Based On L-OPA1 To Explore The Mechanism Of Tonifying Yin And Pulling Square To Regulate Mitochondrial Homeostasis To Prevent Parkinson's Disease

BackgroundParkinson’s disease(PD)is a common and highly prevalent neurodegenerative disease in the middle-aged and elderly population.Its main pathological change is the progressive loss of dopamine(DA)neurons in the substantia nigra of the midbrain,which induces a series of motor and non-motor impairment symptoms that seriously affect the quality of life of patients.Based on the etiopathogenesis of PD,scholars have proposed that its development is closely related to mitochondrial dynamics.Mitochondrial fission proteins and mitochondrial fusion proteins can regulate mitochondrial dynamics,which in turn affects the morphology,structure and function of mitochondria.As a key molecule regulating mitochondrial inner membrane fusion,optic atrophy 1(OPA1)is closely related to the development of PD,but the specific mechanism of action is not fully understood and needs to be investigated in more depth.At present,the clinical treatment of PD mainly adopts symptomatic treatment with levodopa and other drugs,which can improve the symptoms but cannot overcome the disadvantages of high incidence of side effects and reduced efficacy.Traditional Chinese medicine has the advantages of less toxic side effects and stable efficacy,which is widely used in clinical practice.Bu-Yin-Qian-Zheng-Fang(BYQZF)is composed of Da-Bu-Yin-Wan and Qian-Zheng-San,which has the effect of nourishing the liver and kidney,dispelling wind and phlegm,detoxifying and dispersing blood stasis,and is one of the commonly used prescriptions for the treatment of PD.Preliminary studies have shown that BYQZF can not only improve the morphological structure and function of mitochondria in PD cell models,but also regulate the expression of OPA1.However,the relationship between the improving effect of BYQZF and OPA1 and the mechanism of action of BYQZF in regulating OPA1 is not clear.In this study,starting from L-OPA1,a subunit of OPA1,a key molecule for maintaining mitochondrial dynamics,we construct PD cell model with L-OPA1 overexpression and knockdown to reveal the mechanism of action of L-OPA1 in improving the morphological structure and function of mitochondria in PD cell models;to elucidate the molecular mechanism of BYQZF in maintaining mitochondrial mass through L-OPA1,to further reveal the effect of BYQZF on GSK3β/OMA1/L-OPA1 in PD models,to elucidate the targets and links of BYQZF in the prevention and treatment of PD,to provide an experimental basis for the clinical application of BYQZF in the treatment of PD,and to provide scientific connotation for further explaining the correlation between mitochondria and“Yin and Yang”and the syndrome of deficiency of liver and kidney Yin.Objective1.Taking molecules that maintain mitochondrial fission and fusion proteins as the entry point,we reveal the effects of BYQZF on the morphological structure and function of mitochondria and the mechanism of action from the cellular level of PD pathogenesis,and elucidate this formula’s protective effect on mitochondria of PD cells through GSK3β/OMA1/L-OPA1 pathway.2.Taking L-OPA1,a key molecule for maintaining mitochondrial fission and fusion proteins,as the entry point,we revealed L-OPA1 ’s protective effect on the morphological and function of mitochondria in PD cell models,and elucidated the correlation between the protective effect of BYQZF on the morphological and function of mitochondria in PD cells and L-OPA1.3.To explore the regulatory effect of BYQZF on brain GSK3β/OMA1/L-OPA1 pathway in PD animal models and further reveal the link of BYQZF’s role in maintaining mitochondrial dynamics by regulating L-OPA1.MethodsChapter 1 Effects of BYQZF on the morphological structure and function of mitochondria in PD cell model:SH-SY5Y cells were used to construct a PD cell model,and then MPP+and BYQZF interventions were performed.The survival rate of each group of cells was detected by CCK-8 method;the apoptosis rate of each group of cells was detected by flow cytometry;the ultrastructure of each group of SH-SY5Y cells was observed by transmission electron microscopy;Mitochondrial probe was used to observe the morphology of cellular mitochondria by laser confocal scanning microscopy.The mitochondrial morphology of each group of cells was observed under laser confocal scanning microscope;the mitochondrial ATP level and ADP/ATP ratio were detected by luciferase;the mitochondrial membrane potential was detected by JC-1;the mitochondrial ROS level was detected by flow cytometry;the expression level of CytC protein in each group of cells was detected by Western Blot.Chapter 2 Effects of BYQZF on mitochondrial fission and fusion proteins in PD cell model and mechanism of action:Based on chapter 1,Western Blot technique was used to detect the expression of mitochondrial fission proteins DRP1,FIS1 and MFF,mitochondrial fusion proteins MFN1,MFN2,L-OPA1 and OMA1 and GSK3β proteins;Immunofluorescence double-labeling technique was used to observe the fission proteins DRP1 and TOM20,DRP1 and FIS1,DRP1 and MFF,fusion proteins MFN1 and TOM20,MFN2 and TOM20,OPA1 and COX IV were observed for their expression and co-localization levels.Chapter 3 Effects of BYQZF on the morphological structure and function of mitochondria in PD cell model regulated by L-OPA1:L-OPA1 overexpression/knockdown plasmids were constructed and liposome-mediated transfection of SH-SY5Y cells was performed,and L-OPA1 overexpression/knockdown cell models were identified by Western Blot and qPCR techniques.On this basis,MPP+and BYQZF was performed.Cell survival rate and apoptosis rate,mitochondrial morphology,ATP levels,ADP/ATP ratio,mitochondrial membrane potential,ROS levels and CytC expression