Vitamin D Inhibits Mouse Colitis By Targeting The Local Renin-Angiotensin System

Objective: Renin-angiotensin system(RAS)is an important system which regulates body fluid in human.In addition to systemic RAS,there are relatively independent local RAS in various organ tissues,such as digestive tract,skeletal muscle,brain and gonad,which directly regulate the physiological and pathological activities of various organs and tissues through paracrine and/or autocrine methods.The gut is the basis for the absorption of fluids and electrolytes,with abundant smooth muscle tissue,epithelial cells and defense cells which transporting ionic and liquid.As a regulator of humoral balance,RAS regulates some pathophysiological processes targeting the intestine.Inflammatory bowel disease(IBD)is a major chronic disorder of the gastrointestinal tract in human,it is believed that derangements in the complex interplays among genetic,environmental,microbial,and immune factors contribute to these disorders.The gut epithelial barrier consists of a monolayer of epithelial cells and intercellular junctions between adjacent cells,which prevent harmful solutes,microorganisms,toxins,and luminal antigens from entering the body.Disruption or dysregulation of the epithelial junctions and/or excess epithelial cell apoptosis can impair the intestinal barrier function,which leads to translocation of luminal antigens and bacteria into the lamina propria and triggers colonic inflammation.It is well established that gut mucosal epithelial tight junction is regulated by the myosin light chain kinase(MLCK)pathway,with MLCK activation disrupting the tight junction leading to increased permeability.It is also well known that up-regulation of PUMA,a pro-apoptotic protein of BCL2 family,in the gut promotes excess gut epithelial apoptosis,which can also lead to increased mucosal barrier permeability..Vitamin D has a wide range of physiological functions,including calcemic and non-calcemic activities.Insufficient or lack of vitamin D is closely related to IBD.Severe vitamin D deficiency occurs in patients with new ulcerative colitis(UC)and Crohn's disease(DC).If vitamin D is supplemented,the development of IBD can be reduced.Activation of RAS can promote colitis.Ang? promotes colonic mucosal inflammationby promoting intestinal epithelial cell apoptosis and mucosal Th17 response.The RAS is activated in inflammatory bowel disease(IBD),and vitamin D defciency aggravates the development of colitis,but the relationship between the local colonic RAS and vitamin D is unclear with regard to the pathogenesis of IBD.Therefore,the purpose of this study was to investigate whether vitamin D suppresses the local colonic RAS to prevent colonic mucosal inflammation in a mouse model of experimental colitis.Methods: 1.In vivo: C57BL/6 mice were fed with sufficient or deficient Vitamin D feed for 8 weeks,then divided into the following 8 groups: sufficient Vitamin D feed control group(VDS Ctrl),model group(VDS TNBS),losartan-treated low-dose group(VDS TNBS+Llo),losartan-treated high-dose group(VDS TNBS+Hlo);deficient Vitamin D feed control group(VDD Ctrl),model group(VDD TNBS);losartan-treated low-dose group(VDD TNBS+Llo),losartan-treated high-dose group(VDD TNBS+Hlo).The mixture of acetone,olive oil and TNBS was used to presensitize themouseand the mouse colitis model was induced by 2,4,6-trinitro-benzene-sulfonic acid(TNBS)after 8 days.The general behavior and body weight after 3 days of modeling was observed and recorded.The weight loss index and the gross score of the colon were compared between the experimental group and the control group.The level of 25 hydroxyvitamin D₃[25-(OH)D₃]in the serum was determined by Elisa method.The expression of myosin light chain kinase(MLCK)and p53-upregulated modulator of apoptosis(PUMA),Renin,AT1 R,and angiotensin was detected by Western blotting.Real-time PCR was used to detect the expression of Tnfa,Il1 b,Ccl2,Il6,Il17,Il23 a m RNA in mouse colonic mucosa.FITC-dextran permeability test and Ussing chamber experimental techniques were used to detect colonic mucosal permeability.HE staining was used to observe the pathological changes of colon tissue and pathological scores were performed.2.In vitro experiment:HCT116 weretreated with LPS or TNF-?