Mechanism And Protective Role Of 1,25-dihydroxyvitamin D3 In Intestinal Mucosal Barrier In Experimental Intestinal Inflammation Model Induced By DSS

Ulcerative colitis (UC) is an intestinal disease with a high risk of recurrence, occupying the main clinical form of inflammatory bowel disease (IBD). However, the pathogenesis of it is still unclear, and no effective measures are found to prevent and treat it yet. Therefore, it is important to investigate the mechanism of UC.Currently, intestinal commensal bacteria and their products breaking through the mucosal barrier generally induce the local mucosal immune responses. Normally, the intestinal epithelial barrier consists of epithelial cells and intercellular junctions. Tight junction (TJ) is the apical-most intercellular structure in epithelial cells, accounting for the cell-cell adhesion, polarity, and the permeability barrier to paracellular transport of the solutes. The TJ creates a semipermeable barrier, separating different organ compartments. Therefore, maintaining TJ and barrier function may provide potential therapeutic benefits in the treatment of UC.These pro-inflammation cytokines, such as tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ) are potential pathogenic factors for damaging mucosal barrier function and intestinal hyperpermeability. Previous studies have demonstrated that expression of junction complex proteins decreased in the mucosa of patients with IBD. Impaired gut epithelial barrier function may lead to persistent immune response. CD4⁺T cells play a vital role in initiating and shaping this pathologic response. Th17 cell-derived cytokines are supposed to account for protection of the host against various bacteria and fungi, particularly at mucosal surfaces, enhancing the recruitment, facilitating the activation of neutrophils, and stimulating the production of defensins by epithelial cells. On the other hand, evidences were found that uncontrolled and persistent active Th17 cells can cause damages in various organs, as Th17 cells are able to promote the synthesis of inflammatory cytokines, such as IL-1, IL-6, TNF-α, et al.1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active form of vitamin D, has been shown to be a key regulator in many experimental autoimmune diseases. It is also important for maintaining homeostasis in the respiratory tract and skin barrier as well as intestinal mucosa. Additionally, 1,25(OH)₂D₃ acts a key role in immune regulation, and the lack of 1,25(OH)₂D₃ was found to be related with the serum levels of IFN-γ, IL-6, IL-17.In sum, we made such a hypothesis that 1,25(OH)₂D₃ may be involved in the pathogenesis of UC, through maintaining the integrity of the intestinal mucosal barrier and regulating the immune response. The experiment mainly consists of the following four parts:Part 1: Protective effect of 1,25-dihydroxyvitamin D₃ on inflammation of the intestinal mucosa in experimental acute and chronic colitisObjective: To intvestigate the protective role of 1,25(OH)₂D₃ in colonic mucosal inflammation in experimental acute and chronic colitis.Methods: (1) C57BL/6 mice were used for in vivo studies. The acute colitis model was established by administration of DSS (Dextran sodium sulfate, DSS) for 7 days. Mice were randomly grouped as follows: control group (distilled water, n=10); model group (2% DSS, n=10) and 1,25(OH)₂D₃ group (2% DSS and 0.2μg/25 g, n=10). Chronic colitis was induced by 2% DSS drinking from 1 to 5 days, 8 to 12 