Based On The PINK1 Gene, The Protective Mechanism Of Dabuyin Pill And Zhizhengsan On Mitochondria In Parkinson's Cell Model Was Discussed

BackgroundParkinson's Disease(PD)is a progressive degenerative disease of the central nervous system in the middle-aged and elderly people.The main pathological feature is the absence of progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta,resulting in decreased dopamine levels in the striatum.It has a series of movement disorders,such as:resting tremors,muscle rigidity,slow movement and so on.At present,there is no effective way to cure PD.Western medicine mainly treats Parkinson's disease with levodopa replacement therapy,mainly to improve the clinical symptoms of patients,but there will be toxic and side effects such as dyskinesia after prolonged use.Traditional Chinese Medicine(TCM)has the advantages of small toxic and side effects and stable curative effect in the prevention and treatment of PD due to its unique theory and the overall prevention and control methods of the system.At present,it is widely believed that PD is the result of a combination of genes,environment,and aging factors,and its specific pathogenesis remains unclear.It has been confirmed that mitochondrial dysfunction is closely related to the pathogenesis of PD.PINK1 is a relatively common gene involved in the pathogenesis of PD.Mutation can cause mitochondrial morphology and function abnormalities.Normal expression of PINK1 can protect mitochondria from endogenous and exogenous damage,and plays an important role in maintaining normal mitochondrial function and protecting dopaminergic neurons.Da-Bu-Yin-Wan and Qian-Zheng-San(DBYW&QZS)are commonly used in clinical treatment of PD.Our previous studies confirmed that DBYW&QZS can regulate the expression of brain mitochondrial genes and reduce mitochondrial DNA in PD mice to some extent.The injury has a clear protective effect on the mitochondrial function of PD animal model of brain mitochondria and PD cells,and the effect is more significant if DBYW&QZS are combined administrated.Based on this research,PINK]gene was used as an entry point to combine TCM theory with modern molecular biology techniques to further study the role and mechanism of TCM compound at the cellular and molecular levels.ObjectiveThis study uses PINK1 gene regulation as the cure point.PINK1 gene knockdown plasmid was transfected into human neuroblastoma SH-SY5Y cells.Under the construction of MPP+ induced SH-SY5Y cell PD model,this study clarified the relationship between DBYW&QZS protection of mitochondrial morphology and function in PD and the regulation of PINK1 gene knockdown,and revealed a link between PINK1 protein,Parkin protein,and mitochondrial fission/fusion protein closely related to mitochondrial body weight and plasticity.It provides scientific basis for the clinical application of DBYW&QZS in the treatment of PD,and also provides scientific connotation and experimental basis for the theory of traditional Chinese medicine compound,and provides a reliable platform for further utilization of traditional Chinese medicine compound prescription.MethodsStudy 1:Construction of PINK1 knockdown cell model:Experiments were divided into 3 groups:?normal control group;?negative control group;?PINK1 knockdown group.Fluorescence microscopy was used to observe the transfection of SH-SY5Y cells;Flow cytometry was used to detect the transfection efficiency of SH-SY5Y cells;RT-PCR was used to detect the expression of PINK1 mRNA;Western Blot technique was used to detect the expression of PINK1 protein.Study 2:Effect of DBYW&QZS on the mitochondrial morphology and function of PINK1 gene knockdown PD cell model:Divided into 6 groups:?empty plasmid control group;?empty plasmid model group;?empty plasmid treatment group;?PINK1 knockdown control group;?PINK1 knockdown model group;?PINK1 knockdown treatment group.CCK-8 method was used to detect the survival rate of cells in each group;MitoTracker(?)Red CMXRos probe was used to label mitochondria and observed by laser confocal microscopy Mitochondrial morphology and fluorescence intensity;Luciferase assay was used to detect the level of ATP in cells;Tetramethylrhodamine methyl ester(TMRM)was used to detect the mitochondrial membrane potential(??m).Study 3:Effect of DBYW&QZS