| Objective:1.Analyzing differential metabolites of non-target metabolomics to screen biomarkers,to solve the problems of rapid metabolism of nitrous oxide,short detection window,and difficult detection in biological samples,which can provide scientific basis for effective control of nitrous oxide and forensic identification of cases related to nitrous oxide abuse.2.To investigate the mechanism of nitrous oxide abuse-related toxicity by using the combined multi-omics analysis of untargeted metabolomics and microbiology.Methods:1.Animal model preparation and sample collection: C57BL/6 mice were randomly divided into air control group and nitrous oxide experimental group(n=16 each).The mice were placed in a whole-body static exposure device,and set the device conditions.The experimental group mice were divided into two groups,8 mice in each group,and the concentration of nitrous oxide in the container was adjusted to 90000ppm(0.6NL/min for9min)for 28 days,2 times a day,one hour each time;The rats in the air control group were divided into two groups,8 rats in each group,and were exposed to air for 28 days,2 times a day,one hour each time.When 24 hours after the end of exposure,we collected feces,and then orbital blood.The supernatant was obtained by centrifugation,snap frozen in liquid nitrogen,and stored in the refrigerator at-80℃ until testing.2.Metabonomics study of mice exposed to laughing gas: plasma/feces samples were pretreated and detected by ultra-performance liquid chromatography-tandem time-of-flight mass spectrometry(UHPLC-Q-TOF MS)for non-target metabolomics.The raw data were preprocessed,and analyze the differences between groups using statistical software.Then screen differential metabolites,and determine biomarkers,which provided a basis for the identification of laughing gas abuse.The corresponding metabolic pathways were identified by cluster analysis,correlation analysis and KEGG database analysis.3.Microbiome study of mice exposed to laughing gas: genomic DNA was extracted from fecal samples,and the V3-V4 region of bacterial 16 S rDNA gene was amplified by PCR.The products were purified,quantified,and mixed for high-throughput sequencing.The original data were spliced,filtered,and removed to obtain effective data,and then OTUs clustering and species classification analysis were performed to obtain the effective data.Compared the structural differences of the microbiota in each sample and between groups at different taxonomic levels,we can screen the bacteria with significant differences were screened.4.Combined with the results of metabolomics and microbiome,we analyzed the relationships between microflora and metabolites,microflora and metabolites-metabolites from multiple perspectives to further explain the toxic mechanism of laughing gas abuse.Results:1.Metabolomics study of mice exposed to laughing gasKEGG metabolic pathway analysis identified 7 metabolic pathways that might be related to nitrous oxide abuse,including Glycine,serine and threonine metabolism,Phenylalanine,tyrosine and tryptophan biosynthesis,Protein digestion and absorption,African trypanosomiasis,Tryptophan metabolism,Pyrimidine metabolism and Choline metabolism in cancer.KEGG metabolic pathway analysis showed that two metabolic pathways may be related to nitrous oxide abuse,c AMP signaling pathway and Sphingolipid metabolism.A total of 35 significant differential metabolites related to laughing gas abuse were screened from the plasma samples of mice exposed to laughing gas,including lipids and lipid-like molecules,organic acids and their derivatives,and nucleoside analogues.A total of 112 significant differential metabolites related to laughing gas abuse were screened from the fecal samples of mice exposed to laughing gas,including lipids and lipid-like molecules,organic acids and their derivatives,and nucleoside analogues.According to the screening of biomarkers in plasma samples of mice exposed to laughing gas,8 differential metabolites were related to 7 significantly different metabolic pathways.1-stearoyl-2-oleoyl-sn-glycerol 3-phosphocholine(SOPC)and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine(PC(16:0/16:0))were up-regulated.DL-tryptophan,Creatine,Ectoine,Indole,His-ser and Ile-Pro were all down-regulated,affecting multiple metabolic pathways in mice.Finally,DL-tryptophan,creatine,and tetrahydropyrimidine were considered as potential biomarkers of nitrous oxide abuse.2.Microbiome study of mice exposed to laughing gasHigh-throughput sequencing of fecal samples 16 S rDNA showed that there was a significant difference in the intestinal microbiota between the experimental group and the control group.Alpha diversity showed that the species richness of the experimental group was significantly higher than that of the control group,while there was no significant difference in the diversity of the intestinal microbiota.