Effect Of1,25(OH)₂D₃on Activation Of Th1,Th17in Chronic Colitis Mice

Ulcerative colitis (UC), one form of inflammatory bowel disease (IBD),is chronic disease featured by aberrant immune responses to luminal bacteriain genetically susceptible groups. Numerous researches have revealed that theimmune factors play a critical role in its pathogenesis, which relates to theactivation of T helper (Th)1/Th17cells. More and more studies have focusedon the functions of Th1/Th17cells in the intestinal immune responses of UC,which can promote the generation of inflammation and relate to manyautoimmune diseases. It is demonstrated that specific transcription factors thatare activated by various stimulus may mediate immune response of Th1/Th17cells, secreting Th1/Th17cells related factors, mainly including interleukin-17(IL-17), tumor necrosis factor-α (TNF-α), IL-6, and iterferon-γ (IFN-γ), whichhave formidable abilities of raising and activating the neutrophil, contribute tothe process of adjusting the immune response and removing the pathogens,and further induce inflammation. The activation of Th1/Th17cells playsessential roles in the chronic inflammation and may be the main immuneresponse pathway in the onset of UC.Extensive researches have shed new light on the link between UC andvitamin D.1,25-dihydroxyvitamin D3(1,25(OH)₂D₃), the active form ofVitamin D, which has a range of physiological activities, recently has emergedas a direct regulator of immune system function in humans. Because vitamineD receptor (VDR) widely exists in lymphocytes and mononuclear cells, so ithas other biological activities. Vitamin D combined with receptors in themononuclear cells, macrophages and pre-sensitized lymphocytes participatedin the immune response can inhibit the functions of Th cells, especiallyTh1/Th17cells. Insufficiency production of vitamin D will robustly increase the risk of UC. In short, effects of vitamin D on immune suppression have apotential role in the therapy of UC.Objective: To explore the regulatory pathogenesis of1,25(OH)₂D₃toDSS-induced chronic colitis so as to provide an attractive and noveltherapeutic agent for the adjuvant therapy of UC.Methods:⑴Chronic colitis was induced by administration of dextransodium sulfate (DSS) drinking water from day1to day5, day8to day12, day15to day19and day22to day26, and distilled water was given during theremaining time. A total of30mice were randomly assigned to control, DSSand DSS＋VD group (each group=10). The mice in the DSS＋VD groupreceived1,25(OH)₂D₃daily (0.2ug/25g/d) by intragastric administration for14days, and the mice in control and DSS group were given PBS forcomparion.⑵Severity of colitis was assessed by body weight (BW) changes,disease activity index (DAI), colon length, colon histology changes andpathology score.⑶Myeloperoxidase (MPO) was measured in each group.⑷The serum calcium was tested in each group.⑸Cells were extracted fromMLN and then counted one by one.⑹The concentrations of IL-17, TNF-α,IFN-γ and IL-6from MLN monocytes supernatant were tested by ELISA.⑺The pathology changes of colons in each group were detected byHaematoxylin and eosin staining (HE dyeing).⑻The expressions of IL-17,TNF-α, IL-6and IFN-γ in colon mucosa were detected byimmunohistochemistry, western blot and real-time Q-PCR, respectively.Results:⑴The results of severity of chronic colitis showed thatcompared to that in the control group, BW had distinctly greater loss in DSSgroup (15.5%±1.50%vs0.0%±0.0%, P<0.05). However, weight of mice inDSS+VD group restored rapidly (5.2%±0.53%vs19.6%±1.82%, P<0.05).DAI (3.77±0.36vs0.00±0.00, P<0.05) and pathology scores (9.80±1.26vs0.00±0.00, P<0.05) in DSS group were remarkablely higher than that in thecontrol group, the colon length became shorter (4.35cm±0.48cm vs5.98cm±0.44cm, P<0.05), and also the degrees of the congestion and edema of thecolon wall and infiltration of granulocytes into the mucosa were much severer in DSS group (2.50±0.58vs0.00±0.00, P<0.05). While DAI (1.79±0.19vs3.77±0.36, P<0.01) and pathology score (7.00±0.82vs9.80±1.26, P<0.05)evidently decreased in DSS＋VD group, colon length became longer (5.27cm±0.46cm vs5.03cm±0.56cm, P<0.05), and the degrees of the congestionand edema of the colon wall and infiltration of granulocytes into the mucosawere improved (1.50±0.58vs2.50±0.58, P<0.05).