on the mitochondrial quality-related protein in PINK1 gene knockdown PD cell model:Grouping is the same as Study 2.Western Blot technique was used to detect the expression levels of mitochondrial quality related proteins PINK1 and Parkin;Western Blot technique was used to detect the expression levels of fusion protein 1(Mfh1)and fusion protein 2(Mfn2);Western Blot technical detection of mitochondrial fusion-associated protein OPA1 expression;Western Blot technique was used to detect the expression levels of Dynamic related protein 1(Drp1)and mitochondrial fission protein 1(Fisl).ResultsResluts of Studyl:1.Expression of Green Fluorescent Protein and Determination of Cell Transfection Efficiency:More green fluorescent protein expression was observed in the negative control group and the PINK1 knockdown group,and the transfection efficiency was higher after transfection 48 h.2.Expression of PINK1 mRNA and protein:The expression of PINK1 mRNA and protein in the PINK1 knockdown group SH-SY5Y cells was significantly decreased(P<0.05),indicating that the PINK1 gene knockdown cell model can.be successfully constructed.Results of Study 2:1.Cell viability:MPP+ could significantly decrease the cell survival rate of the model group(P<0.05);DBYW&QZS could significantly increase the cell survival rate of the empty plasmid treatment group(P<0.05);but there was no significant difference in the cell survival rate of the PINK1 knockdown treatment group.2.Mitochondrial morphology:MPP+ can reduce the number of mitochondrial grids,morphology factors and increase the number of debris in the model group(P<0.05),and the mitochondrial fluorescence intensity decreases significantly(P<0.05).After treated with DBYW&QZS,the mitochondrial morphogenetic factors and fluorescence intensity in the empty plasmid treatment group were increased(P<0.05);however,there was no significant change in mitochondrial morphogenetic factors and fluorescence intensity in the PINK1 knockdown treatment group.3.Mitochondria function:MPP+ can significantly reduce the mitochondria membrane potential(??m)and ATP level in the model group(P<0.05).Compared with the empty plasmid control group,the mitochondria membrane potential(??m)and ATP level significantly decreased after PINK1 knockdown(P<0.05);After DBYW&QZS treatment,the mitochondria membrane potential(??m)and ATP level were significantly increased in the empty plasmid treatment group(P<0.05).There was no significant difference in the mitochondria membrane potential(??m)and ATP level in the PINK1 knockdown treatment group.Results of Study 3:1.Expression of PINK1 and Parkin protein:MPP+ significantly decreased the expression of PINK1 and Parkin protein in the model group(P<0.05).Compared with the empty plasmid control group,PINK1 knockdown decreased the expression of PINK1 and Parkin protein significantly(P<0.05);After treatment with DBYW&QZS,the expression of PINK1 and Parkin protein in the empty plasmid treatment group was significantly increased(P<0.05);however,the expression of PINK1 and Parkin protein in the PINK1 knockdown treatment group was not significant change.2.Expression of mitochondrial fusion-related proteins Mfn1,Mfn2,OPAl(OPA11 and OPA12)and mitochondrial fission-related proteins Fisl and Drp1:MPP+ could significantly reduce the expression of mitochondrial fusion and fission protein in the model group(P<0.05).Compared with the empty plasmid control group,the knockdown of PINK1 could significantly increase the expression levels of mitochondrial associated fusion proteins Mfn2 and OPA12;After treatment with DBYW&QZS,the expression of mitochondrial fusion protein and fission protein in the empty plasmid treatment group and the expression of mitochondrial fusion protein in PINK1 knockdown treatment group was significantly increased(P<0.05),but there was no significant difference in the expression of mitochondrial fission-related protein in the PINK1 knockdown treatment group.ConclusionsDBYW&QZS had a certain protective effect on the mitochondrial damage of SH-SY5Y cells caused by MPP+,and its mechanism is related to the PINK1 gene.PINK1 may maintain the mitochondrial morphology and function by regulating the expression of Parkin and mitochondrial fission-related and fusion-related proteins to maintain mitochondrial homeostasis.