Essential Function Of1,25（OH）₂D₃in Spleen Immunity Of Chronic Experimental Colitis In Mice

Ulcerative colitis （UC） is a kind of chronic inflammation, whosemechanism is not clear, it is believed that the imbalance of immunologicalreaction is the pivotal part in the pathogenesis. A great deal of researches haveproved that, the foreign antigen active the effect T cells and their subgroups,mediate the T helper （Th）1, Th2and Th17immunological reaction, secretemassive inflammatory factors, such as tumor necrosis factor-α （TNF-α）,interferon-γ （IFN-γ）, interleukin （IL）-6and IL-17, and so on, together withother inflammatory cells and proinflammatory cytokines, induce the innateimmune response. Meanwhile, the antigen presenting cells, including thedendritic cells and the macrophages, transporting the antigens to the associatedlymphoid tissue, for example, the Peyer’s patches and mesenteric lymph nodes（MLNs）, to induce the adaptive immune responses.The spleen is the most important peripheral immune organ, and kinds ofimmune cells settled here, so it is the center of the specific cellular immunityand humoral immunity. It was proved that, the splenic immune cells wereactived in the modle of experimental encephalomyelitis and rheumatoidarthritis, and the level of proinflammatory cytokines increased, which supportthat the abnormal immune reaction is the common characteristic of theinflammatory diseases.1,25-dihydroxyvitamin D₃（1,25（OH）₂D₃） is the active metabolites ofvitamin D, and much is known about the critical role in the skeleton growthand development. Dramatic evidences showed that,1,25（OH）₂D₃was alsoimportant in the differentiation of immune cells, the regulation of immunesystem and central nervous system.1,25（OH）₂D₃and the analogue couldprevent or inhibit the develoment of the disease in the modle of type1 diabetes and rheumatoid arthritis. So we hypothesis that1,25（OH）₂D₃couldprevent and treat colitis by influencing the immune regulation.Objective: To investigate the influence of1,25（OH）₂D₃in the splenicimmune regulation of chronic experimental colitis mice induced by dextransodium sulfate （DSS）.Methods:（1） A total of30mice were randomly assigned into the Control,DSS and DSS+VD group （each group=10）. The Control group drink thedistilled water; the DSS and DSS+VD group received DSS drinking water onday1-5,8-12,15-19,22-26,27and28, distilled water was given during theremaining time. The mice in the DSS+VD group received1,25（OH）₂D₃daily（0.2μg/25g/d） by intragastric administration from the14th day, and the micein the Control and DSS group were given PBS for comparion. The mice wereharvested on the29th day.（2） Severity of the disease was assessed by bodyweight （BW） changes, disease activity index （DAI） and the routine status.（3）The change of splenic morphology, spleen weight, spleen length and thespleen index （SI） were measured in each group.（4） The histopathologychanges of spleen were detected by Haematoxylin and eosin （HE） staining.（5）The mononuclearcells were isolated and cultured, and the percentage ofdentritic cells and macrophages was measured by FACS, and levels of IFN-γ,TNF-α, IL-17and IL-6secreted by the mononuclearcells were detected byenzyme linked immunosorbent assay （ELISA）.（6） The expressions of IFN-γ,TNF-α, IL-17and IL-6in the spleen were detected by immunohistochemistryand Real-time Q-PCR, respectively.Results:（1） Compared to that of the control group, BW was significantlydecreased in the DSS and DSS+VD group （15.50%±1.50%vs0.00%±0.00%, P<0.05）. BW in the DSS+VD group restored rapidly compared to thatin the DSS group （5.20%±0.53%vs19.60%±1.82%, P<0.05）. The DAI inthe DSS and DSS+VD group increased gradually, compared to that in theControl group. While after the treatment, DAI in the DSS+VD groupsignificantly decreased compared with that in the DSS group （3.77±0.36vs1.79±0.19, P<0.001）.（2） There was no morphological change in the spleen of the Control group, the spleen weight and length were0.048g±0.018g and1.15cm±0.06cm, respectively. The DSS group developed splenomegaly, andthe spleen weight were remarkablely increased than that in the Control group（0.146g±0.023g vs0.048g±0.018g, P<0.001）, also the spleen length （1.60cm±0.14cm vs1.15cm±0.06cm, P<0.001）. While, the degree ofsplenomegaly in the DSS+VD group were lessened than in the DSS group, thespleen weight were decreased than that in the DSS group （0.093g±0.016g vs0.146g±0.023g, P<0.005）, and there was no significant deviation in thespleen length between the DSS group and DSS+VD group （1.40cm±0.18cmvs1.60cm±0.14cm, P>0.05）. The SI in the Control group were0.263±0.058, the DSS group were increased significantly （0.787±0.140vs0.263±0.058, P<0.001）, and DAI in the DSS+VD group decreased compared withthat in the DSS group （0.425±0.052vs0.787±0.140, P<0.01）, there was nosignificant deviation between the DSS+VD group and the Control group（0.425±0.052vs0.263±0.058, P>0.05）.