Intestinal Microbiota Dysbiosis And Abnormal Bile Acids Metabolism In Colitis-associated Cancer

ObjectiveColorectal cancer(CRC)has become the third most common cancer and one of the leading causes of death.Patients with a long history of inflammatory bowel disease(IBD)are more likely to develop intestinal cancer,also known as colitis-associated cancer(CAC).Factors involving the development of CAC include intestinal inflammatory response,immune response,alteration of the epigenetic landscape and gut dysbiosis.Intestinal microbiota dysbiosis promotes intestinal disease and also affects bile acid metabolism.Bile acid undergoes deconjugation,oxidation,7α-dehydroxylation,esterification and desulfatation in the presence of intestinal flora,and participates in maintaining cholesterol metabolism,digestion and absorption of nutrients,and metabolic homeostasis.Bile acids can also activate bile acid receptors FXR and TGR5,which have a role in regulating intestinal mucosal barrier,inflammatory response and flora composition.We chose the CAC model to explore the interaction between gut dysbiosis and abnormal bile acid metabolism in the development of CAC,providing new insights to the treatment of intestinal tumors.MethodsSeven-week-old female C57BL/6 mice were divided into control group and azoxymethane/dextran sodium sulphate(AOM/DSS)group,and each group containing 10 mice.After acclimatized 1 week before the experiment,the feces of the two groups were collected for intestinal microbiota analysis.Mice in the AOM/DSS group were intraperitoneally injected with 10 mg/kg AOM on day1,while the control group received intraperitoneal injection of 0.9% sterile isotonic saline.After seven days,AOM/DSS group was given 1.5% DSS in drinking water on days 8–12,27–31 and 46–50,each cycle of DSS followed by 14 days of drinking water.The control group was only provided sterile drinking water.The mice were observed daily for drinking and eating,defecation and general conditions,and weighed weekly.At days 70,each mouse was individually housed in a clean cage to collect feces.Mice were sacrificed in 10 weeks,and the colon length and spleen weight was recorded.The intestine of mice was dissected longitudinally and then the location,number and size of the tumors were observed under a dissecting microscope.Part of the colon was stored in a 10% formalin solution for paraffin embedding.Ileum and the rest of colon tissues were frozen and stored at-80℃ for subsequent experiments.(1)Colon sections(5 μm)were stained with hematoxylin and eosin(HE).An experienced pathologist performed pathological assessments.(2)The expression of inflammatory cytokines IL-1β,IL-6 and TNF-α in the colon was detected by Realtime-PCR.(3)Realtime-PCR was used to detect the expression of tight junction protein ZO-1,Occludin,Claudin1 and Claudin3.(4)Fecal microbiota diversity of the control group and the AOM/DSS group at 0 and 10 weeks was measured using 16 S rRNA high-throughput sequencing.The sequences of samples were clustered to generate operational taxonomic units(OTUs)at 97% identity using Usearch.Classify the representative sequence of each OTU using the 16 S database.Alpha diversity and beta diversity were performed using QIIME.(5)Liquid chromatography-mass spectrometry(LCMS)method was used to measure the fecal bile acids concentration.After pretreatment of each sample,the concentration of cholic acid(CA),chenodeoxycholic acid(CDCA),lithocholic acid(LCA),deoxycholic acid(DCA),α-muricholic acid(α-MCA)and β-muricholic acid(β-MCA)were determined by the peak areas of the external standards and individual bile acids.(6)The mRNA levels of ileum FXR,FGF15 and bile acid transporters ASBT,OSTα and OSTβ were detected by Realtime-PCR.The expression of TGR5 was determined using Realtime-PCR and immunohistochemical staining ResultsIn the AOM/DSS group,mice showed significant weight loss and accompanied with hematochezia during each cycle of DSS treatment.The colon of AOM/DSS group showed multiple tumors,while no hematochezia and tumor formation were observed in the control group.After AOM/DSS treatment,the colon developed severe inflammatory response and the intestinal mucosal barrier was disrupted,manifested by an increase in inflammatory cytokines and a decrease in tight junction proteins.The 16 S rRNA sequencing showed that the richness and diversity were decreased,and the composition of gut microbiota was significantly altered in the CAC model.During CAC development,the abundance of phylum Bacteroidetes decreased and Firmicutes increased.At the genus level,the abundance of pathogens Helicobacter and Streptococcus increased,while the short-chain fatty acids(SCFA)producing bacteria Lachnospiracea_incertae_sedis,Alistipes and Odoribacter decreased.It indicated that the beneficial bacteria decreased and the pathogenic bacteria increased during the development of CAC.More importantly,Clostridium XlV and Lactobacillus,which involving bile acid deconjugation,transformation and desulfatation,also showed lower levels.Fecal bile acid profiles showed a reduced level of free bile acids in line with decreased conversion of primary bile acids to secondary bile acids following AOM/DSS treatment.Considering the bile acid receptors,the ileum FXR-FGF15 axis was down-regulated,and the expression of TGR5 was elevated in the colon tumor tissues of the CAC model.ConclusionsDuring the development of CAC,intestinal gut dysbiosis affected the metabolism and composition of bile acids.The reduced expression of secondary bile acids with anti-inflammatory effects companied with bile acid receptors promotes intestinal tumorigenesis.