| Inflammatory bowel disease(IBD)is a chronic inflammatory disease in intestines.With repeated rectal irritation and abnormal mucosal hyperplasia,the incidence risk of colon cancer increases.IBD includes two types,such as ulcerative colitis(UC)and Crohn’s disease(CD).Patients with UC accompanied by dull abdominal pain,abdominal distension,diarrhea,constipation,and intestinal bleeding.Sustained tissue damage will develop active inflammation into chronic low-grade inflammation,even bowel cancer,seriously wrecking human health.However,the mechanism of IBD has not yet been explained clearly.In the past two decades,IBD’s animal models have been established by changing mucosal immunology and intestinal homeostasis in mice.Their clinical symptoms,pathological changes,and treatment responses are similar to human IBD.During mucosal inflammation,cytokines are released into the epithelial environment and target the junctions and mucus layer,leading to altered barrier function and an increase in intestinal epithelial permeability.Resulted of its antioxidant activity,selenium(Se)and selenium compounds protect the body by maintaining oxidation-reduction homeostasis.3,3’-Diselenodipropionic acid(DSePA)is a laboratory synthesized selenium-organic compound,which was revealed anti-inflammatory and antioxidant activities.It has been used as a clinical radiation protection agent in the chest,abdomen,and whole-body exposured by y-ray.The effection has been demonstrated in previous radiation irradiation experiments,especially in abdominal treatment.DSePA significantly ameliorated the redox homeostasis and Jejunal inflammation.Although the effect of DSePA in other types of intestinal inflammation is rarely reported,we aim to provide new insights into improving ulcerative colitis by considering its effects on mucosal immunity and barrier function.Objective:To investigate the effect of DSePA intervention on dextran sodium sulfate(DSS)-induced colitis in mice,and inflammatory response and intestinal epithelial barriar dysfuction in lipopolysaccharide(LPS)-induced colorectal adenocarcinoma cells.Methods:1.Effect of DSePA on DSS-induced UC in mice and mechanism6-8 week-old male C57BL/6 mice randomly divided into control group(Con),DSePA group(2mg/ml DSePA intraperitoneal injection)and 3 UC model groups.To establishe UC model,the mice drank 2%DSS solution for 5 weeks and divided into DSS group,LSe group and HSe group.LSe and HSe groups were injected intraperitoneally with lmg/ml and 2mg/ml DSePA for 5-weeks,6 times a week,respectively.During the whole experiment,the body weight,food intake,and water intake were recorded regularly.After sacrifice,the colon length,H&E staining,Ki67 staining and AB/PAS staining of colon tissue were measured for histopathological examination.The inflammatory cytokines and Th17 differentiation in colon tissue were analyzed by Western blot.RT-qPCR and ELISA.2.Effect of DSePA on the intestinal epithelial barrier and inflammatory response in colon epithelial cells and mechanismHuman colon adenocarcinoma Caco-2 cells were starved for 12 hours,which was replaced by Roswell Park Memorial Institute-1640(RPMI-1640)medium containing 10%fetal bovine serum(FBS).Different concentrations of DSePA were added to each group,and the cell survival rate was determined by CCK-8.Then,10 μM DSePA was selected as a intervention dose to correspond with 80-90%survival rate.Both of the model group and DSePA intervention group were treated with 100mg/ml LPS solution and interfered with 10 μM DSePA to activate Caco-2 cells for 24h.Then,measure the redox homeostasis,intestinal epithelial barrier and Inflammation-related pathway NF-κB.In addition,Caco-2 cells,co-culturing with human colon adenocarcinoma HT-29 cells,replaced by RPMI-1640 medium containing 10%FBS and starved for 12 hours.HT29-MTX-E12 cells was obtained by sodium butyrate induced HT-29 cells differentiation and limited dilution cloning culture.According to LPS treatment time,cells were divided into Oh,2h,4h,8h,16h and 24h groups,and measured MUC-2.Both model group and drug intervention group were treated with 2mM O-glycosylation inhibitor(Benzyl-α-GalNAc).After 10μM DSePA intervention,co-cultured cells were performed mucus staining and mucus secretion indicators.Results:1.Effect of DSePA on DSS-induced UC in mice and mechanism(1)Establishment of the mice UC model:Compared with the Con group,the mice in the DSS group significantly decreased body weight,and increased the water intake and fecal scores.At autopsy,it was found that colon length was significantly lower in the DSS group than in the Con group.Histological analysis of H&E staining showed that there were a large number of inflammatory cell infiltration,epithelial fragmentation,and extensive loss of crypts and goblet cells in the DSS group.Ki67 staining revealed abnormal epithelial proliferation.AB/PAS staining revealed goblet cells loss and mucus reduction.Thus,the mice UC model was established.(2)The effect of DSePA intervention on general vital signs in mice:Compared with the DSS group,the water intake decreased significantly in the HSe group.Although no diffenrces of body weight were obeserved,the average colon length was increased in the LSe and HSe groups.H&E staining showed that DSePA intervention reformed colon structure.Ki67 staining revealed that DSePA intervention reduced epithelial proliferation.AB/PAS staining revealed that DSePA intervention increased mucus content and maintained the integrity of goblet cells.There was no statistically significant difference between the LSe and HSe groups.(3)The effect of DSePA intervention on the tissue structure and function of mice:Compared with the control group,H&E staining revealed that DSePA intervention improved the colon structure damage in mice.The Ki67 staining also showed that DSePA intervention reduced the abnormal proliferation rate of the epithelium.Additionally,AB/PAS staining demonstrated that DSePA intervention increased mucus content and maintained the integrity of intestinal epithelial goblet cells.There were no significant differences between the two groups receiving DSePA intervention.