Metabolomics-based Study Of Neurotransmitter Alterations In Chronic Alcohol Consumption And Alcohol Withdrawal And Mechanisms Of Memantine Treatment

BackgroundAlcohol has a strong diffusion and distribution capacity in all body fluids and tissues,so alcohol exposure is closely related to changes in metabolic processes in several tissues,and alcohol can enter the central nervous system（CNS）through the blood-brain barrier by interacting with a variety of neurotransmitter systems,such as dopamine（DA）,glutamate,gamma-aminobutyric acid（GABA）,5-hydroxytryptamine,norepinephrine（NE）.The alteration of neurotransmitter systems caused by long-term alcohol exposure is an important cause of alcohol addiction and alcohol withdrawal syndrome（AWS）,and the excitation of the glutamatergic system in the hippocampus caused by sudden withdrawal after long-term drinking can induce the occurrence of alcohol withdrawal symptoms such as anxiety and seizures.ObjectiveThis study explores the neurotransmitter and amino acid profiles in serum induced by alcohol exposure and alcohol withdrawal by constructing an animal model of chronic alcohol consumption and alcohol withdrawal based on targeted metabolomics techniques.The alterations of Glu N1,Glu N2B and Glutamate transporter-1（GLT-1）induced by memantine were also examined by using memantine as a therapeutic drug to explore the alterations of glutamatergic system by memantine.MethodsA chronic alcohol consumption model was constructed using 10 Sprague Dawley（SD）rats who freely drank 20%（w/w）alcohol for 8 weeks.Blood samples were collected from all rats before modeling（week 0）as a control,and blood samples were collected at weeks 1,2,3,4,5,6,7,and 8 of alcohol consumption.After selecting serum samples from weeks 0,1,2,4,6,and 8 for preprocessing,relative quantitative analysis of216 metabolites in rat serum was performed using high-performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）based on developed widely targeted metabolomics method.After the detection was completed,the data was exported to Excel and the metabolites whose missing data proportion was more than 20%were removed.The remaining missing values were filled with the K nearest neighbor algorithm.The data was corrected based on the random forest signal drift of QC samples,and the metabolites whose RSD of QC samples is less than 15%were retained.Then,the total peak area normalization,logarithmic transformation and Pareto scaling were performed.Univariate analysis was performed using the Metaboanalyst platform and multivariate analysis was performed using SIMCA-P software,based on the obtained VIP values>1 and adjusted P values<0.05 by Benjamini-Hochberg method.Finally,the Metaboanalyst platform was used for pathway analysis based on the Kyoto Encylopaedia of Genes and Genomes（KEGG）database,and pathways with an impact value greater than 0.1 were significantly affected.In this study,C57BL/6J mice were used to construct an alcohol withdrawal model,and the study was divided into four groups:a control group,an alcohol withdrawal group,a memantine administration group and a saline group,with five mice in each group.Firstly,in addition to the control group,the other three groups of mice were used to construct an alcohol withdrawal model.The construction method was as follows:in the first week,200μL of 5%（v/v）alcohol solution was administered orally every day,and at the remaining time,5%（v/v）alcohol solution was freely consumed.In the second,third,and fourth weeks,10%（v/v）,20%（v/v）,and 35%（v/v）alcohol solution were administered respectively.Alcohol was stopped for withdrawal on days 6,12,18,and 24.Open field test and elevated plus maze were conducted on days 7,14,21,and 28.The memantine administration group and saline group were intraperitoneally injected with 30 mg/kg memantine and 200μL saline once a day for 7 days starting from the 29^th day.Behavioral tests were conducted on the 36^th day;Collect serum from mice for semi-quantitative targeted metabolomics analysis.The sample preprocessing method and data processing and analysis are similar to the above.Use q RT-PCR to detect the m RNA content of GLT-1,Glu N1,and Glu N2B in hippocampal tissue.ResultsAfter signal drift correction of metabolomics data from chronic alcohol consumption rats,the RSD distribution of most metabolites in QC samples was within30%.After removing metabolites with RSD greater than 15%,82 metabolites were obtained.Based on the VIP value,and adjusted P value,33 differential metabolites were finally screened,including 18 up-regulated metabolites and 15 down-regulated metabolites,among which GABA showed significant up-regulation,while glutamate,DA,5-HT,and aspartate showed significant down-regulation;finally,the pathway analysis revealed that eight metabolic pathways were significantly affected,namely alanine Pathway analysis identified eight metabolic pathways significantly affected,namely alanine,aspartate and glutamate metabolism,riboflavin metabolism,D-glutamine and D-glutamate metabolism,tryptophan metabolism,arginine biosynthesis,biotin metabolism,arginine and proline metabolism,and tyrosine metabolism.After four weeks of alcohol withdrawal induction,the percentage of the open arms entry and the residence time in open arms showed a significant decrease（P<0.05）.After one week of treatment with memantine,the percentage of the open arms entry significantly increased（P<0.05）,and the percentage of the residence time in open arms also increased to a certain extent（P=0.056）,Explain that memantine can alleviate anxiety symptoms caused by alcohol withdrawal.The results of serum metabolomics studies showed that after alcohol withdrawal,valine and threonine were significantly upregulated（Adjusted P<0.05）,while urocanic acid and glutamate were significantly downregulated（Adjusted P<0.05）.After one week of treatment with memantine,glutamate,glycine,serine,and aspartic acid were significantly upregulated（Adjusted P<0.05）.Finally,the q RT-PCR method was used to detect the m RNA content of Glu N1,Glu N2B,and GLT-1.Compared with the control group,the m RNA expression of Glu N1 in hippocampal tissue was increased in the alcohol withdrawal group（P=0.12）and appeared significantly increased in the saline group（P<0.05）,and the m RNA content of Glu N2B was significantly increased in the alcohol withdrawal group（P<0.05）and saline group（P<0.01）were significantly upregulated,and the m RNA expression of Glu N1 was significantly downregulated（P<0.05）and Glu N2B had a tendency to decrease（P=0.16）after memantine treatment compared with the saline group.ConclusionsThe metabolic abnormalities of neurotransmitters such as glutamate in the serum caused by chronic alcohol exposure and alcohol withdrawal are closely related to psychological symptoms such as anxiety,depression,and compulsion,indicating that changes in neurotransmitters caused by alcohol are important reasons for inducing alcohol addiction and alcohol withdrawal symptoms.In addition,excessive activation of NMDA receptors may be an important cause of anxiety symptoms after alcohol withdrawal.Memantine can alleviate anxiety symptoms by reducing the expression of NMDA receptors,indicating that NMDA receptors may be an important therapeutic target for alcohol withdrawal syndrome.At the same time,this study provides a basis for further exploration of the mechanism of Memantine in treating alcohol withdrawal syndrome.