| Background and objectives: Severe acute pancreatitis(SAP)is an inflammatory disease associated with systemic inflammatory response syndrome(SIRS)and multiple organ failure(MOF),with a mortality rate of up to 40%.Intestinal barrier dysfunction plays an important role in its occurrence and development,but the mechanism is still unclear.Bacterial and endotoxin(lipopolysaccharide,LPS)translocation secondary to Intestinal barrier dysfunction plays an important role in the development of SIRS and MOF.In addition,ascitic fluid(AF)contains a large amount of trypsin,amylase,lipase and inflammatory cytokines,bacteria and their metabolites endotoxin,etc.These components aggravate the damage of the pancreas and surrounding tissues,and also destroy the intestines,even promote the occurrence of SIRS and MOF.In SAP,Toll like receptors(TLRs)signal pathways are overactivated in many systems and organs,which are closely related to intestinal barrier dysfunction.The TLRs family includes multiple signaling pathways,among which TLR4/NF-κB is the most thoroughly studied.TLR4 mainly recognizes LPS and initiates signal transduction to mediate inflammation.Early research by our group found that SAP ascites can activate the TLRs signaling pathway in macrophages and mediate the release of various inflammatory mediators.Then,do LPS and AF participate in the occurrence of intestinal barrier dysfunction during SAP by activating the TLRs signaling pathway? Do not know yet.To solve this problem,this article studied the effects of AF and LPS on the barrier function of intestinal epithelial cells and their possible mechanisms.Methods: Ⅰ.Influence of LPS and SAP ascites on intestinal barrier function 1.Construction of Caco-2 and IEC-6 monolayer intestinal epithelial cell barrier models;2.The monolayer intestinal epithelial cell barrier model treated with LPS or biliary SAP ascitic fluid(B-AF)or hypertriglyceridemia SAP ascitic fluid(HTG-AF)or MIX ascitic fluid(MIX-AF)for 48 hours;(1)LPS treatment:(1)control group,(2)LPS group(LPS concentration 50μg/ml);(2)SAP ascites treatment:(1)control group,(2)B-AF group(concentration 10%),(3)HTG-AF group(concentration 10%),(4)MIX-AF group(biliary type + hypertriglyceridemia type)(concentration 10%)3.Assessment:(1)Transmembrane resistance(TEER): measure the transmembrane resistance of the intestinal epithelial cell barrier model with a cell resistance meter at 1,3,6,12,24,and 48 hours after LPS or SAP ascites treatment;(2)Lucifer yellow transmittance: LPS or SAP ascites treatment at 48 hours,detection method: add 20μg/ml fluorescent yellow solution to the upper layer of the intestinal epithelial cell barrier model,and calculate the amount of fluorescent yellow from the upper layer to the lower layer per unit time;(3)Expression of tight junction proteins ZO-1,Occludin and Claudin-1 among intestinal epithelial cells;Ⅱ.The influence of LPS and SAP ascites on TLRs signaling pathway in intestinal epithelial cells Different concentrations of LPS or SAP ascites were treated with Caco-2 cells and IEC-6 cells,and the changes in the expression levels of molecules related to the TLRs signaling pathway were observed.1.Experiment grouping:(1)LPS treatment:(1)control group,(2)LPS group: 0.1μg/ml,1μg/ml,10μg/ml,50μg/ml,100μg/ml;(2)SAP ascites treatment:(1)control group,(2)B-AF group: 1.25%,2.5%,5%,10%,20%,(3)HTG-AF group : 1.25%,2.5%,5%,10%,20%;2.Assessment:(1)Fluorescence quantitative PCR was used to detect the m RNA expression of My D88,IRAK-1 and TRAF6 related molecules in the TLRs signaling pathway in each group;(2)Western Blot detects the expression of TLR4,My D88,IRAK-1,TRAF6, NF-κB p65,IKB-α related to the TLRs signaling pathway in each group;Ⅲ.The effect of LPS and SAP ascites on the expression of SIGIRR in intestinal epithelial cells 1.Experimental groups:(1)control group,(2)LPS group,(3)B-AF group,(4)HTG-AF group;2.Assessment:(1)Fluorescence quantitative PCR was used to detect the levels of IL-37,IL-18Rα and SIGIRR m RNA in the cells of each group;(2)Western Blot detects the level of SIGIRR protein in the cells of each group;Ⅳ.The regulation of SIGIRR on TLRs signaling pathway in intestinal epithelial cells 1.Construction and verification of SIGIRR knockdown Caco-2 cell line Construct three lentiviral vectors containing SIGIRR-RNAi(si-1),SIGIRR-RNAi(si-2)and SIGIRR-RNAi(si-3)and carry out lentiviral packaging(the above experiment was commissioned by Shanghai Jikai Company to construct).Transfect Caco-2 with different MOIs(30,50,70,90),determine the best MOI for transfection efficiency,screen the stable transfected cell line with SIGIRR knockdown by puromycin drug,and detects the expression of SIGIRR m RNA and protein