（3） The histopathology showed thespleen in the Control group had clear white pulp, the splenic sinusoid andsplenic cord disposed regularly. Compared with the Control group, the area ofwhite pulp increased in the DSS group, and the structure was disordered, thegerminal center was unclear; the periarterial lymphatic sheath cell densityincreased; the red pulp engorge and infiltrated with lymphocyte, also with theincreased of glant cells. The white pulp in the DSS+VD group was integrated,there was remote hemorrhage in the red pulp, and the engorge and lymphocytewere decreased compared with the DSS group.（4） The number ofmononuclearcells in the spleen of Control group was63.92×10⁶±7.18×10⁶,and the percentage of macrophages and dentritic cells were2.95%±0.89%and2.38%±0.85%, respectively. The number of mononuclearcells in the DSSgroup significantly increased compared with the DSS group （157.10×10⁶±17.32×10⁶vs63.92×10⁶±7.18×10⁶, P<0.001）, also the the percentage ofthe macrophages （7.65%±1.07%vs2.95%±0.89%, P<0.001） and thedentritic cells （6.45%±0.86%vs2.38%±0.85%, P<0.001）. Themononuclearcells in the DSS+VD group decreased compared to the DSS group （116.56×10⁶±10.63×10⁶vs157.10×10⁶±17.32×10⁶, P<0.005）,and the the percentage of the macrophages （4.98%±0.88%vs7.65%±1.07%,P<0.05） and the dentritic cells （2.68%±0.53%vs6.45%±0.86%, P<0.001）.The levels of IFN-γ, TNF-α, IL-17and IL-6secreted by the mononuclearcellswas detected by ELISA, compared with the Control group, the levels of thecytokines from the unstimulated mononuclearcells in the the DSS group（IFN-γ:1.34ng/mL±0.21ng/mL vs0.26ng/mL±0.05ng/mL, P<0.01;TNF-α:0.36ng/mL±0.05ng/mL vs0.19ng/mL±0.03ng/mL, P<0.01; IL-17:0.37ng/mL±0.05ng/mL vs0.22ng/mL±0.03ng/mL, P<0.01; IL-6:0.16ng/mL±0.03ng/mL vs0.08ng/mL±0.01ng/mL, P<0.01） and the stimulatedmononuclearcells （IFN-γ:8.56ng/mL±0.92ng/mL vs2.89ng/mL±0.51ng/mL, P<0.01; TNF-α:2.87ng/mL±0.29ng/mL vs0.37ng/mL±0.04ng/mL, P<0.01; IL-17:3.66ng/mL±0.49ng/mL vs0.41ng/mL±0.06ng/mL,P<0.01; IL-6:2.17ng/mL±0.33ng/mL vs0.29ng/mL±0.04ng/mL, P<0.01）were all increased. While compared with the DSS group, the4cytokines fromthe unstimulated mononuclearcells in the the DSS+VD group （IFN-γ:0.97ng/mL±0.16ng/mL vs1.34ng/mL±0.21ng/mL, P<0.01; TNF-α:0.26ng/mL±0.02ng/mL vs0.36ng/mL±0.05ng/mL, P<0.01; IL-17:0.29ng/mL±0.05ng/mL vs0.37ng/mL±0.05ng/mL, P<0.01; IL-6:0.12ng/mL±0.03ng/mL vs0.16ng/mL±0.03ng/mL, P<0.01） and the stimulatedmononuclearcells （IFN-γ:6.83ng/mL±0.53ng/mL vs8.56ng/mL±0.92ng/mL, P<0.01; TNF-α:0.73ng/mL±0.06ng/mL vs2.87ng/mL±0.29ng/mL, P<0.01; IL-17:1.91ng/mL±0.23ng/mL vs3.66ng/mL±0.49ng/mL,P<0.01; IL-6:1.23ng/mL±0.26ng/mL vs2.17ng/mL±0.33ng/mL, P<0.01）were all decreased.（5） In the Control group, the expressions of IFN-γ, TNF-α,IL-17and IL-6measured by immunohistochemistry were very little, butsignificantly increased in the DSS group （0.39±0.04vs0.14±0.02, P<0.001;0.48±0.05vs0.10±0.01, P<0.001;0.59±0.06vs0.27±0.02, P<0.001;0.63±0.06vs0.25±0.02, P<0.001）, and decreased in the DSS+VD group （0.17±0.02vs0.39±0.04, P<0.001;0.23±0.02vs0.48±0.05, P<0.001;0.31±0.05vs0.59±0.06, P<0.001;0.32±0.03vs0.63±0.06, P<0.001） compared with that in the DSS group.（6） The levels of IFN-γ mRNA, TNF-α mRNA, IL-17mRNA and IL-6mRNA in the DSS group were significantly higher than thatin the Control group （13.36±1.25vs1.00±0.00, P<0.001;6.21±0.69vs1.00±0.00, P<0.001;5.87±0.61vs1.00±0.00, P<0.001;8.12±0.64vs1.00±0.00, P<0.001）, and the levels of IFN-γ mRNA, TNF-α mRNA, IL-17mRNA and IL-6mRNA decreased in the DSS+VD group compared with thatin the DSS group （7.97±0.73vs13.36±1.25, P<0.001;4.72±0.61vs6.21±0.69, P<0.05;3.02±0.47vs5.87±0.61, P<0.001;4.21±0.52vs8.12±0.64,P<0.001）.（7） The serum calcium in the DSS group was a bit lower than that inthe Control group （2.33±0.06mmol/L vs2.50±0.03mmol/L, P>0.05）, alsolower than that in the DSS+VD group （3.33±0.32mmol/L vs2.33±0.06mmol/L, P>0.05）, there were no significant deviation between the threegroups.Conclusion:1,25（OH）₂D₃could relieve the immune reaction in thespleen of the chronic experimental colitis mice, whose mechanism may beinhibit the activition of immune cells and inflammatory factors.