(100 ng/ml)for 12 h and 20 h,or given 1,25-(OH)D₃ was pretreated for 12 h and then stimulated by LPS or TNF-? for 12 h.Then the cells were collected followed.The m RNA expressions of Ren,Agtr1 and Agt were detected by Real-time PCR.The expressions of Renin,AT1 R and AGT were detected by Western blotting.3.Statistical analysis: SPSS22.0 software was used for statistical analysis.Data values were expressed by Mean ± S.E.M.Statistical comparisons were performed with un-paired two-tailed student's t-test,P<0.05 was considered to be statistically significant.Results: 1.Vitamin D deficiency aggravated ulcerative colitis.Compared to control,TNBS-induced mice VDD mice showed more severe weight loss than VDS mice follwing TNBS instillation.TNBS-treated colons were more shortened and swollen,with no visible fecal pellet formation and developed higher clinical(disease)scores in VDD mice compared with VDS mice.Histological examination showed more severe colonic ulceration and histological damage in VDD mice compared with VDS mice following TNBS induction.The induction of mucosal pro-infammatory cytokines and chemokines(Tnfa,Il1 b,Ccl2,Il6,Il17 and Il23a)was more robust in VDD mice than in VDS mice.In FITC-dextran feeding assays,TNBS-treated VDD mice exhibited greater increases in plasma FITC-dextran concentrations compared with the VDS counterparts,indicating greater increases in gut mucosal permeability in VDD mice following TNBS induction.Direct measurement of colonic mucosal TER using a Ussing chamber confrmed that TNBS markedly reduced colonic TER in both VDD and VDS mice,but the reduction in TER was greater in VDD mice.Western blotting analyses showed that TNBS treatment markedly up-regulated MLCK and PUMA,but suppressed VDR,and these changes were much more robust in VDD mice.2.Vitamin D deficiency increased the expression of RAS in the colon of mouse with ulcerative colitis.The transcripts of mucosal Renin,AT1 R,and AGT(Ren,Agtr1 and Agt)in the colon were markedly induced following TNBS treatment,but VDD mice showed much more robust induction compared to VDS mice.Western blotting analyses confrmed that vitamin D defciency markedly up-regulated the protein expression of Renin?AGT and AT1 R in the colonic mucosa of VDD mice following TNBS treatment.We treated TNBS-induced VDS and VDD mice with losartan,an AT1 receptor inhibitor,to block the Ang?-AT1 receptor signaling.Losartan treatment was able to alleviate the body weight loss in VDD mice as well as VDS mice following TNBS induction,and the preventative effect on VDD mice was comparable to that on VDS mice.FITC-dextran assays showed that TNBS treatment increased mucosal permeability in both VDS and VDD mice,but losartan was able to markedly attenuate the increase in mucosal permeability in VDD mice.Consistently,losartan treatment was able to markedly improve the morphology of the colon and the clinical scores of TNBS-induced VDD and VDS mice,compared to TNBS-induced VDD and VDS mice without losartan treatment.Histological examination confirmed that losartan treatment ameliorated colonic ulceration and infammation in TNBS-induced VDS mice as well as VDD mice,which was refected by the improvement of the semiquantitative histological scores in the mice,even though TNBS has more severe effects on the VDD mice.Moreover,the induction of mucosal infammatory cytokines and the Renin,AGT and AT1 R were markedly suppressed in TNBS-induced VDS and VDD mice following losartan treatment.3.Effect of vitamin D on the expression of RAS in HCT116 cells.LPS or TNF-?(100 ng/ml)pre-stimulated HCT116 cells increased expression of Ren,Agtr1,Agt m RNA after 12 h and 20 h.Renin,AT1 R,AGT and PUMA protein expression also increased.After administration of 1,25-(OH)D₃,the corresponding protein expression of RAS decreased.Conclusion: Both in vivo and in vitro experiments showed that the expression of RAS was increased under inflammatory conditions.Vitamin D deficiency aggravated colitis,and RAS was activated in the colon,which may be related to the reduced inhibition of RAS.The decrease in RAS resulted in a high expression of local RAS in the colonic mucosa.The administration of losartan significantly inhibited the expression of RAS and the colitis was alleviated in mice.Our results suggested that vitamin D could regulate colitis by affecting the local RAS.