(4)The effect of DSePA intervention on the antioxidant capacity of mice:There were not significant differences in serum reduced glutathione(GSH),oxidized glutathione(GSSH),and total glutathione(T-GSH)among groups.Compared with the Con group,the GPX3 and GPX4 proteins expression was significantly up-regulated in the DSS group.Compared with the DSS group,the GPX3 protein expression was significantly up-regulated in the LSe and HSe groups.(5)The effect of DSePA intervention on colitis related cytokines and inflammatory related proteins in mice:Compared with Con group,the inflammatory cytokines interleukin 6(IL-6),interleukin 1β(IL-1β),tumor necrosis factor-a(TNF-α),interleukin 18(IL-18),macrophage marker(F4/80),and inflammatory corpuscle(NLRP3)protein expression were significantly up-regulated in the DSS group.Compared with the DSS group,the expression of F4/80 was significantly decreased in the LSe group,while the expression of all the above proteins were significantly decreased in the HSe group.(6)The effect of DSePA intervention on the colonic epithelial barrier function in mice:Intestinal epithelial barrier proteins ZO-1、Occludin and MUC-2 were significantly lower in the DSS group than in the Con group.After DSePA intervention,all of the proteins expression above were recovered.The expression of MUC-2 protein was significantly up-regulated in the HSe group.(7)The effect of DSePA intervention on the PI3K/Akt signaling pathway in the colon of mice:Compared with the Con group,the cell survival related p-PI3K/PI3K and p-Akt/Akt were significantly increased.Compared with the DSS group,p-PI3K/PI3K was decreased significantly in LSe group,and p-Akt/Akt was decreased significantly in the HSe group.(8)The effect of DSePA intervention on Th17 proliferation in the colon of mice:Compared with the Con group,phosphorylated signal transduction and transcription activating factor 3(p-STAT3)protein expression was significantly up-regulated in the DSS group.The helper T cell 17(Th17)inhibitory protein RORa was significantly decreased.The protein and mRNA expression of interleukin 17A(IL-17A)and interleukin 17F(IL-17F)in intestine tissue were increased significantly.Compared with the DSS group,p-STAT3 protein expression and the p-STAT3/STAT3 was significantly decreased,RORa protein expression was significantly increased,and IL-17F protein and mRNA expression was significantly decrease in the HSe group.In addition,IL-17A mRNA expression was significantly reduced in the LSe and HSe groups.(9)There were no significant differences in these indicators between the DSePA group(DSePA alone)and the Con group.Therefore,DSePA had no impact on the body health under normal physiological conditions.2.Effect of DSePA on the intestinal epithelial barrier and inflammatory response in colon epithelial cells and mechanism(1)The effect of DSePA intervention on the antioxidant capacity of intestinal cells:Compared with the Con group,the activity of glutathione peroxidase(GPx)decreased,reactive oxygen species(ROS)and malondialdehyde(MDA)increased,and IL-6、IL-1β and TNF-α mRNA expression increased in the LPS group.Compared with LPS group,DSePA enhanced GPx activity,inhibited ROS and mRNA expression of MDA,and IL-6,IL-1β and TNF-α.(2)The effect of DSePA intervention on intestinal cell permeability:Compared with Con group,the transmembrane resistance(TEER)of LPS group decreased,and the fluorescein Isothiocyanate Glucan flux(FITC dextran)increased.Compared with the LPS group,the TEER value of the DSePA group significantly increased and the FITC extrapolation flux decreased.(3)The effect of DSePA intervention on intestinal epithelial barrier related proteins:Compared to the control group,in the LPS group,the expression levels of protein and mRNA of Tight junction protein-1(ZO-1),Occludin,and Claudin-1,which are indicators related to epithelial barrier,were significantly reduced.Immunofluorescence staining also showed decreased fluorescence intensity of ZO-1,Occludin,and Claudin-1.However,in the DSePA group,there was a significant upregulation of the expression levels of ZO-1,Occludin,and Claudin-1 proteins and mRNA,and an increase in their fluorescence intensity when compared to the LPS group.(4)The effect of DSePA intervention on the NF-κB signaling pathway of intestinal cells:Compared with the Con group,high mobility group protein B1(HMGB1),toll-like receptor 4(TLR4),myeloid differentiation factor 88(Myd88),and nuclear factor κB(NF-κB)protein expression in the LPS group significantly increased.Compared with the LPS group,DSePA group significantly decreased these proteins expression.(5)The effect of DSePA intervention on the mucous layer of intestinal cells:The expression of MUC-2 protein and mucus secretion revealed by AB staining increased with the increase of LPS treatment time.Compared with the Con group,MUC-2 and VAMP-8 proteins expression in the Benzyl group were significantly decreased,and the fluorescence intensity of calcium ions was decreased.Compared with the Benzyl group,DSePA significantly increased MUC-2 and VAMP-8 proteins expression,while the fluorescence intensity of calcium ions was increased.Conclusion:1.DSePA improved DSS-induced mice UC through inhibiting the secretion of inflammatory cytokines(IL17,IL-6,IL-1β and TNF-α)and Th17 differentiation,increasing the expression and amount od mucoprotion,and maintaining the integrity of intestinal epithelial goblet cells.2.DSePA inhibited LPS-activated HMGB1/TLR4/Myd88/NF-κB pathway,improved oxidative stress and dysfunction of cellular epithelial barrier,and decreased paracellular permeability.DSePA is beneficial for mucus layer reconstruction and intestinal permeability in advanced colitis with severe loss of goblet cells. |
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