Mechanisms Of Mitochondrial Gene Atpif1 In The Development Of High Altitude Polycythemia

High altitude polycythemia?HAPC?is a chronic high altitude disease with the highest morbidity and the greatest harm among the inhabitants of high altitude,most of patients are migrants from plateau,especially those who live at altitude higher than4000 meters for a long time and the highest incidence of HAPC is 85.7%.HAPC can cause multi-system and multi-organ damage,especially cardiovascular system and nervous system,which seriously endangers the health of permanent staff at high altitude,due to the slow blood stasis caused by excessive proliferation of red blood cells and hemoglobin,aggravation of tissue hypoxia,thrombosis and embolism.Relevant studies have found that the occurrence of HAPC is the result of interaction between many genes or genes and hypoxia.Therefore,this reserach assumes to explain the mechanism of HAPC from the perspective of gene,and study the mechanism of Atpif1 gene in the occurrence of HAPC.Mitochondrial adenosine triphosphate inhibitor-1?Atpif1?is an endogenous small molecule polypeptide regulating mitochondrial F1F0-ATP synthase.It is main function is to inhibit ATP hydrolysis,avoid ATP depletion,maintain mitochondrial membrane potential and electrochemical proton dynamic potential energy,and play an important role in energy metabolism regulation.Recent studies have found that mitochondrial Atpif1 is an important regulator of ferrous chelating enzyme activity,which can effectively regulate the synthesis of heme and hemoglobin.But can Atpif1effectively regulate the synthesis of heme and hemoglobin,whether erythrocyte overproliferation is related to the overexpression of it in hypoxic environment?Further study of Atpif1 may further expand the pathogenesis of HAPC and explore new targets or molecular markers for prevention and treatment of HAPC.Objective:1.To observe the expression of genes in HAPC model rats and to reveal the effects of hypobaric hypoxia.2.To observe the effects of proliferation,apoptosis,hemoglobin synthesis and gene expression on K562 cells in hypoxia environment,to understand the effect of hypoxia on K562 cells and to reveal the mechanism of HAPC.3.After atpif1 gene was silenced by siRNA,to observe the proliferation,hemoglobin synthesis and the content changes of related genes in K562 cells.On this basis,the pathogenesis of HAPC was explored and new ideas for clinical treatment were provided.Methods:1.The rat model of HAPC was established after 4 weeks in hypobaric hypoxia chamber for 8 hours and altitude of 5000 m.The changes of hemoglobin content in hypoxic experimental group and normoxic control group were detected.The mRNA expression of Atpif1,NF-?B,Alas2,Fech,Hmox1 genes in cardiopulmonary tissue were detected by qRT-PCR,and protein expression of Atpif1,NF-?B and Alas2 gene was detected by Western Blot.2.K562 cells were divided into hypoxic experimental group and normoxic control group.K562 cells were cultured in hypoxic incubator?2%O₂,5%CO₂,93%N₂,37??and normoxic incubator?21%O₂,5%CO₂,37??,respectively.Cell viability was detected by CCK8 assay,the effect of hypoxia on cell apoptosis was detected by flow cytometry,and the effect of hypoxia on was detected by hemoglobin chloride-induced hemoglobin synthesis in K562 cells.Quantitative qRT-PCR was used to detect the mRNA expression of Atpif1,NF-?B,Alas2,Hmbs and Hmox1,and WB was used to detect the protein expression of Atpif1 and NF-?B.3.Silencing mitochondrial Atpif1 gene in K562 cells by Lipofectamine 3000.Negative control group?NC?and blank control group?Blank?were set up.The transfection efficiency was detected by qRT-PCR and WB.Cell proliferation,hemoglobin synthesis,expression of NF-?B,Alas2,Hmbs,Hmox1,Epor mRNA and the protein expression of NF-?B in K562 cells were also observed.Results:1.After detecting the HAPC models of SD rats constructed in hypobaric hypoxia chamber,we found that compared with the normoxic control group,the weight of the hypoxic experimental group decreased,the hemoglobin increased significantly,and the mRNA expression of Atpif1,NF-?B,Alas2,Fech and Hmox1 in cardiopulmonary tissue increased,and the expression of Atpif1,NF-?B and Alas2protein increased.2.The K562 cells were constructed by hypoxic incubator.Compared with the normoxic control group,the activity of K562 cells in the hypoxic experimental group decreased,cells apoptosis increased,and the content of hemoglobin increased in a time-dependent manner.The expression levels of Atpif1,Alas2,NF-?B,Hmbs and Hmox1 mRNA were up-regulated,and the expression levels of Atpif1 and NF-?B protein were also up-regulated.3.Compared with NC and Blank groups,the experimental group showed that silencing mitochondrial Atpif1 gene could inhibite the proliferation of K562 cells,reduce the elevated hemoglobin and down-regulate the levels of NF-?B,Alas2,Hmbs,Hmox1,Epor mRNA and NF-?B protein in the erythroid differentiation model of K562 cells.Conclusion:1.Constructing a stable animal model of HAPC,the expression level of related genes in HAPC rats was up-regulated.2.Hypoxic intervention could reduce the activity of K562 cells,promote cells apoptosis,hemoglobin synthesis,and increase the mRNA expression and protein expression of genes regulating erythroid differentiation.It also indicated that hypoxic intervention could successfully construct erythrocyte differentiation model.3.After silencing Atpif1 gene,the hemoglobin content in K562 cells decreased,and the levels of NF-?B,Alas2,Hmbs,Hmox,Epor mRNA and NF-?B protein also decreased.Mitochondrial Atpif1 gene might be involved in regulating HAPC together with the signal transduction pathway of NF-?B.