days, 15 to 19 days and 22 to 26 days, and distilled water was given during the remaining time. Mice were randomly grouped as follows: control group (n=10); model group (n=10); 1,25(OH)₂D₃ group (n=10) and 1,25(OH)₂D₃ (0.005μg/d, once a day, dissolved in peanut oil) was given in 1,25(OH)₂D₃ group on day 14 by intragastric adminstration, and the other groups were given the isodose peanut oil, animals were sacrificed at 29^th day; (2) Severity of colitis was evaluated by body weight (BW) changes, disease activity index (DAI), colon length/weight, colon histology changes and pathology score; (3) The ultrastructural features of the colonic ephithelium were observed by scanning electron microscope (SEM).Results: (1) The results of acute colitis showed that BW had significantly greater loss in the model group compared with that in the control group. However, after the treatment of 1,25(OH)₂D₃, mice in the 1,25(OH)₂D₃ group had rapid weight recovery; DAI (3.00±0.41 vs 0.00±0.00, P<0.01) and pathology scores (8.60±1.67 vs 5.00±0.00, P<0.01) in the model group were significantly higher than control group, and colon length was shorter (4.88 cm±0.63 cm vs 6.80 cm±0.27cm, P<0.01); congestion and edema of the colon wall and infiltration into the mucosa superficial layers of granulocytes were much severity in the model group (2.00±0.71 vs 0.00±0.00, P<0.01). DAI (0.00±0.00 vs 0.70±0.41, P<0.01) and pathology score (7.40±0.55 vs 8.60±1.67, P<0.01) significantly decreased in the 1,25(OH)₂D₃ group, and colon length was longer (6.10 cm±0.42 cm vs 4.88 cm±0.63 cm, P<0.01); congestion and edema of the colon wall and infiltration into the mucosa superficial layers of granulocytes were significantly ameliorated (0.60±0.55 vs 2.00±0.71, P<0.01); (2) The results of chronic colitis showed that BW of mice in the model group had significantly greater loss compared with that of control group. However, after the treatment of 1,25(OH)₂D₃, mice in the 1,25(OH)₂D₃ group had rapid weight recovery; DAI in the model group were significantly higher than control group (4.00±0.43 vs 0.00±0.00, P<0.01), congestion and edema of the colon wall (2.17±0.98 vs 0.00±0.00, P<0.01) significantly increased and colonic pathological section showed increased neutrophil, lympholeukocyte infiltration of rectum, proximal colon, terminal ileum (8.80±0.45 vs 5.40±0.55, 9.80±0.45 vs 5.40±0.55, 8.80±0.84 vs 5.40±0.55, P<0.01) in the model group compared with those of the control group. However, after the treatment of 1,25(OH)₂D₃,, DAI, colon length (5.03 cm±0.56 cm vs 5.98 cm±0.44 cm, P<0.01) and pathology score of rectum, proximal colon, terminal ileum (8.80±0.45 vs 5.40±0.55, 9.80±0.45 vs 5.40±0.55, 8.80±0.84 vs 5.40±0.55, P<0.01) were significantly ameliorated compared with that of the model group in chronic colitis model; (3) The ultrastructural features of the colonic ephithelium were observed by SEM. The control group showed that the mucosa was normal without histological lesion, while the model group displayed a severer loss of the mucosa in histological lesions, however, after the treatment of 1,25(OH)₂D₃, areas with histological lesions were significantly ameliorated and crypt openings were enlarged by chance.Conclusions: 1,25(OH)₂D₃ ameliorated intestinal inflammation of the acute and chronic colitis.Part 2: Protective effect of 1,25-dihydroxyvitamin D₃ on intestinal mucosal barrier in experimental acute and chronic colitisMethods: (1) The method of establishing model is the same as the