| BackgroundParkinson’s disease(PD)is a kind of neurodegenerative disease with tremor,myotonia and bradykinesia as the main clinical manifestations,which is more common in the elderly.The degeneration and loss of dopaminergic neurons in substantia nigra pars compacta and the appearance of Lewy body(LB)in neurons are the main pathological features.Epidemiological survey shows that among nervous system diseases,PD has the fastest growth rate(according to the prevalence,disability and mortality).At present,there are more than 6 million people in the world.It is estimated that by 2040,the number will double to more than 12 million.At present,the prevalence rate of PD among people over 65 years old in China is about 1.7%.With the development of aging,the number of PD patients in China will increase to 4.94 million by 2030,accounting for about half of the global PD patients.The growth of incidence rate of PD not only reduces the quality of life of patients and their families,but also brings heavy medical and economic burden to society.The pathogenesis of PD involves mitochondrial dysfunction,neuroinflammation,apoptosis,protein homeostasis disorder and other mechanisms.Inducible kinase 1(PINK1)is a serine/threonine kinase located in mitochondria.PINK1 can regulate its downstream parkin and mitotic fusion protein,and cooperatively regulate the dynamic balance of mitochondria.It plays an important role in maintaining the morphology,structure and function of mitochondria.Mitochondrial dysfunction caused by abnormal gene and protein expression is an important mechanism of PD.So far,no cure for PD has been found.At present,the main treatment is levodopa and other Western drugs,but with the passage of time,this kind of drugs will appear drug resistance and side effects.PD is classified as "tremor syndrome" in modern Chinese medicine.Traditional Chinese medicine has a long history in the treatment of PD.Traditional Chinese medicine can effectively improve the overall state of PD patients with small side effects,and can better improve the quality of life of patients.It is of practical significance to clarify the mechanism of treatment and provide experimental basis for the development and promotion of more extensive use of traditional Chinese medicine in clinic.Our previous study found that Buyin Qianzheng Formula(BYQZF),a combination of Dabuyin pill and Qianzheng powder,can reduce the damage of brain mitochondria and PD cell mitochondria in PD mice,and protect the quality of mitochondria.Based on this,this study will use PD mouse model to further study the regulatory mechanism of BYQZF on mitochondrial quality control in PD brain from the perspective of pink1 gene knockout.ObjectiveObjective to explore the relationship between pink1 gene and the morphological structure and function of brain mitochondria in PD mice,and to explore whether BYQZF can protect DA neurons by regulating the related molecules of brain mitochondria dynamic balance in PD mice through pink1 gene,and to clarify the mechanism of traditional Chinese medicine in the treatment of PD,so as to lay an experimental foundation for better clinical application.MethodChapter 1:Construction of lentivirus(LVs)-mediated brain pink1 RNAi mouse model:firstly,the pink1 RNAi plasmid was constructed and identified;Lentivirus pink1 RNAi packaging was completed,and virus titer and pink1 mRNA and PINK1 expression in mouse brain were detected.The mice were divided into three groups:normal group,negative control virus group and lentivirus pink1 RNAi group.After 2W,3 mice in each group were taken for brain frozen sections and processed accordingly.The expression of green fluorescent protein(GFP)carried by lentivirus packaged pink1 RNAi vector in the left striatum of mice in each group was observed under fluorescence microscope.After 4 weeks,the brain of 3 mice in each group was taken.The mRNA level of pink1 in the brain was measured by quantitative real-time PCR.The protein expression of PINK1 in the left and right brain was detected by Western blot.Chapter 2:Therapeutic effect of BYQZF on lentivirus mediated brain pink1 RNAi PD mouse model:mice were divided into 6 groups,3 in each group:negative control virus group,negative control PD group,lentivirus negative PD Chinese medicine group,lentivirus PINK1 RNAi group,lentivirus pink1-RNAi PD group,lentivirus pink1-RNAi PD Chinese medicine group.The movement coordination ability of mice in each group was evaluated by recording the climbing time and hanging score;The changes of dopamine neurons in substantia nigra were observed by immunofluorescence;High performance liquid chromatography(HPLC)was used to detect the levels of DA,DOPAC and HVA.Chapter 3:Effect of BYQZF on the quality of brain mitochondria of lentivirus mediated brain pink1-RNAi PD mouse model:mice grouping is the same as Chapter 2;The ultrastructural changes of substantia nigra neurons were observed by transmission electron microscope.The ATP level of brain mitochondria was detected by luciferase method,and the activity of respiratory chain complex I was measured by colorimetry.The protein levels of PINK1 and parkin were detected by Western blot;The levels of mitofusin 1(Mfn1),mitofusin 2(Mfn2)and optic atrophy 1(Opa1)were detected;The levels of drp1 and Fis1 were detected.The co localization of PINK1 and Parkin,PINK1 and Tom20,and parkin and Tom20 were observed by immunofluorescence double labeling technique and fluorescence microscope.ResultsChapter11.Construction of lentivirus pink1-RNAi vector:the pink1-RNAi sequence was consistent with the alignment sequence,and the packaging of lentivirus pink1-RNAi was completed.The titer of lentivirus was 5 × 108。