⑵Mice in DSS group hadmuch higher MPO activity than that in the control group (1.30U/g±0.09U/gvs0.18U/g±0.01U/g, P<0.05), and after the treatment, it decreasednotewortily (0.41U/g±0.03U/g vs1.30U/g±0.09U/g, P<0.05).⑶The serumcalcium was tested in each group, but there are no statistics significanesbetween the groups, P>0.05.⑷Relative with control group, cells from MLNraised in DSS group (6.56×10⁶±0.06×10⁶/mL vs1.63×10⁶±0.27×10⁶/mL,P<0.01), while in the DSS+VD group, they desended significantly(3.51×10⁶±0.18×10⁶/mL vs6.56×10⁶±0.06×10⁶/mL, P<0.01). Inflammatorymarkers of IL-17, TNF-α,IL-6and IFN-γ in cell culture supernatants weretested by ELISA. In the control group, the unstimulated concentrations of theIL-17, TNF-α, IL-6and IFN-γ were as followed: IL-17:0.22ng/mL±0.04ng/mL, TNF-α:0.046ng/mL±0.004ng/mL, IFN-γ:0.02ng/mL±0.01ng/mL,IL-6:0.012ng/mL±0.01ng/mL. But after the stimulation of CD3/CD28, theywere significantly increased(IL-17:1.85ng/mL±0.14ng/mL, TNF-α:0.068ng/mL±0.006ng/mL, IFN-γ:1.43ng/mL±0.15ng/mL, IL-6:0.15ng/mL±0.01ng/mL, P<0.01). In DSS group, the expression levels of IL-17, TNF-α,IL-6and IFN-γ significantly increased than those in the control group,(IL-17:0.58ng/mL±0.10ng/mL vs0.22ng/mL±0.04ng/mL; TNF-α:0.17ng/mL±0.02ng/mL vs0.046ng/mL±0.004ng/mL, P<0.05; IL-6:0.14ng/mL±0.01ng/mLvs0.012ng/mL±0.01ng/mL; IFN-γ:0.61ng/mL±0.12ng/mL vs0.02ng/mL±0.01ng/mL, P<0.05), after the stimulation of CD3CD28, theexpression levels of IL-17, TNF-α,IL-6and IFN-γ also significantly increasedthan those in the control group (IL-17:12.23ng/mL±1.03ng/mL vs1.85ng/mL±0.14, ng/mL TNF-α:0.35ng/mL±0.04ng/mL vs0.068ng/mL±0.006ng/mL, IFN-γ:6.67ng/mL±0.46ng/mL vs1.43ng/mL±0.15ng/mL, IL-6: 0.47ng/mL±0.06ng/mL vs0.15ng/mL±0.01ng/mL, P＜0.05). In theDSS+VD group, the expression levels of IL-17, TNF-α,IL-6and IFN-γdecreased compared with that in DSS group (IL-17:0.38ng/mL±0.04ng/mLvs0.58ng/mL±0.10ng/mL, TNF-α:0.13ng/mL±0.01ng/mL vs0.17ng/mL±0.02ng/mL, IFN-γ:0.41ng/mL±0.13ng/mL vs0.61ng/mL±0.12ng/mL, IL-6:0.07ng/mL±0.01ng/mL vs0.14ng/mL±0.01ng/mL, P＜0.05),after the stimulation of CD3CD28while they also significantly decreasedcompared with that of DSS group (IL-17:6.35ng/mL±0.34ng/mL vs12.23ng/mL±1.03ng/mL, TNF-α:0.24ng/mL±0.02ng/mL vs0.35ng/mL±0.04ng/mL, IFN-γ:4.45ng/mL±0.41ng/mL vs6.67±ng/mL0.46ng/mL, IL-6:0.43ng/mL±0.04ng/mL vs0.47ng/mL±0.06ng/mL, P＜0.05).⑸Expressions of IL-17, TNF-α, IL-6and IFN-γ were observed byimmunohistochemistry. In the control group, the expressions of the IL-17,TNF-α, IL-6and IFN-γ were not seen and they were significantly increased inDSS group than those in the control group,(IL-17:0.70±0.10vs0.17±0.05,P<0.05; TNF-α:0.85±0.10vs0.25±0.05, P<0.05; IL-6:0.65±0.07vs0.10±0.01, P<0.05; IFN-γ:0.90±0.10vs0.20±0.05, P<0.05). While theexpression levels of IL-17, TNF-α,IL-6and IFN-γ significantly decreased inDSS+VD group compared with that in DSS group (IL-17:0.40±0.08vs0.70±0.10, P<0.05; TNF-α:0.35±0.08vs0.85±0.10, P<0.05; IL-6:0.30±0.02vs0.65±0.07, P<0.05; IFN-γ:0.45±0.07vs0.90±0.10, P<0.05).⑹Theproteins evels of IL-17, TNF-α,IL-6and IFN-γ in DSS group were higherthan those in control group (IL-17:1.81±0.10vs1.21±0.05; TNF-α:1.79±0.10vs1.04±0.05, P<0.05; IL-6:1.75±0.13vs1.00±0.05, P<0.05; IFN-γ:2.06±0.10vs1.32±0.05, P<0.05), and in DSS＋VD group these cytokinessignificantly decreased compared with those in DSS group (IL-17:1.48±0.08vs1.81±0.10, P<0.05; TNF-α:1.44±0.08vs1.79±0.10, P<0.05; IL-6:1.34±0.11vs1.75±0.13, P<0.05; IFN-γ:1.73±0.07vs2.06±0.10, P<0.05); thecomparion of the mRNA between the DSS group and the control had statisticssignificane (IL-17mRNA:1.91±0.86vs1.00±0.00, P<0.05; TNF-α mRNA:1.94±0.64vs1.00±0.00, P<0.05; IL-6mRNA:2.04±0.02vs1.00±0.00, P<0.05; IFN-γ mRNA:2.11±0.11vs1.00±0.00, P<0.05), and in DSS＋VDgroup these cytokines significantly decreased compared with those in DSSgroup (IL-17mRNA:1.56±0.30vs1.91±0.86, P<0.05; TNF-α mRNA:1.39±0.67vs1.94±0.64, P<0.05; IL-6mRNA:1.33±0.01vs2.04±0.02,P<0.05; FN-γ mRNA:1.72±0.40vs2.11±0.11, P<0.05).Conclusion:1,25(OH)₂D₃can improve the tissue damage caused byinflammation and has a protective effect on the mice of chronic colitis, whosemechanism may inhibit the activition of Th1/Th17cells.