| Objective1. The rats were stereotaxically injected with6-hydroxydopamine (6-OHDA) solution into the left striatum in two-site. The change of Dopamine(DA)and Acetylcholine(Ach) levels were tested in the left striatum of PD rat in different time after operation. The change of the Nrf2nucleoprotein and HO-1protein were observed in the left substantia nigra of midbrain in different time after operation.2. To investigate neuroprotective effect and mechanism of Pabing II Formula on the dopaminergic neurons of the substantia nigra of midbrain in Parkinson’s disease rats. To provide experimental research based on the clinical application of Pabing II Formula.MethodsExperiment1:Male, Sprague-Dawley rats were randomly divided into two groups:sham group and6-OHDA group. Each group was divided into7subgroups randomly:6h,24h,48h,1w,2w,4w,8w after operation. PD rats were induced by6-OHDA twice stereotaxically into the left side of striatum. Sham group were injected with0.02%Ascorbic acid. At different survival times, All animals rotational behavior were detected by recording the number of apomorphine(APO) induced turns. ELISA was used to measure the levels of DA and Ach in the left striatum. HE staining was used to observe the pathomorphological changes of the left substantia nigra of midbrain.Experiment2:Apply with the rat left substantia nigra tissue which were saved in Experiment1. The Nrf2nucleoprotein protein and HO-1protein levels were detected by western blot at different survival times.Experiment3:Method of making model with experiment1. Male, Sprague-Dawley rats were randomly divided into three groups:normal group, sham group and6-OHDA group. The successful models of PD were randomly divided into four groups:model group, Pabing Ⅱ Formula low dose group(8. Og·kg-1), Pabing II Formula medium dose group(16.0g·kg-1) and Pabing Ⅱ Formula high dose group(32.0g·kg-1). The treatment lasted for4weeks. All animals rotational behavior were detected by recording the velocity of apomorphine (APO) induced turns. ELISA was used to measure the level of DA and Ach levels in the left striatum. The Nrf2nucleoprotein and HO-1protein levels of the left substantia nigra tissue were detected by western blot, and the mRNA levels of Nrf2and HO-1were determined with RT-PCR.Experiment4:Method of making model with Experiment1. Male, Sprague-Dawley rats were randomly divided into three groups:normal group, sham group and6-OHDA group. The successful models of PD were randomly divided into two groups:model group, Pabing Ⅱ Formula therapy group(32.0g·kg-1). The treatment lasted for4weeks. To observe mobile latent period by grid experiment. the pathomorphological changes of left subatantia nigra were observed by Nissl staining. Nrf2and HO-1expression were detected by immunohi stochemi stry.ResultsExperiment1:1. Rotational behavior:The rotation rate induced by APO had no significant differences at different points in sham group(P>0.05). The rotation rate had no significant different between6h and24h after6-OHDA injection (P>0.05), Rotation rate in6-OHDA group increased significantly in a time-dependent manner compared with sham group(P<0.05), it reached the highest point at4w, There were no significant differences in rotation rate between4w and8w(P>0.05).2. DA and Ach level:At each time point of sham group, The DA and Ach levels were no significant difference(P>0.05). The DA levels in6-OHDA group were decreased at6h, Compared with sham group at6h, the difference were statistically significant(P<0.05), and decreased gradually in the following time, but there were no difference in DA levels between4w and8w(P>0.05). Compared with sham group, The Ach levels of6h and24h were no significant differences(P>0.05). and increased gradually in the following time, but there were no significant differences in Ach levels between4w and8w(P>0.05).3.HE staining:Under light microscope, The sham group in the left substantia nigra dopaminergic neurons morphology in the intact, cells were arranged relatively orderly;the nucleus was round or cone-shaped, large and clear;nerve fibers are arranged in rows, with compact structure. A small amount of neurons mild swelling at6h after6-OHDA injection. A large number of dopaminergic neurons swelling at24h,48h and1W. The nuclei are pale and vesicular, Some dopaminergic neurons is surrounded by4-6oligodendrocytes forming satellite phenomenon. Swelling of dopaminergic neurons decreased and nucleolus offset or rupture in2w. The number of neurons were reduced with the time. Many nuclei have become pyknotic and karyolysis. An example of neuronophagia in which a neuron is surrounded by microglial cells.Experiment2:The western blot shows that the levels of Nrf2nucleoprotein and HO-1protein are low expression in sham group, different time have no significant changes (P>0.05). The levels of Nrf2nucleoprotein increased from6h, and the highest level at1w, and had begun to decrease again by2w in6-OHDA group. The levels of HO-1protein increased from24h, reached the peak at1w, and had begun to decrease again by2w in6-OHDA group. There were statistically significant between the sham group and each6-OHDA group(P<0.01).Experiment3:1. Rotational behavior:Rotation rate in6-OHDA group increased significant differences compared with normal group and sham group(P<0.05). There were no significant differences in rotation rate in model group and Pabing Ⅱ formula