Study On Immune Mechanism Of DSS - Induced Experimental Colitis In Mice

The two major clinically defined forms of inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are chronic remittent or progressive inflammatory conditions that may affect the entire gastrointestinal tract and the colonic mucosa, respectively, and are associated with an increased risk for colon cancer. IBD is common in developed countries, the incidence rate rise in china in recent years. The pathogenesis of IBD is not fully defined, but is clearly multifactorial, resulting from multiple genes, developmental and environmental factors, which together result in a dysregulated innate and adaptive mucosal immune response.Under normal physiological conditions, lamina propria contains numerous inflammatory T cells and regulatory T cells which inhibit inflammation, the two T cells are in a state of dynamic equilibrium. If the balance is broken, such as inflammation effect is too strong or regulating effect too weak, may lead to intestinal inflammation. Therapy for IBD mainly is 5-amino salicylic acid, glucocorticoid and immunosuppressive and biological agents such as anti-TNF-a monoclonal.According its clinical symptoms, IBD belongs to "diarrhea" and "dysentery" in traditional Chinese medicine (TCM). Sishen pill has the function of warming kidney and invigorating spleen, warming yang to melt wet, inducing astringency and relieving diarrhea. Sishen pill often is used as a basis prescription for treating IBD caused by asdthenic splenonephro-yang, but immune mechanism of treating IBD is not clear. In this study, we observe the therapeutic effect of Sishen pill and its immune mechanism by the acute and chronic colitis induced by DSS in mice.The effect of Sishen pill treat acute and chronic colitis induced by DSSAcute colitis was induced by feeding mice with 4%(wt/vol) DSS dissolved in drinking water. After 1 day mice were administrated with Sishen pill (2.25 g/kg) or Gegenqinlian pellet (6.5 g/kg) for 8 days. Chronic experimental colitis was induced by multiple cycle administration of 3%(wt/vol) DSS drinking water on days 1-5,8-12,15-19, and 22-26. After 12 day mice were administrated with Sishen pill (2.25 g/kg) or Gegenqinlian pellet (6.5 g/kg) for 16 days. Changes in disease activity index (DAI) together with histological scores were evaluated, and myeloperoxidase (MPO) in colonic tissue was detected. Both Sishen pill and Gegenqinlian pellet therapy significantly reduced DAI and histological scores of mice in acute DSS colitis. They also alleviate inflammatory cell infiltration of colonic tissue in the model. Although Gegenqinlian pellet has fast effect on DAI in actue DSS model, Sishen has better effectiveness. Sishen pill therapy significantly reduced DAI and histological scores of mice in chronic DSS colitis, but Gegenqinlian pellet show no obvious effect in the model. Amelioration efficacy of Sishen pill on mice acute colitis and chronic colitis induced by DSS were obtained. Gegenqinlian pellet only ameliorate acute colits induced by DSS. The effectiveness of Sishen pill is slightly better than Gegenqinlian pellet, but there is no significant difference between two prescriptions.Effect of Sishen Pill on CX3CR1, CD103, IL-23 expression in experimental colitis induced by DSS in miceAcute colitis induced by DSS in mice significantly increased the expression of CX3CR1, CD 103 and IL-23 in tissues. Sishen pill after treatment can increase the expression of CD103, but no obvious effect of the expression of CX3CR1 and IL-23. The expression of CX3CR1, CD103 and IL-23 also significantly increased in chronic colitis induced by DSS in mice, but the extent of increase of CX3CR1 and IL-23 decrease. There is no significant effect of Sishen Pill on CX3CR1 and CD103 expression in chronic colitis, but the expression of IL-23 was decreased.Effect of Sishen Pill on inflammatory T cells in experimental colitis induced by DSS in miceTh1 and Th17 are mainly inflammatory effector T cells in the intestinal mucosa. IFN-y is characteristic cytokine of Th1, and IL-17A is characteristic cytokine of Th17. IFN-y production level from colon organ culture supernatant and CD4+ T cells of expressing IFN-y in LPMC increased significantly in acute DSS model mice compared with control normal mice. IL-17A level from