Based On DRP1 To Explore The Mechanism Of Buyin Qianzheng Prescription To Regulate Parkinson's Disease Mitochondrial Homeostasis

BackgroundParkinson's Disease(PD)is an neurodegenerative disease,which is second to Alzheimer's Disease.The main pathological changes of PD are dopaminergic neuron degeneration,deletion and Lewy body formation.The clinical symptoms of PD are mainly manifested as progressive motor impairments.The pathogenesis of PD is not clear,and it may be related to environmental toxins and genetic defects.More and more studies have shown that mitochondrial dysfunction plays a very important role in the pathogenesis of PD.Mitochondria are dynamic organelles.The dynamic balance of mitochondrial division and fusion plays an important role in maintaining mitochondrial morphology,function and cell activity.Among them,Dynamic related protein 1(DRP1)is the key molecule to regulate mitochondrial division.A large number of studies have shown that DRP1-mediated mitochondrial breakage is closely related to PD,but the specific regulatory and control mechanism are not fully understood,and further research is still needed clarify.At present,traditional Chinese medicine treatment of PD is widely used in clinics,which can effectively improve the symptoms of patients,with certain effects and small side effects.Therefore,the discussion and in-depth study of the mechanism of action of traditional Chinese medicine have important guiding significance and value in clarifying the modern scientific connotation of clinical application of traditional Chinese medicine in the treatment of PD and the development of new drugs.Bu-Yin-Qian-Zheng-Formula(BYQZF)is one of the commonly used prescriptions for clinical treatment of PD,which is composed of Da-Bu-Yin-Wan(DBYW:Rehmannia glutinosa 9 g,Phellodendron chinense 6 g,Anemarrhena 6 g,Turtle Board 6 g)and Qian-Zheng-San(QZS:White Aconite 6 g,Zombie Silkworm 6 g,Whole Scorpion 6 g).The Chinese medicine in the two prescriptions is also commonly used in clinical treatment of PD drug.The previous studies of the research group have confirmed that BYQZF has a certain protective effect on the mitochondrial function of PD mice and cell models.On the basis of this research,this study directly uses the key molecule DRP1 that maintains mitochondrial homeostasis as an entry point,using gene knockdown and overexpression techniques at the celluar and overall animal levels to explore the role of BYQZF in the regulation of mitochondrial homeostasis in the pathogenesis of PD and further reveal its role Targets and linksObjective1.At the cell level in vitro,we will explore the effect of DRP1 gene regulation on PD cell mitochondrial homeostasis,and clarify BYQZF's regulation mechanism of PD cell mitochondrial division/fusion homeostasis and its correlation with DRP1.2.At the overall animal level,we will explore the effect of BYQZF on Drpl inhibited dopamine neurons and mitochondria in PD mice,and further reveal the role of BYQZF in protecting dopamine neurons and mitochondrial function.Methods1.Cell experiment part(including experiment 1 to experiment 4):Experiment 1:The effect of BYQZF on mitochondrial morphology and function of DRP1 knockdown PD cell model:Construct DRP1 knockdown plasmid liposome-mediated transfection of SH-SY5Y cells to establish and identify DRP1 knockdown cell model.On this basis,1-Methyl-4-phenylpyridinium ion(1-methyl-4-phenylpyridinium ion,MPP+)was used to construct a PD cell model and the Chinese medicine BYQZF intervention was performed.The CCK-8 method and Annexin V-PE were used to detect cells Survival rate and apoptosis rate,using MitoTracker(?)Red CMXRos probe labeling technology,laser confocal scanning microscope to observe the mitochondrial morphology of cells,JC-1 method to detect mitochondrial membrane potential;luciferase method to detect mitochondrial ATP levels and ADP/ATP ratio;MitoSOX red fluorescence detection of relative ROS levels in mitochondria.Experiment 2:The effect of BYQZF on the mitochondrial homeostasis of DRP1 knockdown PD cell model:On the basis of experiment 1,Western Blot technology was used to detect mitochondrial fission/fusion related proteins including:DRP1,mitochondrial fission 1(Mitochondrial fission protein 1,FIS1)and mitochondrial fission factor(MFF)and mitochondrial fusion protein 1(Mitofusin 1,MFN1),fusion protein 2(Mitofusin 1,MFN2)and optic atrophy protein(Optic atrophy 1,OPA1)expression;Immunofluorescence dual-label technology was used to detect the co-localization relationship between cells DRP1 and TOM20,DRP1 and MFF,and DRP1 and FIS1,MFN1 and TOM20,MFN2 and TOM20,and OPA1 and Complex ?.Experiment 3:The effect of BYQZF on mitochondrial morphology and function of PD cell model with overexpression of DRP1:First construct the DRP1 overexpression plasmid,and transfect SH-SY5Y cells with liposome-mediated method to establish and identify the cell model of DRP1 overexpression.Modeling,intervention and testing indexes are the same as experiment one.Experiment 4:The effect of BYQZF on mitochondrial homeostasis of PD cell model with overexpression of DRP1:The method of constructing plasmid and modeling is the same as Experiment 3,and the intervention and detection indexes are the same as experiment two.2.Animal experiment part(including experiment 5 to experiment 6):Experiment 5:The protective effect of BYQZF on dopamine neurons in brain of PD mice inhibited by