| BackgroundParkinson's disease(PD)is a neurodegenerative disease characterized by the degeneration and loss of dopaminergic neurons(DA)in the substantia nigra pars compacta.The clinical symptoms of PD are mainly manifested as progressive motor impairments,The prevalence of PD increases with age in China,accounting for about half of the global cases.The disease brings huge economic burden to PD families.In China,the economic burden of PD patients significantly reduces the quality of life of PD patients and their caregivers.The risk of death is about twice that of the whole PD patients,which indicates that PD seriously endangers human health.Therefore,it is urgent to find an ideal method to treat PD.The etiology and pathogenesis of Parkinson's disease are not yet clear,but mitochondrial dysfunction can cause PD,which has been widely recognized.Both environmental and genetic factors can cause mitochondrial dysfunction.The mutation of PINK1 gene and protein functional defect can cause autosomal recessive PD.PINK1 protein is mainly located in mitochondria.A large number of existing studies have shown that PINK1 and downstream Parkin proteins constitute the PINK 1/Parkin pathway,which can regulate mitochondria1 division/fusion,but the specific regulatory mechanism remains to be studied.Currently,there is no effective cure for PD.Chinese medicine based on the overall concept and the theoretical system of syndrome differentiation and treatment can play a therapeutic role against multiple targets of PD,which can significantly improve the motor and non-motor symptoms of PD patients,improve the living quality of PD patients,and has unique advantages with small adverse reactions.Therefore,to seek a more effective method to treat PD,it is of great significance to further explore the mechanism of traditional Chinese medicine and.Our previous studies confirmed that Buyin Qianzheng Formula can protect mitochondrial function in PD mice.Based on the previous researches,this study focus PINK1 as the core,further explored the protect effects of Buyin Qianzheng Formula in PD pathogenesis process on the quality of the mitochondria.ObjectiveIn this study,molecular biology and other techniques were used to investigate the neuroprotective effect and the molecular mechanism of pink]on the quality of dopaminergic neurons and mitochondria in PD.This study further revealed the correlation between the protective effect of traditional Chinese medicine Buyin Qianzheng Formula on the function of mitochondria and the regulation of mitochondrial dynamic balance by PINK 1 of PD dopaminergic neurons and mouse brain,so as to seek the therapeutic targets of traditional Chinese medicine for the prevention and treatment of PD and provide scientific basis for the clinical application of traditional Chinese medicine for the prevention and treatment of PD and the development and application of traditional Chinese medicine.MethodsThis study is divided into two parts:cell experiments and animal experiments.The first part is cell experiments,including experiment 1 to experiment 3.Experiment 1:Construction of SH-SY5Y cell model overexpressed with pink1 gene.Plasmids with pink1 gene overexpressing were constructed,amplified,extracted and sequenced,and then transfected into SH-SY5Y cells.Flow cytometry were used to show the transfection efficiency.qPCR and Western Blot were applied to identify the expression of pink1 gene and PINK1 protein.Experiment 2:The effect of Buyin Qianzheng Formula on mitochondrial morphology and function of PD cell model with pink1 overexpression.CCK-8 method was used to detect the survival rate of cells in each group.After dyeing with MitoTracker(?)Red CMXRos(MTR)probe,laser confocal microscope was used to detect the mitochondria morphology and activity.The level of mitochondrial ATP was detected by luciferase method.After staining with Tetramethylrhodamine methyl ester(TMRM)probe,laser confocal microscope was used to observe and detect mitochondrial membrane potential.Experiment 3:The effect of Buyin Qianzheng Formula on mitochondrial fission and fusion in PD cell model with overexpression of pink1 gene.The expression of PINK1 and Parkin proteins was detected by Western Blot.The co-localizations of Parkin with PINK1 and Parkin with mitochondria(labeled with MTR)were detected by immunofluorescence double-labeling technique.Western Blot analyses were performed to detect the expression of mitofusinl/2(Mfnl/2),optic atrophy 1-like protein(OPA1)and dynamin-related protein 1(Drp1)and fission 1(Fisl).The second part is animal experiments,including experiment 4 to experiment 7.The methods were as follows:Experiment 4:Establishment of C57BL/6J mice model overexpressed with pink1 in the brain.Reconstructed adenovirus(rAAV)overexpressed with pink1 and control adenovirus were constructed using genetic engineering technology,and brain stereotactic injection was performed.The expression of EGFP was observed by laser confocal microscopy,and the pink1 mRNA and Pinkl protein were detected by qPCR and Western Blot.Experiment 5:The neuroprotective effect of Buyin Qianzheng Formula on PD mice model with pink1 overexpression in the brain.Pole test and grasping test were performed to detect behavior.The number of TH positive neurons stained by immunofluorescence in the substantia nigra was observed by laser confocal microscopy.The content of dopamine and its metabolites in forebrain was determined by HPLC.Experiment 