to verify the knockdown effect by q-PCR and Western Blot.2.The influence of SIGIRR on TLRs signaling pathway in Caco-2 cells(1)Experimental grouping:(1)Blank group: negative control cells SIGIRR low Caco-2(2)LPS group: negative control cells +LPS SIGIRR low Caco-2 + LPS(3)B-AF group: negative control cells + B-AF SIGIRR low Caco-2 + B-AF(4)HTG-AF group: negative control cells + HTG-AF SIGIRR low Caco-2 + HTG-AF(2)Detection indicators: fluorescence quantitative PCR was used to detect the expression of TRAF6 and IRAK-1 m RNA related to the TLRs signaling pathway in each group.Result: Ⅰ.The influence of LPS and SAP ascites on intestinal barrier function 1.The influence of LPS and SAP ascites on the resistance of the intestinal epithelial cell barrier model(1)After LPS treatment,the resistance value of Caco-2 and IEC-6 cell barrier models was continuously measured: the resistance value of Caco-2 cell barrier model decreased significantly at the first hour of LPS treatment(P<0.05),within 48 hours The resistance value continued to decrease with time and there was a statistical difference(P<0.05);the IEC-6 cell barrier model decreased at the 3rd hour of LPS treatment,but there was no statistical difference(P>0.05)at the 6th hour The resistance value was significantly reduced with statistical difference(P<0.01),and continued to decrease with the prolonging of the treatment time(P<0.05).(2)After SAP ascites treatment,the resistance value of Caco-2 cell barrier model was continuously measured:(1)B-AF treatment: the resistance value decreased significantly in the first hour of treatment(P<0.05),and showed a continuous downward trend within 48 hours(P<0.05);(2)Treatment of HTG-AF: the resistance value decreased from the 3rd hour with statistical difference(P<0.01),and it showed a continuous downward trend within 48 hours(P<0.01);(3)Mixed ascites treatment: the resistance value decreased statistically from the 3rd hour(P<0.05),and it showed a continuous downward trend within 48 hours(P<0.01);(4)Comparison of different ascites: There was no significant difference in resistance value reduction between 1 hour and 48 hours after treatment(P>0.05).The TEER of the 3 hour,6 hour,12 hour,and 24 hour HTG-AF group was lower than that of the B-AF group Obviously(P<0.05),and MIX-AF is between B-AF and HTG-AF.2.The influence of LPS and SAP ascites on the transmittance of fluorescent yellow in the intestinal epithelial cell barrier model (1)LPS treatment: The LPS treatment group significantly increased the permeability of Caco-2 and IEC-6 cell barrier models of fluorescent yellow significantly higher than that of the control group(P<0.05,P<0.01);(2)SAP ascites treatment: Whether it is B-AF,HTG-AF or a mixture of the two,the permeability of Caco-2 cell barrier model fluorescent yellow is significantly higher than that of the control group(P<0.05);at the same time;,The permeability of Caco-2 cell barrier model fluorescent yellow to the HTG-AF treatment was significantly higher than that of the B-AF treatment group(P<0.05).3.The effect of LPS and SAP ascites on tight junction protein between intestinal epithelial cells(1)Western Blot results showed that the expression of tight junction proteins ZO-1,Occludin,and Claudin-1 in the LPS treatment group was significantly lower than that in the control control group(P<0.001);after treatment with B-AF or HTG-AF,It was also significantly lower than the control group(P<0.001);and the expression of ZO-1 in the HTG-AF treatment group was significantly lower than that of the B-AF treatment group(P<0.05),while Occludin and Claudin-1 had no significant difference(p> 0.05);(2)Cellular immunofluorescence results showed that the expression of ZO-1 in the LPS treatment group was significantly lower than that in the control group(P<0.001);the B-AF and HTG-AF treatment group were significantly lower than the B-AF treatment group(P < 0.05),and the HTG-AF treatment group was significantly lower than the B-AF treatment group(P<0.05);Ⅱ.Effects of LPS and SAP ascites on TLRs signaling pathway in intestinal epithelial cells 1.The effect of LPS on TLRs signaling pathway in intestinal epithelial cells:(1)Effects on Caco-2 cells:(1)At the m RNA level,the expression of TRAF6 and IRAK-1 in the LPS treatment group was significantly higher than that in the control group(P<0.05),and there was no significant difference in the expression of My D88(P>0.05).(2)At the protein level,the expression of TLR4 and TRAF6 in the LPS treatment group was significantly higher than that in the control group(p<0.05),the expression of My D88 was not significantly different(P>0.05),and the expression of IKB-α in the LPS treatment group was significantly lower than that in the control group(p <0.05).