Intestinal Dysbiosis Of Short Bowel Syndrome And Its Role In Intestinal Failure Associated Liver Disease

The short bowel syndrome(SBS)is caused by congenital defects or surgical resection,which leads to a shortened intestinal remnant and is characterized as severe malnutrition,water-electrolyte disturbance,and malabsorption of micronutrients.SBS can be divided into three types based on the anatomy:end-jejunostomy(type I),jejuno-colic anastomosis(type ?),and jejuno-ileal anastomosis(type ?).Most SBS patients are totally or partly dependent on parenteral nutrition(PN)to meet the daily energy requirements,however,long-term dependence on PN may lead to lots of complications,such as intestinal failure associated liver disease(IFALD)and catheter-related bloodstream infection.On the other hand,small bowel resection itself causes severe physiological disturbances and contributes to the development of IFALD.Therefore,the goals of managing SBS patients include facilitating intestinal adaption and enteral diet advancement,accelerating the process of weaning off PN,and prevention and reduction of complications.Currently,intestinal rehabilitation therapies mainly focus on promoting intestinal growth and enhancing absorptive functions,slowing transit and decreasing diarrhea,reducing gastrointestinal secretion,and surgical procedures to lengthen small intestine.However,little is known about the alterations in intestinal microbiota after small bowel resection,especially the intestinal microbiota signatures of adult SBS patients.Intestinal microbiota has been considered to play an important role in fermenting and absorbing undigested carbohydrate,regulating the biosynthesis and transport of bile acid,improving the intestinal immune functions and maintaining the integrity of intestinal harrier.Study of pediatric SBS patients demonstrated that intestinal dysbiosis could delay the process of intestinal adaption,destroy the compensation of intestinal functions,prolong the use of PN,and finally lead to poorer clinical outcomes.This suggests that clarification of the changes of intestinal microbiota and their effects on intestinal functions in SBS patients may provide new targets for the therapy of SBS.Both SBS and PN contribute markedly to the occurrence of IFALD,of note,SBS is regarded as an independent risk factor of IFALD.Although numerous studies have explored the relationships between PN and IFALD,little is known about how SBS itself is involved in the development of IFALD.Animal study showed that SBS piglet models,which had never received PN,exhibited altered compositions of bile acids in colonic content and portal serum,with an increase of primary bile acids and a decrease of secondary bile acids.As we know,after being metabolized by specific intestinal microbes,bile acids in the gut can further negatively regulate their hepatic biosynthesis by activating farnesoid X receptor(FXR)in terminal ileum.As a result,we hypothesize that the changes of bile acid metabolism in gut caused by intestinal dysbiosis of SBS may disturb hepatic bile acid biosynthesis and finally contribute to the development IFALD.In this thesis,we will first analyze the fecal microbiota signatures of adult patients with different types of SBS by 16S rRNA gene sequencing.Next,we will produce rat models with type II and type III SBS to profile intestinal microbial compositions in different intestinal positions and at different times after small bowel resection.The relationships between gut microbiota of SBS and functions of remnaning intestine will also be studied.Finally,through investigating the relative abundance of specific microbes responsible for bile acid metabolism,the compositions of intestinal bile acids,and the expression levels of genes involved in biosynthesis,transport and regulation of bile acid,we will explore the mechanism by which SBS gut dysbiosis contributes to the development of IFALD.This study may provide new perspectives on intestinal rehabilitation and prevention of IFALD in SBS patients.This thesis is divided into three parts.Part ?:Fecal microbiota signatures of adult patients with short bowel syndromeObjectives:To investigate the fecal microbiota signatures of adult patients with different types of SBS.Methods:We enrolled adult SBS patients who were hospitalized in our department from November 1,2015 to November 30,2016.Healthy controls were recruited from outpatients coming for routine medical examinations.Fecal samples were collected from all the participants,and genomic DNA was extracted for the amplification of V4-V5 region of 16S rRNA genes.The PCR products were then sequenced and data were analyzed.Results:Ten SBS patients meeting the inclusion criteria were finally recruited into this study,five with type ? SBS and five with type III SBS.Another five healthy volunteers were matched as controls.All participants were divided into three groups,type ? SBS group,type ? SBS group and control group.There were no differences in gender,age and body mass index among three groups,and