The Effect And Mechanism Of Mitochondrial DNA Copy Number Variation On Ischemic Stroke

Background and PurposeIschemic stroke(IS)refers to a kind of disease with corresponding neurological dysfunction due to stenosis or occlusion of cerebral feeding arteries(carotid artery and vertebral artery)and insufficient cerebral blood supply.The pathological mechanism of IS is extremely complicated.Following cerebral ischemia,a series of cascades of damage such as oxidative stress,energy disorder,excitotoxicity,acidosis,inflammatory response,calcium dyshomeostasis,and apoptosis will be produced in nerve cells,eventually leading to central nervous system dysfunction.Mitochondria are the main site of oxidative phosphorylation and ATP synthesis in cells,and it is also the center of energy metabolism in the body.When IS occurs,the mitochondrial balance mechanism is broken,and related signal pathways are activated,which eventually leads to cascade damage to the nerve cells and dysfunction of central nervous system.Therefore,taking mitochondria as an entry point to explore IS can not only explain the pathogenesis of IS from a new perspective,but also provide a basis for future treatment strategies of IS.Mitochondria are the main site for the generation of reactive oxygen species(ROS).Mitochondrial DNA(mtDNA)has no histone protection,lacks antioxidant mechanisms and effective repair systems,and is vulnerable to attack by ROS,which in turn causes mtDNA mutations and mitochondrial dysfunction.Owing to a large amount of unsaturated fatty acids,the antioxidant capacity is weak in the brain tissue.Oxidative stress damage through the whole pathological process of IS.Our team tested ROS oxidation products hydrogen peroxide(H2O2),catalase(CAT),superoxide dismutase(SOD),and malondialdehyde(MDA)in the early stage,and found that significant oxidative stress reaction exists in the acute phase of IS.H2O2 and CAT were independent risk factors and protective factors of IS respectively.It has been reported that oxidative stress can change the copy number of mtDNA and affect the function of mitochondria.The number of mtDNA in the mitochondrial genome is called mtDNA copy number.Each mitochondrion contains 2-10 mtDNA copy number,and the normal mtDNA copy number of each somatic cell is about 103-104.mtDNA copy number has a certain specificity in different types of tissue cells.At present,the relationship between oxidative stress and mtDNA copy number in patients with IS is rarely reported.Displacement loop region(D-loop)of mtDNA is the only non-coding region in human mitochondrial genome,with about 1122 bp,accounting for 6%of all mtDNA molecules.It participates in and regulates the replication and transcription of mtDNA.As the binding site between mtDNA and mitochondrial membrane,D-loop region,which is rich in A and T bases,is sensitive to oxidative stress.The ratio of base substitution is 6-8 times higher than that of other regions of the mitochondrial genome,which is likely to cause gene mutations and further affect mtDNA replication and transcription,causing changes of mtDNA copy number.In summary,this study proposes the following hypothesis:activation of oxidative stress and high mutations in the mitochondrial D-loop region will cause mtDNA copy number variation in IS patients,and participate in the pathological process of IS by affecting the function and stability of mitochondrial respiratory chain.Therefore,this study was divided into three parts:In the first part,the mtDNA copy number of patients with IS and the normal control was tested to explore the variation level of mtDNA copy number in the IS population,and to explore the correlation between mtDNA copy number of patients with IS and oxidative stress and mitochondrial D-loop region mutation.The second part was to simulate the pathological process of cerebral ischemia/reperfusion in IS to construct an oxygen-glucose deprivation and reperfusion injury(OGD/R)model,verify the population results in a cell model,and find the genes that regulate mtDNA copy number changes in IS.In the third part,the knockdown-overexpression cell model of TFAM gene was constructed,and the influence of mtDNA copy number variation on mitochondrial function was explored by testing the enzyme activity of mitochondrial respiratory chain complex,mitochondrial membrane potential and ATP production.In conclusion,this study explored the role of mtDNA copy number variation in the occurrence and development of IS and its related mechanisms from two aspects of population samples and in vitro cell model,providing new evidence and new ideas for the pathological mechanism of the occurrence of IS and the prevention and treatment of the population.Materials and MethodsPart ? Study on mtDNA copy number variation in patients with ischemic stroke1.Subjects:Case group:From April 2018 to August 2018,101 patients with IS who were hospitalized in the First Affiliated Hospital of Henan University of traditional Chinese medicine and diagnosed with age?65 years old.Control group:101 healthy individuals matched with IS group in gender and age were selected,excluding those with family history of hypertension,cardiovascular disease and diabetes.2.DNA was extracted from the peripheral blood of the subjects,and the mtDNA copy number levels of IS patients and normal controls were detected by quantitative real-time PCR(qPCR)Part ? Study on mtDNA copy number variation and its regulatory mechanism in OGD/R cell model1.PC 12 cells were used to construct an OGD/R model,the cell viability levels at