Dynamic Changes Of Amino Acid Content In Cerebrospinal Fluid Of Diabetic Cognitive Impairment

Objective:In the early stage of our research group,we used non-target metabolomics to screen the differential metabolites in cerebrospinal fluid of diabetic(DM)cognitive impairment rats,which showed that the disease was accompanied by amino acid metabolism disorder.Therefore,based on target metabonomics,this experiment dynamically monitors the amino acid content in cerebrospinal fluid of diabetic rats,comprehensively analyzes the metabolic changes of amino acids at multiple time points,screens out differential amino acids and enriches related metabolic pathways,discusses the pathogenesis from the molecular level,and provides experimental basis for finding new targets for disease prevention and treatment.Methods:The rat model of diabetes was established by intraperitoneal injection of 60 mg kg-1 streptozotocin(STZ),Glucose oxidase method was used to measure fasting blood glucose for 72 hours.Taking the blood glucose value≥16.70 mmol·L-1 as the modeling standard,the fasting blood glucose,body weight and water consumption of rats were dynamically monitored to investigate the stability of the model during the experiment;80 days after modeling,the social behavior of diabetic rats was investigated.The learning and memory ability of diabetic rats was tested by Morris water maze on 84 days.Cerebrospinal fluid was collected by cerebellomedullary cistern method at 0 d and 35 d,60 d and 94 d after modeling.Based on target metabonomics technology,23 kinds of amino acids in cerebrospinal fluid were determined by UHPLC-MS/MS technology.Principal component analysis(PCA)and cluster analysis were used to screen out the differential amino acids,and the related pathways of the differential amino acids were analyzed through MetaboAnalyst/KEGG website.Results:The modeling rate of DM model group was 91.67%.Compared with the control group,the fasting blood glucose and water consumption in the model group increased significantly(p<0.01),while the body weight decreased significantly(p<0.01).In social behavior,compared with the control group,the contact times and contact time between the model group rats and the device rats were significantly reduced(p<0.01,p<0.05).In Morris water maze test,compared with the control group,the escape latency of the model group rats during the 5th-9th and 11th training of the location navigation was significantly prolonged(p<0.01,p<0.05),and the times of platform crossing of the model group rats were significantly reduced(p<0.01).The results of target metabonomics showed that compared with the control group,the contents of amino acids in cerebrospinal fluid of the model group had no significant change at 0 d(p>0.05).At 35 d,60 d and 94 d,the contents of L-phenylalanine,L-tryptophan,L-methionine,L-tyrosine,L-threonine,L-lysine,L-serine,3-methyl-L-histidine and L-histidine decreased significantly(p<0.01,p<0.05),the content of L-valine increased significantly(p<0.01).At 60 d and 94 d,the contents of 4-hydroxyproline and 1-methyl-L-histidine decreased significantly(p<0.01,p<0.05).After 60 days,the content of L-glutamic acid decreased significantly(p<0.05).On the 94th day,the contents of L-proline and L-ornithine increased significantly(p<0.01,p<0.05).The results of PCA and cluster analysis showed that the amino acid contents of the model group and the control group were significantly different on the 60th day.Combined with the amino acids with differences on the 94th day,11 downregulated amino acids(L-phenylalanine,L-tryptophan,L-methionine,etc.)and 1 up-regulated amino acid(L-valine)were selected.Among them,L-valine,4-hydroxyproline,1-methyl-Lhistidine and 3-methyl-L-histidine may be closely related to cognitive impairment,and six metabolic pathways may be related to the pathogenesis of cognitive impairment,including phenylalanine,tyrosine and tryptophan biosynthesis,valine,leucine and isoleucine biosynthesis,phenylalanine metabolism,histidine metabolism,cysteine and methionine metabolism,glycine,serine and threonine.Conclusion:The contents of some amino acids in cerebrospinal fluid changed significantly at different time points after 35 days of rat modeling,and 11 down-regulated amino acids and 1 up-regulated amino acid were screened out.Among them,L-valine,4hydroxyproline,1-methyl-L-histidine and 3-methyl-L-histidine may be closely related to cognitive impairment.Metabolic pathways involving different amino acids suggest that DM rats may induce oxidative stress,inflammatory reaction and abnormal neurotransmitters,which may lead to central nervous system diseases,and then lead to cognitive decline.