| Background and Aims:The small intestinal bacterial overgrowth(SIBO)has been associated with a variety of chronic diseases.SIBO is characterized by nonspecific gastrointestinal symptoms such as abdominal distention,abdominal pain,diarrhea,and constipation,and is easily confused with or associated with other organic diseases.As a result,SIBO is often overlooked,leading to inadequate understanding of SIBO.Gut microbiota is closely related to host metabolism and immunity,and intestinal epithelial cells may be the key link in the interaction between host and gut microbiota.Currently,it is not clear how metabolism and immunity affect small intestinal microbiota.We sought to investigate the related factors and gut microbial characteristics of SIBO,to evaluate the relationship among diet,metabolism,inflammation,and SIBO,and to explore the pathogenesis of SIBO.Material and Methods:1.Study on the related factors of SIBO in middle-aged and elderly patients in gastroenterology clinicThis study enrolled male middle-aged and elderly patients(age 50 or older)who visited the gastroenterology clinic of the Second Medical Center of the PLA General Hospital.A lactulose breath testing was used to diagnose SIBO.A survey of gastrointestinal symptoms using gastrointestinal symptom rating scale(GSRS)questionnaires,prior medical history,abdominal surgery history and laboratory data was conducted.2.Gut microbiota and intestinal inflammation in patients with SIBO16S rRNA sequencing was performed on the mucosal biopsies of duodenum,ileum,and sigmoid colon and stool samples collected from some of the SIBO+and SIBO-subjects mentioned above.Bio-Plex was used to detect the levels of cytokines in intestinal mucosal biopsies.3.Association between hypercholesterolemia and SIBOStepwise adjusted binary regression analysis was used to construct association models between hypercholesterolemia and SIBO based on the population as chapter 1.4.Mechanism of SIBO induced by HFDMice were divided into 3 groups fed with HFD,HFHCD and SD,respectively.Each diet group was randomly divided into 2 groups,with 6W and 12 W as the observation end points,respectively.Serum cholesterol was detected by automatic biochemical analyzer.QPCR was used to quantify the number of bacteria in the small intestinal contents.16 S rRNA gene sequencing was performed on the ileum and small intestinal contents.Caco-2 cells were cultured and treated with FFA,FFA+ cholesterol,cholesterol and solvent control.Total RNA of each group was collected for m RNA microarray to screen DEGs.QPCR was used to detect the expression of DEGs in the ileum samples of mice,as well as the expression of antimicrobial peptides.Results:1 Study on the related factors of SIBO in middle-aged and elderly patients in gastroenterology clinic1.1 The prevalence of SIBO in the study population was 70.04%.1.2 The proportion of hypercholesterolemia and history of gastroenterectomy in SIBO+ group was higher than that in SIBO-group(P < 0.05).1.3 The scores of feeling of incomplete evacuation,abdominal distention,increased flatus and total score of the SIBO+ group were significantly higher than those of the SIBO-group(P < 0.05).1.4 Multivariate logistic regression analysis showed that hypercholesterolemia,history of gastrointestinal surgery and albumin level were correlated with SIBO(P < 0.05).2 Gut microbiota and intestinal inflammation in patients with SIBO2.1 Compared with the SIBO-group,the microbiota richness values of the mucosa of the ileum and sigmoid colon were significantly decreased(P<0.001 and P < 0.05,respectively)and the microbiota diversity of the ileum mucosa was significantly reduced(P<0.001)in the SIBO+ group.2.2 PCo A analysis showed that the mucosal microbiota of the duodenum,ileum,and sigmoid colon were structurally different in the SIBO+ group compared to SIBO-group(P<0.05,P<0.01,and P<0.01,respectively).2.3 Four genera(Lactobacillus,Prevotella＿1,Dialister,and norank＿f＿＿Ruminococcaceae)showed similar changes among the mucosal samples of the duodenum,ileum,and sigmoid colon in the SIBO+ subjects compared to the SIBO-subjects.2.4 A signature consisting of