Metabolomics Study On Hippocampus And Serum Of Hypoxic Preconditioning Mouse

BackgroundOxygen is essential for the maintenance of life activities,a variety of reasons of oxygen supply or utilization disorder,can lead to hypoxia.Brain is the most sensitive tissue of the body to hypoxia,because it is extremely dependent on aerobic metabolism.long or short time of severe hypoxia can cause central nervous system dysfunction,and even pathological changes.Therefore,how to prevent hypoxic injury to the brain,to find the measures of tolerance to hypoxia for the body,which are very meaningful to enhance the ability to adapt to hypoxic environment and the prevention or treatment of clinical hypoxic disease.Hypoxic preconditioning(HPC),motivated by the repetitive exposure of organism,organ,tissue,and cell to hypoxia is a strategy for the intrinsic cytoprotection,widely existing in the heart,brain,kidney,liver,intestine,and other organs.As an endogenous protective mechanism,the discovery of this phenomenon reveals new possible mechanisms to enhance the ability to adapt to hypoxia.Previous study in our lab reveals: 1)HPC present significant protective effect in mice hippocampal neuron against hypoxia,and can improve study and memory ability of mice during hypoxia.2)The hypoxia tolerance time is increased by exposed times to hypoxia in acute repetitive HPC mice.With repeated exposure of mice to hypobaric hypoxia,the tolerance time of the mice in the 2nd,3rd and 4th runs were 2,4 and 6 times longer than that of lst run respectively.We speculate that some anti-hypoxia active substances in body increase after repeat exposure to hypoxia.These substances show dose-effect relationship in hypoxia tolerance.3)We find that both protein and non-protein component of the homogenate of HPC mice hippocampus can improve hypoxia tolerance of NGF induced PC12 cell,it is indicated that this homogenated play an important role to prevent the damage of neuron induced by hypoxia,and micromolecule metabolite may be participate in this process.Metabolomic is the scientific study of metabolome.The metabolome represents the collection of all metabolites in a biological cell,tissue,organ or organism,which are the end products of cellular processes.As the termination of genomic,transcriptomic and proteomic,metabolomic can reflect the physic status of body more directly.This study aims to observe the dynamic change of metabolomic using HPC mice model.Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOFMS)is used to identify the difference of metabolome in hippocampus and serum between HPC and control animal.This study can provide theory background in hypoxia protection research.Method:Part 1: Metabolomic research on the hippocampus of HPC mouse1.30 male BALB/C mice were randomly divided into 3 group,normoxic control group(H0),hypoxia exposure 1 times group(H1),repeated hypoxia exposure 4 times group(HPC),according to reported acute repeated hypoxia HPC mice model was built.The hippocampus and serum samples were collected,metabolite profiling was performed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOFMS)in conjunction with univariate and multivariate statistical analyses;2.We select SOD activity,MDA and lactic acid concentration as indicators,compare the difference between groups in hippocampus.Part 2: Metabolomic research on the serum of HPC mouse.1.In accordance with the same animal group and copy method of HPC mouse model.Spate serum from each group,using UPLC-Q-TOFMS to screen different metabolite and execute metabolic pathway enrichment analysis based on MetaboAnalyst 3.0 database;2.We select SOD activity,MDA and lactic acid concentration as indicators,compare the difference between groups in serum.Result1.A total of 37 metabolites were identified 37 metabolites was identified,which have difference between the three groups.Dopamine,Adenosine,Phenylalanine,Taurine are significant increased in hippocampus of HPC group compares to H0 group.5-methylcytidine,Glutamate,Glutamine,Acetylcholine,Cytidine,NADH,2-Hydroxyglutarate,Uric acid,LysoPCs,Palmitic acid,Linoleic acid and Arachidonic increased in H0 group,and decreased in HPC group.Tryptophan,Sphingosine and Xanthine decreased in H0 and HPC group.Pathways involved by these substances also changed significantly,including Phenylalanine,tyrosine and tryptophan biosynthesis,Taurine and hypotaurine metabolism,Glutathione metabolism,Glyoxylate and dicarboxylate metabolism,Arachidonic acid metabolism,Linoleic acid metabolism,Tyrosine metabolism,Glycerophospholipid metabolism,Alanine,aspartate and glutamate metabolism,Tryptophan metabolism,Phenylalanine metabolism and TCA;2.SOD activity in hippocampus increased in HPC group,compare to H0 and H1 group.The MDA and lactate acid level increased in both H1 group and HPC group,but the increasing extent in HPC is less than H0 group.There is significant statistic difference between each two groups;3.10-15 difference metabolites were screened from serum under positive and negative ion pattern.In these indicators,the level of Valine,Isoleucine,Phenylalanine,Tyrosine and Taurine increased in HPC group continuously.The level of Carnitine,Methionine,Arachidonic acid,LysoPCs and Uric acid increased H1 group,and decreased in HPC group.The pathway of Phenylalanine,tyrosine and tryptophan biosynthesis,Taurine and hypotaurine metabolism,Linoleic acid metabolism and Arachidonic acid metabolism are changed simultaneously;4.SOD activity in serum increased with adding hypoxia exposure time.There is significant difference among HPC,H1 and control group.The serum MDA and lactate acid level increased in both H1 group and HPC group,but the increasing extent in HPC is less than H0 group.There is significant statistic difference between each two groups.Conclusion1.Metabolomic in HPC serum and hippocampus has significant changes.The common changed pathway includes Phenylalanine,tyrosine and tryptophan biosynthesis,Taurine and hypotaurine metabolism,Arachidonic acid metabolism,Linoleic acid metabolism,Phenylalanine metabolism and Tyrosine metabolism.The pathway of Glutathione metabolism and Alanine,aspartate and glutamate metabolism was significantly different only in the hippocampus,up-regulation /inhibition of the key metabolic enzyme in those pathways may enhance the ability of anti-hypoxia in the CNS and the body;2.The change of the glutathione and uric acid et al which can effectively clear free-radicals and biochemical test results together indicate that against oxidative stress damage may be the potential targets of the nerve protective effect to hypoxia of HPC;3.Combined with serum and hippocampal metabolic data,changes in energy metabolism model involved in the formation of hypoxic preconditioning against hypoxic injury.