| Part1 Study of the mechanism of Bacteroides vulgatus involved in the pathogenesis of ulcerative colitis by regulating bile acid metabolismObjectivesUlcerative colitis(UC)is a chronic inflammatory disease of the colorectal mucosa,the exact etiology of which is unknown.Many studies have confirmed that the intestinal flora is one of the important factors in the pathogenesis of UC,and studies have reported differences in the microbial composition of the gut between UC patients and healthy individuals,especially in terms of microbial diversity and the relative abundance of specific bacteria.Many studies have focused on the important role played by certain species of the Bacteroidetes in the pathogenesis of UC.Bacteroidetes vulgatus(B.vulgatus),a member of the Bacteroidetes,have been found to be closely associated with the development of colonic inflammation,but little is known about how they drive the inflammatory response in the host.Therefore,in this study,we constructed a mouse model of B.vulgatus infection to study its effects on colonic inflammation and alterations in the bile acid metabolic pool in mice,and further investigated the specific mechanisms by which B.vulgatus-mediated alterations in bile acid metabolism are involved in the progression of UC.MethodsAt the clinical level,fecal from 80 UC patients and 80 normal subjects were selected and 16s rDNA was used to detect differences in flora composition and structure.At the animal level,B.vulgatus was gavaged with antibiotic pretreated mice and DSS-induced pseudo-sterile mice for 4 weeks.After colonization,colon pathology,inflammatory factors and barrier proteins were measured to assess the effect of B.vulgatus on the degree of colon inflammation in mice;targeted metabolomics was used to detect changes in the composition and content of fecal bile acids in mice in the B.vulgatus gavage group and normal control group,and to screen for differential bile acids in pseudo-sterile mice,and to detect colon pathology,inflammatory factors and barrier proteins in mice.Differential analysis of RNA expression profiles in colon tissues of mice from the B.vulgatus intervention and normal control groups using transcriptome sequencing.qRT-PCR,Western blot,and immunohistochemical techniques were used to detect the expression levels of FXR in the colon of mice in the gavage group and the normal control group of B.vulgatus.The FXR knockout mouse model was constructed,and the FXR knockout pseudo-sterile mice were gavaged with B.vulgatus for 4 weeks,and the mouse colonic pathology,inflammatory factors and barrier proteins were examined.At the cellular level,a co-culture model of colonic epithelial cells T84 and B.vulgatus was constructed to detect the expression levels of inflammatory factors and barrier proteins.Changes in the expression of inflammatory factors and barrier protein levels by T84 cells were examined after supplementation with differential bile acids.Changes in the expression levels of secreted inflammatory factors and barrier proteins by T84 cells were assessed after knockdown of FXR by general mimicry.Finally,the downstream genes involved in the regulation of FXR as a transcription factor were detected using CUT&Tag technology and intersected with transcriptome sequencing results to screen candidate target genes.ResultsAt the clinical level,we found a significant enrichment of B.vulgatus in fecal specimens from UC patients.Animal level showed that the body weight of mice decreased,the DAI score and pathology score increased significantly,the expression levels of inflammatory factors TNFα,IL-1β and IL-6 increased significantly,and the expression of intestinal barrier protein Occludin was significantly down-regulated after gavage with B.vulgatus and the above results suggested that B.vulgatus could induce colonic inflammation in mice.Next,DSS-induced mice were gavaged with B.vulgatus and found to have increased colonic inflammation in DSS mice gavaged with B.vulgatus compared with the DSS group,suggesting that B.vulgatus promotes the degree of DSS-induced colonic inflammation in mice.Targeted bile acid metabolomics analysis of fecal bile acid changes in mice after B.vulgatus intervention.Principal component analysis showed that the fecal bile acid composition was well differentiated between the B.vulgatus gavage group and the control group;cluster heat map analysis revealed that the levels of NorCA and HDCA were significantly up-regulated and the levels of 12-ketoLCA,CA,UDCA,α-MCA,β-MCA,TCA and T-α-MCA were significantly down-regulated in the feces of mice in the B.vulgatus gavage group.Among the above differential bile acids,we found that T-α-MCA,HDCA,CA,NorCA,and UDCA differed significantly between the two groups through VIP screening.Combining with literature reports,we finally selected UDCA to be instilled back into pseudo-sterile mice intervened by B.vulgatus,and found that UDCA supplementation in B.vulgatus gavaged mice significantly reduced colonic inflammatory injury,significantly decreased expression of inflammatory factors TNF-α,IL-6,and IL-1β,and significantly up-regulated expression of barrier protein ZO-1,tentatively suggesting that B.vulgatus exerted pro-inflammatory effects through regulating UDCA levels.The transcriptome sequencing results