Vitamin D Treatment Of Colitis Efficacy And Immune Regulation In Rats

Background:Inflammatory bowel disease (IBD) is a group of subacute and chronic inflammatory gastrointestinal disease with unknown etiology. There are two major distinct forms of IBD, ulcerative colitis (UC) and Crohn’s disease (CD). It’s now generally agreed that CD is a Thl mediated disease. Vitamin D has recently been found playing an important role in autoimmune diseases, especially in Thl mediated autoimmune diseases. Vitamin D can regulate the expression of TLRs on APCs. TLRs signaling pathway can affect the differentiation of naive T cells, and change the proportion of Th cells in local tissue. There are some groups found that the colonic inflammation can be eased by adding1,25(OH)2D3to experimental IBD. But until now there aren’t any reports about using vitamin D3in experimental IBD. And no report was found about using different doses of vitamin D in treating IBD.Objective:To investigate the therapeutic and immunoregulatory effects of vitamin D3and1,25(OH)2D3on TNBS-induced colitis in rats.Methods:A total of54SD rats were randomly divided into nine groups (6rats in each group):normal control, TNBS group,5-ASA group (0.4g/kg/d),1,25(OH)2D3group (0.2ug/kg/d),1,25(OH)2D3plus5-ASA group, large dosage vitamin D3group (7500IU/d), large dosage vitamin D3group plus5-ASA group, small dosage vitamin D3group (1800IU once), small dosage vitamin D3group plus5-ASA group. Experimental colitis was induced by enema administration of TNBS (100mg/kg). Rats were treated by lavage everyday for the following9days. The degree of inflammation of each rat was measured by DAI score, macroscopic lesion, histological colonic damage and the activity of myeloperoxidase (MPO) in the excised colonic tissues. TLR4and Foxp3were determined by immunohistochemistry. TLR9mRNA was determined by immunohistochemistry and reverse transcript PCR.Results:1. DAI score, macroscopic lesion score, histological colonic damage score and the activity of MPO in the excised colonic tissues were elevated in all groups which were treated with TNBS than normal control group(P<0.01). All the groups which were given treatment have lower score of DAI, macroscopic lesion and histological colonic damage, but no statistically significant was seen. All the treated groups have less activity of MPO than TNBS group (P<0.01). There were no significant difference between the treated groups in DAI score, macroscopic lesion score, histological colonic damage score and the activity of MPO. Large dosage vitamin D3group and large dosage vitamin D3plus5-ASA group had elevated serum calcium than other groups (P<0.01). Large dosage vitamin D3group also had elevated serum creatinine than other groups (P<0.05).2. There were little TLR4and TLR9positive cells in epithelial, lamina propria and submucosa of colon tissue in normal control group. The number of TLR4and TLR9positive cells were greatly elevated in rats which were treated by TNBS (P<0.01). Foxp3positive cells were mainly in lamina propria and submucosa of colon tissue. The normal control group showed little Foxp3positive cells. And the TNBS treated groups showed a lot more Foxp3positive cells (P<0.01), mainly in nucleus of lymphocytes.5-ASA group had less TLR4, TLR9and Foxp3positive cells than TNBS group (P<0.05). The group treat with vitamin D3or1,25(OH)2D3whether plus5-ASA or not showed less numbers of TLR4(P=0.313), TLR9(P<0.05) and Foxp3(P<0.01) positive cells than TNBS group. But the difference between each vitamin D treated groups was slim and had no statistically significant. The TLR9mRNA was poorly expressed in normal control group. There were no statistically significant difference between nine groups according to the grayscale of TLR9/β-actin (P=0.111).Conclusion:1.5-ASA, vitamin D3and1,25(OH)2D3can relieve the inflammation in TNBS induced colitis. Using vitamin D3or1,25(OH)2D3in conjunction of5-ASA can not relieve the inflammation furthermore. The anti-inflammation effect of vitamin D isn’t dose-dependent. Using high dose of vitamin D3can cause hypercalcemia and following high creatinine.2. The normal rat colon has little TLR4, TLR9and Foxp3positive cells.3. The expression of TLR4, TLR9and Foxp3are greatly up-regulated in TNBS induced colitis. Treating with5-ASA can down-regulate the expression of TLR4, TLR9and Foxp3. Treating with vitamin D3or1,25(OH)2D3can reduce the number of TLR9and Foxp3positive cells in colon compared with TNBS group. However the number of TLR4has little difference. Using vitamin D3or1,25(OH)2D3in conjunction of5-ASA don’t bring big difference on the expression of TLR4, TLR9and Foxp3in colon. There is no significant difference in expression of TLR4, TLR9and Foxp3between large dose vitamin D3group and small dose vitamin D3. The mRNA of TLR9shows little difference in nine groups.