| Background and aimInflammatory bowel disease(IBD)is a chronic and nonspecific bowel disease with unknown etiology.Clinically,the treatment effects of patients with IBD are not ideal,which is prolonged and easy to relapse,which seriously affects the patients‘quality of life.Therefore,it is urgent to seek effective therapeutic methods to relieve IBD for patients.The current etiological hypothesis of IBD is following,Hosts with susceptible genes suffer intestinal flora disorder,intestinal mucosal barrier damage,and immune dysfunction,ultimately leading to persistent intestinal inflammation under the influence of environmental factors.Therefore,The "functional elements"that regulate intestinal flora,promote intestinal mucosal barrier repair,and maintain immune balance to counter the process of inflammatory responses,may mitigate IBD.Bacteroides fragilis strain ZY-312 is a symbiotic bacterium,which is isolated from the feces of a healthy infant.Bacteroides fragilis strain ZY-312 is belonging to the genus Bacteroides,with negative Gram staining,no flagella,no spore,and mandatory anaerobic.The identification and safety assessment of Bacteroides fragilis strain ZY-312 have been completed in the early stage.The previous study from our team found that Bacteroides fragilis strain ZY-312 regulated intestinal flora,and promoted intestinal epithelial cell proliferation in an antibiotic-associated diarrhea model and an enterocolitic enteritis model in rats.However,the efficacy and mechanism of Bacteroides fragilis strain ZY-312 in IBD remains unclear.Based on the above,The study aims to investigate the effects of Bacteroides fragilis strain ZY-312 in Dextran sodium sulfate(DSS)-induced colitis and the related molecular mechanism.Methods1.Efficacy evaluation of Bacteroides fragilis strain ZY-312 in DSS-induced colitis mouse model(1)C57/BL6 mice were constructed DSS-induced colitis model and gavaged with Bacteroides fragilis strain ZY-312.Then the therapeutic effect and intestinal inflammation were evaluated according to weight loss,disease activity index,the shorten of colon,and histopathologic associated index.(2)Detection of colonic mucosa proliferation indexes:Colonic mucus and the goblet cell density were evaluated through periodic acid schiff reaction(PAS).The levels of PCNA,cleaved Caspase-3 and Caspase-3 were detected by western blot(WB)and qPCR.The level of Ki-67 in colonic mucosa was evaluated by immunohistochemistry(IHC).The fecal flora was detected by 16srRNA.2.The mechanism of Bacteroides fragilis strain ZY-312 promoting the regeneration of colonic mucosa(1)The screening and verification of Bacteroides fragilis strain ZY-312-regulated proliferative pathway in colonic mucosa:The proliferative pathway was screened by the KEGG pathway analysis of protein microarray from colon tissue,and verificated by WB and IHC in vivo.The level of the proliferative pathway in colonic organoids was detected by immunofluorescence in vitro.(2)The DSS-induced colitis model was constructed in gene knockout mice with the administration of Bacteroides fragilis strain ZY-312,Then the therapeutic effect and intestinal inflammation were evaluated according to weight loss,disease activity index,the shorten of colon,and histopathologic associated index.Colonic mucus and the goblet cell density were evaluated by Alcian Blue.The levels of Ki-67,PCNA,Caspase-3,and cleaved Caspase-3 in colonic mucosa were evaluated by WB and IHC.The level of PCNA and cleaved Caspase-3 in colonic organoids was detected by immunofluorescence after adding pathway inhibitor.The fecal flora was detected by 16srRNA.3.Exploration of upstream factors related to the activation of proliferation pathway under Bacteroides fragilis strain ZY-312 administration(1)Cytokine detection:The levels of cytokines were detected by inflammatory microarray,and verified through ELISA and IF.(2)The detection of immune cells for cytokine secretion:the CLP-derived immune cell populations for cytokine secretion were detected by flow cytometry(FCM).(3)The DSS-induced colitis model was constructed in gene knockout mice with the administration of Bacteroides fragilis strain ZY-312,Then the therapeutic effect and intestinal inflammation were evaluated according to weight loss,disease activity index,the shorten of colon,and histopathologic associated index.Colonic mucus and the goblet cell density were evaluated by Alcian Blue.The levels of Ki-67,PCNA,Caspase-3,and cleaved Caspase-3 in colonic mucosa were evaluated by WB and IHC.The level of PCNA and cleaved Caspase-3 in colonic organoids was detected by immunofluorescence after adding cytokine inhibitor.The fecal flora was detected by 16srRNA.Results1.Bacteroides fragilis strain ZY-312 relieved DSS-induced colitis in miceThe results showed that Bacteroides fragilis strain ZY-312 reduced the weight loss,disease activity index,colon shortening and