| Rationales:Depression is a common psychopathological state or mood disorder syndrome.The serious harm to human life and the inability of existing antidepressant drugs force us to understand the pathological mechanism of depression from a new perspective.A reasonable depression model is the key to studying the pathogenesis of depression.The in vitro cell model is widely used in the study of depression mechanisms because of its stability and reliable results.Cortical ketone(CORT)-induced PC12 cell damage is based on depression.The in vitro depression model established by the theory of thalamus-pituitary-adrenal axis activation has the advantages of being easily available and sensitive to external stress,and can more realistically reflect the state of the body's nervous system under external stimulation,so this study intends to adopt Corticosterone-induced PC12 cell damage model is used as an in vitro cell model to study depression.The research group found that the pathogenesis of depression and doxorubicin nephropathy are related to energy metabolism disorders in the early stage of the research group.What are the specific metabolic pathways and specificities of depression leading to energy metabolism disorders? Metabolomics technology cannot solve the above two problems,because metabolomics can make a systematic response to subtle metabolic changes and identify potential biomarkers,but metabolomics detects the complex metabolism of compounds at the end of biological reactions in the body after the sum,and each type of substance involved in the body's energy metabolism has multiple metabolic pathways,the results of metabolomics cannot be accurately determined to specific metabolic pathways.Stable isotope tracing technology can target atoms in the metabolic pathway of the targeted atomic labeling,using mass spectrometry analysis,through the tracer atoms to track the movement of the labeled compound in the body,fromthe isotope peak of the intermediate product distribution to judge specific metabolic pathways,and to accurately track the trace of the activity of trace compounds in the body can just make up for the defects of metabolomics,and is particularly suitable for the study of metabolic pathways and metabolic mechanisms.In this study,corticosterone-induced PC12 cell damage model was used as the research object,adriamycin-induced glomerular podocyte damage model was used as the comparison object,and 13C6-glucose was used as the tracer.The detection technology optimizes the time,concentration and sample preparation method of stable isotope incubation cells.At the same time,it combines the metabolomics technology to study the metabolic characteristics of the depression cell model in the energy metabolism network and clarify the specific mechanism of energy metabolism disorder.Objective:1.Based on the high-resolution LC-MS analysis technology,the cell sample pretreatment method was investigated.Based on the successful replication of the two in vitro cell models,the LC-MS metabolomics technology was used to study the two in vitro cell models to initially explore energy metabolism different metabolites on the glucose metabolism pathway in the network,find the energy metabolism disorder mechanism existing compared with the normal group,and clarify the energy metabolism pathway disorder status of each model.2.Using LC-MS analysis technology,taking the total ion current map and the ratio of isomer signal abundance of key intermediate metabolites as reference indicators,the optimal concentration of13C6-glucose stable isotope tracer introduced into the cell and the incubation time of the cell were clarified.3.Based on the results of metabolomics research,the energy metabolism precursor 13C6-glucose was used as a stable isotope tracer to analyze the metabolic pathways of isotopes in two in vitro cell models,and found that the specific energy metabolism disorder caused by depression sexual metabolism pathways explain the energy metabolism disorder mechanism of depression,and provide a reference for the development of antidepressant drugs based on the regulation of energy metabolism.Method:1.Based on the LC-MS analysis method,five sample pretreatment methods for PC12 cells were investigated.The five pretreatment methods were: 33% methanol quenching-ultrasonic disruption of cells-70%acetonitrile reconstitution,33% methanol quenching-ultrasound+repeated freeze-thaw cell disruption-70% acetonitrile reconstitution,33%methanol quenching-ultrasonic cell disruption-70% methanol reconstitution,80% acetonitrile quenching-ultrasonic cell disruption-70%acetonitrile reconstitution,80% acetonitrile quenching-ultrasonic disruption of cells-70% methanol reconstitution.At the same time,based on the successful replication of the two in vitro cell models,metabolomic technology was used to conduct metabolic studies on the two cell models to find differential metabolites and key metabolic pathways.In order to use tracer technology to further clarify the energy metabolism-related The mechanism of action provides the basis.2.The introduction of 13C6-glucose while the cells are in an exponential growth