β-diversity analysis showed that there were significant differences in the microbial flora between the experimental group and the control group,and the between-group difference was greater than the within-group difference.From the perspective of community structure and distribution,Firmicutes,Bacteroidota,Actinobacteriota,Proteobacteria,and Deferribacteres were dominant in the gut microbiota of mice.LEFSe and STMPA statistical methods were used to screen the difference in community structure between the experimental group and the control group at different taxonomic levels.The proportion of species abundance of Muribaculacea,Ruminococcaceae,Gordonibacter,Sva0081 sediment group,Erysipelatoclostridium and Mycoplasma in the experimental group were significantly higher than those in the control group.The species abundance ratios of Rikenellaceae,Tannerellaceae,Turicibacter,Parabacteroides,Bacillus,Rothia,Streptomyces and Bosea in the experimental group were significantly lower than those in the control group,which is possible that nitrous oxide abuse may affect the abundance of significantly different bacteria and lead to changes in the composition of intestinal microorganisms.Through functional prediction analysis,we found that the effects of intestinal bacteria on the metabolic pathways of mice were closely related to Carbohydrate Metabolism,Amino Acid Transport and Metabolism,Energy Metabolism,Nucleotide Metabolism,Metabolism of Cofactors and Vitamins,and Cellular Processes and Signaling,which was basically consistent with the above discussion and metabolomics results.Conclusion:1.Nitrous oxide exposure affects the normal metabolic profile of mice,and affects many metabolic pathways related to Amino acid metabolism in plasma samples,such as Glycine,serine and threonine metabolism,Phenylalanine,tyrosine and tryptophan biosynthesis,Protein digestion and absorption,African trypanosomiasis and Tryptophan metabolism.They disrupt the homeostasis of the gut,leading to an imbalance of the immune system in the gut and gastrointestinal symptoms such as nausea,and also cause nervous system damage,such as paresthesia,motor weakness,short-term memory impairment,and learning disabilities.Disorders in Glycine,serine and threonine metabolism,Pyrimidine metabolism,and Choline metabolism in cancer also lead to limited DNA synthesis,disrupted methylation of choline accumulation,and symptoms associated with megaloblastic anemia.The two metabolic pathways,Sphingolipid metabolism and c AMP signaling pathway in fecal samples were interfered,which not only affected important cell behaviors such as cell growth,differentiation,proliferation,apoptosis and metabolism,produced neurotoxicity and damaged nervous system toxicity,but also caused intestinal microbial disorders and destroyed the homeostasis of intestinal flora.This may be the cause of neurological damage such as subacute combined spinal cord lesions and peripheral neuropathy in clinical practice,further explaining the toxic effects of nitrous oxide abuse.2.Based on the non-target metabolomics data analysis of plasma samples of nitrous oxide abuse,combined with the analysis of significant differential metabolites and metabolic pathways,DL-tryptophan,Creatine,and Ectoine can be considered as biomarkers for nitrous oxide abuse.3.Nitrous oxide exposure affects the abundance of significantly different bacterial flora in mice,which leads to changes in the composition of intestinal microorganisms.On the one hand,it participates in Amino acid metabolism and other metabolic pathways,and affects signal transduction between cells in the nervous system.On the other hand,it is closely related to the health status of the body,activates the immune regulation and protection mechanism of the body,and plays an important role in cardiovascular diseases and related metabolic and immune responses.Combined with the analysis of fecal metabonomics data and fecal microbiome data,it can be found that the abuse of laughing gas affects the composition and abundance of intestinal microorganisms in mice exposed to laughing gas,and the changes in the composition and abundance of microbial communities destroy the homeostasis of intestinal flora.Therefore,it is believed that there is a close relationship between the changes in the abundance of intestinal flora and metabolism.Changes in the composition and abundance of gut microbiota in the microbiome lead to changes in differential metabolites in fecal samples,which further interfere with metabolic pathways in mice. |
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