Trichinella Spiralis Specific Antigens Applying In Inflammatory Bowel Disease By Immune Intervening

Inflammatory bowel disease (IBD) is an agnogenic autoimmune disease,which can include ulcer catarrh (UC) and Crohn's disease (CD). It's also an enteric chronic recurrent disease. Study shows that autoimmune disorder with disturbance of Thl/Th2 are the main cause of IBD. Recently,environment and genetic factors boost incidence rate of IBD. The environmental factors account for the susceptibility of genetic individual agent affects genetic individual which appears functional disorder disease of immunization system. IBD is most prevalent in developed country but rare in the developing countries. However, the people in developing areas immigrants in developed region displayed higher incidence of IBD. These results indicate that environment factors play key role in IBD. ?Hygiene hypothesis'to IBD explains that IBD usually happens in the people lack of intestinal mucosa stimulation. Study showed that intestinal nematode infection ameliorates the incidence of IBD. Our previous study demonstrated that IBD model mice infected with T.spiralis elevate therapeutic effect of IBD. Although parasite infection is prospective to the treatment of IBD, the biological risk of helminth infection in conventional concept spurn the therapeutic method. Persistent infection of live parasite could make people as fixed parasite host, which may affect the gastrointestinal function. Considering all these reasons, the patients couldn't psychologically accept parasite treatment. Therefore, it is unsuitable to use parasite infection as a therapeutic method for IBD patience. In our current study, we get the extraction of parasite and use it to treat IBD. The result shows that the extraction displayed the same therapeutic efficacy compared to live worm; it avoids the insecurity factors and is easily accept by the IBD patients. It avoids the unfavorable factors of the live helminth to choose T.spiralis immunologically competent as a therapeutic agent, more importantly, the competent of T.spiralis displayed the immune response effect of the live helminth against IBD, from an ethical point, and this is met with the requirement of the clinical treatment of biological agents.In this study, we used the recombinant highly reactive antigen from trichinella spiralis to vaccinate IBD mice model. The genes of antigens were screened by the constructed cDNA library of trichinella spiralis in different life cycle. The genes include ZH68 adult Trichinella antigen gene (serine protease), WM5 muscle larvae of Trichinella antigen gene (serine protease inhibitor), WN10 antigen gene of Trichinella spiralis newborn larvae (cysteine protease inhibitor), and T668 newborn larvae of Trichinella antigen gene (serine protease). The soluble protein expression vector was constructed and the recombinant protein was purified. The method is asfollowing: recombination proteins with His label (pET-22b-T668, pET-22b-ZH68, pET-22b-WM5 and pET-22b-WN10) were isolated using nickel agarose gel FF color spectrum. The BALB/c mice were immunized with the protein (T668, ZH68, WM5 and WN10) that was prepared for injection by emulsifying with Freund's adjuvant under asepsis condition,. Control group mice were vaccinated with Sodium Chloride. After the 3nd immunization, the animals were exposed TNBS to induce IBD disease model. In the following days, various parameters, such as mean weight, survival rate, DAI, macroscopic appreciation of colon and score of microscopic damage, inflammation target MPO and SOD active detection, were observed to evaluate the therapeutic effect of four kinds of dissolubility protein in mice IBD model. The expression level of IFN-γ, IL-12, IL-4, IL-5, IL-10, IL-17 and TGF-βin intestinal mucosa, mesenteric lymph node and spleen was measured by real-time quantitative RT-PCR. Next, The secreted level of IFN-γ, IL-12, IL-4, IL-5, IL-10, IL-17 and TGF-βwas detected using ELISA assay.Thus the dynamic relationship between intestinal immunization and cellular immune function was explored. Finally, the cell population changing of CD4+ CD25+ FoxP3+ Treg in spleen was checked by Flow Cytometer. We explained the immune regulated roles of Treg from cellular level andobjectively elucidated the mechanism of of recombination protein against IBD mice.The results demonstrated that effective ingredients of Trichinella spiralis have potential therapeutic effects on IBD mice model. The mice immunized with the two proteins displayed higher mean weight, higher survival rate and weaker clinical inflammation symptoms compared with Sodium Chloride injected group (p<0.05), and the score of DAI was obviously decreased (p<0.05). On the 3rd to 7th day, the colon injury degrees of treated mice from the macro and micro levels were significantly attenuated compared with the negative control group (p<0.05). Briefly, for the colon of treated mice, it showed fewer damage on mucous membrane, thickener on the bowel wall, narrower adhesion range, less ulcer closed to normal tissue, smaller area of inflammatory cells, less edema, obviously decreased MOP value (p<0.05). The SOD result displayed its value of treated mice is obviously lower compared with the sodium chloride injected group after 3rd treatment and its SOD got the similar level after 7nd treatment (p>0.05). The real time PCR result displayed the expression levels of IFN-γ, IL-12 and IL-17mRNA in IBD disease mice treated with ZH6 or T668 recombinant protein were significantly decreased compared with control group companied with higher level of IL-4, IL-10 and TGF-βmRNA in colon tissue (p<0.05). In spleen tissue of treated IBD disease mice, the expression levels of IL-17 and IL-10 mRNA were obviously down regulated (p<0.05), and the expression quantity of other cytokines was no statistically significant differences between two groups (p>0.05). The ELISA result demonstrated that the secreted level of IL-10 and TGF-βby colon LPMC is up-regulated on the 3rd day and later 7th day immunization with ZH68 and T668 recombinant protein to mice (p<0.05), and the related levels of IFN-γ, IL-17 and IL-12 by LPMC in protein treated mice were down regulated compared with sodium chloride treated group (p<0.05), the levels of IL-12 and IL-4 secreted by LPMC were similar to control group after 7th day or 3rd day respectively, the production of IL-4 by LPMC was up regulated after 7th day treatment with the related protein compared with sodium chloride treated group (p<0.05). The expression levels of IL-4, IL-10 and TGF-βin spleen were significantly increased in IBD mice on 3rd and after 7th day treatment with ZH68 or T668 recombinant protein compared with control group (p<0.05) companied with down regulated level of IL-17 (p<0.05), we didn't check the any different level of IFN-γafter 3rd treatment and IL-12 on 7th treatment between two groups (p>0.05), the levels of IFN-γand IL-12 produced by spleen lymphocytes after 7th day treatment and 3rd day treatment were obviously decreased in protein treated group compared with model group. The RT-PCR and ELISA results showed that the related immune parameters displayed no statistically significant differences between WM5 and WN10 recombinant protein immunization group and model group, and the cytokines with difference in another two protein treated group demonstrated opposite changing trend in the protein treated mice compared with the control group. The Flow Cytometer analysis results exhibited that the percentage of CD4+ CD25+ FoxP3+ Treg cells in CD4+ lymphocytes in ZH68 and T668 recombinant protein immunized IBD mice obviously decreased compared with model group (p<0.05), while the ratio CD4+ CD25+ FoxP3+ Treg cells to CD4+ lymphocytes of WM5 and WN10 recombinant protein treated IBD mice was no obviously changed compared with model group, and no significantly changing was observed during the disease peak for 3 days (p<0.05).The study shows that the special effective antigen of Trichinella spiralis displayed potential therapeutic effect in curing the IBD model mice induced by TNBS. All datum support that the special antigens contribute to correct the Th1/Th2 immune imbalance in IBD model mice, suppress the immune response, and regulate the cellular and humoral immune reaction by playing strong immune adjust function. Taken together, all this factors make the imbalances immune in IBD mice recovery.