| Part One Alterations of Gut Microbiota in Cholestatic Infants and Their Correlation with Hepatic FunctionObjective: Cholestasis is a major hepatic disease in infants,with increasing morbidity in recent years.Accumulating evidence has revealed that the gut microbiota(GM)is associated with liver diseases,such as non-alcoholic steatohepatitis,cirrhosis,and hepatocellular carcinoma.However,GM alterations in cholestatic infants and the correlation between the GM and hepatic functions remain uninvestigated.Therefore,this study we compare the liver function and gut microbiota between cholestasis infants and healthy control infants,we aimed to: evaluate bacterial correlation and their contribution to hepatic function;identify GM biomarkers for non-invasive diagnosis of IC;elucidate GM discrepancy among patients with IC owing to di erent causes.These findings enhance our understanding of the pathogenic mechanism of dysbiotic GM,and provide a solid theoretical basis for GM intervention for the treatment of IC.Methods: We collected infants with cholestasis in hospital and healthy infants with physical examination in outpatient from December 2016 to January 2018.They were divided into two groups: healthy control group(H Group)and infant cholestasis group(IC group).Their sex,age(month),delivery mode,feeding mode and clinical diagnosis were collected,blood was collected to detect liver function,stool was collected to analyze gut microbiota by 16 S r DNA the difference of gut microbiota between group H and group IC,the correlation between liver function and intestinal flora were analyzed.The biomarkers were screened and verified by random forest model,and the gut microbiota function was predicted by PICRUST.Results:1.A total of 37 healthy infants(H group)and 43 cholestatic infants(IC group)were enrolled for stool sample collection.2.All the samples predominantly showed the following genera: Bifidobacterium,Bacteroides,Enterococcus,Blautia,Roseburia,and Faecalibacterium.The diversity in the bacterial community was significantly lower in the IC group than that in the H group(P = 0.013).The feeding pattern of the infants had an impact on the difference in GM between the H and IC groups(P = 0.009,PERMANOVA analysis).3.After determining the top 10 abundant genera of microbes in the IC and H groups,we found that 13 of them were differentially enriched,including Bifidobacterium,Bacteroides,Streptococcus,Enterococcus,and Staphylococcus.As compared with the H group,the IC group had a more complex GM co-occurrence network featured by three core nodes: Phyllobacterium,Ruminococcus,and Anaerostipes.In addition,the positive correlation between Faecalibacterium and Erysipelatoclostridium(r = 0.689,P = 0.000,FDR = 0.009)was not observed in the IC patients.Using the GM composition,the cholestatic patients can be distinguished from healthy infants with high accuracy [AUC>0.97],where in Rothia,Eggerthella,Phyllobacterium,and Blautia are identified as valuable biomarkers.4.Using KEGG annotation,we identified 32 functional categories with significant difference in enrichment of the GM of IC patients,including IC-enriched functional categories that were related to lipid metabolism,biodegradation and metabolism of xenobiotics,and various diseases.In contrast,the number of functions associated with amino acid metabolism,nucleotide metabolism,and vitamins metabolism was reduced in the IC patients.5.We also identified significant correlation between GM composition and indicators of hepatic function.Megasphaera positively correlated with total bilirubin(r = 0.455,P = 0.002)and direct bilirubin(r=0.441,P=0.003),whereas γ-glutamyl transpeptidase was positively associated with Parasutterella(r = 0.466,P = 0.002)and negatively related to Streptococcus(r =-0.450,P = 0.003).Part Two Intestinal microecological changes in rats with subacute cholestasis induced by ANITObjective: The etiology of cholestatic liver disease is complex,and it is a chronic process.Through the establishment of subacute cholestasis SD rat model,we studied the changes of intestinal flora before and after cholestasis and during the course of disease progression,and correlation of intestinal flora with liver function and liver fibrosis index,in order to explore its mechanism from the perspective of Microecology and to provide basic data for targeted treatment of microecological agents.Methods: SD rats were randomly divided into two groups: normal control group(N group,n = 8 rats)and subacute cholestasis model group(M group,n = 28 rats).At the first,seventh,fourteenth