Study On The Effect Of Probiotics And Farnesoid X Receptor On Colitis And Its Mechanism

Part 1 Effect of VSL#3 and S.Boulardii on Intestinal Microbiota In Mice with Acute ColitisObjective To investigate the effects of probiotics(VSL#3,S.Boulardii)on intestinal flora of mice with DDS-induced acute colitis.Methods C57BL/6 mice were administered with 2.5%dextran sulfate sodium for 5 consecutive days to develop the acute colitis model except for the blank control group.Meantime,Mice were treated with drinking water(DSS model group),VSL#3(1.5×109 CFU),or S.Boulardii(5×107CFU)by gavage for 7 days respectively,and mice were sacrificed 2 days after the model of colitis was established.The fecal specimens before gavage(day 0),in the middle of experiment(day 4),and the end of gavage(day 7)and the intestinal mucosa after sacrifice were collected to analyze the differences between these four groups by 16s rDNA sequencing method.Results Compared with the DSS model group,VSL#3 group showed a decrease in disease activity index(DAI)and histological scores,and there was no significant change in the S.Boulardii group.Fecal microbiota:In the middle of experiment,the alpha diversity of DSS model group,VSL#3 group and S.Boulardii group were lower than that of the blank control group(p=0.0135,p=0.0018,p=0.0151).After the end of gavage,the diversity of the VSL#3 group was lower than that of the blank control group(p=0.025),and the difference between any other two groups was not statistically significant.Mucosa-adherent microbiota:biodiversity of DSS model group,S.Boulardii group were lower than the blank control group(p=0.031,p=0.0437),while biodiversity of VSL#3 group was higher than DSS model group and S.Boulardii group(p=0.0394,p=0.0426).Compared with the blank control group,the DSS model group showed an increase in Bacteroides and a decrease in Lactobacillus.Abundance in the genus Turicibacter and Odoribacter increased in intestinal microbiota of mice with acute colitis,while VSL#3 inhibited them.Conclusion VSL#3 alleviates inflammation in DSS-induced colitis of mice.Both VSL#3 and S.Boulardii can affect intestinal microbiota.Compared with healthy mice,mice with colitis showed a reduced diversity of microbiota both in feces and in intestinal mucosa.VSL#3 increases biodiversity of mucosal microbiota in mice with acute colitis,while it does not increase biodiversity of fecal microbiota.Genera such as Turicibacter and Odoribacter increase in mice with acute colitis,and these genera can be inhibited by VSL#3.Part 2 Role of Farnesoid X Receptor in Improving Colonic Inflammation and Inhibiting Colonic Tumor Formation in Mice by VSL#3Objective To investigate whether VSL#3 affects FXR expression in acute colitis model mice and whether FXR is involved in inhibition of colon cancer formation by VSL#3.Methods Twenty C57BL/6 mice were randomly divided into blank control group,VSL#3 group,DSS group,and DSS+VSL#3 group.The latter two groups were subjected to 2.5%DSS for 5 days.VSL#3 group and DSS+VSL#3 group were gavaged with 1.5×109 CFU VSL#3 per day for 7 days.FXR mRNA expression in colon was detected by qPCR,and its expression in nuclear protein extraction were determined by Western blot.FXR knockout mice were constructed and validated.Fifteen WT mice and fifteen FXR knockout mice were divided into six groups:WT blank control group,WT model group,WT VSL#3 group,and KO blank control group,KO model group,KO VSL#3 group.Except for two blank control groups,the other 4 groups were induced by intraperitoneal injection of AOM and 1 round of 2.5%DSS to develop a model of colitis-associated colorectal cancer(CRC).Two VSL#3 group was gavaged with 1.5×109 CFU/d VSL#3,and the blank groups and the model groups were gavaged with drinking water.Mice were sacrificed after 12 weeks and tumor burden was measured.TNFa,IL-α and IL-1β mRNA expression in the colon were detected by qPCR.Results In acute colitis model experiment:There was no significant difference of FXR expression in mRNA levels between blank control