A Study Of The Role And Mechanism Of MMP-7 In Ulcerative Colitis

BackgroundInflammatory bowel disease(IBD),which includes ulcerative colitis(UC)and crohn's disease(CD),is a group of chronic nonspecific diseases of the digestive tract with unknown causes.Cytokines,proteolytic enzymes and other inflammatory mediators are involved in the tissue damage and mucosal healing of the disease,and intestinal mucosal healing is closely related to the complications,hospitalization rate and prognosis degree of IBD,especially UC.Matrix metalloproteinase 7(MMP-7),as one of the proteolytic enzymes,is involved in the processes of extracellular matrix remodeling,abnormal immune regulation,mucosal healing,and tumor invasion,but the role of MMP-7 in UC colon abnormal immune response is still unclear.It was found that ligand-dependent activation of peroxisome proliferator-activated receptor y(PPARy)down-regulated the formation of MMP-7 in the colon tissues of mice in tumors and dextran sulfate sodium(DSS).However,whether the main inflammatory substances in the pathogenesis of UC,such as interleukin-1?(IL-1?)and tumor necrosis factor-?(TNF-?),affect the expression of MMP-7 and whether the over-expression of MMP-7 affects the levels of cytokines related to mucosal healing and the mucosal healing process of UC.ObjectiveBy verifying the expression level and localization of MMP-7 in the colon tissues of UC patients,this experiment preliminarily understood the relationship between MMP-7 and the degree of UC inflammation.By constructing a monolayer cell model by Caco-2 cells,the expression of MMP-7,ZO-1,Occludin,MLCK and p-MLC were detected after intervention with inflammatory factors,and the relationship between MMP-7 level and inflammatory cytokines and mucosal barrier molecules was preliminarily explored.Overexpression of MMP-7 was used to detect whether this cytokine affected the expression of molecules related to mucosal healing,providing a basis for the diagnosis and prognosis of UC.Methods1.Collection of patient tissue specimens:a total of 33 UC patients,from May 2018 to October 2019,who were hospitalized or treated in outpatient clinics were collected,including 7 mild cases,14 moderate cases,and 12 severe cases.Another 20 cases of colonic carcinoma were collected and no infiltration of carcinoma cell were observed at the incision margin.The specimens were all more than 5 cm away from the paracancer and were confirmed by professional pathologists.These patients have not received glucocorticoids,immunosuppressants and biologic agents in the past 1 month.2.A logarithmic Caco-2 were selected and inoculated with 4×105/ml cells in 12-well transwell chamber to construct an intestinal epithelial barrier model,we measured the level of TEER relative value after intervention with a mixture of IL-1?and TNF-? proinflammatory factors for 10 ng/ml for 48 h respectively and the addition of Rosiglitazone for 10 ?mol/L in the above drug intervention group.3.No less than 1×107 cells were collected and treated according to the above treatment methods for 48 h,the expression levels of mRNA or protein such as MMP-7,ZO-1,Occludin,MLCK and p-MLC were detected by fluorescence quantitative PCR or Western Blot.4.Single cell layer was laid by 4×104/well cells.According to the above intervention conditions,the immunofluorescence localization expression of tight junction protein ZO-1 and Occludin cells was observed under laser confocal microscopy.5.MMP-7 overexpressed stable cell lines were constructed,and the expression of mucosal healing related molecule SDC-1 in MMP-7 overexpressed Caco-2 cells was detected by Western Blot.6.SPSS 24.0 software was used to analyze and process the data.The mean±standard deviation was used to describe the enumeration analysis data,and the composition ratio between groups was tested by ? 2 test.After homogeneity of variance or normality test,independent sample t test or rank sum test were used for comparison between two or more groups.The test standard was ?=0.05,P<0.05 showed statistically significant differences.Results1.Hematoxylin-eosin(HE)staining of colon tissues in patients with UCThe colonic epithelium of UC patients is damaged to different degrees,accompanied by the loss of intestinal mucosa and submucosa,and even the loss of lamina propria glandular structure.Different degrees of infiltration of inflammatory cells,fusion of recess structure,deformation,disappearance,etc.can be seen in the colon tissues of UC.Under the microscope,a large number of inflammatory cells can be seen in the mucosa and submucosa,which is closely related to the degree of UC.2.The expression of MMP-7 in the colon tissues of UC patientsA small number of MMP-7 positive cells were observed in the negative control group and the mild UC group,and the staining intensity was weak.In the moderate to severe UC group,a large number of stained brownish yellow or dark yellow positive areas were found in the colon tissues,which were mainly concentrated in the damaged intestinal tissues in the epithelial cells at the edge of ulcer,and the positive expression and localization of MMP-7 were also found in some inflammatory cells.The score of the positive expression of MMP-7 in the colonic tissues of moderate to severe UC was higher than that of the control group and the mild UC group,the difference was statistically significant(P<0.05),but there was no statistical difference between the negative control group and the mild UC group(P>0.05).3.Single-layer epithelial barrier permeability of Caco-2 cellsDifferent groups at different times(12,24 and 48 h)between the TEER analysis found that the relative ratio of inflammatory factor were 0.613±0.015?0.507±0.021 and 0.457±0.015,the relative ratio of rosiglitazone were 0.800±0.026?0.660±0.026 and 0.600±0.020.The TEER in the negative control group did not change significantly over time,but the TEER changes in the inflammatory factor group decreased significantly,while the ratio changes in the rosiglitazone group were in between.4.Expression of MMP-7?ZO-1?Occludin mRNA was detected by fluorescence quantitative PCRThe relative expression of MMP-7 mRNA in the inflammatory cytokines group was higher than that in the negative control group and the rosiglitazone group,and the difference was statistically significant(P<0.05).However,the mRNA levels of tight junction protein ZO-1?Occludin mRNA in the inflammatory factors group were lower than those in the normal control group and the rosiglitazone intervention group,with statistically significant differences(P<0.05).5.Immunofluorescence localization of tight junction protein ZO-1 and OccludinUnder the confocal laser microscope,ZO-1 and Occludin excitation light were red.In the negative control group,the fluorescence distribution of ZO-1 and Occludin protein was clearly visible along the cell membrane,with strong fluorescence and smooth edges,delineating the outline of endothelial cell paving stones and close connections between cells.In the inflammatory cytokines group,the fluorescence intensity of the compact junction protein was weak,and there were cracks in the connections between the cells.In the rosiglitazone group,the fluorescence intensity,integrity,contour and fluorescence signal of cell connections were all clearer than those in the inflammatory factor group,but still lower than the normal control group.6.The expression of MLCK and p-MLC was detected by Western BlotThe expression levels of MLCK and p-MLC in the inflammatory cytokines group were higher than those in the negative control group and the rosiglitazone group,with statistically significant differences(P<0.05).7.Expression of MMP-7 and intestinal healing of related proteinWestern Blot results showed that the expression of SDC-1 in the negative control group was higher than that in the MMP-7 overexpression Caco-2 group,and the difference was statistically significant(P<0.05).Conclusions1.The expression of MMP-7 in colon tissues of patients with UC was enhanced.2.Inflammatory cytokines IL-1? and TNF-? promote the expression of MMP-7,disrupt the expression of intestinal tight junction protein,and activate the MLCK/p-MLC signaling molecular pathway.3.The mucosal destruction of MMP-7 is related to the decrease of SDC-1.