and 21 st day of model establishment,rats in group M were given ANIT(75mg / kg)by gavage once to make subacute cholestasis model.Rats in group N were fed with normal food.On the 3rd,9th,16 th and 23 rd day after the start of modeling,7 rats in group M and 2 rats in group N were killed respectively.The liver function indexes(ALT,AST,TBIL,DBIL,TBA,ALP,γ-GT)and liver fibrosis indexes(HA,PCIII,c IV,LN)were detected by blood sampling;the liver tissues were stained with HE,the pathological changes of liver were observed under microscope(x200),and the fecal were collected for 16 S r DNA analysis of Gut Microbiota.Results:1.On the 3rd,9th,16 th and 23 rd day,the liver function and liver fibrosis index(HA)in M group were significantly higher than those in N group(P < 0.05).2.Analysis of intestinal flora: there was no difference in species diversity of intestinal flora abundance between group M and group N,and there was difference in species composition between the two groups by NMDS analysis.At the level of phylum,Bacteroides was the main intestinal flora in group M and Firmicutes in group N.At the genus level,the abundance of Bifidobacteria,Lactobacilli and Ruminococcus in Gut Microbiota of group M was significantly lower than that of group N(P < 0.05),and that of Paracharacteroids and Prevotella were significantly higher than that of group N(P < 0.05);at the 23 rd day,the abundance of Bifidobacteria,Lactobacilli and Paracharacteroids in Gut Microbiota was significantly lower than that of group N(P < 0.05),and that of group N was significantly higher than that of group n(P < 0.05).The Ruminococcus of intestinal flora in group M were different at two time points on the 9th and 23 rd day of modeling;lefse analysis: at genus level,there were statistical differences among Bifidobacteria,Ruminococcus and Prevotella.3.Through linear correlation analysis,it was found that Bifidobacterium and Lactobacillus had negative correlation with ALP,AST,ALT and TBA of liver function,Ruminoccus had negative correlation with TBA and ALP of liver function,Parabaracteroids had positive correlation with TBIL,TBA,ALP,AST and ALT of liver function,and Prevotella had positive correlation with TBA(P < 0.05).Part Three Study on the mechanism of different intervention measures to protect the liver and to influence the intestinal flora in rats with ANIT induced subacute cholestasisObjective: Based on the new therapeutic concept of "gut liver axis" applied to cholestatic liver disease and the results of our previous studies,we designed different interventions including probiotics,UDCA and their combination to observe the intervention effect of subacute cholestasis,and whether they can affect bile acid metabolism and intestinal microecology? related mechanisms?Methods: SD rats were randomly divided into 5 groups :normal control group(N group,n = 16),UDCA intervention group(A group,n = 22),LGG intervention group(B group,n = 22),combined intervention group(C group,n = 22),disease observation group(M group,n = 22).Based on the second part,the model of subacute cholestasis was made in group A,B,C and M.Rats in each group were given corresponding drugs on the third day of modeling(UDCA 100 mg / kg in group A,LGG 2.5ml in group B,UDCA 100 mg / kg + LGG 2.5ml in group C,No additional intervention in group M).Four rats were killed on the first day,the seventh day,the fourteenth day and the twenty-first day of the intervention respectively.The liver function and the index of liver fibrosis were examined.The pathological changes of the liver were observed。The Western blot was used to detect protein expression of the FXR protein and CYP7A1 protein in liver,FXR protein and ASBT protein in ileum.On the 7th and 21 st day of intervention,the intestinal contents were collected and the composition of intestinal flora was analyzed by 16S-r DNA。Results:1.UDCA intervention had little effect on liver function,LGG intervention could reduce liver function injure(ALT,AST,TBA,TBIL,DBIL,ALP)(P < 0.05).There was no synergistic or antagonistic effect between LGG and UDCA,and there was no interaction between LGG/UDCA/LGG+UDCA and time.2.HA in groups A,B,C and M were significantly higher than that in group N(P < 0.05).There was no interaction between UDCA and LGG on liver fibrosis(P > 0.05).At the first week,HA level of group C was significantly lower than that in group A,B and M,and at the third week,HA level in group B was lower than that in group C and M(P < 0.05).3.At the first,second and third week of drug intervention,the expression of ASBT