group,DSS group and DSS+VSL#3 group.However,FXR expression in nuclear protein extraction was significantly lower in DSS group than that of blank control group and DSS+VSL#3 group.FXR expression in nuclear protein extraction was similar in blank control group and VSL#3 group.In colitis-associated CRC model experiment:Tumor burden in WT VSL#3 group was lower than WT model group(p=0.0546)whereas there was no difference of colonic tumor formation between KO VSL#3 group and KO model group.Compared with WT model group,WT VSL#3 group had decreased IL-6 and IL-1β in the colon,but the difference was not statistically significant,and there was no significant change of TNFα.TNFa,IL-α,and IL-1β were similar in KO VSL#3 group and KO model group.Conclusion Our results indicate that inflammation reduces nuclear FXR expression,while VSL#3 increase nuclear FXR expression in colitis,and VSL#3 prevents colon carcinogenesis through FXR.Part 3 Effects of Farnesoid X Receptor Activation on Colitis and Its Potential MechanismObjective To investigate the effects of farnesoid X receptor activation on intestinal inflammation in colitis model mice and human colonic cancer cell lines,and the expression of FXR in clinical specimens of UC patients.Methods Animal experiment:C57BL/6 mice were divided into five groups.Except for the blank control group,the other 4 groups of mice were subjected to 2.5%DSS for 5 days.Then,the experimental groups were gavaged with INT-747 for 2 days and 4 days,respectively,and the control groups were gavaged with 1%methylcellulose for 2 days and 4 days,respectively.Body weight and colon length of the mice were measured and the histological scores of the mice in each group were evaluated.The transcriptome of mice colon tissue was sequenced based on Illumina sequencing platform,and qPCR was used to verify the expression level of chemokines and cytokines.The levels of TNFa and IL-6 in serum were detected by ELISA.The LPMCs of the colonic mucosa of the experimental group and the control group were isolated,and the difference of immune cell phenotypes was identified by flow cytometry.Cell experiment:Caco-2 and HT-29 cells were treated with FXR agonists,GW4064,and NF-κB p65 phosphorylation was detected by Western blot after TNFa stimulation.Clinical validation:Specimens of normal colonic mucosa from non-IBD patients and biopsy specimens from mild to moderate UC were collected during colonoscopy.Severe inflammatory specimens of the colon were taken from surgical specimens of UC patients who had undergone colorectal resection in our hospital.The expression of FXR in intestinal epithelium of each group was evaluated by immunohistochemistry.Results Compared with the control mice,mice gavaged with INT-747 for 4 days had higher body weight,increased colon length,and lower colon histology score,whereas mice gavaged with INT-747 for 2 days did not have significant differences.Sequencing and qPCR results showed that compared with its control group,levels of IL-6,CSF3,CXCL1,MCP-1,IL-1β,CXCL12 were significantly decreased in mice treated with INT-747 for 2 days.Serum TNFa levels decreased significantly in mice gavaged with INT-747.Flow cytometry showed that FXR activation decreased the ratio of CD11b+cells,monocytes,and macrophages in LPMCs.FXR agonist inhibited the phosphorylation of NF-κB p65 in human colonic cell line.Clinically,the expression of FXR in intestinal epithelium with severe inflammation was significantly lower than that of normal epithelium.Clinically,FXR in mild and moderate UC mucosa was lower than that in normal mucosa(p>0.05).FXR in severe UC mucosa was significantly lower than that in normal mucosa(p<0.05).Conclusion Activation of FXR alleviate colonic inflammation,inhibit the expression of various chemokines and cytokines,and regulate multiple immune cell subsets in LPMCs.One potential mechanism is that FXR can inhibit NF-κB pathway activation.The expression level of FXR in colonic epithelium is negatively correlated with the degree of inflammation in ulcerative colitis.