Effects Of Gabexate Mesylate On Gut Microbiota And Metabolomics In Septic Rats

Sepsis is a life-threatening organ dysfunction caused by the body’s dysregulation of infection,threatening the lives and health of millions of people around the world each year.Existing sepsis treatment mainly included fluid resuscitation,rapidly effective antibiotics,and supportive care.Due to the heterogeneity and complexity of sepsis pathophysiology,there has been no specific therapy for sepsis to date.The gut microbiota of normal organisms participated in the formation of the intestinal immune barrier to prevent the colonization and translocation of intestinal pathogens.However,the composition of the gut microbiota in sepsis could change within hours,manifested by loss of microbial diversity and predominance of pathogenic microorganisms.Studies have shown that changes in gut microbiota composition are associated with changes in immune responses and airway homeostasis,and gut microbiota dysbiosis could also increase susceptibility to sepsis.Metabolomics is the qualitative and quantitative analysis of small molecule metabolites involved in various biochemical reactions.In recent years,many studies have used metabolomics to detect and analyze the changes of metabolites in blood and urine in sepsis.These studies carried out metabolomic analysis from the pathogenesis of sepsis,screening of early diagnostic markers,and drug efficacy.This provides theoretical support for the complex pathogenesis of sepsis,and a reliable basis for the development and application of personalized medicine for sepsis.Gabexate Mesilate（GM）is a non-peptide proteolytic enzyme inhibitor with strong inhibitory effects on protease and plasmin.Pretreatment of lipopolysaccharide（LPS）rats with GM could significantly reduce the elevation of serum inflammatory factors,and alleviate lung,liver and renal insufficiency.This study hypothesized that GM might alter sepsis gut microbiota and fecal metabolomics to attenuate sepsis-induced acute lung injury.A rat model of sepsis was induced by cecal ligation and puncture（CLP）,and the gut microbiota and metabolomics of the sham control（SC）group,CLP group and GM administration group were compared.The correlation between differential microbiota and differential metabolites was analyzed,and then mortality of rats 24 hours after CLP,and the lung damage were observed to explore the effect of GM on gut microbiota and metabolomics,and the potential of GM in mitigating sepsis-induced acute lung injury.Part Ⅰ Effects of gabexate mesylate on gut microbiota in septic ratsMethod1.Adult male SD rats aged 6-8 weeks were randomly divided into SC group,CLP group,and intraperitoneal injection of GM group 1 hour after CLP.The establishment of the CLP model was based on our previous research reports.Colonic feces of surviving rats in each group were collected 24 hours after CLP.2.DNA was extracted from the fecal samples of rats in each group,and 16S rRNA high-throughput sequencing was performed.Operational taxonomic units（OTUs）clustering and taxonomic analysis were performed on the obtained valid data with 97%consistency.3.Use R software to analyze the differences between a diversity and β diversity groups.Metastats analysis was conducted to compare the microbiota of SC,CLP and GM groups,and LEfSe was used to analyze two or more groups to find the gut microbiota with statistical differences between groups.In addition,correlation analysis was performed between the differential microbiota and the function of the microbiota predicted by Tax4Fun.Result1.660,616,and 850 OUTs were identified in SC group,CLP group,and GM group,respectively.And 495 OUTs were shared by the three groups.The predominant phyla in SC group and GM group were Firmicutes,and the predominant phyla in CLP group were Proteobacteria and Verrucomicrobiota.And the ratio of Firmicutes to Proteobacteria was significantly reduced in the CLP group.The dominant bacterial genera in SC group and GM group were Lactobacillus and Ruminococcus.The dominant genera of CLP were Escherichia shigella and Akkermansia.2.The a diversity of SC group and GM group was higher than that of CLP group.PCoA analysis showed that samples from SC group and GM group tended to cluster together,indicating that the two groups had similar species composition,but were quite different from the CLP group.3.There were three different genera between CLP group and SC group,namely Lactobacillus,Romboutsia and Escherichia shigella.There were 5 different genera between the CLP group and the GM group,namely Lactobacillus,Pygmaiobacter,Erysipelotrichaceae_UCG₀03,Escherichia shigella and Akkermansia.The only difference between SC group and GM group was Parasutterella.4.The genera of the GM group and the CLP group were jointly constructed into an interaction network.Compared with the CLP group,the GM group had a higher correlation between the genera.Tax4Fun analysis showed that 18 pathways（such as cell motility and secretion,glycan biosynthesis and metabolism,etc.）were differentially enriched between the CLP and GM groups.In addition,Spearman correlation analysis of differential microbiota and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway showed that Proteus,Muribaculum,Escherichia shigella,Akkermansia were positively correlated with Transport,Porphyrin and chlorophyll metabolism,Pathways