| Helicobacter hepaticus(H.hepaticus)is a pathogen of mice,which can infect the gastrointestinal tract and hepatobiliary system of mice and can cause chronic hepatitis and colitis.H.hepaticus is mainly transmitted through the fecal-oral route and colonizes in the cecum and colon of mice,infecting intestinal epithelial cells and causing inflammation.Immunodeficiency mice infected with H.hepaticus are often used as animal models to study inflammatory bowel disease.Inflammatory bowel disease is a widespread intestinal disease.The colitis model caused by the infection with H.hepaticus is of great significance for the study of inflammatory bowel disease.CdtB is the main virulence factor of H.hepaticus,which can cause the cell cycle arrest and promote inflammation.Therefore,this study aims to prepare a CdtB mutant strain of H.hepaticus and to provide a reference for studying the mechanism of CdtB in colitis induced by H.hepaticus infection.Using H.hepaticus as a template to amplify the upper and lower homology arms of CdtB,and insert chloramphenicol resistance genes in the middle to construct a pcDNA-ΔCdtB homologous recombination plasmid.Chloramphenicol was used as a screening antibiotic to select the correct strain.The plasmid was electrotransfected into H.hepaticus competent cells,and the homologous recombination results of the mutant strain was verified by PCR and sequencing after 4-5 rounds of passage.The correct recombinant strains were stored,and identified with wild-type H.hepaticus for bacterial morphology,growth curves,biochemical characteristics,stress resistance,and biofilm,etc.The results showed that the two groups of strains all grew in films,the growth rate was not significantly different,and the absence of CdtB did not affect the biological characteristics of H.hepaticus.6-week-old B6.129P2-IL10tmlCgn/J mice were infected with wild-type H.hepaticus(WT H.hepaticus)and CdtB mutant strains(H.hepaticus ΔCdtB).The mice were necropsied at 12 w and 18 w,and the length of colon was measured.The proximal,middle and distal sections of colon tissue were fixed for histology and the rest samples were stored at-70℃.The pathological changes of colitis in mice caused by WT H.hepaticus and H.hepaticusΔCdtB strains were examined and scored under microscope after HE staining.The RNA of proximal colon was extracted according to pathplogical score and cytokine expression was detected by qRT-PCR.The protein of proximal colon was used to detect IL-6 protein content,NO content,and JAK/STAT signaling pathway.GraphPad Prism 8 was used to analyze the differences of related indicators,cytokines and protein expression levels in each group.The results showed that compared with the ΔCdtB strain,the infection of wild type H.hepaticus to mice for 18 weeks caused more severe clinical manifestations and pathological changes,and the weight trends of the mice was significantly slow down,accompanied with the symptoms of prolapse,watery stool,and shortening of the colon length;Microscopic examination revealed colonic epithelial damage with inflammatory cell infiltration.CdtB did not affect the colonization efficiency of H.hepaticus,meaning the pathological changes were not related to the number of bacteria.CdtB can up-regulate the transcription levels of IL-1β,TNF-α,IL-6,and iNOS in the colon,and increase the protein expression of IL-6.In addition,CdtB phosphorylates Jak2 and Stat3 proteins and activates the IL-6/Jak2/Stat3 signaling pathway.Taken together,these data indicate that CdtB promotes the progress of inflammation,and enhances the inflammatory ability of H.hepaticus by activating the IL-6/Jak2/Stat3 signaling pathway,which plays an important role in the pathogenesis of H.hepaticus. |
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