low dose group before and after treatment (P>0.05), Rotation rate in Pabing Ⅱ formula medium dose group and high group decreased significantly compared with before treatment (P<0.05). Rotation rate in Pabing II formula high dose group decreased significantly compared with Pabing II formula medium group.2. There is no difference in Nrf2nucleoprotein and HO-1protein in left substantia nigra between normal group and sham group (P>0.05). Compared with normal group, the Nrf2nucleoprotein and HO-lprotein in left substantia nigra tissue were significantly increased(P<0.01), and the mRNA levels of Nrf2and HO-1were increased in model group(P<0.05); Compared with model group, the Nrf2nucleoprotein and HO-1protein in left substantia nigra tissue were significantly increased (P<0.01), and the mRNA levels of both Nrf2and HO-lwere also upregulated in Pabing Ⅱ formula low dose group, media dose group and high dose group(P<0.05), Pabing Ⅱ formula high dose group were significantly differences compared with the low dose group and media dose group(P<0.01).3.DA and Ach level:Compared with normal group, The DA and Ach levels in sham group had no statistical difference (P>0.05), The DA levels of model group dropped significantly(P<0.05), The Ach levels of model group increased significantly (P<0.05). There is no marked difference in DA levels between model group and Pabing Ⅱ formula low dose group(P>0.05), The DA levels of medium and high dose group increased significantly(P<0.05), The Ach levels of low, medium and high dose group dropped significantly (P<0.05). Compared with low and medium dose group, the Ach levels of high dose group dropped significantly(P<0.05).4.The T-AOC content in the left striatum was no significant difference between sham group and normal group (P>0.05), Compared with the normal control group, there were significant differences in model group, Pabing Ⅱ formula low, medium and high group (P<0.05). Compared with the model group, Pabing Ⅱ formula in each treatment group can obviously increase the T-AOC content (P<0.05), and the effect of high dose group was obviously.Experiment4:1. Body weight Measurement:After4weeks treatment, No differences in body weight were observed between normal group and sham group (P>0.05). Compared with normal group, weight of model group and Pabing Ⅱ Formula therapy group decreased significantly(P<0.05). Compared with model group, weight of Pabing Ⅱ Formula therapy group increased significantly(P<0.05).2.Grid experiment:Compared with normal control group, The sham group mobile latent period was not statistical differnce(P>0.05); The model group and Pabing Ⅱ Formula therapy group mobile latent period were longer than the normal group, the difference was significant (P<0.05);After treatment, the PD rats in Pabing Ⅱ Formula therapy group mobile latent period were lower than model group, difference was significant(P<0.05).3. The normal group and sham group in the Substantia nigra DA neurons were structural forms, and for large and medium-sized cone cells. Cytoplasm is blue, the nissl body is purple blue color uniform distributed around the nucleus. Model group in the Substantia nigra DA neurons take seriously reduce the number of neurons, the nucleus were pycnosis or offset, Complete neuron number decreased significantly. 4. The total TH positive neurons of the substantia nigra of the left side in normal group and sham group had no statistical difference (P>0.05). The total TH positive neurons of the substantia nigra of left side in model group and Pabing II Formula therapy group significantly decrease compared with normal control group (P<0.01), Compared with model group, Pabing Ⅱ Formula therapy group was obviously increased(P<0.05).5. Immunohistochemistry experiments showed that the Nrf2protein of normal control group and sham group were expressed only in cytoplasm, in which there were no positive brown staining in the nucleus. The value of IOD of Nrf2protein in cytoplasm was no difference between normal group and sham group(P>0.05). The Nrf2protein of model group was expressed in cytoplasion and nucelus Pabing II Formula therapy group was expressed in cytoplasion and nucelus, while the expression of nucelus Nrf2obviously, Compared with model group, the value of IOD of Nrf2positive expression increased obviously (P<0.05). The low expression of HO-1protein was in sham group. Compared with model group, the value of IOD of HO-1expression in sham group had no evident difference (P>0.05). The value of IOD of HO-1expression of model group increased compared with normal group (P<0.05). The value of IOD of HO-1expression of Pabing Ⅱ Formula therapy group increased compared with model group (P<0.05).Conclusion1. After6-OHDA injection, the level of DA decreased and Ach increased in the left striatum of PD rats.2. After6-OHDA injection, the Nrf2nucleoprotein is activated. Pabing Ⅱ formula can enhance antioxidation ability. The underlying mechanism might be associated with promoting the Nrf2nucleoprotein and Nrf2mRNA, then advancing the expression of antioxidant gene in the downstream such as HO-1protein and HO-1mRNA. The effects were more obvious in Pabing Ⅱ formula high dose group.3. Pabing Ⅱ Formula’s neuroprotective effect may attenuate the6-OHDA induced oxidative stress through upregulation of Nrf2and HO-1and regulation of DA and Ach levels. |
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