Interaction Between Bile Acids Metabolisms And Gut Microbiota Controls The Progression Of Intestinal Adenoma

Aims Colorectal cancer(CRC)is a leading cause of cancer deaths around the world.The main precancerous lesion of CRC is colorectal adenoma.Adenomatous polyposis coli(Apc)is mutated in almost 90%-95% of sporadic human colorectal cancers,which is one of the earliest events in the adenoma-carcinoma sequence.High fat diet(HFD)promotes the bile acids synthesis by liver,resulting in increased bile acids level in gut lumen.Excess bile acids escape the hepatoenteral circulation and access the colon.Recent researches suggests that HFD can change the composition of microbiota and induce intestinal dysbiosis and internal environment disturbance.The primary bile acids in humans,cholic acid(CA),is modified to the secondary bile acids in intestine by bacterially facilitate 7?-dehydroxylation reaction.However,the interplay of bile acids,gut microbiota and CRC is not clear.The aim of this study is to determine the role of the crosstalk between bile acids and gut microbiota in intestinal carcinogenesis.Methods Part 1: To investigate the effect of CA in intestinal carcinogenesis,4 weeks old Apcmin/+ mice were divided into two group: CA group(diet with 0.4% CA supplemented)and the control group(the control diet).After 12 weeks,the mice were sacrificed.The total intestine tissue was collected and intestinal tumors were categorized according to different sections and sizes.Tissue sections were used to hematoxylin and eosin(H&E)and immunohistochemical(IHC)staining.And immunofluorescence double staining,real-time PCR and IHC were used to assess the activation of mononuclear-phagocyte system in tumor microenvironment.Tumor associated Wnt signaling pathway were examined by IHC.Part 2 After 12 weeks,FITC-D was given by gavage before sacrificing,and then FITC-D concentration in serum was measured by extracting the eyeball blood.The feces were collected for 16 S r RNA sequencing analysis and bile acids concentrations tests.The concentrations of SCFAs in cecal contents were measured by gas chromatography(GC)method.Realtime PCR and IHC were used to analyze the function of tight junction,goblet and paneth cells.The inflammation level of intestinal tissue was analyzed via real-time PCR.Part 3 Next in order to prove the key role of intestinal bacteria,Apcmin/+ mice were fed with a 0.4% CA diet and a daily dose of antibiotics cocktail(500 mg of ampicillin,250 mg of vancomycin,500 mg neomycin and 250 mg of metronidazole).After 12 weeks,the mice were sacrificed,the intestinal tumor and inflammation were observed.Part 4 To further elucidate the mechanisms of the effects of CA-induced dysbiosis on intestinal carcinogenesis,we tested the key factors of IL-6/STAT3 signaling pathway by realtime-PCR and IHC,and we also investigated the effects of fecal bacteria liquids from the control,CA-treated,CA-Abx treated mice on IL-6/STAT3 signaling in IMCE cell line via Western blot.Results Part 1(1)The total tumor number in CA-diet mice was almost two fold compared with that in the control group.CA promoted intestine tumor development mainly in the middle and distal portions of the small intestine.The numbers of all sizes of tumors in small intestine(<1mm,1-2mm,>2mm)and large tumors(>2 mm)in the colon were also increased.Moreover,supplement with CA in diet also led to intestinal carcinogenesis.In distal small intestine,HGIN including intramucosal carcinoma were detected in 80%(8/10)of CA-diet mice,whereas age-matched Apcmin/+ mice on a normal diet developed only adenoma with or without LGIN.Increased cell proliferation(Ki-67)and ?-catenin expression levels in CA-diet mice were also revealed.(2)The inflammation factors of intestinal tissue,the expressions of MCP-1 and F4/80 also increased in CA group.And CA treatment promoted the recruitment of tumor-associated macrophages and polarization of M2 macrophages according to immunofluorescence double staining.(3)CA activated Wnt signaling pathway,downregulated the E-cadherin level,and up-regulated the ?-catenin and Cyclin D1 level.Part 2(1)CA induced the intestinal dysbiosis,presented by increased abundance of pathogens(Prevotella,Escherichia coli,Desulfovibrio and some opportunistic pathogens such as Akkermansia and Bacteroides)and decreased abundance of probiotics(Lactobacillus and SCFA producing bacteria).The diversity of the OTUs in CA group,expressed as Shannon and Simpson indices,was lower than that of the control group,suggesting a lower diversity in CA group compared with control group.(2)And the concentrations of SCFAs in cecal contents were significant decreased in CA-diet mice.(3)The concentration of FITC-D in serum was significantly higher than the control group,suggesting an impairment of gut barrier function.In particular,our findings showed decreased m RNA expression of ZO-1,Claudin 3,Claudin 7 and MUC2.Periodic acid schiff(PAS)and MUC2 staining showed that CA attenuated the number of goblet cells.Gene expression of cryptdin and defensin,Paneth cell staining of lysozyme were decreased in CA-diet mice,suggesting an impairment of Paneth cell function(4)The relative m RNA expression levels of interleukin-6(IL-6),interleukin-1?(IL-1?)and tumor necrosis factor-?(TNF-?)were up-regulated by CA treatment.(5)Increased intestinal bile acid flow as a result of CA diet dramatically increased the fecal concentrations of both the highly the administered CA and bactericidal DCA.Part 3 The tumor numbers in all sections of small intestine and colon were decreased in CA-Abx group.No HGIN was found in distal small intestine and colon after antibiotics cocktails treatment.And the intestinal inflammation also decreased significantly compared with the control group.Part 4 IHC revealed a strong expression of p-STAT3 in intestine tissues in CA group,while there was no significantly difference in STAT3 expression.We also detected the expression of IL-6,p-STAT3 and STAT3 in intestinal tissue in CA-Abx group,and found that increased expression of IL-6 and p-STAT3 were significantly abolished by CA-Abx treatment.When IMCE cells were incubated with fecal bacteria liquid from CA-diet mice,STAT3 signaling and its downstream regulators such as Cyclin D1,cMyc,BCL-XL and VEGF were also activated.In comparison,treatment of IMCEs with fecal bacteria liquids from the control and CA-Abx mice could not lead to STAT3 signaling activation.ConclusionThe present study shows that the progression of intestinal adenoma Apcmin/+ mice model was deeply influenced by the interplay between bile acids and gut microbiota controls.The increase of some inflammation-related bacteria,such as Akkermansia,might be potential promoters in intestinal carcinogenesis.Decreased protective SCFAs,increased bile acids and activated STAT3 signaling pathway were all involved in tumor malignant progression.