different OGD/R treatment time points were detected by CCK8 to determine OGD/R modeling time.2.Total DNA was extracted from PC 12 cells,and mtDNA copy number levels in OGD/R group and control group were detected by qPCR.3.Total proteins were extracted from cells of OGD/R group and control group,and the expression level of HSP60 protein was detected by Western blot.4.Total RNA was extracted from cells,and reverse-transcribed into cDNA.qPCR was used to detect the relative expression levels of mRNA of TFAM,POLG,TWNK and PGC-1? genes involved in the regulation of mtDNA replication and transcription in cells of OGD/R group and control group.Part ? Construction of TFAM gene knockdown-overexpression cell model to investigate the effect of mtDNA copy number variation on mitochondrial function1.We constructed TFAM gene knockdown-overexpression plasmid to transfect into HEK 293T cells,extracted RNA and protein from the cells,and detected the plasmid construction effect and transfection efficiency by qPCR and Western blot.2.The activity of the mitochondrial respiratory chain complex I,II,III and IV in different transfection groups was detected by using the mitochondrial respiratory chain complex activity detection kit.3.Mitochondrial membrane potential detection kit(JC-1)was used to detect the changes of mitochondrial membrane potential in different transfection groups.4.ATP detection kit was used to detect the levels of ATP production in cells of different transfection groups.ResultsPart ? Study on mtDNA copy number variation in patients with ischemic stroke1.The level of mtDNA copy number in the IS group was significantly lower than that in the normal control(P<0.05).Gender stratification results showed that the mtDNA copy number of IS patients in male group was significantly lower than that in normal control group(P<0.05),while there was no statistically significant on mtDNA copy number between IS patients in female group and control group(P>0.05).Age stratification results showed that the mtDNA copy number of IS patients in the>50 years old group was significantly lower than that in the normal control group(P<0.05),while there was no statistically significant between the IS group and the control group in the?50 years old group(P>0.05).2.The results of correlation analysis showed no correlation between oxidative stress indexes of H2O2,CAT,SOD,MDA and mtDNA copy number.3.The mutation ratio of m.T195C mutation in D-loop region of mitochondrial genome was significantly different between IS group and control group(P<0.01).The mtDNA copy number of IS patients with D-loop region mutations was significantly lower than that of IS patients without D-loop region mutations(P<0.05).Statistical results of a single site showed that the mtDNA copy number of IS patients with m.A16215G or m.C16355A mutation was significantly lower than that of IS patients without D-loop region mutations(P<0.05).Part ? Study on mtDNA copy number variation and its regulatory mechanism in OGD/R cell model1.Cell viability test results at different time points after OGD/R treatment showed that cell viability was reduced to 60%after 4 h of OGD,and cell viability was reduced to the lowest after OGD 4 h/R 4 h.OGD 4 h/R 4 h was the optimal modeling time for the model of OGD/R.2.The mtDNA copy number of cells in the OGD/R group was significantly lower than that in the control group(P<0.05),which was consistent with the results of the population study.3.There was no difference in HSP60 protein expression between OGD/R group and control group(P>0.05).4.The relative mRNA expression level of TFAM gene in the OGD/R group was significantly lower than control group(P<0.05),and the relative mRNA expression levels of POLG,TWNK and PGC-1? were not statistically significant between the two groups(P>0.05).TFAM gene may regulate mtDNA copy number variation in OGD/R cell model.Part ? Construction of TFAM gene knockdown-overexpression cell model to investigate the effect of mtDNA copy number variation on mitochondrial function1.After transfection of HEK 293T cells with TFAM gene knockdown and overexpression plasmids,the expression levels of TFAM gene and protein were significantly decreased or increased in different groups(P<0.05),and the mtDNA copy number mutant cell model was successfully constructed.2.In transfected knockdown TFAM plasmid(sh-TFAM)cells,the activity of mitochondrial respiratory chain complex ? and ? was decreased significantly(P<0.05);however,in transfected overexpressed TFAM plasmid(OE-TFAM)cells,the activity of respiratory chain complexes ? and ? was increased(P<0.05).3.Compared with the control group,the mitochondrial membrane potential significantly decreased in the sh-TFAM group(P<0.05),and significantly increased in the OE-TFAM group(P<0.05).4.Compared with the control group,the ATP generation level was significantly decreased in the sh-TFAM group(P<0.05),and significantly increased in the OE-TFAM group(P<0.01).Conclusions1.There is mtDNA copy number variation in IS patients.The mutations of m.A16215G and m.C16355A in the D-loop region of mitochondrial genome can cause the decrease of mtDNA copy number in IS patients.2.The decrease of TFAM gene expression level in the OGD/R cell model is associated with mtDNA copy number variation.3.The TFAM gene regulates mtDNA copy number variation,thereby affecting the the activity of the mitochondrial respiratory chain complex,membrane potential levels and ATP production,eventually leading to mitochondrial dysfunction and participating in the pathological damage of IS.