| BackgroundThe body damage caused by plateau environment has become one of the major public health problems at present.The Qinghai-Tibet Plateau plays an extremely important role in national defense strategy and economic construction.The hypoxia hypobaric environment in the Plateau seriously damages human health.Plateau hypoxia acclimatization refers to non-genetic changes that occur after entering the plateau for a period of time to adapt to the plateau environment.It is of great significance to rapidly promote the acclimatization of plateau hypoxia and reduce the damage of plateau hypoxia to the body.The mitochondria are the center of cell oxygen consumption and energy metabolism,and their functional changes are closely related to oxygen utilization and energy production.Mitochondrial DNA(mtDNA)has a high mutation rate and copy numbers,which plays an important role in regulating mitochondrial function.mtDNA mutation may be a key factor in regulating hypoxia acclimatization at high altitude.However,it is not known what the mtDNA variation characteristics of people acclimatized to hypoxia at high altitude are and whether it can promote hypoxia acclimatization at high altitude by regulating the mtDNA variations.Coenzyme Q10 exists widely in human tissues and plays an important role in mitochondrial oxidative phosphorylation.Oxidative stress and mtDNA changes are the key factors affecting plateau hypoxia acclimatization,and coenzyme Q10 has antioxidant function and can protect mtDNA by reducing the level of reactive oxygen species.Therefore,coenzyme Q10 may be the key drug to promote plateau hypoxia acclimatization.ObjectivesThe purpose of this project is to provide potential solutions for solving the public health problem of body damage caused by plateau environment,to provide possible methods for promoting rapid acclimatization of the human body to high-altitude environment,and to provide scientific and powerful guidance for people entering plateau environment.Specifically,it is divided into:1.Collect the clinical information and peripheral blood samples of individuals with different altitude acclimatization status and then use the Next Generation Sequencing methods to accurately detect peripheral blood plasma mtDNA and obtain sequencing data;2.Through the analysis of peripheral blood plasma mtDNA sequencing data,find the mtDNA variation characteristics related to hypoxia acclimatization at high altitude;3.On the basis of the mtDNA variations which promote hypoxia acclimatization at high altitude,initially explore the drug to promote hypoxia acclimatization at high altitude.MethodsThe Han people with poor plateau acclimatization and the matched Han people with good plateau acclimatization were selected from the 95 th Military Hospital,and the plain healthy control population were selected from the Xijing Hospital.Then collect the data and the peripheral blood samples of objects of the study and accurately obtain the mtDNA sequencing data of the peripheral blood of the subject through extracting DNA,constructing library,targeted capturing mtDNA and mtDNA precise sequencing.The quantity and frequency of mtDNA mutations in peripheral blood were analyzed after the quality control test of sequencing data;The fragmentation sizes,copy number levels,gene mutation sites and haploid differences were analyzed;On the basis of these results,the promotion effect of coenzyme Q10 on hypoxia acclimatization at high altitude was preliminarily explored.Construct the mouse models of plain control,plateau control and oral coenzyme Q10 plateau.Then we detect the cardiac function,myocardial injury,cardiomyocyte apoptosis,myocardial mitochondrial reactive oxygen species level and myocardial mtDNA copy number in mice.Results1.The processing method,time and storage temperature of plasma samples are crucial to the accurate detection of mtDNA.The storage of blood or plasma samples at room temperature(RT)or 4℃ will affect the detection of mtDNA.The difference was statistically significant differences(P<0.001).The storage time of blood or plasma at-80℃ had no significant effect on mtDNA detection,and the difference was not statistically significant(P=0.654,P=0.592).2.The operations which were under the same person’s different batches and the same conditions had no effect on the accurate detection of mtDNA in peripheral blood plasma or blood cells,and the difference was not statistically significant(P>0.05).3.The frequency and number of plasma mtDNA mutations in the plain healthy control group,good altitude acclimatization group and poor altitude acclimatization group were significantly higher than those in blood cells.The difference was statistically significant(P<0.01).The number and frequency of plasma mtDNA mutations in the good altitude acclimatization group or poor altitude acclimatization group were significantly higher than those in the plain healthy control group.The difference was statistically significant(P<0.01).While there was no significant difference in the number and frequency of plasma mtDNA mutations between the well acclimatized and poorly acclimatized groups at high altitude(P=0.634,P=0.161).4.There were differences in plasma mtDNA fragmentation levels among the three groups.The level of plasma mtDNA fragmentation in the good plateau acclimatization group was higher than that in control group,and the level of plasma mtDNA fragmentation in the poor plateau acclimatization group was further increased.The plain healthy control group vs the good plateau acclimatization group(P=0.0097),the good plateau acclimatization group vs the poor plateau acclimatization group(P=0.0089).5.Compared with the plain healthy control group,the mtDNA copy number level of the good plateau acclimatization group was higher(P<0.0001),while the mtDNA copy number level of the poor plateau acclimatization group was lower(P=0.002).6.The frequency of mutations at G7697 A,T3394C,T16519 C,T5543C and other sites and the level of haploid D and M9 mutations in plasma mtDNA of the good plateau acclimatization group increased,which suggests that they may be a favorable factor for high altitude hypoxia acclimatization.While the mutation frequency of A4576 G,A3397G,G3552 A,T16172C and other sites and the level of haploid M7 and B mutations in the plasma mtDNA of the poor plateau acclimatization group disorder increased.The difference is statistically significant(P<0.0001).7.After oral administration of coenzyme Q10,the cardiac function of mice exposed to hypoxia at high altitude was significantly improved compared with that of control mice,the myocardial injury was alleviated,the level of myocardial cell apoptosis and ROS was decreased,and the level of mtDNA copy number was increased.The difference was statistically significant(P<0.01).Conclusions1.Processing time and storage temperature of peripheral blood samples are important for accurate detection of plasma mtDNA,while freezing time and different batches of treatment have no significant impact on the experimental results.2.The number and frequency of mtDNA mutations in peripheral blood plasma are more than those in blood cells and in plateau population than in plain population.3.The hypoxia environment at high altitude leads to the increase of mtDNA fragmentation level4.The mtDNA copy number of the good plateau acclimatization group is higher than that of he plain healthy control group,while the mtDNA copy number of the poor plateau acclimatization group is lower.5.Mt DNA fragmentation level,mtDNA copy number variation level,mtDNA gene mutation and haploid are closely related to altitude acclimatization,which may be the promotion or susceptibility factor of altitude acclimatization.6.Coenzyme Q10 may promote hypoxia acclimatization at high altitude by regulating mitochondrial level. |
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