revealed that there were 1914 differentially expressed genes between the gavage group and the normal control group,of which 1009 differentially expressed genes were up-regulated and 905 differentially expressed genes were down-regulated.The KEGG pathway enrichment analysis revealed that the above differentially expressed genes were mainly involved in various biological processes such as cell adhesion,inflammatory response and bile acid secretion.FXR is one of the most characteristic bile acid receptors,qRT-PCR,Western blot and immunohistochemical assays revealed that the expression level of FXR in mouse colon was significantly down-regulated after gavage by B.vulgatus,confirming that B.vulgatus down-regulates FXR expression level by regulating specific bile acids.Further,the FXR knockout mouse model was constructed,and the FXR knockout mice pretreated with antibiotics were gavaged with B.vulgatus.It was found that B.vulgatus transplantation induced colonic inflammation in mice compared with wild-type mice,while after knocking out FXR,there were no significant differences in colonic histological scores and expression of inflammatory factors TNF-α,IL-6,and IL-1β between the transplanted B.vulgatus and control FXR mice.The above results tentatively suggest that B.vulgatus-mediated alterations in bile acids play a pro-inflammatory role in the development of colonic inflammation through the FXR axis.At the cellular level,a co-culture model of colonic epithelial cells T84 and B.vulgatus was constructed,and it was found that the relative levels of TNF-α,IL-6 and IL-1β were significantly increased,and the relative expression levels of barrier proteins ZO-1,E-Cadherin and Claudin 1 were significantly down-regulated after co-culture of T84 cells with B.vulgatus.In contrast,the expression levels of cellular inflammatory factors TNF-α,IL-6 and IL-1β were significantly decreased and the expression levels of barrier proteins ZO-1,E-Cadherin and Claudin 1 were up-regulated after simultaneous supplementation with ursodeoxycholic acid,which was consistent with the results of in vivo experiments in mice.After knockdown of FXR,there was no significant difference in TNF-α expression levels in co-cultured colonic epithelial cells of B.vulgatus,further confirming that B.vulgatus plays a role in colonic inflammation by regulating the FXR axis.Finally,in order to investigate the mechanism of action of B.vulgatus-mediated induction of colonic inflammation,RNA expression profiles were analyzed differently between the colon tissues of mice in the B.vulgatus intervention group and the normal control group using transcriptome sequencing technology.We used CUT&Tag technology to detect the downstream genes involved in the regulation of FXR as a transcription factor,and took the intersection with RNA-seq results to screen 21 candidate target genes,which will be followed by an in-depth study of FXR and candidate target genes.ConclusionsB.vulgatus affects the FXR by modulating changes in ursodeoxycholic acid,which in turn induces colonic inflammation.Part 2 Comparison of the effificacy of endoscopic submucosal dissection and transanal endoscopic microsurgery in the treatment of rectal neuroendocrine tumors≤2 cmObjectivesCurrently,complete tumor resection is considered the most effective treatment for rectal neuroendocrine tumors(NETs).Endoscopic submucosal dissection(ESD)and transanal endoscopic microsurgery(TEM)are recommended for rectal NETs ≤2 cm,but it is not clear which method is better.Thus,we evaluated the efficacy of ESD and TEM in the treatment of rectal neuroendocrine tumors(NETs)≤ 2 cm.MethodsWe conducted a single-centre retrospective cohort study between 2010 and 2021 of rectal NETs≤2 cm in 114 patients with long-term follow-up data who were divided into ESD(n=55)and TEM groups(n=59).Our study assessed differences between groups in the complete resection rate of lesions,recurrence rate,surgical complications,procedure time,and length of hospital stay.ResultsThe co-primary outcomes were the complete resection rate of lesions and the recurrence rate.Compared to that in the ESD group,the complete resection rate was significantly higher in the TEM group(91.5%vs.70.9%,p=0.005).The median follow-up time was 22 months in our study,and the follow-up outcomes suggested that the rates of recurrence were 1.8%(1/55)and 6.8%(4/59)in the ESD and TEM groups,respectively,with no significant difference between the two groups.The secondary outcomes of the evaluation were surgical complications,procedural time,and length of hospital stay.The rate of complications(gastrointestinal bleeding and perforation)was low in both the ESD(7.3%,4/55)and TEM(5.1%,3/59)groups.No difference in hospitalization duration was observed between the two groups in our study.However,the procedure time was significantly shorter in the ESD group than in the TEM group(27.5 min vs.56 min,p<0.001).ConclusionsAlthough the rate of complete resection in the TEM group was higher than that in the ESD group,there was no difference in recurrence rates between the two modalities during long-term follow-up.Depending on the qualities of the available hospital resources in the area,one of the two approaches can be adopted. |
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