The Study Of Duodenal Microbiome And Probiotics Intervention Mechanisms In Liver Cirrhosis

Liver cirrhosis is the intermediate stage in the process of chronic liver disease progression.In the early stage,the patients have no obvious symptoms because of liver compensatory function.In the later stage,patients have liver function damage and portal hypertension as the main manifestations with multiple systemic involvements.Studies have shown there is a significant imbalance of intestinal flora in patients with liver cirrhosis,which is closely related to the severity of cirrhosis.Liver cirrhosis disease exacerbation and other complications caused by bacterial translocation and endotoxemia is the leading cause of death.Small intestinal bacterial overgrowth is a key factor in the development of bacterial trasnslocation in patients with cirrhosis.However,at present,the sample of gut microbiota in patients with cirrhosis is mainly feces,and the structure and function of the small intestinal flora is not yet clear.Studies show that improving gut microbiota dysbiosis can protect liver function,reduce moribidty and mortality of severe liver disease.However,the causal relationship of how the intestinal microflora affects the progression of cirrhosis is not clear,and there are huge differences in physiological functions among different probiotic strains,and there is a lack of microecological regulators that can specifically attenuate liver fibrosis.Therefore,this study intends to use high-throughput sequencing method to analyze the composition of the cirrhotic duodenal flora,to identify star bacterial taxa closely related to the progression of cirrhosis,and then to compare and analyze five different probiotic strains in the occurrence and development of cirrhosis to screen out the strain with significant protective effects on fiborsis and reveal its potential mechanisms.Methods:The study was conducted in two parts.First,we compare the structural characteristics and functional genetic differences of duodenal mucosal micorbiota in patients with HBV-related liver cirrhosis,primary billary cirrhosis（PBC）and healthy controls by 16s rRNA high-throughput sequencing and to explore the effect of different treatment on the flora.Then,we applied five probiotic strains Lactobacillus salivarius LI01,Pediococcus pentosaceus LI05,Lactobacillus rhamnosus GG,Clostridium butyricum,Bacillus licheniformis on CCl4 and phenobarbital induced cirrhosis rats by gavage once daily for 13 weeks.The effects of different probiotic strains on attenuating liver fibrosis were compared by detecting liver function level,fibrosis severity,profibrogenic genes,inflammatory cytokines and Toll-like receptors expression,plasma endotoxin,cytokine and chemokine levels,gut barrier function and gut microbiota changes.Results:The study revealed that cirrhotic patients were colonized by remarkable different duodenal mucosal microbiota in comparison with controls.At the genus level,Veillonella,Megasphaera,Dialister,Atopobium,and Prevotella were found enriched in cirrhotic duodenum.And the duodenal microbiota of healthy controls was enriched with Neisseria,Haemophilus,and SRI genera incertae sedis.On the other hand,based on predicted metagenomes analyzed,gene pathways related to nutrient absorption（e.g.sugar and amino acid metabolism）and transporters were highly abundant in cirrhosis duodenal microbiota,and functional modules involved in bacterial proliferation and colonization（e.g.bacterial motility proteins and secretion system）and replication,recombianation and repair proteins were overrepresented in controls.When considering the etiology of cirrhosis,two operational taxonomic units（OTUs）,OTU₂3（Neisseria）and OTU₃6（Gemella）,were found discriminative between HBV-related cirrhosis and primary biliary cirrhosis.At the genus level,SRI genera incertae sedis was found significantly higher in patients with endoscopic treatment than those without（0.23%versus 0.03%,P = 0.04）,Staphylococcus was moderate decreased in treated group than in untreated group.Cloacibacterium was significantly reduced in patients on PPIs（0.03%versus 0.15%,P = 0.03）.Patients on PPIs were found to have moderate higher relative abundance of Dialister than those without（0.20%versus 0.06%,P = 0.054）.The probiotics Lactobacillus salivarius LI0 and Pediococcus pentosus LI05 prolonged the survival time,protected the weight loss and delayed the formation of ascites on cirrhotic rast.Moreover,probiotic LI01 and LI05 reduced liver damage,protected liver function,inhibted hepatic stellate cell activation and extracellular matrix secretion,attenuated hepatic and systemic inflammatory response,reduced plasma endotoxin and regulated Toll-like receptor family（TLR2,TLR4,TLR5,TLR9）gene expression to improver the progression of liver fibrosis.In addition,probiotic strain LI01 and LI05 can significantly reduce bacterial translocation rate,improve intestinal mucosal barrier function,increase gut β-defensin-1 gene expression,protect microvilli structure and enhance tight junction protein ZO-1 expression.This is closely related to the improvement of gut microbiota.Probiotic strain LI01 and LI05 increased the microbial richnss and changed the microbial diversity of fungal community.There is a dysbiosis of gut microbiota on cirrhotic rats manifested as increased abundance of potentially pathogenic bacteria and opportunistic infectoious fungi,and reduced colonization of normal flora and probiotics abundance.Elusimicrobium,Paraprevotella,Prevotella,enriched in probiotic groups,and Oscillospira,Oscillibacter,Flavonifractor,and Escherichia/Shigella,enriched in positive control NS group,may be the gut core flora after probiotic intervention,and play an important role in regulating inflammation levels and liver fibrosis progression.Conclusion:There is a significant dysbiosis of duodenal mucosa microbiota in patients with liver cirrhosis.The deviation of duodenum microbiota might be related to alterations of oral microbiota and duodenal microenvironment.Also,when considering the etiology of cirrhosis,PBC and HBV have slight difference of gut microbiota,indicating the effect mediated by immunity or bile acids between host and microbiota.The duodenal microbiota is primary determined by cirrhosis itself and only slight association can be observed between duodenal microbial alterations and endoscopy varices treatment or PPIsThe long-term regulation of gut microbiota by probiotics can affect the progression of liver cirrhosis.The administration of L.salivarius LI01 or P.pentosaceus LI05 can improve the gut flora composition,resulting in the restoration of gut barrier function.These changes reduce liver inflammation level and fibrogeneic factor,and ultimately improve the progression of liver fibrosis in cirrhotic rats.The current study potentially provides new insights into the treatment of liver cirrhosis via selection of appropriate probiotic strains to manipulate the gut microbiota.