histopathologic associated index compared with the DSS group(mice with PBS administration).2.Bacteroides fragilis strain ZY-312 promoted colonic mucosa regenerationCompared with the DSS group,Bacteroides fragilis strain ZY-312 promoted the expression of Ki-67 and PCNA,and mucus secretion in colonic mucosa,increased the density of goblet cells,and reduced cleaved Caspase-3 and Tunel-related apoptosis levels in colonic mucosa.Bacteroides fragilis strain ZY-312 promoted the expression of PCNA,and decreased cleaved Caspase-3 level in colonic organoids from the co-culture model.The 16srRNA results showed that Bacteroides fragilis strain ZY-312 up-regulated the proportion of Firmicutes,and decreased the proportion of Proteobacteria.Bacteroides fragilis strain ZY-312 promoted colonic mucosa regeneration through regulating intestinal microbiota.3.Bacteroides fragilis strain ZY-312 promoted colonic mucosa regeneration through motivated the STAT3 signaling pathwayThe KEGG pathway analysis of protein microarray showed that Bacteroides fragilis strain ZY-312 up-regulated JAK-STAT pathway in colitis.Furthermore,The WB and IHC results showed that Bacteroides fragilis strain ZY-312 mainly up-regulated the expression of STAT3 phosphorylation(pSTAT3)in colonic mucosa.And immunofluorescence results showed that Bacteroides fragilis strain ZY-312 promoted the expression of pSTAT3 in colonic organoids.In order to verify the role of the STAT3 signaling pathway for colonic mucosa regeneration under Bacteroides fragilis strain ZY-312 administration.we constructed the intestinal epithelial STAT3 specific knockout(Stat3ΔIEC)mice colitis model,and gavaged mice with Bacteroides fragilis strain ZY-312.The results showed that compared with wild-type mice(Stat3fl/fl),Bacteroides fragilis strain ZY-312 did not reduced the weight loss,disease activity index,colon shortening and histopathologic associated index in Stat3ΔIEC mice.Further,the expression of proliferative indexes were further detected,and the results showed that Bacteroides fragilis strain ZY-312 did not increase the PCNA,Ki-67 levels,or mucus density,either decrease the cleaved Caspase-3 level or TUNEL-positive cells density in Stat3ΔIEC mice compared with Stat3fl/fl mice.The immunofluorescence results showed that Bacteroides fragilis strain ZY-312 did not elevate PCNA expression,or decrease the cleaved Caspase-3 level in the colonic organoids once adding STAT3 inhibitor.The 16srRNA results showed a similar microbiota composition between the DSS+ZY-312 group and the DSS group in Stat3ΔIEC mice.4.Bacteroides fragilis strain ZY-312 elevated the level of IL-22 in colon tissueThe upstream factors involved in activation of STAT3 phosphorylation were explored by inflammatory microarray.The ELISA and IF results showed that Bacteroides fragilis strain ZY-312 mainly up-regulated the expression of IL-22 in colon tissue.Next,we constructed IL-22 knockout(IL-22-/-)mice colitis model,and gavaged mice with Bacteroides fragilis strain ZY-312.The results showed that Bacteroides fragilis strain ZY-312 did not reduced the weight loss,disease activity index,colon shortening and histopathologic associated index in IL-22-/-mice compared with wild-type mice(IL-22+/+).Further,the expression of proliferative indexes were further detected,and the results showed that Bacteroides fraigilis strain ZY-312 did not increase the PCNA,Ki-67 levels,or mucus density,either decrease the cleaved Caspase-3 level or Tunel-positive cells density in the IL-22-/-mice compared with IL-22+/+mice.The immunofluorescence results showed that Bacteroides fragilis strain ZY-312 did not elevate PCNA expression,or decrease the cleaved Caspase-3 level in the colonic organoids once adding IL-22 neutralizing antibody.The 16srRNA results showed a similar microbiota composition between the DSS+ZY-312 group and the DSS group in IL-22-/-mice.5.Bacteroides fragilis strain ZY-312 promoted ILC3 to secrete IL-22IL-22 is mainly derived from ILC3 and CD4+T cells.Therefore,the study mainly detected the cell percentage of ILC3 and CD4+T cells that secreting IL-22.The flow cytometry results showed that Bacteroides fragilis strain ZY-312 mainly promoted ILC3 to secrete IL-22.Besides,The inflammatory microarray results showed that Bacteroides fragilis strain ZY-312 up-regulated the expression of IL-7,which promoted the development and maturation of ILC3.Conclusion1.Bacteroides fragilis strain ZY-312 relieved DSS-induced colitis in mice.2.Bacteroides fragilis strain ZY-312 promoted colonic mucosa regeneration through motivating STAT3 phosphorylation(pSTAT3).3.Bacteroides fragilis strain ZY-312 elevated the level of IL-22 in colon tissue.4.Bacteroides fragilis strain ZY-312 promoted ILC3 to secrete IL-22 through innate immunity mode,thus activating STAT3 phosphorylation to promote colonic mucosa regeneration. |
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