-dopa group were higher than normal group,and there was statistical difference between GSH+Tet group and GSH group,between GSH+L-dopa+Tet group and L-dopa group(P＜0.05);The level of MAO-B in GSH+Tet group was significantly decreased compared with other groups and there was statistical difference with GSH group and L-dopa group(P＜0.05);The HVA/DA in GSH+Tet group is the lowest among all groups.2.The Effects of Glutathione Combined with Tetrandrine on substantia nigra mitochondrial complex I and oxidative stress in PD RatsObjective:To observe the effects of Glutathione combined with Tetrandrine on substantia nigra mitochondrial complex I and striatum oxidative stress in PD RatsMethods: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 activity of Complex I,GSH in nigra and the contents of MDA,ROS in striatum were assayed with kits.Results:(1)The levels of GSH,complex I in nigra of GSH+Tet group were significantly increased compared with other treatment groups except for normal group,and there is statistical difference compared with GSH group(P＜0.05);The levels of GSH, complex I in nigra of GSH+Tet +L-dopa group were significantly increased compared with L-dopa group(P＜0.05).(2)The levels of MDA and ROS in striatum from GSH+Tet group were significantly decreased compared with other groups except for normal group, and there is statistical difference compared with GSH group(P＜0.05);The levels of MDA and ROS in striatum of GSH+Tet+L-dopa group were significantly decreased compared with L-dopa group(P＜0.05).3.The Effects of Glutathione Combined with Tetrandrine on dopaminergic neuron apoptosis of PD RatsObjective:To observe the effects of Glutathione combined with Tetrandrine on dopaminergic neuron apoptosis of PD RatsMethods: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 immuno histochemical stain of tyrosine hydroxylase in dopaminergic neuron and did poaching, postfix,dehydration,embedding and made ultra-thin section.At last,The morphology of dopaminergic neuron cell apoptosis was observed by electron microscope.Results:(1)There was intermediate and advanced stage morphology of L-dopa group dopaminergic neuron apoptosis.(2)There were earlier and intermediate stage dopaminergic neuron apoptosis morphology of GSH group,GSH+Tet group and GSH+L-dopa+Tet group;and there were obviously earlier stage morphology of dopaminergic neuron apoptosis in GSH+Tet group and GSH+L-dopa+Tet group.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 L-dopa,GSH+Tet and GSH +L-dopa+Tet could improve the biological behavior and increase DA and HVA in PD model rats and GSH+L-dopa+Tet group was the highest.The rotational behavior of post-treament PD model had negative correlation with the levels of DA in PD rats striatum.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;Both GSH+Tet and GSH+L-dopa+Tet could play protective effect on mitochondrial complex I,and the former better.3.GSH combined with Tet could delay dopaminergic neuron apoptosis in PD rats,and play protective effect on dopaminergic neuron;L-dopa could possibly accelerate dopaminergic neuron apoptosis. |
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