The Influence Of High Salt Diet On Mice Intestinal Immunity And TNBS-induced Colitis

BackgroundThe main ingredient of salt is sodium chloride(NaCl),salt provide human with Na+and Cl-ions,which is necessary to human health.However,the daily salt intake of most people is significantly exceeds their physiological needs.The health hazards of high salt intake has acpuired widespread concern,it is clear that excessive salt intake could increase the risk of cardiovascular-related diseases,and studies suggest that high salt intake affect the kidney,skeletal system,and metabolic function.Interestingly,incresing studies suggest that excessive salt intake can affect the immune system and may even increase the risk of autoimmune diseases.Intestine is an important part of the digestive system,which playing a key role in the food digestion and nutrients absorption.However,the intestine is also an important immune organ,it serve a pivotal role in local and systemic immunity.Intestinal lamina propria contains abundant T helper 17 cells(Th17)and regulatory T cells(Treg),Th17 cells promote inflammation and even increase the risk of autoimmune diseases,whereas Treg cells inhibit inflammation and reduce the risk of autoimmune diseases,Thl7/Treg balance play a critical tole in maintaining the balance of intestinal immunity.The antigens and various other elements of food may be directly or indirectly contact with the intestinal lymphocytes,and many studies have found that different dietary habits could change the intestinal immunity.However,whether long-term intake of high salt food could change the balance of intestinal immunity is unclear.Inflammatory bowel disease(IBD)is a chronic recurrent intestinal autoimmune disease,it cause a huge threat to human health and bring a great economic burden to the society.The animal colitis model is the main tool for studying the pathogenesis of IBD,for example,tranitrobenzenesulfonic acid(TNBS)-induced mice colitis is a classical model for studying IBD.According to previous studies,immune factors are an important regulatory factor of IBD,for instance,Th 17 cells and their related cytokines play a key role in the pathogenesis of IBD.In recent years,the world incidence of IBD has increased,poor eating habits and the prevalence of Western diet may be associated with the high incidence of IBD.According to previous studies,excessive fat and meat intake could increase the risk of IBD,but high dietary fiber intake and eat more fruits and vegetables could significantly reduce the risk of IBD.However,whether excessive salt intake could affect the incidence of IBD is unclear.In summary,the salt intake of most people exceeds physiological need,and excessive salt intake could affect the immune system.Intestine is not only a digestive organ but also an important immune organ,and IBD is an autoimmune disease which mainly involve in the intestine.However,whether excessive salt intake would affect the intestinal immunity or the incidence of IBD is not clear.Therefore,this study established the high salt diet(HSD)and normal diet diet(ND)mice model to study th effects of excessive salt intake on mice intestinal immunity,and established TNBS-induced colitis mice model to study the ralationship between excess salt intake and IBD.Part 1:High salt diet disrupts mice intestinal Th17/Treg balanceAims:To investigate the effects of excess salt intake on mice intestinal immunity by animal experiments.Methods:1)HSD and ND mice models were established with food and water in different salt content.2)Lymphocytes in intestinal related lymphoid tissues were isolated by density gradient centrifugation and mechanical grinding.3)We analyzed the immune difference of lymphocytes from intestinal related lymphoid tissues between HSD and ND mice by flow cytometry,enzyme-linked immunosorbent assay,suspension array technology,fluorescence quantitative PCR and associated cell function test.Result:1)HSD increases the percentage of IL-17A+ lymphocytes in mice intestinal lamina propria.2)Most IL-17A+ cells of the intestinal lamina propria were Th17 cells,and HSD further increase the proportion of Thl7 cells.3)HSD increased the Th17 responses in both small and large intestinal lamina propria,and HSD significantly promoted the expression of Th17-related cytokines and transcription factor in the intestinal lamina propria.However,HSD did not significantly affect the Th17 responses of other intestinal related lymphoid organs other than the intestinal lamina propria.Besides,HSD does not affect the IFN-y expression of-CD4+TCR(3+ cells,CD8 T cells and natural killer cells.4)Although,HSD did not change the percentage of Treg cells,HSD significantly inhibit secretion of IL-10 and the suppressive function of Treg cells.Part 2:High salt diet aggravates TNBS-induced mice colitisAims:To evaluate the effect of excessive salt intake on TNBS-induced mice colitis,and elucidate whether excessive salt intake is a potential risk factor for IBD.Methods:1)We established HSD and ND mice models with food and drinking water in different salt content.And combined special diet with TNBS treatment,we established TNBS-induced colitis in HSD or ND mice.2)Differences in the severity of TNBS-induced colitis were evaluated by the mice weight loss,survival rate,colon inflammation score and pathological staining.And we analyzed the lymphocyte of colonic lamina propria by flow cytometry.The level of Th17-related cytokines and the collagen fibers in colon were detected by enzyme-linked immunosorbent assay and fluorescence quantitative PCR.The function of IL-17A in cytotoxicity of lymphocytes against the intestinal epithelial cells was evaluated by in vitro cell co-culture experiments.Results:1)We found HSD aggravated both TNBS-induced acute and chronic mice colitis.2)In TNBS-induced mice colitis,HSD enhance the Th17 response of the colon lamina propria,and HSD increase the expression of Th17-related cytokines in colon tissue.3)In vitro cell co-culture experiments,we found that IL-17A enhance the cytotoxicity of lymphocytes against the intestinal epithelial cells.Conclusion:This study found that excessive salt intake would disrupt the intestinal Th17/Treg balance,manifested as enhancing the intestinal lamina propria Th17 response,reducing the suppressive function of Treg cells.Moreover,excessive salt intake can aggravate the TNBS-induced acute and chronic mice colitis,and this effect is associated with enhanced colonic Th 17 response and increased expression of Th 17-associated cytokines.These results suggest that high salt diet can disrupt the balance of intestinal immunity and may increase the risk of IBD.This study suggested that reduce the daily salt intake may have a great clinical significance for the prevention and treatment of IBD.