Study Of Gut Microbiota And Cerebrospinal Fluid Metabolomics In Epilepsy

Objective:Epilepsy is a common neurological disease,which is caused by abnormal discharge activities in brain.The etiologic mechanism of many epileptics has not been understood.The purpose of this study was to explore the etiologic mechanism of epilepsy by the analysis of gut microbiota and cerebrospinal fluid metabolomics in epileptics.16 S can identify bacterial genus high-throughput at the molecular level.The compositions and the relative abundances of gut microbiota in epileptics were compared with those in healthy people to investigate different bacteria.Culturomics is a method that can isolate and identify bacterial strains with increasing the culture conditions in vitro.The second purpose is to investigate the metabolic changes of cerebrospinal fluid in epileptics based on untargeted metabolomics gas chromatography-mass spectrometry(GC-MS).Methods:1.From March 2019 to September 2019,12 patients were included as the epileptics group(A)with definitive diagnosis in the first affiliated hospital of Dalian medical university,and 14 healthy people were the control group(C)at the same time.Fecal samples of subjects were collected.All fecal samples were inoculated directly in culture medium,and cultured in blood culture bottles at the same time.Then the stains were isolated and purified to be identified by matrix-assisted laser desorption/ ionization tine of flight mass spectrometry(MALDI-TOF-MS),then compared the detection rates of bacteria between the two groups.Illumina Nova Seq PE250 16 S r DNA detection platform was used to conduct fecal samples flora sequencing.We tried to describe the diversity of gut microbiota,species abundances in phylum,family and genus of two groups,and to search for significantly different species based on LEf Se analysis and rank-sum test statistical methods.2.From April 2019 to September 2019,23 patients were included as the epileptics group(ES)with definitive diagnosis in the first affiliated hospital of Dalian medical university,and 13 patients were included as the control group(NS)who had not appeared epileptic seizures.Cerebrospinal fluid of the subjects were collected.The metabolites of the two groups were identified by untargeted metabolomics GC-MS.The significantly different metabolites were screened by the combination of one-dimensional test(t test)and multidimensional test(OPLS-DA analysis),and the differential metabolic pathways were further screened by enrichment analysis.Results:1.Culturomics: Escherichia,Klebsiella and Streptococcus were the mainly detected bacteria in group A and C.Escherichia coli was detected in all samples.Compared with group C,the detection rates of bacillus cereus and clostridium perfringens were higher in group A,and the detection rates of klebsiella pneumoniae and enterococcus faecalis were lower.2.16 S r DNA: Compared with group C,there was no significant difference in the Alpha diversity in group A(p>0.05),and there was a difference in the Beta diversity(R=0.018,P=0.267)in the results of 16 S r DNA sequencing.On the level of phylum,the relative abundances of Firmicutes and Bacteroidetes in group A were higher than those in group C,while those of Proteobacteria,Actinobacteria and Verrucomicrobia were lower.On the family level,the relative abundances of Bacteroidaceae,Lachnospiraceae and Veillonellaceae of group A were higher than those of group C,while those of Enterobacteriaceae and Prevotellaceae were lower.The relative abundances of Bacteroides,Faecalibacterium and Megamonas were higher in group A,while those of Escherichia/Shigella and Prevotella were lower.LEf Se analysis showed that Coprobacter and Anaeroglobus were significantly reduced in group A(LDA SCORE>2).The analysis of rank-sum test showed that there were 11 different OTUs between group A and C.After species annotation,the relative abundances of Butyricicoccus,Blautia and Paraprevotella were significantly higher in group A,while those of Clostridium IV,Coprabacter and Escherichia/Shigella were significantly lower(p<0.05).3.Metabolomics: The results of metabolomics showed that there were 146 metabolites selected from cerebrospinal fluid of 36 subjects.30 top metabolites were showed in heatmap.According to the requirement of p<0.05 in one-dimensional test,there were three different metabolites,including glycine 2,alpha-ketoisocaproic acid 1 and xylose 1.According to the requirement of VIP>1 in multidimensional test,there were 56 different metabolites,21 of those satisfied the requirement of VIP>1.5.To sum up,there were 3 metabolites satisfied the requirement of p<0.05 and VIP>1 between the two groups.Compared with group NS,the expressions of alpha-ketoisocaproic acid 1 and xylose 1 were up-regulated and the expression of glycine 2 was down-regulated in group ES.The results of enrichment analysis showed that the eight metabolic pathways were different,five of them including glycine,serine and threonine metabolism,glyoxylate and dicarboxylate,glutathione metabolism,valine,leucine and isoleucine degradation,and primary bile acid biosynthesis were significantly different between group ES and group NS.Conclusions:1.The composition of gut microbiota in epileptics was different from those in healthy people.The relative abundances of Coprobacter,Anaeroglobus,Clostridium IV and Escherichia/Shigella were lower in the gut microbiota of epileptics,while those of Butyricicoccus,Paraprevotella and Blautia were higher.2.Cerebrospinal fluid metabolites and metabolic pathways in epileptics were different from control group.The expressions of alpha-ketoisocaproic acid 1 and xylose 1 were up-regulated and the expression of glycine 2 was down-regulated in epileptics,resulting in the significant disturbance of 5 metabolic pathways.3.Both the changes in gut microbiota and the abnormal metabolism of the central nervous system were involved in the pathogenesis of epilepsy.