| BackgroundInflammatory bowel disease(IBD)is a is a chronic intestinal Inflammatory diseases,including Crohn’s disease(CD)and ulcerative colitis(UC).In the process of inflammatory bowel disease(IBD),intestinal mucosal barrier function is impaired,antigen translocation stimulates immune cells,and produces a large number of inflammatory cytokines and mediators.Inflammatory cytokines affect the expression of tight junction protein,destroy the junction complex and increase the permeability of intestinal epithelium.The Tumor necrosis factor-alpha(TNF-α)plays a very important role in this process.The study found that the TNF-α in the patients with inflammatory bowel disease is abnormally increased and is associated with the increase of intestinal barrier permeability,and the permeability of the intestinal barrier in these patients is significantly reduced after the use of TNF-alpha inhibitors.At present,the specific mechanism of TNF-α induced increased intestinal barrier permeability is still unclear.A tight junction,also known as a closed band,is a tightly connected structure between cells,through which liquid molecules cannot pass.It is mainly composed of three kinds of intact membrane proteins,including occlusal protein occludin,claudins,and adhesion molecules(JAMs),and peripheral cytoplasmic proteins(ZO-1,ZO-2,ZO-3).Among them,claudin-1 is a very important one,which plays an indispensable role in the maintenance of barrier function and the integrity of tight junctions.CUG-binding protein 1(CUGBP1)is a member of the CELF family of RNA binding proteins.Its function is to regulate the expression of target genes by binding to target mRNA to change the stability of mRNA and regulate its translation.CUGBP1 can specifically recognize the binding of CUG rich response elements in target mRNA and regulate the expression of target genes.The CUG response element is usually located in the mRNA 3’-UTR,which binds to CUGBP1 mainly by promoting the degradation of target mRNA,inhibiting its translation and regulating its expression.Some studies have also found that CUGBP1 can bind to the coding region of target gene to regulate the expression of target gene.CUGBP1 exists mainly in the nucleus of the cell and can be regulated by translocation into the cytoplasm and binding to the target gene.Multiple factors can regulate the distribution of CUGBP1 in cells.ObjectivesThe aim of this study was to explore the molecular mechanism by which TNF-α down-regulates the expression of tight junction protein(Claudin-1)through CUGBP1/Claudin-1,thereby disrupting the structure and function of intestinal tight junction.Methods and ResultsIn this experiment,we first established the acute ulcerative colitis model of mice with dextran sodium sulfate(Dextran Sulfate Sodium Salt,DSS).The results showed that mice in DSS treatment group had weight loss,dilute stool and blood stool.The histological analysis of colonic part of mice with decreased activity and HE staining showed that the expression of Claudin-1 in DSS treated group was significantly lower than that in normal control group.Detection of FD-40(FITC–dextran 40 kDa)in mice blood by full wavelength fluorescence labelling system.The content of the mice in the DSS treatment group was higher than the control group,indicating that the barrier permeability of the DSS mice increased.In the cultured human colon cancer cell(Caco2),the expression of Claudin-1 mRNA and Claudin-1 and CUGBP1 protein levels were detected by RT-PCR,WB and Immunofluorescence.The study found that TNF-α increased the permeability ofcells and impaired its barrier function.TNF-α reduced the expression of tight junction protein(Claudin-1)by promoting the degradation of Claudin-1 mRNA.CUGBP1 can bind to Claudin-1 mRNA,and Claudin-1 decreases by overexpression of CUGBP1.Moreover,TNF-α can stimulate the extranuclear cells of CUGBP1 and increase its cytoplasmic level,thus promoting the binding of CUGBP1 and Claudin-1 mRNA.After transfection of CUGBP1 siRNA to inhibit CUGBP1,TNF-α can partially reverse the inhibitory effect of TNF-α on the expression of Claudin-1 protein.ConclusionsDSS treatment resulted in significant ulcerative colitis,weight loss,dilute stool,bloody stool,intestinal mucosal damage,increased permeability and decreased Claudin-1 expression of tight junction protein.TNF-α can inhibit the barrier function of Caco2 cells and inhibit the expression of Claudin-1 by promoting the degradation of Claudin-1 mRNA.TNF-α can regulate the localization of RNA binding protein CUGBP1 in cells and promote its binding to Claudin-1 mRNA.TNF-α inhibits the expression of tight junction protein Claudin-1 by promoting cytoplasmic translocation of CUGBP1,promotes cell permeability and impairs its barrier function.This may be one of the reasons for the development of IBD. |
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