first part; (2) Mesenteric lymph nodes (MLN) were homogenated to observe whether E. coli bacterial were found in each group; (3) Serum acid hydrogen fluorescein isothiocyanate-dextran (FITC-D) was observed by fluorescence spectrophotometer, and the levels of lipopolysaccharide (LPS) were detected in serum by limulus lysate test; (4) The expressions of zo-1,occludin and claudin-1 proteins were detected by immunohistochemistry, immunofluorescence, western blot and real-time Q-PCR, respectively.Results: (1) Serum levels of the FITC-D and LPS in the model group significantly increased (FITC-D: 933.4±133.5 vs 4266.8±326.6, P<0.01; LPS: 0.119±0.05 vs 0.224±0.06, P<0.01) compared with those of the control group, while the levels of the FITC-D and LPS remarkedly deseased in 1,25(OH)₂D₃ group compared with that of the model group (FITC-D: 253.4±222.9 vs 4266.8±326.6, P<0.01; LPS: 0.138±0.06 vs 0.224±0.06, P<0.01); (2) Bacterial translocation was remarkedly increased in the model group (80%) compared with the control group (0%), while bacterial translocation to mesenteric lymph nodes decreased in 1,25(OH)₂D₃ group (40 %); (3) The results of immunohistochemical and immunofluorescence staining showed that the expressions of the zo-1, occludin and claudin-1 proteins in colonic tissues significantly decreased in the model group compared with that of the control group, while they significantly increased in 1,25(OH)₂D₃ group compared with that of the model group; (4) The results of western blot and real-time Q-PCR revealed that the proteins and mRNA expressions of zo-1, occludin and clandin-1 significantly decreased in the model group compared with that of the control group (Proteins: 0.60±0.05 vs 1.37±0.04, 0.11±0.01 vs 0.25±0.03, 0.44±0.04 vs 0.95±0.04, P<0.05; mRNA: 0.46±0.03 vs 1.00±0.07, 0.36±0.02 vs 1.00±0.04, 0.85±0.05 vs 1.00±0.05, P<0.01), however, after the treatment of 1,25(OH)₂D₃, the proteins and mRNA expressions of zo-1, occludin and clandin-1 significantly increased in 1,25(OH)₂D₃ group compared with those of the model group in acute colitis (Proteins: 1.31±0.15 vs 0.60±0.05, 0.23±0.03 vs 0.11±0.01, 0.93±0.01 vs 0.44±0.04, P<0.05; mRNA: 0.97±0.04 vs 0.46±0.03, 0.98±0.01 vs 0.36±0.02, 1.00±0.04 vs 0.85±0.05, P<0.01). The expressions of zo-1, occludin, and clandin-1 proteins (zo-1: 0.98±0.03 vs 1.56±0.02, 0.34±0.03 vs 0.76±0.020, 0.24±0.02 vs 0.65±0.03, P<0.01;occludin: 1.48±0.03 vs 1.56±0.03, 1.64±0.02 vs 1.76±0.03, 1.44±0.03 vs 1.65±0.02, P<0.01; claudin-1: 1.10±0.03 vs 1.36±0.03,1.04±0.02 vs 1.66±0.03,1.44±0.03 vs 1.85±0.02, P<0.01) and mRNA (zo-1: 0.83±0.05 vs 1.03±0.05, 0.60±0.04 vs 1.01±0.05, 0.69±0.03 vs 1.02±0.05, P<0.01; occludin: 0.90±0.04 vs 1.01±0.05, 0.93±0.04 vs 1.03±0.05, 0.90±0.05 vs1.04±0.05, P<0.01; claudin-1: 0.77±0.02 vs 1.02±0.05, 0.55±0.02 vs 1.01±0.05, 0.66±0.03 vs1.04±0.05, P<0.01) in the rectum, proximal colon, terminal ileum significant decreased in the model group compared with those of the control group in chronic colitis, while after the treatment of 1,25(OH)₂D₃, the expressions of zo-1, occludin, and clandin-1 proteins (zo-1: 1.35±0.02 vs 0.83±0.05, 0.63±0.02 vs 0.34±0.03, 0.55±0.02 vs 0.24±0.02, P<0.01; occludin: 1.45±0.02 vs 1.48±0.03, 1.63±0.02 vs 1.64±0.02, 1.56±0.02 vs 1.44±0.03, P<0.01; claudin-1: 1.25±0.02 vs1.10±0.03, 1.43±0.02 vs 1.04±0.02, 1.65±0.02 vs 1.44±0.03, P<0.01) and mRNA in the rectum, proximal colon, terminal ileum significant increased in 1,25(OH)₂D₃ group compared with those of the model group