Effect of Mycobacterium tuberculosis Ag85B protein on ulcerative colitis in mice and its mechanism
Objective To explore the effect and mechanism of immunological effects induced by the protein of Mycobacterium tuberculosis Ag85B on ulcerative colitis(UC)in mice,so as to provide a reference for the clinical application and further study of Ag85B.Methods The eukaryotic expression plasmid pcDNA 3.1(+)Rv1886c was constructed and transfected into CHO-K1 cells,and the expression of Ag85B protein was detected.The UC model was prepared by female C57BL/6J mice through drinking 3％dextran sulfate sodium(DSS)for 7 days.Six normal mice were randomly selected as control group,and UC model mice were randomly divided into UC group and Ag85B treatment group,with six in each group.Mice in the treat-ment group were intramuscu larly injected with pcDNA 3.1(+)Rv1886c(100 μg/mouse),and the UC group was injected with the same amount of pcDNA 3.1(+)empty plasmid,once a week,three times in total.On the 21 d after the initial immuniza-tion,the blood samples of mice were collected from the orbit,the spleen was taken,the intestinal tube was separated and the colon was intercepted.The colon was measured for the length,observed for the tissue structure by HE staining,and detected for the expression of zonula occludens-1(ZO-1)and occludin by Western blot.The serum proinflammatory cytokines TNF-α,IL-1β and IL-18 were detected by ELISA.The ratio of follicular helper T cell(Tfh)and follicular regulatory T cell(Tfr)in mouse spleen was measured by flow cytometry.Results The eukaryotic expression plasmid pcDNA 3.1(+)Rv1886c was confirmed to be constructed correctly by double digestion and sequencing.The relative molecular mass of Ag85B protein expressed was about 35 000,consistent with the size expectation.Compared with UC group,the colon length of Ag85B treat-ment group increased(t=3.26,P＜0.01),the colonic mucosa was relatively intact,the inflammatory exudation was reduced,and the protein expression of ZO-1 and occludin in colon tissue increased(t=10.86 and 9.22,respectively,each P＜0.05);the expression of TNF-α,IL-1β and IL-18 in serum decreased(t=3.25,3.11 and 2.86,respectively,each P＜0.05),and the proportion of Tfh decreased(t=3.70,P＜0.05),while the proportion of Tfr increased(t=5.24,P＜0.01).Conclusion The immune effect induced by Ag85B protein can relieve UC induced by DSS,which may be related to the regulated Tfh/Tfr balance.
Mycobacterium tuberculosisUlcerative colitis(UC)Ag85BEukaryotic expression plasmidIntestinal barrierFollicular helper T cell(Tfh)Follicular regulatory T cell(Tfr)
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