colon organ culture supernatant, CD4+T cells of expressing IL-17A and characteristic transcription factor RORyt of Th17 increased significantly as well. These data indicate the enhanced Th1 and Th17 reaction in acute colitis induced by DSS. After Sishen pill treatment, IL-17A production level, CD4+T cells of expressing IL-17A in LPMC, and RORyt in intestinal mucosa decreased significantly compared with acute colitis model group; but there is no obvious influence on IFN-y and CD4+T cells of expressing IFN-y in LPMC. The data show the Sishen pill treatment effect is associated with its inhibition of Th17 response.IFN-y production level from colon organ culture supernatant and CD4+T cells of expressing IFN-y in LPMC increased significantly in chronic DSS colitis model. Although IL-17A production level, CD4+T cells of expressing IL-17A in LPMC, and RORyt still increased significantly compared with the control group in chronic DSS colitis, the rate of increase is lower than acute model. After administration of Sishen pill can significantly reduce the number of CD4+T cells of expressing IL-17A in LPMC, but no obvious effect on level of IL-17A, IL-17A+T cells and RORyt. The results show that the Sishen pill had no effect on Th17, but had inhibitory effect on Th1 which may be due to indirect actions.IL-22, another cytokine of Th17, show analogous change trend compare with IL-17A in actue and chronic colitis induced by DSS. Interestingly, Sishen pill promote IL-22 production level from colon organ both acute and chronic colitis, which is inconsistent with Sishen pill on IL-17A. This phenomenon may be related to the more cells to produce IL-22.Effect of Sishen Pill on Th17 plasticity in experimental colitis induced by DSS in miceTh17 plasticity is that Th17 co-express characteristic cytokine such as IFN-y from Thl or IL-10 from Treg while the expression IL-17A. There is significant increase in IFN-γ+Thl7 compare with the control group in acute DSS colitis. Although IFN-y+Thl7 also increased significantly in chronic DSS colitis, the rate of increase is lower than acute model, and IL-10+Thl7 increased significantly compared with control group. Sishen Pill could increase significantly IL-10+Thl7 in acute DSS colitis, but had no significant effect on IFN-γ+Th17, so that IFN-γ+Th17/IL-10+Th17 decreased significantly compared with the control group. Sishen pill decreased IFN-y+Th17 and IL-10+Th17 at the same time, and so therefore has no significant effect on IFN-γ+Th17/IL-10+Th17 compare with model group in chronic DSS colitis. Therefore, therapeutic effect of Sishen pill on actue DSS colitis is closely related to IFN-y+Th17/IL-10+Th17 ratio adjustment.Effect of Sishen Pill on DSS Treg cells in experimental colitis induced by DSS in miceTreg is an important regulatory T cells to control ntestinal mucosal inflammation, IL-10 is a characteristic cytokine secretion of Treg. Treg also can secrete IL-35 and TGF-β. Although IL-35 production level from colon organ culture supernatant and IL-10+T cells in LPMC increased significantly, the feature of Treg increase is not obvious in acute DSS colitis. Administration of the Sishen pill had no significant effect on cytokines and characteristic transcription factor Foxp3 of Treg.In chronic DSS colitis, IL-10 production, IL-10+Treg, and Foxp3 expression increased significantly in model group, which show that the mechanism of controlling inflammation depending IL-10 had been established. The Sishen pill could further promote IL-10 and IL-35 production, increase IL-10+Treg and Foxp3. Therefore, Treg reaction could be amplified after Sishen pill administration in chronic colitis, thus contributing to the recovery of inflammation.In summary, Sishen pill has significant curative effect on acute and chronic colitis induced by DSS in mice. The immune mechanisms of Sishen pill in acute colitis is related to the inhibition of Thl7 reaction and promote regulatory Th17 generation. The immune mechanisms of Sishen pill in chronic acute colitis mainly promotes the expression of IL-10 in Treg generation, inhibition of the Thl response in chronic colitis may be through the promotion of Treg. In acute and chronic colitis, Sishen pill shows increasing the production of IL-22 which may contribute to repair the epithelium and alleviate inflammation.