(2)Effects on IEC-6 cells:(1)At the m RNA level,the expressions of My D88,TRAF6 and IRAK-1 in the LPS treatment group were significantly higher than those in the control group(p<0.05);(2)At the protein level,TLR4,My D88 The expression of in the LPS treatment group was significantly higher than that in the control group(p<0.05).2.The effect of SAP ascites on TLRs signaling pathway in intestinal epithelial cells:(1)Effects on Caco-2 cells:(1)At the m RNA level,the expression of My D88,TRAF6 and IRAK-1 in the B-AF and HTG-AF treatment groups were significantly higher than those in the control group(P<0.05);(2)At the protein level,the expressions of TLR4,My D88,TRAF6 and p-NF-κB p65 were significantly higher in the B-AF and HTG-AF treatment groups than the control group(p<0.05);(2)Effects on IEC-6 cells:(1)At the m RNA level,the expression of My D88 and TRAF6 in the B-AF treatment group was significantly higher than that in the control group(P<0.05),and there was no significant difference in the expression of IRAK-1(P> 0.05);The expression of My D88,TRAF6 and IRAK-1 in the HTG-AF treatment group was significantly higher than that of the control group(P<0.05);(2)At the protein level,the expression of TLR4 and My D88 in the B-AF and HTG-AF The treatment group was significantly higher than the control group(p<0.05).Ⅲ.The influence of LPS and SAP ascites on the level of SIGIRR in intestinal epithelial cells(1)IL-37 and IL-18Rα are the upstream molecules of SIGIRR and are closely related to SIGIRR activation.Compared with the control group,LPS or B-AF treatment can significantly reduce the level of IL-37 m RNA,while HTG-AF can significantly reduce the level of IL-18Rα m RNA;(2)Compared with the control group,LPS or B-AF treatment had no significant effect on SIGIRR m RNA level(P>0.05),and HTG-AF treatment could significantly reduce SIGIRR m RNA level(p<0.01);(3)Compared with the control group,treatment of LPS,B-AF or HTG-AF can significantly reduce the level of glycosylated SIGIRR protein(p<0.01,p<0.001,p<0.05),while there is no significant effect on non-glycosylated SIGIRR protein(P>0.05),B-AF can reduce the level of non-glycosylated SIGIRR protein(p<0.05),and HTG-AF can increase the level of non-glycosylated SIGIRR protein(p<0.001).Ⅳ.The regulation of SIGIRR on TLRs signaling pathway in intestinal epithelial cells 1.The effect of LPS and SAP ascites on the expression of IL-37 and IL-18Rα,the upstream molecules of SIGIRR in intestinal epithelial cells: IL-37 m RNA expression in the LPS and B-AF treatment group was significantly lower than that of the control group(p<0.001),the expression of IL-18Rα m RNA in the HTG-AF treatment group was significantly lower than that in the control group(p<0.05).2.The effect of LPS and SAP ascites on the expression of SIGIRR m RNA in intestinal epithelial cells: The expression of SIGIRR in the HTG-AF treatment group was significantly lower than that in the control group(p<0.01),while there was no significant difference in the LPS or B-AF treatment group(P>0.05).3.The effect of LPS and SAP ascites on the expression of SIGIRR protein in intestinal epithelial cells: The expression of glycosylated SIGIRR in the LPS,B-AF or HTG-AF treatment group was significantly lower than that of the control group(p<0.05);The expression of glycosylated SIGIRR in the B-AF treatment group was significantly lower than that in the control group(p<0.05),and in the HTG-AF treatment group,it was significantly higher than that in the control group(p<0.001).2.The influence of SIGIRR on TLRs signaling pathway in Caco-2 cells IRAK-1 m RNA expression in SIGIRRlowCaco-2 was significantly higher than NC-Caco-2(P<0.05),TRAF6 m RNA expression was not significantly different between the two groups(P>0.05);after LPS or B-AF treatment,IRAK-1 m RNA expression in SIGIRRlowCaco-2 was significantly higher than that of NC-Caco-2(P<0.05),and there was no significant difference in the expression of IRAK-1 between the two after treatment of HTG-AF(P>0.05).After treatment of LPS,B-AF and HTG-AF,the expression of TRAF6 in SIGIRRlowCaco-2 was significantly higher than NC-Caco-2(p<0.05).Conclusion: 1.Both LPS and SAP ascites can cause intestinal barrier dysfunction,in which HTG-AF is more toxic than B-AF.2.Excessive activation of TLRs signaling pathways in intestinal epithelial cells may be one of the mechanisms by which LPS and SAP ascites destroy the barrier function of intestinal epithelial cells.3.The excessive activation of TLRs signals in intestinal epithelial cells may be related to the inhibition of the negative regulator SIGIRR. |
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