the clinical characteristics were well matched between two SBS groups.There was a significant decrease in fecal microbial diversity in both types of SBS patients(P<0.05).The Shannon and Simpson index of fecal microbiota was positively correlated to the length of remant small intestine in SBS patients,with the correlation coefficients of 0.733(P<0.05)and 0.891(P<0.01),respectively.For type II SBS patients,the relative abundance of Proteobacteria and its family Enterobacteriaceae was significantly greater than that of other two groups(P<0.01),while the relative abundance of Firmicutes(P<0.05)and Bacteroidetes(P<0.01)was markedly lower than that of healthy controls.Lactobacillaceae and Prevotellaceae were more dominant in type III SBS patients than controls(P<0.001)and type II SBS patients(P<0.01).Commensal bacterial genera from Bacteroidaceae,Lachnospiraceae,and Ruminococcaceae dramatically reduced in SBS patients.The PN duration of SBS patients was positively correlated to the amount of Enterobacteriaceae(r = 0.701,P<0.05),but negatively correlated to Lactobacillus(r =-0.659,P<0.05).Conclusions:The fecal microbial diversity decreased in both types of SBS patients,and the compositions of fecal microbiota were significantly different among three groups.Type ?SBS was characterized by Proteobacteria,and type ? SBS was featured by Lactobacillus.The PN duration of SBS patients was significantly related to certain bacteria in fecal microbiota.Part ?:Intestinal dysbiosis of rat models with short bowel syndrome and its effect on intestinal inflammation and intestinal barrier functionObjectives:To profile the intestinal microbial compositions of SBS rats in different intestinal positions and at different times after small bowel resection,and to investigate the relationships between SBS gut dysbiosis and intestinal inflammation as well as intestinal barrier function.Methods:Thirty Sprague-Dawley(SD)rats weighted 200-250g were randomly assigned to three groups:type ?SBS,type ? SBS and sham.Rat models of SBS were produced by resection of 75%small intestine with(type ?)or without(type II)ileocecal junction remnant.Each group was divided into two subgroups after surgery,and rat models were raised for 2 or 4 weeks.Descending colonic and cecal(except type II SBS)contents were collected for 16S rRNA gene sequencing.Colon samples were collected to detect the mRNA expression of interleukin-8(IL-8),IL-18 and tumor necrosis-a(TNF-a).Immunohistochemistry staining method was used to analyze the protein expression of zona occludens protein-1(ZO-1)and Occludin in colon and terminal ileum(except type II SBS)samples.Pearson correlation analysis was used to test the correlation between intestinal microbial diversity and expression levels of intestinal inflammatory factors and tight junction protein.Results:(1)The changes of intestinal microbiota in same type of rat models were similar at week 2 and week 4.The microbial a diversity of both colonic and cecal contents markedly decreased in type ? and ? SBS rats(P<0.05),and the ? diversity was also statistically significant among groups(P<0.001,colonic microbiota;P<0.01,cecal microbiota).a)Colonic microbiota:Compared with sham and type ? SBS rats,type ? SBS rats had a higher relative abundance of Proteobacteria(P<0.01),and a lower relative abundance of Bacteroidetes(P<0.01).At week 4,the proportion of Firmicutes was greater in type ? SBS rats than other two groups(P<0.01),but the amount of Bacteroidetes was lower in type III SBS rats than sham(P<0.01).Genera capable of producing short chain fatty acids(SCFAs)were dramatically deficient in both types of SBS rats,and genera from Enterobacteriaceae were significantly predominant in type II SBS rats.b)Cecal microbiota:The relative abundance of Firmicutes was significantly greater(P<0.05),while the proportion of Bacteroidetes was markedly lower(P<0.05)in type ? SBS rats.The beneficial bacteria and microbes involved in bile acid metabolism decreased,but opportunistic pathogens increased in type III SBS rats.(2)At week 2,the expression levels of intestinal inflammatory factors and tight junction protein were comparable between SBS and sham rats.At week 4,the mRNA expression of IL-8,IL-18,and TNF-a in colonic mucosa increased significantly in type II and III SBS rats,and was negatively related to Shannon index of colonic microbiota,with the correlation coefficients of-0.486,-0.438,and-0.458,respectively(P<0.05).Meanwhile,the expression of ZO-1 and Occludin in colonic and terminal ileum mucosa significantly decreased in SBS rats,and was positively related to Shannon index of colonic or cecal microbiota,with the correlation coefficients of 0.364 and 0.554 in colon(P<0.05),and,0.508 and 0.467 in terminal ileum(P<0.05),respectively.Conclusions:Dysbiosis was detected both in colonic and cecal contents of SBS rats.Moreover,decreased microbial diversity and altered microbial compositions occurred early after small bowel resection and existed for a long time.The reduction of microbial diversity could cause