To Explore The Effect Of Dabuyin Pill And Qianzheng Powder On The Mitochondrial Quality Of PD Cell Model From The Perspective Of Parkin Gene

BackgroundParkinson's disease(PD)is the second most common chronic neurodegenerative disease after Alzheimer's disease(AD),Which main clinical manifestations are resting tremor,stiffness and slow movement,characterized by the degeneration and depletion of dopaminergic neurons(DA),in the substantia nigra pars compacta.It is generally accepted that PD may be the result of a combination of genes,environment and aging factors.It has been confirmed that biological change,such as mitochondrial morphological abnormalities,dysfunction and kinetic imbalance,is one of the main pathogenesis of PD.Parkin is a genetic susceptibility gene of Parkinson,s disease,the mutation of which leads to PD and has a close relationship with sporadic PD.Currently,there are already a large number of studies have shown that Parkin plays an important role in regulating the quality of mitochondria,and its effects on mitochondria morphology and function has become research hotspots.Currently,there is no effective way to cure PD.Most treatments towards PD are focused on the alternative treatment of DA,however,long-term use of which can cause toxic side effects.Traditional Chinese Medicine(TCM),because of its unique theoretical system and the overall concept,have a certain effect and advantages in improving clinical symptoms.Da-Bu-Yin-Wan and Qian-Zheng-San(DBYW&QZS)are commonly used clinical decoctions in treating PD in China.Former studies suggested that to a certain extent,DBYW and QZS individually can regulate the expression of brain mitochondrial gene in PD animal model,improve the symptoms of PD animal model,and protect the function of dopaminergic neurons in the brain.The effect is more significant if DBYW and QZS are combined administrated.Based on the basis of former research,taking Parkin as the core,the study is to explore the effect of DBYW&QZS on the protection of mitochondrial function and the mechanisms from the cellular and molecular level.ObjectiveIn this study,MPP+-induced SH-SY5Y cell PD model was established to explore the effect of Chinese medicine combined prescription on mitochondrial morphology and function of PD model cells and the correlation with PINK/Parkin pathway.Then,Parkin overexpression plasmid was transfected into human neuroblastoma SH-SY5Y cells.Experiments are designed to investigate the protective effect and molecular mechanism of DBYW&QZS modulating mitochondria quality in PD cell models,which aims to help to provide a scientific basis for PD,Provide experimental basis and scientific connotation for traditional Chinese medicine compound,lay the foundation for the further utilization of Chinese medicine compound.MethodsStudy 1:Effects of DBYW&QZS on mitochondria and PESNK1/Parkin expression in PD cell model:Cultured SH-SY5Y cell,and then treated with MPP+ and DBYW&QZS.The experiment was divided into 3 groups:?control group;?model group;?TCM group.Experimental Detection:? Apply CCK-8 to test cell viabilities;(3Use Laser scanning eonfocal microscopy to observe morphology and activity of mitochondria stained with MitoTracker(?)Red CMXRos;?Analyze Mitochondria membrane potential(??m)by staining cells with tetramethylrhodamine methyl ester(TMRM).?Apply ATP assay kit to detect cell ATP level;?Perform Immunofluorescence assay to detect the expression of PINK1?Parkin protein;?The positioning relationship of Parkin,PINK I and mitoehondria(TOM20)was evaluated by three-color immunofluorescent staining.Study 2:Establishing Parkin overexpression cell model:Construct Parkin overexpression plasmids,then plasmid DNA was transformed into SH-SY5Y cell.The experiment was divided into 3 groups:?control group;?negative control group;? Parkin overexpression model group.Experimental Detection:?The transfection of SH-SY5Y cells was observed by fluorescence microscopy;?RT-PCR technique was used to detect the expression of Parkin at mRNA level;?Westernblot technique was used to detect the expression of Parkin at protein level.Study 3:Effects investigatation of DBYW&QZS on the mitochondrial quality of Parkin overexpressing PD cells:Based on the construction of Parkin gene overexpression plasmid and culture of SH-SY5Y cells and transfection,the cells were then treated with MPP+and DBYW&QZS.The cells were divided into 6 groups:?negative control group;?negative model group;?negative DBYW&QZS treatment group;?Parkin overexpression group;?Parkin overexpression model group;?Parkin overexpression DBYW&QZS treatment group.Experimental Detection:?Apply CCK-8 to test cell viabilities;?Use Laser scanning confocal microscopy to observe the morphology of mitochondria stained with MitoTracker-Red CMXRos;?Analyze Mitochondria membrane potential(??m)by staining cells with tetramethylrhodamine methyl ester(TMRM)·?Apply ATP assay kit to detect cell ATP level;? Perform Immunofluorescence assay to detect the expression of PINKI protein and Parkin protein;?The eo-localization of Parkin,PINK1 and mitochondria(TOM20)was evaluated by three-color immunofluorescent staining.? Use Westernblot technique to detect the expression of Mitofusin 1(Mfn1),Mitofusin 2(Mfo2),Dynamic related protein 1(Drpl)and mitochondrial fission protein 1(Fisl)at protein levels,which are associated with mitochondrial quality and remodeling.ResultsResluts of Studyl:1.Cell viability and mitochondria morphology:Compared with control group,the cell survival rate of SH-SY5Y cells decreased significantly in PD model group(PO.O1)mitochondrial grid reduced,mitochondrial debris accumulated and F-factor and mitochondria activity decreased(P<0.05);Compared with model group,DBYW&QZS improved cell