protein in group A,B and C was lower than that in group M,the expression of ASBT protein in group C was higher than that in group A and B,the expression of FXR protein in group C was higher than that in group M,the expression of FXR protein in group A and B was higher than that in group M and C,and the expression of FXR protein in group C was higher than that in group M(P < 0.05);At the first and third week of drug intervention,the expression of CYP7A1 protein in the liver of group A,B and C was lower than that of group M(P < 0.05).4.NMDS analysis showed that the species composition of groups A,B and C was similar,and the species composition of groups M,N and drug intervention groups(A,B and C)was comparable.There was no significant difference in species diversity of intestinal microflora abundance at each time point(P > 0.05).The GM of rats in group N was mainly composed of Firmicutes and Actinobacteri,while in group A,B,C and M,Bacteroides was the dominant.The abundance of Actinomycetes in group B and group M was significantly lower than that in group N(P < 0.05),and that in group C was significantly higher than that in group A and group B(P < 0.05,LDA > 4).On the 7th day of drug intervention,the abundance of Bifidobacteria in group A,B and M was significantly lower than that in group n(P < 0.05,LDA > 4),and that in group B and C was significantly higher than that in group A and m(P < 0.05).The abundance of Bifidobacteria in group B was higher than that in group A,C and m on the 23 rd day of drug intervention.5.By linear correlation analysis,we found that Bifidobacterium had negative correlation with ALP,AST,ALT and TBA(P < 0.05),Lactobacillus had negative correlation with ALP(P < 0.05),Ruminococcus had negative correlation with AST、ALT and ALP(P < 0.05),Parabacteroides had positive correlation with TBIL、TBA、ALP、AST、ALT(P < 0.05)。Conclusions:1.The Gut Microbiota of cholestasis infants was different from that of healthy infants.The diversity in the bacterial community was significantly lower in the IC group than that in the H group.IC group had a more complex GM co-occurrence network featured by three core nodes: Phyllobacterium,Ruminococcus,and Anaerostipes.Rothia,Eggerthella,Phyllobacterium,and Blautia are identified as valuable biomarkers to distinguish IC infants and healthy infants with high accuracy.There is a significant correlation between GM composition and liver function indicators.2.The model of subacute cholestasis in rats was successfully made in our study,which is consistent with the pathological process of clinical subacute or chronic cholestasis liver disease.3.There are dynamic changes of gut microbiota before and after cholestasis and during the process disease progression in the model rats of subacute cholestasis.It is a kind of adaptive change,and the flora structure tends to be stable with time.4.The decrease of Bifidobacteria,Ruminococcus and the increase of Prevotella of intestine in early cholestasis model rats,can be used as the characteristic bacterial group marker and biomarker of early cholestasis.5.The linear correlation analysis showed that the abnormal increase of some metabolism indexes of liver function was related to the decrease of some bacteria in intestine,such as Bifidobacterium,Lactobacillus and the increase of Parabacteroides.6.LGG can improve the synthesis and metabolism of liver in cholestasis model rats,reduce the HA of liver fibrosis in the early stage of disease7.The possible mechanism that LGG can improve the liver function of cholestasis model rats,is to directly or indirectly promote the expression of FXR protein in liver and ileum,inhibit the expression of CYP7A1 protein in liver,so as to inhibit the production of bile acid;inhibit the production of ASBT protein in intestine,so as to reduce the reabsorption of bile acid,alleviate cholestasis and further damage of bile acid to liver.At the same time,HA was reduced.8.LGG intervention can promote the increase of intestinal beneficial bacteria such as Bifidobacteria,strengthen the structure of intestinal flora,reduce the imbalance of intestinal flora,and play a protective role on the liver by improving the intestinal habitat.9.The protective effect of UDCA on liver and intestinal microflora in ANIT induced cholestasis rats has not been shown in this study.10.The application of UDCA combined with LGG has no synergistic or antagonistic effect on liver function and liver fibrosis index,which needs to be confirmed by large sample study. |
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