Protective Effects Of Lactobacillus Against Sepsis And Its Influence On Microbiota And Metabolome Profile Of Serum And Fecal In Mice

BackgroundsSepsis is one of the leading causes of mortality and morbidity in ICU patients especially the elders.A hostile gut environment can always be found in patients with sepsis.Intestinal epithelium dysfunction and microbiota imbalance induced by critical illnesses would result in increased translocation of bacteria to the blood,which contributes to the formation and aggravation of sepsis.Lactobacillus,the living microorganisms colonizing in the intestine bring benefits to the host through rebalancing microbiota,modulating intestinal barrier function and enhancing the immunity system.Currently,a growing number of articles have indicated the potential of some lactobacillus in some diseases,such as diarrhea,allergic diseases,inflammatory bowel disease.However,most of these articles focus merely on evaluating the prevention and treatment effects of lactobacillus on diseases.Only a few of researches tried to explain the precise molecular mechanism of beneficial effects of these microorganisms.Our research intended to investigate the protective effects of lactobacillus rhamnosus GG(LGG)on the occurrence and development of sepsis induced by cecal ligation and puncture(CLP),and elaborate the possible mechanism from regulation of LGG on gut barrier function,intestinal microecology and metabolism.Partl Protective effects of LGG against CLP-induced SepsisObjective:Observing the effect of LGG on the survival rate,inflammatory cytokines and intestinal pathological changes in septic mice and investigating whether LGG has protective effects on intestinal barrier function in sepsis.Methods:Mice were orally gavaged with LGG(high,medium,and low does group)(LGG+Septic),or normal saline(Control Septic group and Sham group)4 weeks prior to CLP.Sham mice underwent sham laparotomy.The LGG does improve the 7 day survival rate most significantly was used as standard LGG does in follow up tests.Serum albumin(Alb)and C-reactive protein(CRP)were detected.Detecting serum Interleukin-6(IL-6),Tumor necrosis factor-α(TNF-α),Interleukin-22(IL-22)and Interleukin-2(IL-2)level using enzyme-linked immunosorbent assay(ELISA).Blood from the mice were cultured 24 h for bacteremia analysis.The colon tissues were sectioned and stained with hematoxylin and eosin(H&E)for histological observation in order to examine the pathology of intestinal injury.The tissues were then stained with Ki67 and TUNEL for observation of cell proliferation and apoptosis.At last,immunohistochemical staining for occludin were done to evaluate the tight junction between intestinal epithelial cells.Results:(1)LGG reduced mortality in septic mice.The survival rate of LGG+septic mice was statistically improved compared with the control septic mice in a dose-dependent pattern.(2)LGG pretreatment increases Alb and decreases CRP level.Serum levels of IL-22,IL-2 were decreased with LGG treatment,and LGG had no effects on TNF-a and IL-6 expression.(3)Septic mice pretreated with LGG exhibited significantly decreased bacteremia,alleviated colon barrier injury,increased epithelial cell proliferation,decreased epithelial cell apoptosis and tight junction proteins expression.Conclusion:Early administration of LGG reduces mortality through attenuating inflammatory responses,increasing gut barrier integrity and function,and prevention of systemic bacteremia.Part2 Effects of LGG on microbial diversity in CLP-induced septic miceObjective:Observing the effect of LGG on intestinal microbiota in sepsis and analysing the changes of intestinal microbiota in mice with sepsis.Methods:Mice(LGG+Septic,Control Septic and Sham)were orally gavaged LGG or normal saline and went CLP operation as before,fecal samples were collected at 24 h after CLP operation.The fecal bacteria composition was investigated by high-throughput sequencing of the V3-V4 region of the 16S rDNA gene.Results:(1)LGG pretreatment improved richness and diversity of intestinal microbiota in septic mice.(2)The PCoA clustering plots revealed a significant separate clustering in microbiota structure between three groups.Bacteria related with energy consumption like Bacteroidetes,opportunistic infection like Proteobacteria,Staphylococcaceae and Enterococcaceae,LPS productor like Enterobacteriaceae and facultative anaerobe like Bacteroidaceae,Erysipelotrichaceae increased in septic mice.Whereas,bacteria related with energy harvest like Firmicutes,intestinal barrier function regulator like Akkermansia,hepatic function regulator like Coprococcus and Oscillospira,obligate anaerobes like Prevotellaceae decreased in septic mice.With LGG treatment,beneficial bacteria increased and harmful bacteria decreased.Conclusion:LGG treatment increased diversity of gut microbiota and expression of beneficial bacteria,Akkermansia and Prevotellaceae can be used as potential probiotics for the treatment of sepsis.The results elaborated the possible mechanism on treatment of sepsis by regulating intestinal microbiota.Part3 Effects of LGG on metabolic profiles in CLP-induced septic miceObjective:Observing the relationship between sepsis and intestinal microbiota from metabolic level,analysing the effect of LGG on metabolism of sepsis.Mthods:The analysis of serum and fecal samples were performed using an Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)to identify biomarkers related to sepsis.Results:(1)Through supervised partial least-squares discriminate analysis(PLS-DA),there was a distinguished classification among all three groups.(2)In serum samples,phosphatidylcholines(PCs)were increased and lysophosphatidylcholines(LPCs)that contained polyunsaturated fatty acids were decreased in septic mice,whereas saturated fatty acid LPCs reveal no significant difference between septic and sham mice.In addition,the metabolic profile in the LGG+septic was much closer to that of sham mice compared with control septic mice.In fecal samples,LPCs,arachidonic acid,retinyl phosphate and diacylglycerols(DAG)were increased,parts of cholic acid and L-Phenylalanine fragment was decreased significantly in control septic mice compared with sham mice.After LGG treatment,no statistical difference existed between LGG+septic mice and shams.Conclusions:LGG treatment normalized the key metabolites in serum and fecal.The metabolites were mainly involved in lipid and bile acid metabolism.Dysbiosis was closely related with abnormality of fecal metabolic profiles.LGG treatment normalized global metabolic profiles of serum and fecal effectively.