survival rate(P<0.01),F-factor(P<0.05)and mitochondria activity(PO.05).2.Mitochondria function:Compared with control group,the mitochondria membrane potential(??m)dropped(P<0.01)and ATP level decreased(P<0.05)in other two groups;compared with the model group,the ATm of the DBYW&QZS treatment group was significantly increased P<0.01),but the level of ATP was not obvious.3.PINKI protein and Parkin protein expression:Compared with control group,MPP+treatment can largely reduce the relative fluorescence intensity of PINK1 protein and Parkin protein(P<0.01)5 while the expression of PINK1 protein and Parkin protein was significantly increased in DBYW&QZS treatment group(P<0.01).4.Co-localization of Parkin,PINK1 and mitochondria:Compared with control group,the co-localization coefficients of Parkin and TOM20(P<0.05),Parkin and PINK1(P<0.01),and PINK1 and TOM20(P<0.05)in PD model group decreased,while increased in DBYW&QZS treatment group(P<0.05),Results of Study 2:After the extraction of Parkin overexpression plasmid,more expression of enhanced green fluorescent protein(EGFP)was observed in the transfected group compared with the normal control group;Compared with the normal control group and negative control group,the mRNA and protein expression of Parkin was significantly increased,after transfection with Parkin overexpression plasmid(P<0.01).Resluts of Study 3:1.Cell viability and mitochondria morphology:Compared with negative control group,the cell survival rate of SH-SY5Y cells increased significantly in AWn overexpression group(P<0.05),the cell survival rate and mitochondria activity decreased significantly in both negative model group and Parkin overexpression model group(P<0.01);Compared with negative model group,the cell survival rate(P<0.01),F-factor(P<0.05)and mitochondria activity(P<0.01)increased in negative DBYW&QZS treatment group(P<0.05);Compared with Parkin overexpression group,cell survival rate(P<0.01),F-factor(P<0.05)and mitochondria activity(P<0.01)decreased in Parkin overexpression model group;Compared with Parkin overexpression model group,cell survival rate,F-factor and mitochondria activity increased in Parkin overexpression DBYW&QZS treatment group(P<0.01).2.Mitochondria function:Compared with negative control group,the ??m(P<0.01)and ATP level(P<0.05)increased in Parkin overexpression groups,the ??m(P<0.01)and ATP level(P<0.05)decreased in negative model group;Compared with negative model group,the??m(P<0.01)and ATP level(P<0.05)increased in negative DBYW&QZS treatment group;Compared with Parkin overexpression group,the ??m(P<0.01)and ATP level(P<0.05)decreased in Parkin overexpression model group(P<0.05);Compared with Parkin overexpression model group and negative DBYW&QZS treatment group,the ??m(P<0.01)and ATP level(P<0.05)increased in Parkin overexpression DBYW&QZS treatment group.3.The expression of PINK1 protein and Parkin protein:Compared with negative model group and Parkin overexpression model group,the expression of PINK1 protein and Parkin protein was significantly decreased P<0.01),and compared with negative groups,the expression of PINK1 protein and Parkin protein was significantly increased in Parkin overexpression groups;After DBYW&QZS treatment,the expression of PINK1 protein and Parkin protein increased in negative DBYW&QZS treatment group and Parkin overexpression DBYW&QZS treatment group(P<0.01),and the expression of PINK1 protein in Parkin overexpression DBYW&QZS treatment group was higher than that in Parkin overexpression model group,but there was no significant difference.4.Colocalization of Parkin,PINK1 and mitochondria:Compared with Parkin overexpression model group,the co-localization of Parkin and PINK1 increased in Parkin overexpression DBYW&QZS treatment group(P<0.05),the differences between the other groups were not significant.5.Expression of mitochondrial fission proteins and fusion proteins:Compared with negative control group and Parkin overexpression group,the expression of mitochondria]fission protein Fisl,Drpl(P<0.05),fusion protein Mfnl(P<0.01),Mfn2 were decreased in negative model group and Parkin overexpression model group(P<0.05);Compared with negative model group,the expression of Fisl protein and Mfn2 protein was enhanced in negative DBYW&QZS treatment group(/P<0.05);Compared with Parkin overexpression model group,the expression of Mfn2 protein was enhanced in Parkin overexpression DBYW&QZS treatment group(P<0.05):The differences between the other groups were not significant.Conclusions1.DBYW&QZS displays protective effects on mitochondria by improving MPP+induced PD model SH-SY5Y cells,mitochondria morphology,maintaining mitochondrial network structure,improving the membrane potential of mitochondria,but has no significant effect on the synthesis of ATP,the mechanism may be related to PINK1/Parkin pathway.2.Using liposome transduction technology,the Parkin overexpression plasmid was successfully transfected into SH-SY5Y cells by lipo 3000 transfection reagent,and the cell model of gene overexpression was successfully established,which laid the foundation for further experiments.3.Parkin gene overexpression can improve the activity of SH-SY5Y cells and protect the mitochondrial morphology and function of mitochondria,have a protective effect on MPP+ caused by SH-SY5Y cells and mitochondrial damage,in particular,can signiflcantly improve mitochondrial activity and membrane potential.4.DBYW&QZS can enhance the protective effect of Parkin gene overexpression,on MPP+-induced cell and mitochondrial damage.The mechanism may be to maintain the quality of mitochondria by promoting PINKI to recruit Parkin to mitochondria and regulating the dynamic balance of mitochondrial fission and fusion proteins.