An Untargeted Metabolomics Approach Reveals Biomarkers And Metabolic Pathways Of Liver Tissue In Hepatitis B Virus And Alcohol-induced Mouse

Background In recent years,alcoholic liver disease(ALD)accounting for 0.9% of global mortality is a major liver disease threating human health.Meanwhile,Hepatitis B virus(HBV)infection and its associated complications maintain a major threat to global public health.However,the underlying interaction between hepatitis B virus carrier and ALD is considered to be an increasing potential risk.The metabolic changes among the population is unknown.Qualitative analysis of its metabolites was carried out to search for medical biomarkers and the most significant differential metabolic pathways,which can be used for disease mechanism research,drug target discovery and cancer risk assessment.Methods In this project,we constructed a new HBV infection combined with ALD mouse model,in animal models,We collect Liver tissue homogenate easily to perform,Ultra High Performance Liquid Tandem Chromatography Quadrupole Time of Flight Mass Spectrometry(UHPLC-QTOFMS)were investigated the hepatic metabolic profiles and Screening of biomarker in liver used Multivariate Analysis.Results Several metabolites involved in mouse model were identified,which was glycine,L-Glutamate,L-Alanine,L-Valine,L-Pyroglutamic acid,Monomethyl glutaric acid,Pyruvaldehyde,N-Acetyl-L-alanine.5-oxoproline indicated the most conspicuous difference between the HBET group and ET group,when statistically carried out by biomarker analysis.Moreover,after HBV or alcohol treatment,clear separation among groups was well achieved.Metabolic pathway analysis showed that these different metabolites participated in seven pathways: Glutathione(γ-glutamyl-l-cysteinylglycine,GSH)metabolism;Nitrogen metabolism;Riboflavin metabolism;Valine,leucine and isoleucine biosynthesis metabolism;Glycine,serine and threonine metabolism;Arachidonic acid metabolism;D-Glutamine and D-glutamate metabolism.Based on the metabonomic view,the highest impact pathway was Glutathione metabolism and Vitamin C metabolism.Conclusions The identified 5-oxoproline may act as a crucial biomarker for distinguish ethanol feeding group and ethanol feeding combined HBV-carrier group.Glutathione metabolism and Vitamin C metabolism was the most commonly associated metabolic differences in the 7 metabolism pathways between the ethanol feeding group and ethanol feeding combined HBV-carrier group.Objective The purpose of this experiment was to observe the effect of different alcohol concentrations on the number,activation and apoptosis of T-lymphocyte subsets at different time points when co-cultured with human peripheral blood T lymphocytes in vitro.Method Mononuclear cells were isolated from the peripheral blood of healthy people by density gradient centrifugation.PBMC activation was stimulated with phytohaemagglutinin(PHA)and co-cultured with 3% and 5% alcohol.After co-culture for 12 h,18 h,36 h,48 h,flow cytometry detected the activation,function and apoptosis of lymphocytes in each group.In addition,we established a mouse model of Alcoholic liver disease,prepared a single cell suspension from the spleen,performed flow cytometry analysis,and compared the results of the test in vitro and in vivo under the action of alcohol.Results 1)PBMC was co-cultured with alcohol for 12 hours,and our data showed that the number of lymphocytes stimulated by alcohol did not change significantly for less than 5% alcohol concentration within 12 hours.However,compared with the blank control,CD8+ Tcells in the PHA group were activated,and the 5% alcohol inhibited activation of CD8+ Tcells(P<0.05).2)PBMCs were co-cultured with alcohol for 18 hours,we observed ethanol-treated in combination with PHA had significantly decreased the number of Lymphocytes compared to the pure PHA group(P<0.05).Ethanol-treated in combination with PHA had reduced the expression of CD69(P<0.05).3)PBMCs were Co-cultured with alcohol for 36 hours,alcohol with PHA group significantly reduced the number of lymphocytes compared with PHA group(P<0.05).4)PBMC co-cultured with alcohol for 48 hours,compared with the pure PHA group,the lymphocytes of the alcohol plus PHA group almost disappeared.5)The data of apoptosis showed the number of apoptotic cells was remarkably increased with the prolonged time of alcohol culture PBMC and was significantly increased from 3% to5% in a concentration-dependent manner.6)Compared with control group,the percentage of CD3 + T lymphocyte,CD8 + T lymphocyte and CD8 CD69 in spleen of ALD mice was significantly decreased.Conclusion Alcohol could inhibit PHA-activated T lymphocytes when co-cultured with 3% alcohol and 5% alcohol in vitro.As the time to culture the cells is prolonged,alcohol significantly reduced the number of lymphocytes until it disappeared,which one of the reasons was alcohol could promoted T lymphocyte apoptosis in a concentration-dependent manner.