were detected in the same way as in Chapter 1.Chapter 4 Effects of BYQZF on mitochondrial fission and fusion proteins in PD cell model regulated by L-OPA1:On the basis of chapter 3,DRP1,FIS1 and MFF expressions,MFN1,MFN2 and L-OPA1 expressions were detected in the same way as in Chapter 2.The expressions and co-localization level between cells DRP1 and TOM20,DRP1 and MFF,DRP1 and FIS1,MFN1 and TOM20,MFN2 and TOM20,OPA1 and COX IV were detected in the same way as in Chapter 2.Chapter 5 Effects of BYQZF on GSK3β/OMA1/L-OPA1 in PD animal model and its mechanism:Animal models of PD were constructed by MPTP,and intervention with BYQZF.Immunofluorescence technique was used to observe the expression of TH-positive neurons in the substantia nigra of mice.The ultrastructure of substantia nigra neurons was observed under transmission electron microscope.JC-1 method to detect mitochondrial membrane potential.Western Blot technology was used to detect DRP1,FIS1 and MFF expressions,MFN1,MFN2 and L-OPA1 expressions,OMA1 and GSK3β expressions in the brain of each group of mice;Immunofluorescence dual-label technology was used to detect the expressions and co-localization level between cells DRP1 and TOM20,DRP1 and MFF,DRP1 and FIS1,MFN1 and TOM20,MFN2 and TOM20,OPA1 and COX IV in brain substantia nigra in each group of mice.Results and analysis1.Results of chapter 1:BYQZF can significantly improve the damage of mitochondrial morphology and function,inhibit cell apoptosis,increase the mitochondrial activity,morphological factors,aspect ratio and average number of the network branches,ATP level and mitochondrial membrane potential,and reduce the ADP/ATP ratio ROS level and the generation of CytC in PD cell model.2.Results of chapter 2:BYQZF significantly increased the expressions of MFN1,MFN2 and L-OPA1;decreased the expressions of DRP1,FIS1,MFF and GSK3β,OMA1 proteins;increased the expressions and co-localization level MFN1 and TOM20,MFN2 and TOM20,OPA1 and COX IV;decreased the expressions and co-localization level of DRP1 and TOM20,DRP1 and FIS1,DRP1 and MFF.3.Results of chapter 3:The mitochondrial activity,morphological factors,aspect ratio and average number of the network branches,ATP level and mitochondrial membrane potential increased,ADP/ATP ratio,ROS level and CytC production decreased;cell survival rate increased and apoptosis rate decreased in the L-OPA1 overexpression medicine group compared with L-OPA1 overexpression model group.Compared with the negative medicine group,the cell survival rate was increased and the apoptosis rate was decreased.Compared with the L-OPA1 knockdown model group,the mitochondrial morphological factors,mitochondrial membrane potential increased,ADP/ATP ratio and CytC production decreased,cell survival rate increased in the L-OPA1 knockdown medicine group.Compared with the negative medicine group,the cell survival rate,the mitochondrial activity,morphological factors,aspect ratio and average number of the network branches,ATP level and mitochondrial membrane potential increased,ADP/ATP ratio,ROS level and CytC production and apoptosis rate decreased in the L-OPA1 knockdown medicine group.4.Results of chapter 4:The expressions of MFN1,MFN2 and L-OPA1 were significantly increased in the L-OPA1 overexpression medicine group compared to the negative medicine group and the L-OPA1 overexpression model group;the expressions of DRP1,FIS1 and MFF was significantly decreased;the expressions and co-localization level of fusion proteins in mitochondria was increased,and the expressions and co-localization level of fission proteins in mitochondria was decreased.Compared with the L-OPA1 knockdown model group,the expressions and co-localization level of DRP1,FIS1 was significantly decreased in the L-OPA1 knockdown medicine group,and the fusion protein was not significantly changed;Compared with the negative medicine group,the expressions and co-localization level of MFN1,MFN2 and L-OPA1 in mitochondria was decreased,and the expressions and co-localization level of DRP1,FIS1 and MFF in mitochondria was increased in the L-OPA1 knockdown medicine group.5.Results of chapter 5:BYQZF can significantly improve the number of TH positive neurons in the substantia nigra,the damage of ultrastructure and the mitochondrial membrane potential in brain neurons of PD mice,increase the expressions of MFN1,MFN2 and L-OPA1,decrease the expressions of GSK33,OMA1 and DRP1,FIS1 and MFF in brain neurons of PD mice.Increase the expressions and co-localization level of MFN1 and TOM20,MFN2 and TOM20,OPA1 and COX IV;decrease the expressions and co-localization level of DRP1 and TOM20,DRP1 and FIS1,DRP1 and MFF in brain substantia nigra in each group of mice.Conclusions1.The regulation of mitochondrial dynamics in PD models by BYQZF is closely related to L-OPA1,which can improve the morphological structure and function of mitochondria and exert protective effects on DA neurons by increasing the expression of mitochondrial fusion proteins and co-localization levels with mitochondria,and decreasing the expression of mitochondrial fission proteins and co-localization levels with mitochondria.The molecular biological mechanism of BYQZF,which has the effects of nourishing liver and kidney(treating the root cause),dispelling wind and phlegm,and relieving spasm(treating the symptoms),is probably related to L-OPA1 regulating the balance of "yin and yang"of mitochondrial fusion and reducing mitochondrial damage caused by liver and kidney deficiency.2.The link between the role of BYQZF in maintaining mitochondrial dynamics to protect DA neurons may be related to ameliorating the impairment of the morphological structure and function of mitochondria in PD models by inhibiting the expressions of GSK3β and OMA1 and thus reducing the hydrolysis of L-OPA1.