| Background and purposeTraumatic brain injury(TBI)is a brain injury caused by direct or indirect external force and can be divided into primary injury and secondary injury.With the development of society,the incidence of global TBI is increasing year by year.WH0 estimates that by 2020,TBI will become the third leading cause of death and disability worldwide.The pathogenesis of TBI is complicated.At present,clinical diagnosis and treatment mainly depends on the patient's clinical manifestations and imaging findings,but the method of diagnosis and treatment has great limitations,and it is still a difficult point in clinical diagnosis and treatment.In the field of forensic science,the identification of TBI is mainly the determination of damage degree and the inference of damage time.The traditional identification method is mainly pathological morphology examination.However,the accuracy of this method is not enough.There is no accurate judgment method at present,so it is also one of the difficulties in forensic identification.Therefore,it is important to study the pathophysiological mechanism of TBI and find biomarkers that can be used to formulate treatment plans,determine the extent of injury,and infer the time of injury.Metabolomics is one of the hot spots in the field of medical research in recent years.It can directly reflect the changes of metabolites in organisms and explain the physiological and pathological state of the body.Now it has been widely applied to much Important fields such as pathophysiology,gene omics,nutrition environmental science and microbiology pharmacology.In this research,a rat brain injury model was established,then,a comprehensive metabolomics analysis on brain tissue and blood of the TBIand control rats was carried out based on the platform of ultrahigh performance liquid chromatography coupled to a high-resolution Orbitrap mass spectrometer(UHPLC-Q Exactive),which aimed at discovering characteristic biomarker for the diagnosis of craniocerebral injury,the degree of damage judgment and the inference of injury time.Method1 Sample collection and preparation: We purchased 100 healthy SD rats(SPF-class250g-300 g,male and female)from the Experimental Animal Center of Zhengzhou University.The rats were randomly divided into eight groups.The expriment group only used the Feeney method to make the rat brain injury model.The control group were consistent with the experimental group,only did not take the striking measures.The brain tissue and blood of the experimental group were collected at 1h,6h,12 h,24h,3d,7d and 14 d after the injury.The samples were extracted with polar and non-polar components using an organic solvent.The supernatant was lyophilized and placed in a-80 degree refrigerator for ultra-high liquid chromatography-high resolution mass spectrometry.2 metabolomics analysis: Ultrahigh performance liquid chromatography coupled to a high-resolution Orbitrap mass spectrometer was used to detect the polar and non-polar components of the sample,respectively,to obtain the metabolomics raw data files of brain tissue and blood in the control and experimental groups.Raw data files were imported into SEVIE 2.1 for pre-processing,such as peak identification,peak alignment,baseline correction,and peak area normalization,followed by peak table data.We imported peak and table data from the experimental and control groups into OSI/SMMS to identify differential metabolites and qualitatively analyze them.SIMCA-P 13.0 was used to perform pattern discriminant analysis on qualitative data,analyze different metabolic profiles of brain tissue and blood between experimental and control groups,and then select the different metabolites.Finally,the pathway analysis of metabolites with significant difference was processed via the Metabo Analyst database.Result1 Compared with the control group,the rats in the experimental group showed increased heart rate,shallow breathing and even short-term apnea,and then recovered spontaneously.2 This study successfully constructed the OPLS-DA model for the brain tissue and blood of the control and experimental groups(1h?6h?12h?24h?3d?7d?14d),and screened and identified 43 differential metabolites.The study found that there are 26 differential metabolites in brain tissue,of which 14 differential metabolites are up-regulated and 12 are decreased.There are 17 differential metabolites in the blood,9 of which are up-regulated and 8 are decreased.3 The pathway analysis showed that 5 metabolic pathways were mainly involved by the differential metabolites between the TBI group samples and the control group samples: Phenylalanine,tyrosine and tryptophan biosynthesis,Arachidonic acid metabolism,Tyrosine metabolism,Arginine and proline metabolism,Purine metabolism.ConclusionIn this research,the metabolomics approach based on the platform of ultrahigh performance liquid chromatography coupled to a high-resolution Orbitrap mass spectrometer(UHPLC-Q Exactive)revealed the detail information of metabolic profiles of TBI 1h?6h?12h?24h?3d?7d?14d group and control group of brain tissue and blood samples,screening and identifying 43 differential metabolites,of which there are 26 differential metabolites in brain tissue and 17 differential metabolites in the blood.This study found that compared with the control group,rats with brain injury may have disorderes of Phenylalanine,tyrosine and tryptophan biosynthesis,Arachidonic acid metabolism,Tyrosine metabolism,Arginine and proline metabolism,Purine metabolism,and associated with the pathological mechanism of brain injury.The results of this study may contribute to the diagnosis of craniocerebral injury,the judgment of the degree of brain injury and the time inference of brain injury. |
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