| BackgroundIrritable bowel syndrome(IBS),a kind of functional disease,is characteristic of the symptoms of stomachache related to defecation,the change of fecal character and habit following vegetative nerve functional disturbance like anxiety and depression.Diarrhea-predominant irritable bowel syndrome(IBS-D)is the most common subtype in clinical.Currently,there exists low-grade inflammation and immunologic derangement in the gut IBS-D patients,directly related to the intestinal permeability change.Recently,the mechanism of change in intestinal epithelial barrier function related to MCs in IBS-D is hot issue worldwidely.Mast cells are widely exists in the intestinal mucosa lamina propria,participated in the permeability change.MCs is activated by the outside antigen and nerves,degranulating and releasing a variety of active substances.Proteases such as tryptase can cause the intracellular signal transduction by activating Protease-activated receptor 2(PAR-2)receptor on basolateral membrane of intestinal epithelial cell,resulting to the changed expression and redistribution of tight junction proteins(TJs)in apical membrane.Based on the previous research,the study aims to observe the interrelation of colonic mucosa mast cells and intestinal epithelial barrier function in patients with IBS-D.Then with co-culturing,we tyr to make it clear that the interaction between MCs and intestinal epithelial cell Caco-2.Chang’an Ⅱ decoction and Tongxieyaofang are used in the study to observe its effect in co-culturing model.Aims:Using an entry point on with MCs activation,we try to explore the interrelation of colonic mucosa mast cells and intestinal epithelial barrier function in patients with IBS-D.Then,based on the result,we try to set up the modle of intestinal epithelial barrier damages resulted from the co-culture with MCs and tryptase,and observe the effect of Chang’an Ⅱ decoction and Tongxieyaofang.Method:一、Clinical sections:The study include 6 IBS-D patients and 6 healthy people for control group.Their colonic mucosa biopsy are used to detect the level of MCT and Claudin-1 proteins with immunofluorescence.二、Experimental section1.C48/80 activates HMC-1 to degranulate,and intervened by Chang’an Ⅱdecoction and Tongxieyaofang.Detecting tryptase concentration in the supernatant with ELISA.2.(1)Caco-2 is chultured in Transwell plate for differentiation 21 days to establish a complete intestinal epithelial barrier.(2)Co-culture with activated HMC-1 and Caco-2 for 24h.short circuit current measure transepithelial electrical resistance(TER)of the intestinal epithelial cells,confocal immune fluorescence to observe occludin expression of intestinal epithelial cells.(3)Chang’an II decoction and and Tongxieyaofang intervene barrier damage in co-cultivation by measuring TER and observe occludin expression with confocal immune fluorescence.3.(1)The optimal concentration is used to induce intestinal epithelial barrier damages.TER,FITC-dextran permeability and transmission electron microscopy are made to evaluate the model.(2)After Chang’ an ⅡI decoction and Tongxieyaofang intervening,TER and FITC-dextran permeability were evalued.Detecting the express of Occludin,Claudin-1 and PAR-2 proteins with Western Blot,and detecting the express of Occludin,JAM-A and PAR-2 mRNA with Real-Time PCR.Results:一、Clinical sections:There are increased number of MCs and increased expression of MCT closed to intestinal epithelial in colonic mucosal in IBS-D,While Claudin-1 proteins expressed less.The level of MCT and the number of MCs are lower in healthy control with high level Claudin-1 proteins.二、Experimental section1.ELISA:The concentration of tryptase in cells supernatant increased over time(P<0.05).The differences of the different concentrations of C48/80 are not obvious(P>0.05);Chang’an Ⅱ decoction and Tongxieyaofang can inhibit HMC-1 degranulation to release tryptase to different degree(P<0.05)2.(1)After Caco-2 differentiation for 21 days in Transwell plate,TER maintaining stable,and FITC-dextran Papp reaching the demand,differentiatied intestinal epithelial cells could be used in following study.(2)Co-culture with activated HMC-1 and Caco-2 can result in the decreasing value of TER(P<0.05)and the fluorescence intensity of occludin.(3)Chang’ an Ⅱ decoction and Tongxieyaofang medicated serum can rise the TER(P<0.05),but there no difference in low dose group(P>0.05);Fluorescence intensity of occludin in Chang’an Ⅱ-H,Chang’an Ⅱ-M and Tongxieyaofang group have different degrees of enhancement.The tight junction structure is clear.3.(1)The determination of Tryptase concentration:Along with the increase of the concentration,TEER become lower.But the sudden rise in 800 ng/ml at 12h,and after 24h close to the normal.FITC-dextran permeability increases with the increase of Tryptase concentration,except 800 ng/ml with lower permeability.As shown by transmission electron microscope,the microvilli on the surface of intestinal epithelial cells fracture and are deformity with different concentrations stimulating,incomplete tight junctions,widened gap junctions and decreased desmosomes.(2)The effect of Chang’an Ⅱ decoction and Tongxieyaofang:The TEER in TCM medicated serums’intervention group were higher than model group;Increased dose of Chang’ an Ⅱdecoction decrease the FITC-dextran permeability(P<0.05),the Papp in Tongxieyaofang group is also significantly lower than model group(P<0.05).th Western Blot the expression of Occludin,Claudin-1,PAR-2 protein in the model group reduced significantly(P<0.05),Chang’an Ⅱ decoction and Tongxieyaofang can increase the expression(P<0.05);With Real-Time PCR the expression of Occludin,JAM-A and PAR-2 mRNA between each group had no statistical significance(P>0.05)Conclusion:1.The increased number of MCs closed to colonic mucosal epithelial may be related to the low level of Claudin-1 proteins in IBS-D.2.Chang,an II decoction and Tongxieyaofang serum can restrain the process of HMC-1 degranulation and reduce the release of tryptase.3.Intestinal epithelial barrier damage model can be successfully established by co-culturing with activated HMC-1 by C48/80 and Caco-2 cell.4.Chang’ an II decoction and Tongxieyaofang serum could improve intestinal epithelial barrier damage induced by co-culturing and tryptase. |
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