severe colon inflammation and intestinal barrier dysfunction.Part ?:Mechanism of intestinal failure associated liver disease arising from intestinal dysbiosis in rat models with short bowel syndromeStudy 1:Relationship between intestinal dysbiosis and intestinal failure associated liver disease in rat models with short bowel syndromeObjectives:To observe the changes of live function and hepatic histology in SBS rats,and to explore the relationship between SBS intestinal dysbiosis and the development of IFALD.Methods:Twelve SD rats weighted 200-250g were randomly divided into two groups:type ? SBS and sham.Rat models of SBS were produced by resection of 75%small intestine with ileocecal junction remnant.All rats were killed at day 28 after surgery.Blood from inferior vena cava was collected to detect the levels of transaminase and bilirubin;Masson,Oil Red O,and Sirius Red staining was performed to evaluate the hepatic histology;cecal contents were collected for measurement of the amount of bile acids and for 16S rRNA gene sequencing;expression levels of FXR,SHP(small heterodimer partner)and FGF15(fibroblast growth factor 15)in terminal ileum,and enzymes for hepatic bile acid synthesis including Cyp7a1(cholesterol 7?-hydroxylase)and Cyp8b1(sterol 12?-hydroxylase)were determined.Results:The levels of direct and total bilirubin and alanine transaminase significantly increased in SBS rats(P<0.05).The fibrosis,hepatocyte ballooning,and fat droplet accumulation were severer in livers of SBS rats than sham.Compared with sham rats,the relative abundance of bacteria responsible for bile acid metabolism,including Bacteroides(P<0.01),Clostridium(P<0.05)and Bifidobacterium(P<0.01),was much lower in SBS rats,which led to an increase of primary bile acids including cholic acid(CA,P<0.001)and ?-muricholic acid(?MCA,P<0.01),and a decrease of secondary bile acids including deoxycholic acid(DCA,P<0.001),?-muricholic acid(?MCA,P<0.01)and lithocholic acid(LCA,P<0.05).The activation of FXR(P<0.05),SHP(P<0.01)and FGF15(P<0.001),which negatively regulated hepatic synthesis of bile acid,significantly decreased in terminal ileum of SBS rats.Protein(P<0.05)and mRNA(P<0.01)expression levels of Cyp7al and Cyp8bl significantly increased in livers of SBS rats.Conclusions:SBS rats exhibited IFALD evidenced by live function and hepatic histology test.The lower proportions of bacteria involved in bile acid metabolism in gut of SBS rats led to a reduction of secondary bile acids,which decreased the activation of FXR-FGF15 signaling in terminal ileum and further resulted in a higher expression of enzymes responsible for hepatic bile acid biosynthesis.Finally,the over-synthesized bile acids led to liver cholestasis and IFALD.Study 2:Effects of intestinal secondary bile acids on intestinal failure associated liver disease in rat models with short bowel syndromeObjectives:To further investigate the effects of intestinal secondary bile acids on IFALD in SBS rats and to confirm the conclusions in Study 1.Methods:Resection of 75%small bowel was performed on eighteen SD rats weighted 200-250g to produce type III SBS rat models.These SBS rat models were randomly assigned to three groups:Saline,DCA,and Antibiotic.At day 23-27 after surgery,SBS rats in each group were given oral gavage of saline(0.5mL/d),DCA(100mg/Kg/d),or antibiotic cocktail(ampicillin,100mg/Kg/d;neomycin,100mg/Kg/d;metronidazole,100mg/Kg/d;streptomycin,100mg/Kg/d;vancomycin,50mg/Kg/d),respectively.At day 28 after surgery,SBS rats were killed,cecal contents were collected to determine the amount of DCA;Oil Red O staining was performed to evaluate liver histology;expression levels of FXR,SHP and FGF15 in terminal ileum,and Cyp7a1,Cyp8bl,FXR and SHP in liver were analyzed.Results:Compared with Saline group,levels of DCA were significantly higher in cecal contents of SBS rats in DCA group(P<0.05)and lower in Antibiotic group(P<0.05).Meanwhile,expression levels of FXR,SHP,and FGF15 markedly increased in terminal ileum of SBS rats in DCA group(P<0.05),but significantly decreased in Antibiotic group(P<0.05).The altered activation of FXR-FGF15 signaling led to a decreased expression of Cyp7al and Cyp8bl in livers of SBS rats in DCA group(P<0.05),while an increased expression of these two proteins in Antibiotic group(P<0.05).For hepatic histology,the fat droplet accumulation was relieved in SBS rats of DCA group,but severer in Antibiotic group.Although expression levels of FXR in livers were significantly lower in DCA group than other two groups(P<0.05),no differences were detected in expression of SHP in livers among three groups.Conclusions:The increased levels of secondary bile acids in intestine,such as DCA,could enhance the activation of FXR-FGF15 signaling in terminal ileum of SBS rats,which further negatively regulated the expression of Cyp7al and Cyp8b1 in livers and contributed to the relief of IFALD.This confirms the conclusion in Study 1 that SBS intestinal dysbiosis causes IFALD through reducing the levels of secondary bile acids in gut.