Study On The Treatment Mechanism Of Mice Dextran Sulfate Sodium-induced Experimental Colitis With Human Interleukin10Secreted By Transformed Bifidobacterium

Objectives: Ulcerative colitis (UC) is a type of chronic intestinal inflammatory disease, and itsetiology and pathogenesis have not been fully elucidated. Bifidobacterium is the most importantphysiological beneficial bacterium in the intestinal tract of the body, which has a goodtherapeutic effect on UC due to its regulation of intestinal flora balance in UC patients. Cytokineinterleukin-10(IL-10) is a kind of cytokines that negatively regulates inflammation, which canreduce the intestinal inflammatory cascade amplification reaction of UC and relieve the intestinalinflammation of UC. This experiment will continue studying the experimental effects ofBL-hIL-10established in previous experiments (transforming a hIL-10-containing plasmid into B.Longum) on the treatment of UC of mice and then discuss the possible mechanisms related to thetherapeutic effect of BL-hIL-10on UC.Methods:(1) The pBADXs-hIL-10shuttle plasmids were shifted into Bifidobacterium Longum(NCC2705) by the electroporation method to construct BL-hIL-10bacterial strain which canstably express bioactive hIL-10protein.(2) UC mouse model was induced by5%dextran sulfatesodium (DSS). The mice were divided into5groups randomly: control (normal) group, model(colitis) group, Bifidobacterium treatment (BL) group, empty plasmid Bifidobacterium treatment(BL0) group and BL-hIL-10treatment group. Except the normal group, all of the mice in otherexperimental groups were allowed to drink5%DSS freely for7days of modeling beforediscontinuation of modeling drug. Interventional therapy was given to the mice in the treatmentgroups respectively for7days before the mice were killed by breaking neck, and thenexperimental tests were performed.①Record the disease activity index (DAI) of mice every day,take colon tissues from each group for pathological assessment by HE staining and analyze thespleen index (SI) of mice.②Take mice spleens and detect the Treg/Th17ratio by flowcytometry, measure the expression level of cytokine IL-17, IL-10, IL-23, IL-6and TGF-β1inmice serum and colon mucosa by ELISA.③Detect the expression of nuclear protein HIF-1αand cell plasma protein mTOR and STAT3in the colon tissue of mice by western blot test.Results:(1) The BL-hIL-10bacterial strain that can stably express hIL-10factor wassuccessfully screened out.(2) BL-hIL-10, BL and BL0could reduce the DAI and the SI of spleentissue in UC mice, as well as relieve the inflammatory state of colon tissue.(3) BL-hIL-10couldremarkably down-regulate the Th17proportion in the splenic lymphocytes of UC mice, increasethe Treg proportion, obviously elevate the expression of anti-inflammatory cytokine IL-10andTGF-β1in serum and colon tissue, and meanwhile remarkably lower the expression ofproinflammatory cytokine IL-6, IL-17and IL-23.(4) BL-hIL-10could remarkably down-regulatethe expression of nuclear protein HIF-1α and cell plasma protein mTOR and STAT3in the colontissue of UC mice.(5) The therapeutic effect of BL-hIL-10group on UC mice was evidentlysuperior to that of BL and BL0groups.Conclusions: BL-hIL-10can stably express bioactive hIL-10protein and inhibit “the localhypoxia—mTOR—HIF-1α—Th17pathway” as well as the “IL-6—STAT3—HIF-1α—Th17pathway”, and then rectify the Treg/Th17imbalance in UC mice. BL-hIL-10can relieve the localintestinal and systemic inflammatory reactions of UC mice through improving the level ofanti-inflammatory cytokins and reducing that of proinflammatory cytokines. Therefore,BL-hIL-10, which possesses the dual advantages of both Bifidobacterium and IL-10, has a notable therapeutic effect on UC. Moreover, it has simple and convenient administration route,without obvious toxic and side effects on the body, and its synthetic process is simple and cheap,thus it is expected to become a new generation of probiotics which can treat UC effectively.