in chronic colitis(zo-1: 0.94±0.02 vs 0.83±0.05, 0.67±0.01 vs 0.60±0.04, 0.74±0.01 vs 0.69±0.03, P<0.01; occludin: 0.94±0.02 vs 0.90±0.04, 0.94±0.03 vs 0.93±0.04, 0.94±0.01 vs 0.90±0.05,P<0.01; claudin-1: 0.84± 0.02 vs 0.77±0.02, 0.77±0.02 vs 0.55±0.02, 0.74±0.01 vs 0.66±0.03, P<0.01).Conclusions: 1,25(OH)₂D₃ could ameliorate the mucosa damage caused by inflammation, and it may be associated with the decrease of intestinal barrier permeability and the damage of mucosal barrier structure and function.Part 3: The effects of 1,25-dihydroxyvitamin D₃ on the function of CD4 + T cell in the chronic experimental colitisObjective: To investigate the effects of 1,25(OH)₂D₃ on the immunologic function of CD4⁺T in DSS-induced chronic colitis.Methods: (1) The changes of spleen length and spleen weight were observed rapidly after the specimens were harvested; (2) The positive rate of mononuclear cells in the spleen were deteced by Flow cytometry after the mononuclear cells were isolated in spleen, then the cells number was counted under microscope; (3) The mononuclear cells in colon, mesenteric lymph node were isolated and the cells number was counted respectively; (4) Real time Q-PCR was used to detect the mRNA expressions of inflammatory cytokines including TNF-α, IFN-γ, IL-6 and IL-17 in colon tissues.Results: (1) The Spleen length and weight significantly increased in the model group compared with that of the control group (Spleen length: 1.37 cm±0.27 cm vs 1.22 cm±0.12 cm, P>0.05; Spleen weight: 0.09 g±0.02 g vs 0.06 g±0.02 g, P>0.05), while the above index did not significantly decrease after the administration of 1,25(OH)₂D₃(Spleen length: 1.32 cm±0.12 cm vs 1.37 cm±0.27 cm, P>0.05; Spleen weight: 0.084 g±0.02 g vs 0.09 g±0.02 g, P>0.05); (2) The results of cells counting indicated that the number of mononuclear cells from spleens significantly reduced in 1,25(OH)₂D₃ group(76.80×10⁶±18.60×10⁶/mL vs 108.00×10⁶±20.30×10⁶/mL, P<0.01) and the positive rates of immune cells of CD₃, CD4, CD56R significantly decreased after the administration of 1,25(OH)₂D₃(36.58 %±0.70 %, 19.80 %±0.73%, 14.40 %±0.70 %, 26.82 %±0.47 %, P<0.01); (3) The number of mononuclear cells from MLN (2.52×10⁶/mL±0.74×10⁶/mL vs 3.42×10⁶/mL±0.40×10⁶/mL, P<0.01) and colon (0.32×10⁶/mL±0.14×10⁶/mL vs 0.88×10⁶/mL±0.08×10⁶/mL, P<0.01) significantly reduced in 1,25(OH)₂D₃ group compared with that of the model group; (4) The results of real-time Q-PCR indicated that the mRNA levels of TNF-α, IFN-γ, IL-6 and IL-17 significantly reduced in 1,25(OH)₂D₃ group compared with that of the model group(5.48±0.08 vs 9.30±0.07, 7.74±0.11 vs 14.38±0.08, 3.28±0.11 vs 6.38±0.08, 4.54±0.10 vs 7.38±0.07, P<0.01).Conclusions: The administration of 1,25(OH)₂D₃ could relieve the damages of chronic colitis in mice, which may be related to the decrease of positive rates of immune cells from spleens and intestinal tissues, and the inhibition of inflammatory cytokines from immune cells.Part 4: The protective effect of 1,25-dihydroxyvitamin D₃ on epithelial barrier in Caco-2 and the mechanisms thereofObjective: To investigate the protective effect of 1,25(OH)₂D₃ on epithelial barrier disruption in human intestinal epithelial cells and the related mechanisms.Methods: (1) Caco-2 cell monolayers were incubated without or with 2% DSS in the absence or presence of 10^-9, 10^-8, 10^-7 M of 1,25(OH)₂D₃, The cells were grouped as follows: Control group; DSS group; 1,25(OH)₂D₃10^-9 group, 1,25(OH)₂D₃10^-8M group, 