| Parkinson's disease (PD) is a chronic progressive neurodegenerative movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Clinical manifestations of this complex disease include motor impairments involving resting tremor, muscular rigidity, bradykinesia, postural instability. Studies indicated that mitochondrial dysfunction is one of the main reason for dopaminergic neuronal degeneration and losing. A growing number of studies have shown that structure and function abnormalities of complex I are closely related to mitochondrial dysfunction.Despite extensive research, the disease remains poorly understood and there is still no effective treatment.The current medications are efficient in the early stage of the disease but with the continuous use of the pro-dopaminergic medication may become less effective and cause motor complications. Growing attention has been paid on traditional Chinese medicine because of its unique theoretical system, overall control methods, as well as the exact efficacy for the prevention and treatment of PD.According to the understanding of TCM for the etiology and pathogenesis of Parkinson's disease (Vibration Syndrome), the main therapeutic principles are to relieve spasms by expelling wind and resolving phlegm, subdue yang by nourishing liver and kidney. Based on this principle, the Qianzheng Powder and Dabuyin Pill are classic formula in clinic.To elucidate the effect and mechanism of Qianzheng Powder and Dabuyin Pill, we observed the changes of dopamine neurons in SNpc, the activity of mitochondrial complex I and the expression of its related mitochondrial gene in MPTP-induced PD mice.This study is consisted of two parts:(1) To investigate the neuroprotective effect of Dabuyin pill, Qianzheng powder and combined fomular on dopaminergic neurons.we observed the change of PD mice's behavior, dopaminergic neurons, dopamine in the striatum and its metabolites. (2) To explore the curative mechanism of traditional Chinese medicine to PD mice, we observed the influence of Qianzheng Powder and Dabuyin Pill on the activity of mitochondrial complex I and the expression of complex ?-related mitochondrial gene and protein in PD mice.In experimental research,male C57BL/6J mice were injected intraperitoneally with 0.3%MPTP,20mg/kg or 30mg/kg daily and continuous injecting for 5 days, to induce Parkinson's disease model.The experimental animals were randomly divided to five groups:normal control group, model control group, Qianzheng powder group, Dabuyin pi 11 group and the combined formula group. Mice were killed on the 56th day after first injecting with MPTP.The main experimental results are as follows:1. Effects of Qianzheng powder?Dabuyin pill and combined fomular on the behavior of PD mice.Mice showed movement disorder and postural instability after intraperitoneal injection of MPTP. Assay of pole gasping of each groups had no significant difference.2. Effects of Qianzheng powder?Dabuyin pill and combined fomular on pathology of the substantia nigra of PD miceThe results indicated that the number of neurons of the substantia nigra decreased and many forms slender, dyeing deep neurons appeared in model group as compared with normal group. Compared with model group, the number of neurons of the substantia nigra inceased and forms slender, dyeing deep neurons decreased in three Chinese medicine treatment group.3. Effects of Qianzheng powder?Dabuyin pill and combined fomular on the number of TH dopaminergic neurons of PD miceIn the experimental studying, ABC immunohistochemistry technique is used and the TH dopaminergic neurons were counted in every group. The number of TH neurons in SNpc decreased significantly in model group (p<0.01); The number of TH neurons significantly increased in three treatment groups as compared with model group (p<0.05)4. Effects of Qianzheng powder?Dabuyin pill and combined fomular on ultrastructure of brain mitochondria of PD miceThe results showed that some neurons of the substantia nigra creased, nuclei shrinked and mitochondria disorder or disappear in model group.The ul trastructure of neurons showed that nucleus membrane structure is clear and mitochondria attenuated injury in three treatment groups as compared with model group.5. Effects of Qianzheng powder?Dabuyin pill and combined fomular on the levels of monoaminergic neurotransmitters in PD mice brainThe level of HVA?DOPAC and DA in model groups is obviously lower than normal group (p<0.05). Compared with model group, DA increased significantly in combined fomular group (p<0.05).The level of HVA and DOPAC in three treatment groups corresponded with that in model guoup. DA increased significantly in combined fomular group as compared with Dabuyin pill group (p<0.05)6. Effects of Qianzheng powder?Dabuyin pill and combined on the activities of brain mitochondrial complexs of PD mice.Compared to the normal group, the activities of mitochondrial complex ?, III and IV of model group has been significantly decreased (p<0.05); compared with model group, Dabuyin Pill treatment showed significantly reversion of MPTP-reduced complex ?,? activities (p<0.05), the combine formula reversed complex ????? activities (p<0.05),7. Effects of Qianzheng powder?Dabuyin pill and combined fomular on MPTP-induced damaged mtDNA of PD mice.Compared with normal group, the damage parameter of brain mtDNA increased significantly in model group(p<0.01); Compared with model group, mtDNA damage parameter reduced significantly in Dabuyin pill group and combined fomular group (p<0.05).8. Effects of Qianzheng powder?Dabuyin pill and combined fomular on the expressions of brain ND1?ND4 and ND6mRNA in PD miceThe results showed that the expressions of brain ND1 mRNA and ND4mRNA was significantly lower in model group as compared with normal group, (p<0.05)the expression of ND4mRNA no significant change. Compared with model group, the expression of ND1mRNA improved in Dabuyin pill group and combined fomular group and the expression of ND4mRNA improved in the three treatment groups (p<0.05). There were no significant differences in the expression of ND6mRNA bewtween the three treatment groups and model group.9. Effects of Qianzheng powder?Dabuyin pill and combined fomular on the expressions of mitochondrial complex I protein subunitNDl, ND4 and ND6 in PD miceAlthough no statistically significant, results from Western blot analysis showed that the expression of ND1 declined in model group as compared with normal group. There were no significant differences in the expression of ND1 bewtween the three treatment groups and model group. Since the protein content was lower and quality problems of the antibody, detected Bands of ND1 and ND6 did not reach statistical number after repeated experiments.Conclusions:?Dabuyin pill, Qianzheng powder and combined fomular could have a neuroprotective effect on dopaminergic neurons in substania nigra. ?Chinese herbal medicine Dabuyin pill, Qianzheng powder could protect dopaminergic neurons and maintain the mi tochondrial function by regulating mitochondrialgene. ?Chinese herbal medicine Dabuyin pill, Qianzheng powder and combined fomular couldn't affect the expression of protein subunits of complex I. |
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