Studies On The Effects Of High-fat Diet On Mouse Experimental Colitis In The Presence Or Absence Of Vitamin D Or Its Analogs

Objectives:One key feature of inflammatory bowel disease?IBD?is dysfunction of the intestinal epithelial barrier.Tumor necrosis factor-??TNF-??is highly expressed in the colon of IBD patients,and TNF-?can alter the tight junction structure and damage the intestinal epithelial barrier by up-regulating the myosin light chain kinase?MLCK?pathway.High-fat diet?HFD?is known to promote the development of IBD by inducing intestinal epithelial barrier dysfunction.HFD up-regulates the expression of TNF-?and NADPH oxidase,and increases the production of reactive oxygen species?ROS?.However,the molecular mechanism whereby HFD promotes IBD remains not completely clear.Epidemiological studies have suggested that vitamin D-deficiency is a potential risk factor for IBD.Treatment with a vitamin D analog,paricalcitol?PAL?,can effectively inhibit the activation of the MLCK signaling pathway,thereby protecting the intestinal epithelial barrier.However,the effect of vitamin D treatment on the development of HFD-induced colitis is not known.The goals of this study are to further explore the mechanism whereby HFD promotes experimental colitis and to assess the therapeutic effects of vitamine D analogs on the development of HFD-induced colitis in mice.Methods:1.Study the effect of HFD on colitis in mice.?1?Establishment of an HFD model:C57BL/6 mice were fed low-fat diet?LFD?or HFD for 4 weeks.Body weight,triglyceride and cholesterol levels were measured.The liver was assessed by H&E staining.?2?Effects of HFD on colitis:2,4,6-trinitro-benzene-sulfonic acid?TNBS?was used to induce colitis in C57BL/6 mice after LFD and HFD feeding for 4 weeks.Colitis severity was assessed by body weight loss,clinical score and histological score.Colonic pro-inflammatory cytokines were quantified by real-time RT-PCR.?3?Effects of HFD on colonic epithelial barrier:The permeability of the colonic epithelial barrier was measured using FITC-dextran.Colonic transepithelial electrical resistance?TER?was measured by Ussing chamber.Colonic epithelial tight junction was assessed by transmission electron microscopy?TEM?.Tight junction proteins in colonic mucosa were analyzed by Western blot and immunostaining.?4?Effects of HFD on colonic oxidative stress:ROS production in colonic epithelial and lamina propria cells was measured using commercial kits.The expression of Gp91,a key subunit of NADPH oxidase,was measured by Western blot,and the mitochondrial morphology was examined by TEM.2.Study the effect of vitamine D analogs on colitis in HFD-fed mice.?1?Effects of PAL on colitis in HFD-fed mice:PAL was given by daily intraperitoneal injection at the end of the third week of HFD feeding,and colitis was induced by TNBS.Body weight loss,clinical score and histological score were measured.Colonic pro-inflammatory cytokines were quantified by real-time RT-PCR.?2?Effects of PAL on the colonic epithelial barrier:The permeability of the colonic epithelial barrier was measured using FITC-dextran.The tight junction proteins were analyzed by Western blotting.3.Study the effects of long-chain fatty acid and vitamin D on intestinal epithelial barrier using in vitro cell cultures.?1?Effects of fatty acid on oxidative stress and colonic epithelial junctions:HCT116 cell monolayers were treated with TNF-?and palmitic acid?PA?,a major long-chain saturated fatty acid,for 12 hours,or HCT116 cells were pretreated with ROS inhibitor for 1 hour before being treated with TNF-?and PA for 12hours.ROS in the cells was measured using commercial kits.Mitochondrial membrane potential was assessed by flow cytometry.Tight junction proteins were measured by Western blotting.?2?Effects of vitamine D on intestinal epithelial barrier:HCT116 and Caco-2 cells were pretreated with 1,25?OH?₂D₃ for 12 hours before being treated with TNF-?and PA for 12 hours,TER of colonic epithelial cells was measured in Ussing chamber.Tigh junction proteins were assessed by immunostaining.Results:1.Effects of HFD on colitis in mice.?1?Establishment of an HFD model:Compared with the LFD-fed mice,body weight,triglyceride and cholesterol in the plasma and colon in HFD-fed mice were elevated,and liver lipid droplets were significantly increased.?2?HFD exacerbated colitis:Following TNBS induction,HFD-fed mice exhibited more severe decreases in body weight and greater clinical scores and histological scores compared to LFD-fed mice.The expression of Il1b,Tnfa,Ifng,Il6,Il17,Il23p19 and Mcp1 was higher in HFD-fed mice than in LFD-fed mice.?3?HFD promoted colonic epithelial barrier dysfunction:Compared with LFD-fed mice with colitis,the permeability,TER and tight junction open strcture in the colonic epithelium of HFD-fed mice with colitis were significantly increased,and the expression of ZO-1 and occludin proteins were significantly decreased,whereas TNF-?,MLCK,P-MLC,and claudin-2 proteins expression were increased significantly.?4?HFD exacerbated oxidative stress in mice with colitis:Compared with LFD-fed mice with colitis,ROS in colonic epithelial and lamina propria cells and gp91 protein expression were also significantly increased,and mitochondrial length was increased significantly in HFD-fed mice with colitis.2.Effects of of vitamine D analogs on colitis in HFD-fed mice.?1?PAL inhibited colitis in HFD-fed mice:Treatment with PAL decreased body weight loss and increased clinical scores and and histological scores in HFD-fed mice with colitis.The expression of Il1b,Tnfa,Ifng,Il6,Il17,Il23p19 and Mcp1 was suppressed by PAL treatment in HFD-fed mice with colitis.?2?PAL prevented colonic epithelial barrier dysfunction in mice with colitis:Treatment with PAL reduced colonic permeability,increased occludin protein and decreased TNF-?,PUMA,MLCK and p-MLC proteins in HFD-fed mice with colitis.3.Effects of long-chain fatty acid and vitamin D on intestinal epithelial barrier using in vitro cell cultures.?1?Fatty acid aggravated oxidative stress and barrier dysfunction induced by TNF-?in colonic epithelial cells:PA increased cellular ROS production,reduced mitochondrial membrane potential,increased MLCK,p-MLC,claudin-2 and gp91 and decreased occludin proteins expression induced by TNF-?.ROS inhibitor significantly reduced ROS,prevented the reduction of mitochondrial membrane potential,decreased MLCK,p-MLC,claudin-2,gp91 proteins expression and increased occludin expression in colonic epithelial cells treated with PA and TNF-?.?2?Vitamin D protected colonic epithelial barrier in a high fat environment:1,25?OH?₂D₃ treatment increased TER and ZO-1 protein expression in colonic epithelial cells treated with PA and TNF-?.Conclusion:1.HFD feeding promotes colonic epithelial barrier dysfunction by inducing oxidative stress and aggravates colitis in mice.2.Vitamine D analogs prevents colonic epithelial barrier dysfunction and alleviates colitis in HFD-fed mice.3.Cell experiments varify that high fatty acid increases cellular oxidative stress and further promotes colonic epithelial cell barrier dysfunction induced by TNF-?,and vitamine D blocks colonic epithelial barrier dysfunction induced by high fatty acid in vitro.