in cancer and other pathways.Lactobacillus,Erysipelotrichaceae_UCG-003 and Transport,Porphyrin and chlorophyll metabolism,Pathways in cancer and other pathways were negatively correlated.SummaryThe relative abundance of Escherichia shigella and Akkermansia in sepsis rats decreased,and the relative abundance of Lactobacillus increased,which partially improved the gut microbiota imbalance after GM application.Part Ⅱ Effects of Gabexate Mesylate on Metabolomics in Sepsis RatsMethods1.The collected fecal samples of each group were used for non-targeted metabolomics detection.The metabolites were chromatographically separated using liquid chromatography-mass spectrometry（LC-MS）,and secondary information of the mass spectrometry data was collected.2.Process the raw data to obtain a data set containing mass-to-charge ratios,retention times,and peak intensities.Data were normalized and metabolites were annotated.3.Metabolite changes between groups were analyzed using unsupervised principal components analysis（PCA）.Orthogonal partial least-squares discrimination analysis（OPLS-DA）was used to demonstrate the metabolic differences between the two groups.One-way ANOVA and Tukey’s post hoc test were used to analyze data for differences between groups.Pathway analysis of metabolites was performed using MetaboAnalyst 3.0 software.In addition,correlation analysis was performed on the differential microbiota and metabolites between groups.4.The mortality rate of rats in each group was counted 24 hours after CLP operation.The collected lung tissue of each group were stained with Hematoxylin and eosin（HE）and the pathological changes were observed.Result1.There were significant changes of metabolomics in septic rats.PCA showed that the SC group was closer to the GM group and was significantly separated from the CLP group.OPLS-DA showed that CLP group and SC group,CLP group and GM group could be divided into different groups according to their metabolic differences and the distinction was significant.2.Using MetaboAnalyst 3.0 and KEGG database to analyze the different metabolite pathways between CLP group and GM group,the results show that GM mainly regulates Sphingolipid metabolism,Histidine metabolism,Steroid biosynthesis,Glycerophospholipid metabolism,Primary bile acid biosynthesis and other metabolic pathways.3.There are correlations between key genera and differential metabolites in metabolic pathways.In positive ion mode,CLP-enriched Akkermansia was positively correlated with Chenodeoxycholic acid 3-sulfate,Cholesterol,LysoPE（16:1（9Z）/0:0）,LysoPE（16:0/0:0）,LysoPE（15:0/0:0）,PC-M6,and negatively correlated with 15-KETE,Choline,Sphinganine.Escherichia shigella enriched in CLP was positively correlated with Chenodeoxycholic acid 3-sulfate,Calystegin A3,[10]-Gingerdione,and 15-KETE was negatively correlated.CLP-rich Proteus was positively correlated with Chenodeoxycholic acid 3-sulfate,Cholesterol,LysoPE（16:0/0:0）,LysoPE（15:0/0:0）,[10]-Gingerdione and negatively correlated with 15-KETE.In negative ion mode,CLP-enriched Akkermansia was positively correlated with Quinolinic acid,and negatively correlated with Leukotriene B4,9,10-DHOME,Deoxycholic acid,L-Cysteine,Phenylacetylglycine,N-Acetylvaline,Alpha-linolenic acid.CLP-enriched Escherichia shigella was positively correlated with Quinolinic acid,5a-Tetrahydrocorticosterone,and negatively correlated with Leukotriene B4,9,10-DHOME,m-Coumaric acid.CLP-rich Proteus was positively correlated with Quinolinic acid,5a-Tetrahydrocorticosterone,Cortisol,and negatively correlated with Leukotriene B4,9,10-DHOME,Deoxycholic acid,N-Alpha-acetyllysine,Oleoyl glycine,m-Coumaric acid,and Alpha-linolenic acid.In addition,GM-rich Lactobacillus was positively correlated with Leukotriene B4,9,10-DHOME,N-Alpha-acetyllysine,N-Acetylvaline,Phenylacetylglycine,Oleoyl glycine,and negatively correlated with Quinolinic acid.4.The mortality rate of rats in CLP group was significantly higher than that SC and GM groups.In addition,the results of HE staining showed that the inflammatory infiltration,edema and structural damage of lung tissue in the CLP group were significantly worse than the SC and GM groups.SummaryGM mainly regulates Sphingolipid metabolism,Histidine metabolism,Steroid biosynthesis,Glycerophospholipid metabolism and Primary bile acid biosynthesis pathways to alter the metabolic profile of the CLP group.There were correlations between key genera and differential metabolites on metabolic pathways.CLP-rich（Akkermansia,Escherichia shigella and Proteus）were positively correlated with LysoPE（15:0/0:0）,Quinolinic acid,GM-rich Lactobacillus was negatively correlated with LysoPE（15:0/0:0）and Quinolinic acid.CLP-rich（Akkermansia,Escherichia shigella and Proteus）were negatively correlated with Leukotriene B4,9,10-DHOME.GM-rich Lactobacillus was positively correlated with Leukotriene B4,9,10-DHOME.In addition,GM could also reduce the lung tissue damage caused by sepsis and improve the survival rate of sepsis rats.ConclusionGM could affect the changes of Lactobacillus,Akkermansia,Escherichia shigella and other genera,regulate relative abundance of LysoPE（15:0/0:0）,Quinolinic acid,Leukotriene B4,9,10-DHOME and other differential metabolites,and attenuate acute lung injury caused by sepsis,which may provide a potential mechanism for the application of GM in sepsis treatment.