four genera in the duodenal mucosa,or three genera in the ileac mucosa,or six genera in the mucosa of the sigmoid colon exhibited predictive power for SIBO(area under the curve = 0.9,0.93,and 0.87,respectively).2.5 The levels of IL-6 of ileal mucosa in the SIBO+ group were significantly higher than that in the SIBO-group(P<0.05).3 Association between hypercholesterolemia and SIBOHypercholesterolemia was an independent risk factor for SIBO.4 Mechanism of SIBO induced by HFD4.1 After 6W feeding,the levels of serum TC and LDL-C of HFD group and HFHCD group were significantly higher than those of SD group(all P<0.01).While the bacterial quantity of small intestinal contents of the three groups were not significantly different(P>0.05).4.2 After 12 W feeding,the levels of serum TC and LDL-C and the bacterial quantity of the small intestinal contents of HFD and HFHCD group were significantly higher than those of SD group(all P<0.05).4.3 After 12 W feeding,the ileal mucosa-associated microbiota richness values in HFD and HFHCD groups were significantly lower than that in SD group(all P<0.05).4.4 After 12 W feeding,the composition of ileal mucosa-associated microbiota of HFD group,HFHCD group,and SD group was significantly different(P<0.01).4.5 Effects of lipid intervention on gene transcription in intestinal epithelial cells4.5.1 Compared with group A(control group),48 genes were up-regulated and149 genes were down-regulated in group B(FFA group).Compared with group A,175 genes were up-regulated and 260 genes were down-regulated in group C(FFA+ cholesterol group).Compared with group A,93 genes were up-regulated and 324 genes were down-regulated in group D(cholesterol group).4.5.2 KEGG pathway enrichment analysis of DEGs showed that the DEGs in group B,C,and D were all enriched in PPAR pathway compared with group A.4.5.3 PPAR pathway genes FABP6,FABP1,CYP8B1,APOC3,SCD and MMP1 were identified by comparing PPAR pathway with the information of DEGs in group B,C,and D.It was found that the expression of these 6 genes was decreased in all the three intervention groups,and the expression was lowest in group C.4.6 The gene expression of CYP8B1,MMP1 and APOC3 in ileum of mice4.6.1 After 6W feeding,the gene expression of CYP8B1 of ileum in HFD group was significantly decreased than that in SD group(P<0.05).The gene expressions of CYP8B1 and MMP1 of ileum in HFHCD group were significantly decreased than those in SD group(P<0.01 and P<0.05,respectively).4.6.2 After 12 W feeding,the gene expressions of CYP8B1 and MMP1 of ileum in HFD group were significantly decreased than those in SD group(P<0.001 and P<0.01,respectively).The gene expressions of CYP8B1,MMP1 and FABP6 of ileum in HFHCD group were significantly decreased than those in SD group(P<0.01,P<0.001,and P<0.01,respectively).4.7 Antimicrobial peptide gene expression in ileum4.7.1 After 6W feeding,the gene expression of DEFA5 of ileum in HFHCD group was significantly decreased than that in SD group(P<0.05).4.7.2 After 12 W feeding,the gene expressions of MMP7 and DEFA5 of ileum in HFD group were significantly decreased than those in SD group(P<0.01 and P<0.01,respectively).The gene expressions of MMP7,DEFA5 and REG3γ of ileum in HFHCD group were significantly decreased than those in SD group(P<0.01,P<0.001,and P<0.05,respectively).Conclusions:1.The prevalence rate of SIBO was 70.04% in middle-aged and elderly people who visited the gastroenterology clinic excluded organic gastrointestinal diseases,so we should pay attention to SIBO in the department of gastroenterology.2.Some mucosa-associated microbial signatures were identified and certain degree of ileac mucosa inflammation was detected in SIBO patients,which may guide future investigation on the pathogenesis of SIBO as well as potential therapeutic targets.3.Hypercholesterolemia was an independent risk factor for SIBO.4.Dietary lipids may increase ileal bacterial colonization by down-regulating the PPAR pathway in ileum epithelium,which in turn affects the expression of ileal antimicrobial peptide genes,resulting in SIBO. |
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