phase.According to the total ion chromatogram and the isomeric signal abundance ratio of key intermediate metabolites,the stable isotope concentration(5,10,15,20,25 m M)and tracer time(12,24,36,48 h).3.Disordered energy metabolism pathway discovery and specificity:13C6-glucose is used as a stable isotope tracer,and the isotope data processing module “the workflow Stable Isotope Labeling with Metabolika Pathways and ID” in LC-MS and Compound Discoverer 3.0software is used.To clarify the metabolic process of labeled precursors in the energy metabolism network.Results:1.By groping through five pretreatment methods,the peak area of some intermediate metabolites(fumaric acid,lactic acid,malic acid,pyruvic acid,glucose)on the energy metabolism pathway was used as an indicator,combined with the LC-MS total ion flow diagram to determine The optimal pretreatment conditions were first: quenched with 33%methanol,and then disrupted the cells with ultrasound(ultrasound for 5 s,pause for 9 s,for 15 min),and finally reconstituted with 70% acetonitrile.Through metabolomics,23 different metabolites of PC12 cell damage model caused by corticosterone were found,involving 14 metabolic pathways,and 21 different metabolites of glomerular podocyte injury model caused by adriamycin,involving 15 metabolic pathways,among the two in vitro cell models,there are 8 differential metabolites,which are glucose,pyruvate,lactic acid,glutamic acid,leucine,taurine,4-hydroxybutyric acid,and alanine.Nine items are pyruvate metabolism,tricarboxylic acid cycle(TCA cycle),glycolysis/gluconeogenesis,glutamine and glutamate metabolism,alanine,aspartic acid and glutamate metabolism,ammonia Acyl-t RNA biosynthesis,taurine metabolism,glycine,serine and threonine metabolism,arginine biosynthesis.Many different metabolites and multiple metabolic pathways are distributed in the energy metabolism network.2.Based on the total ion chromatogram and the isomeric signal abundance ratio of key intermediate metabolites,the stable isotope concentration and tracer time were investigated,and the optimal stable isotope concentration was 10 m M and the optimal incubation time was determined for 24 h.3.Through stable isotope tracing studies,it was found that the two in vitro cell models have some metabolic differences in the energy metabolism network.For example,in the model of PC12 cell damage induced by corticosterone,the signal abundance of glucose M+3 and malate M+3 accounted for a significant proportion Higher than the blankgroup,indicating that the gluconeogenesis pathway is activated and is activated by increasing the flux of pyruvate to malate,and pyruvate M+3and pyruvate are found in the model of adriamycin-induced glomerular podocyte injury.The proportion of malic acid M+3 signal abundance was significantly lower than that of the blank group,indicating that the gluconeogenesis pathway was inhibited and was reduced by reducing the flux of pyruvic acid to malic acid.In addition,the proportion of M+4signal abundance of malic acid,fumaric acid and succinic acid in the model of PC12 cell damage induced by corticosterone was significantly lower than that in the blank group.The M+2 of fumaric acid in the model of adriamycin-induced glomerular podocyte injury The proportion of M+2 signal abundance of succinic acid was significantly higher than that of the blank group,while the proportion of M+4 signal abundance of malate in the model group was significantly lower than that of the blank group,indicating that the two in vitro cell models.The tricarboxylic acid cycle metabolism is inhibited.The key node of the tricarboxylic acid cycle disorder in the model of adriamycin-induced glomerular podocyte damage may occur in the metabolic pathway of the conversion of fumaric acid to malic acid,while corticosterone causes PC12 cell damage.The model leads to the disorder of its tricarboxylic acid cycle may occur in the upstream pathway of fumaric acid.4.Through stable isotope tracing technology,we also found that the metabolism of the two cells has some similarities.For example,in two in vitro cell models,adenine base synthesis is reduced and adenine nucleoside triphosphate(ATP)level is reduced;by observing urine to the extent that nucleotides such as pyrimidine and guanine were labeled with stable isotopes,it was found that the adenylate metabolism pathways of both in vitro cell models were inhibited,which was consistent with the results of cell viability and lactate dehydrogenase release rate test.Conclusion:This study first explored the energy metabolism disorder pathwaysof two in vitro cell models through metabolomics,and then studied the specific mechanism of energy metabolism disorder caused by corticosterone-induced PC12 cell damage model by stable isotope tracing technology: tricarboxyl in the model group the acid cycle,gluconeogenesis,glycolysis,and adenine nucleoside triphosphate production pathways are disrupted,such as the tricarboxylic acid cycle,glycolysis,and glycolysis are inhibited.The gluconeogenesis pathway is converted to apples by increasing pyruvate acid flux to activate. |
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