1,25(OH)₂D₃10^-7M group; (2) The permeability function of cell monolayers were measured by the transepithelial electrical resistance (TEER) and FITC-D. (3) Ultrastructural features of Caco-2 cells monolayers were observed by a transmission electron microscope (TEM); (4) The expressions of TJ protein and mRNA in Caco-2 cells were detected by western blot and real-time Q-PCR, respectively.Results: (1) TEER and FITC-D permeability results showed that the model of intestinal epithelial barrier was successfully established with Caco-2 cells in vitro; (2) FITC-D permeability results showed that DSS increased the FITC-D permeability, compared with that of the vehical group (51.83±2.48 vs 5.5±1.05, P<0.01), while the administration of 1,25(OH)₂D₃ (10^-9 M, 10^-8 M, 10^-7 M) afforded cytoprotection against DSS-induced monolayer hyper-permeability of Caco-2 cell monolayer (18.5±1.05 vs 51.83±2.48, 8.17±1.17 vs 51.83±2.48, 18.5±1.05 vs 51.83±2.48, P<0.01). The results of TEM indicated that structures of tight juctions in DSS group were destroyed while the TJ were regained with a different degree after the administration of 1,25(OH)₂D₃; (3) Immunofluorescence microscopy assay showed that the localization of TJ proteins zo-1, occludin and claudin-1 were integrity and presented as a high fluorescence signal in vehical group; At 2 h after administration of DSS, the fluorescence intensity were gradually low and the expressions of zo-1, occludin and claudin-1 significantly decreased; while the expressions of zo-1, occludin and claudin-1 significantly increased after the administration of 1,25(OH)₂D₃; (4) The results of western blot revealed that the proteins expressions of zo-1, occludin and clandin-1 significantly decreased in the DSS group compared with that of the vehical group (0.80±0.16 vs 1.51±0.20, 0.99±0.13 vs 1.66±0.14, 0.76±0.09 vs 1.79±0.11, P<0.01), however, after the treatment of 1,25(OH)₂D₃10^-8 M, the proteins expressions of zo-1, occludin and clandin-1 significantly increased in 1,25(OH)₂D₃ group compared with those of the DSS group in caco-2 cell (1.20±0.12 vs 0.80±0.16, 1.47±0.13 vs 0.99±0.13, 1.68±0.11 vs 0.76±0.09, P<0.01). After the treatment of 1,25(OH)₂D₃10^-9 M, 1,25(OH)₂D₃10^-7 M the proteins expressions of occludin and clandin-1 significantly increased in 1,25(OH)₂D₃ group compared with those of the DSS group in caco-2 cell, P<0.01; however, the proteins expressions of zo-1 is not increased in 1,25(OH)₂D₃ group compared with those of the DSS group in caco-2 cell. The results of real-time Q-PCR revealed that the mRNA expressions of zo-1 mRNA, occludin mRNA and clandin-1 mRNA significantly decreased in the DSS group compared with those of the vehical group (0.67±0.11 vs 1.00±0.00, 0.63±0.07 vs 1.00±0.00, 0.70±0.07 vs 1.00±0.00, P<0.01), however, after the treatment of 1,25(OH)₂D₃10^-8 M, the mRNA expressions of zo-1 mRNA, occludin mRNA and clandin-1 mRNA significantly increased in 1,25(OH)₂D₃ group compared with those of the DSS group in caco-2 cell (0.84±0.08 vs 0.67±0.11, 0.84±0.08 vs 0.63±0.07, 0.9±0.08 vs 0.70±0.07, P<0.01), after the treatment of 1,25(OH)₂D₃10^-9 M, 1,25(OH)₂D₃10^-7 M the mRNA expressions of zo-1 mRNA, occludin mRNA and clandin-1 mRNA significantly increased compared with those of the DSS group in caco-2 cell, P<0.01.Conclusions: DSS could cause epithelial barrier disruption and injury, while 1,25(OH)₂D₃ ameliorated the DSS-induced epithelial barrier disruption via upregulating tight junction expression in Caco-2 cells.