6:The effect of Buyin Qianzheng Formula on mitochondrial morphology and function of PD mice model with pink1 overexpression in the brain.The ultrastructure of neurons in the substantia nigra was observed by electron microscopy.The activity of mitochondrial complex I was determined by colorimetry.The content of brain ATP was determined by luciferase method.JC-1 method was used to detect the level of mitochondrial membrane potential.Experiment 7:The effect of Buyin Qianzheng Formula on brain mitochondrial fission and fusion in PD mice model with overexpression of pink1 gene in the brain.The expression of PINK1 and Parkin proteins related to mitochondrial quality was detected by Western Blot.The co-localization of Parkin and TOM20,the co-localization of PINK 1 and TOM20 protein were detected by immunofluorescence double labeling.Western Blot was used to detect the expression levels of Mfn1.Mfn2 and OPA1,as well as the expression levels of mitochondrial fission proteins Drpl and Fis1.Results1.Construction of pink1 overexpression plasmid was completed.Compared with the control group,green fluorescent proteins were detected in both pink1 overexpression group and negative control group.The expression of pink1 mRNA and PINK1 protein were significantly increased in the pink1 overexpression group.This indicated that the pink1 overexpressed SH-SY5Y cell model was successfully constructed.2.MPP+insult decreased survival rate of SH-SY5Y cells,fragmented mitochondria,and decreased mitochondrial activity,ATP content,and mitochondrial membrane potential.Overexpression of pink1 and Buyin Qianzheng Formula can antagonize these damages caused by MPP+,and the combination of the two can enhance the antagonism.3.Both Buyin Qianzheng Formula and pink1 overexpression showed antagonistic effect to the changes caused by MPP+,including the enhanced co-localizations of Parkin protein with MTR and PINK1,the decreased expression of mitochondrial fusion proteins Mfn 1/2,and the increased expression of mitochondrial fission proteins Drp1.Besides,the antagonistic effects were enhanced by the combination of Buyin Qianzheng Formula and pink1 overexpression.In addition,the prescription of Buyin Qianzheng Formula can obviously antagonize the decreased expression of OPA1 and increased expression of Fisl caused by MPP+,but the effect of pink1 overexpression was not obvious.4.Construction of pink1 overexpression adenoviruses was completed.After the stereotactic injections of adenoviruses,the striatum of mice in both the pink1 overexpression group and the negative control group showed significant green fluorescence protein,and the expressions of pinkl mRNA and PINK1 protein of the mouse brain in pink1 overexpression control group were significantly increased compared with the control group and the negative control group.This indicated that the C57BL/6J mice overexpressed with pink1 in the brain were successfully constructed.5.The overexpression of pink1 and the prescription of Buyin Qianzheng Formula significantly antagonized the MPTP-induced motor deficits in C57BL/6J mice,the decreased of the number of TH neurons in substantia nigra and the decreased content of dopamine in the forebrain.In addition,the combination of the two can significantly improve the content of DOPAC,a metabolite of dopamine in the forebrain decreased by MPTP.6.The overexpression of pink1 and Buyin Qianzheng Formula can obviously antagonize the mitochondrial fragmentation,and the decrease of mitochondrial membrane potential and ATP content of the brain caused by MPTR.In addition,Buyin Qianzheng Formula can significantly antagonize MPTP,resulting in an increase in the activity of mitochondrial complex I,and the combination of the two showed the best effect.7.Both overexpression of pink1 and Buyin Qianzheng Formula could significantly improve the expression of PINK 1 protein and Parkin protein in C57BL/6J mice brain decreased by MPTP,and the co-localizations of PINK]and Parkin proteins with TOM20(located in mitochondrial outer membrane)induced by MPTP,and the combined effect of the two was better.Both the overexpression of pink1 and Buyin Qianzheng Formula can significantly upregulate the expression of Mfnl/2,and decrease the expression of Drpl and Fis1 that changed by MPTP.Besides,Buyin Qianzheng Formula can significantly upregulated the expression of OPA1.The combination of the two had the best regulatory effects on the expression of Mfn1/2 and OPA1.Conclusions1,In vitro and in vivo,both the pink1 overexpression and the traditional Chinese medicine Buyin Qianzheng Formula can increase the expression of PINK 1 protein and then regulate the PINK 1/Parkin pathway and the dynamic balance of mitochondrial fission and fusion,ensure the quality of mitochondria,and protective the MPP+insulted SH-SY5Y cells and the MPTP lesioned DA neurons.Besides,the effect of traditional Chinese medicine Buyin Qianzheng Formula is better than the overexpression of pink1 in some aspects.2.The mechanism of Buyin Qianzheng Formula in the treatment of PD is closely related to pink1 gene.Buyin Qianzheng Formula may regulate PINK1/Parkin pathway and expression of mitochondrial fusion and fission proteins by upregulating the expression of PINK1 protein,so as to improve the morphology and function of mitochondria,protect the quality of mitochondria and exert the best neuroprotective effect to prevent and treat PD. |
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