Study On The Effect And Anti-inflammation Mechanism Of Metabolites Secondary Bile Acids Of Gut Microbiota In Klebsiella Pneumoniae Liver Abscess

Part1 Fecal transplantation contributres to host defennse against Klebsiella pneumoniae liver abscess Object : Klebsiella pneumoniae liver abscess(KPLA)is a serious liver infectious disease accompanied by severe complications,high disability and mortality.Faced with the prominent bacterial drug resistance problem in clinic,doctors and researchers are trying to find new and effective methods to treat KPLA.As an important mediator of host immune homeostasis,gut microbiota is closely related to liver diseases such as hepatitis B,liver cancer and alcoholic fatty liver.Therapies that restore intestinal flora homeostasis,for example fecal microbiota transplantation(FMT),have been reported to improve the prognosis of some infectious diseases in clinical and animal studies.As part of liver disease,we hope to explore whether FMT exerts a similar effect on the occurrence and development of KPLA.Methods:(1)Time of flight mass spectrometry was used to verify the source of the strain,and bacterial DNA was extracted.PCR products were amplified and the virulence gene was displayed by 1% gel electrophoresis.Finally,the hypermyxoid phenotype of the strain was verified by string test.(2)The KPLA mouse model was constructed by using Klebsiella pneumoniae(K.p.)infected with C57BL/6J male mice.The successful mouse model was verified by the performance,the formation of liver abscess and H?E staining after infection.(3)Changes of gut microbiota were analyzed by 16 S r RNA sequencing technology in mice before and after K.p.invasion.(4)Multiple antibiotics were added to the drinking water to construct microbiota depletion(MD)mice,which was verified by 16 S r RNA sequencing technology.(5)MD mice were infected with K.p.,and the survival of the mice was observed,the clinical symptoms and liver injury were evaluated,and the levels of inflammatory indexes and biochemical indexes ALT and AST were detected.(6)MD+KPLA mice were transplanted with healthy intestinal flora,and the changes of liver injury degree,survival and inflammation level were observed.Results:(1)The modeling strain was K.p.with three virulence genes of mag A,rmp A and aerobacterin,and it was a clinical isolate with a hypermyxoid phenotype.(2)KPLA mouse model has been successfully constructed by using this strain.The mice infected with the bacteria showed symptoms such as reduced eating,shortness of breath,increased secretions from canthus,bristly hair or lethargy.The liver of KPLA mice was dissected and removed.The liver surface of KPLA mice was characterized by abscesses of different sizes and similar to speckles.H?E staining showed vacuolar deformation of hepatocyte cytoplasm with numerous infiltrated inflammatory cells along the edges.(3)Significantly changes in composition of gut microbiome were observed after the K.p.infected.The abundance of Ruminococcaceae and Lactobacillusaceae were significantly reduced,and the level of Enterohepataceae was significantly increased.(4)The multiple antibiotics successfully exhausted most of the bacteria in MD mice,which could replace the sterile mice for subsequent experiments.(5)After K.p.invaded mice,the bacterial clearance ability and survival rate decreased,and the levels of liver injury and inflammatory factors increased.(6)FMT improved the survival and liver injury,and normalized the high level of inflammatory factors in MD+KPLA mice.Conclusion: K.p.invasion led to significant changes in the composition of the intestinal flora,and restoring the homeostasis of the gut microbiota could significantly reduce liver damage and inflammation in MD+KPLA mice.Part2 Based on targeted metabolomics to analysis of bile acid profiles in KPLA mice Object:The development of metabolomics technology makes it possible to measure the changes of metabolites in the body during the occurrence and development of diseases comprehensively and accurately.KPLA may be delayed due to atypical clinical manifestations during diagnosis,so early clinical diagnosis is particularly important.In addition,we found that K.p.infection led to the basic remodeling of intestinal flora in mice,most notably,the abundance of rumen bacteria involved in the synthesis of secondary bile acids(SBAs)was significantly reduced.Based on the above aspects,we will explore whether the reduction of SBAs can be observed during the pathogenesis of KPLA and whether this metabolite can be used as a biomarker for the early diagnosis of KPLA.Methods:(1)Bile acids in feces and serum were analyzed by targeted metabolomics technique in KPLA and healthy mice.(2)Serum inflammatory factors and biochemical levels were detected in KPLA mice and healthy controls by Elisa.(3)Spearman analysis of correlation between SBAs,inflammatory factors and biochemical indicators.Results:(1)Compared with the Control group,DCA and LCA were decreased in serum and feces of KPLA mice.(2)Compared with the Control group,serum inflammatory indexes(IL-1?,IL-6 and TNF-?)and biochemical indexes(ALT and AST)were increased in KPLA mice.(3)DCA in serum was negatively correlated with IL-1?,IL-6and TNF-?(r =-0.612,P < 0.01;r =-0.692,P < 0.01;r =-0.697,P < 0.01).LCA in serum was negatively correlated with IL-6(r =-0.611,P < 0.01);Conclusion: The low expression of SBAs in KPLA mice is negatively correlated with inflammatory factors level.Part3 SBAs alleviate liver injury and inflammation in KPLA mice by inhibiting NF-?B inflammatory signaling pathway Object:Recent evidence suggested that SBAs play a beneficial role in a variety of diseases,especially an anti-inflammatory and protective role in infectious diseases.Although previous studies have suggested that SBAs may have anti-inflammatory effects,further experimental validation is needed.In addition,the anti-inflammatory mechanism of SBAs in KPLA is also unknown.This section focuses on the verification of the above aspects.Methods:(1)The effect of SBAs was revealed by analyzing the survival rate,bacterial load,histopathology and inflammatory factors of KPLA mice by oral administration of LCA and DCA.(2)WB was detected the levels of key proteins in the NF-?B pathway after SBAs stimulation,including p-I?B?,NF-?B p50 and NF-?B p65.(3)The expression level of G protein-coupled bile acid receptor(TGR5)was detected by q RT-PCR,IHC and WB.Results:(1)Oral administration of SBAs improved the survival rateand liver pathology of K.p.infected mice,and reduced the bacterial load and the inflammatory factors level.(2)SBAs down-regulated the expression of key proteins in the NF-?B inflammatory signaling pathway,including the phosphorylation of I?B? and nuclear translocation of NF-?B p50 and NF-?B p65.The protective effect of SBAs may be dependent on high expression of TGR5.Conclusion: SBAs downregulate the NF-?B inflammatory signaling pathway through TGR5,protecting the liver and inhibiting inflammation in KPLA.