DRP1:Using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)to construct PD mouse model and conduct DRP1 inhibitor The intervention of Mdivi-1 and Chinese medicine BYQZF.Rod climbing and hanging experiments were used to detect the behavior of mice;immunofluorescence technique was used to observe the number of positive neurons in the substantia nigra tyrosine oxidase(Tyrosineoxidase,TH)of mice;high-performance liquid electrochemistry method was used to detect DA and its metabolism in the brain of mice Product content.Experiment 6:The effect of BYQZF on mitochondria in brain of PD mice inhibited by DRP1:On the basis of experiment 5,the ultrastructure of substantia nigra neurons was observed under electron microscope.The content of ATP in rat brain was determined by luciferase method.In vivo ROS fluorescence assay reagent was used to detect the ROS level of rat brain mitochondria.Western Blot was used to detect the expression of related proteins of mitochondrial division/fusion in the brains of mice.The co-localization relationships between DRP1 and TOM20,DRP1 and MFF,DRP1 and FIS1,and MFN1 and TOM20 and MFN2 and TOM20 in the substancia nigra of mice were detected by immunofluorescence double-label technique.Results1.Resluts of Studyl:The expression of DRP1 mRNA and protein in the cells of the DRP1 knockdown group was significantly reduced;the survival rate of SH-SY5Y cells caused by MPP+was reduced,the morphological factors of mitochondria,aspect ratio,average number of branches and activity of the network were reduced,and mitochondrial membrane potential and ATP As content decreases,the ADP/ATP ratio and relative ROS levels increase.BYQZF and DRP1 knockdown can antagonize these damages caused by MPP+,and the combination of the two enhances this effect.2.Resluts of Study2:BYQZF and DRP1 knockdown can significantly improve the expression of mitochondrial fusion proteins MFN1 and OPA1 caused by MPP+;it can also inhibit the increased expression of mitochondrial cleavage proteins DRP1,FIS1 and OPA1;it can reduce the translocation of DRP1 protein to mitochondria and DRP1 Mitochondrial fission caused by synergy with FIS 1 and MFF;this effect is enhanced when the two are treated together.In addition,BYQZF can significantly increase the expression of MFN2 protein caused by MPP+,but the effect of DRP1 knockdown alone is not obvious.3.Resluts of Study3:The expression of DRP1 mRNA and protein in cells of the DRP1 overexpression group was significantly increased;the protective effect of BYQZF on PD model cells and mitochondria was the same as in Experiment 1.The survival rate of DRP1 overexpression decreased more than that of MPP+modular cells,and the mitochondrial activity and morphology Factors,aspect ratio and average number of branches on the network are further reduced,mitochondrial membrane potential and ATP levels are more significantly reduced,ADP/ATP ratio and relative ROS content are more significantly increased;Chinese medicine improves PD model cells and mitochondria after DRP1 overexpression.The effect has been reduced.4.Resluts of Study4:The regulation of BYQZF on the expression and translocation of mitochondrial fusion/division protein in PD model cells is the same as experiment two;after overexpression of DRP1,the expression of DRP1,FIS1 and MFF of MPP+modular cells is more significantly increased,and the fusion proteins MFN1 and MFN2 The expression of OPA1 and OPA1 decreased more obviously;it can further promote the translocation of DRP1 protein to mitochondria after modeling and the mitochondrial fission caused by the synergy between DRP1 and FIS1,MFF;after overexpression of DRP1,traditional Chinese medicine expressed and translocated the mitochondrial division/fusion of PD model cells The regulatory effect has been reduced.5.Resluts of Study5:DRP1 inhibitors Mdivi-1 and BYQZF can significantly antagonize the coordinated movement disorder of C57BL/6J mice caused by MPTP,the number of substantia nigra TH-positive neurons and the decrease of DA content in the forebrain;the antagonistic effect of the two combined increases.6.Resluts of Study6:Mdivi-1 and BYQZF can obviously antagonize the damage of mitochondria and the decrease of ATP content in brain neurons of PD mice caused by MPTP;inhibit the increase of mitochondrial relative ROS level;Mdivi-1 and BYQZF have little effect on PD in vivo The regulation effect of mouse mitochondrial division/fusion protein expression and translocation is the same as that of in vitro cell experiment two;the combination of the two increases this effect.Conclusions1.Knocking down or inhibiting DRP1 can play a protective role in PD model mitochondria by regulating the dynamic balance of mitochondrial fission fusion,and overexpression of Drp1 will increase the susceptibility of PD cells to neurotoxins,suggesting that mitochondrial morphology and function in PD are abnormal and DRP 1-mediated mitochondrial excessive division is involved.DRP1 is an important molecule that regulates the change of PD model mitochondrial morphology and function.2.The protection and regulation of the Chinese medicine BYQZF on the PD model mitochondrial dynamic imbalance is closely related to DRP1.It can reduce the translocation of DRP1 protein to mitochondria after modeling and increase the mitochondrial fission caused by the cooperation with FIS1 and MFF.Protein expression on mitochondria is reduced to improve mitochondrial morphology and function,and play a protective role in neurons.