2.Construction of mouse brain lentivirus pink1-RNAi model:no fluorescent expression of GFP was found in the striatum of normal mice,but GFP was significantly expressed in the striatum of lentivirus negative mice and lentivirus pink1-RNAi mice.Compared with the normal group,there were no significant changes in the mRNA and protein of PINK1 in the brain of the negative control virus group;Compared with the negative control virus group,the mRNA and protein of PINK1 in the brain of the lentivirus PINK1 RNAi group were significantly decreased;There was no difference in protein expression between the left and right brain of the above three groups,suggesting that the lentivirus mediated mouse brain pink1-RNAi model was successfully constructed.Chapter21.Behavior of mice in each group:compared with the negative control virus group,the climbing time of mice in the negative control PD group was significantly longer,and the hanging score was lower;BYQZF can improve the motor coordination disorder of PD mice;The climbing time and hanging score of lentivirus pink1-RNAi group were also prolonged.Compared with the mice in the lentivirus pink1-RNAi group,the mice in the lentivirus pink1-RNAi PD group had longer pole climbing time and lower hanging score;There was no significant change in behavior of mice after intervention with BYQZF.2.The number of TH+ neurons in substantia nigra of mice in each group:compared with the negative control virus group,the number of TH+neurons in the negative control PD group was significantly reduced;After BYQZF intervention,the number of TH+ neurons increased;The number of TH+neurons in lenti viral pink1-RNAi group was also decreased.Compared with the lentivirus PINK1 RNAi group,the number of TH+ neurons in the lentivirus pink1-RNAi PD model group was also significantly reduced;The number of TH+neurons in substantia nigra of mice did not increase significantly after the intervention of traditional Chinese medicine.3.The levels of DA and its metabolites in the brain of mice in each group:compared with the negative control virus group,the levels of DA,DOPAC and HVA in the lentivirus negative PD model group were significantly decreased;After the intervention of BYQZF,the levels of DA and DOPAC increased significantly;The levels of DA and DOPAC in lentiviral pink1-RNAi group were also significantly decreased.Compared with the control group,the levels of DA,DOPAC and HVA in the brain of PD group were significantly decreased;BYQZF intervention did not improve the above indicators.Chapter 31.Ultrastructure of substantia nigra neurons in each group:compared with the negative control virus group,the ultrastructural damage of substantia nigra neurons in the negative control PD group was more obvious;BYQZF intervention can improve the injury to a certain extent;The ultrastructure of substantia nigra neurons in lentivirus pink1-RNAi group was also damaged.Compared with the lentivirus pink1-RNAi group,the ultrastructure of substantia nigra neurons in the lentivirus pink1-RNAi PD group was more severe;BYQZF did not significantly improve the injury.2.Brain mitochondrial function:compared with the lentivirus negative control group,the mitochondrial ATP level and respiratory chain complex I enzyme activity in substantia nigra of the negative control PD group were decreased;After intervention,BYQZF can improve mitochondrial function;The level of ATP and the activity of complex I enzyme in brain mitochondria were also decreased in the lentiviral pink1-RNAi group.Compared with the mice in the lentivirus pink1-RNAi group,the ATP level and complex I enzyme activity of brain mitochondria in the lentivirus pink1-RNAi PD group were lower;BYQZF did not significantly improve the injury.3.The expression of mitochondrial quality related proteins in each group:compared with the negative control virus group,the expression of PINK1 and parkin decreased,the expression of Mfn1,Mfn2 and Opal decreased,the expression of Drp1 and Fis1 increased,and the co localization coefficients of PINK1 and Parkin,PINK1 and Tom20 and parkin and Tom20 increased in the negative control PD group;After BYQZF intervention,the expression of PINK 1 and parkin increased,the expression of mitochondrial fusion related proteins increased,the expression of mitotic related proteins decreased,and the co localization coefficients of PINK1 and Parkin,PINK1 and Tom20,and parkin and Tom20 decreased.Compared with the lentiviral pink1-RNAi group,the expression of PINK1 and parkin in the lentiviral pink1-RNAi PD group decreased,the expression of mitochondrial fusion related proteins decreased,and the expression of related mitotic proteins increased.The co localization coefficients of PINK1 and Parkin,PINK1 and Tom20,and Parkin and Tom20 increased;BYQZF did not significantly improve the expression of related proteins and co localization with mitochondria.Conclusion1.4W could successfully construct a low expression model of PINK1 in mouse brain mediated by slow virus.2.the experiment further confirmed that BYQZF,a Chinese medicine,could protect DA neurons from the middle brain from the negati ve side(knockdown of the pink1 gene in mice).The mechanism may be to maintain the dynamic balance of mitochondria by regulating the expression of brain PINK1 protein,downstream Parkin and mitochondrion mitochondrion fusion protein in mice,so as to repair the mitochondrial morphological structure and functional damage caused by PD.These results indicate that the mechanism of BYQZF in the treatment of PD is closely related to PINK1.This study provides a further scientific basis for the study of action and mechanism of byqzf in the treatment of PD. |
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