Study On Disruption Of Platelet Activating Factor And The Protective Effect Of Intestinal Trefoil Factor On Intestinal Epithelium Mechanical Barrier In Vitro

ObjectiveIn addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system.Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Complete intestinal barrier is comprised of 1.The mechanical barrier is comprised of continual epithelial of gut mucosal;2.Cellular and humoral immunity provided by lymphoid tissue of intestinal tract;3.Normal and massive commensal intestinal bacterial clusters,preventing the over proliferating and permanent planting in gut mucosal of pathogenic microorganism.The mechanical barrier is comprised of mucus,microvilli,epithelial cells and junctions among them and other particular constructions,which is the structural foundation of intestinal mucosal barrier.Not only the morphous change of epithelial cells but also the wideness of junction gap and the increasing of opened cell-cell junctions could affect the function of intestinal epithelial barrier and increase the permeability.The disruption of barrier integrity has been implicated in the pathogenesis of a wide variety of gastrointestinal and systemic disorders such as inflammatory bowel disease,liver failure,acute severe pancreases and multiple organ system failure.In addition to these,many acute and chronic gastrointestinal and systemic disorders in paediatrics such as sepsis,eczema,food allergies,celiac enteropathy,type 1 diabetes, asthma,and so on were also implicated in,Hense studying the mechanisms of high permeability and to explore the effectual treatment become the goal of this experiment.The increase of intestinal epithelial barrier permeability implicated in several factors,including initiatal causative agent as infection or ischemia and secondum inflammatory mediator.In recent years,the roles of platelet activating factor(PAF)to impairment of gastrointestinal mucosa has been paid increasing attention.PAF can be produced by intestinal epithelial cells and constitutively present in normal small intestinal tissue regulating the mucosal permeability.A large body of evidence suggests that PAF is involved in the pathogenesis of intestinal injury in various diseases.Moreover,PAF at doses insufficient to cause bowel necrosis triggers the expression of cytokines,and activates transcription factors in the intestine.So PAF is regarded as central amplify mediator and key factor of increasing of intestinal epithelial barrier permeability.The mechanisms of PAF-related epithelial breakdown are unclear,but regulation of paracellular pathways,especially via rearranging cytoskeleton F-actin and changing interepithelial tight junction(TJ)proteins such as Occludin,Claudin-1,ZO-1 is likelyto play a significant role.Therefore,we detect the effect of PAF on intestinal epithelial F-actin and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells to study the mechanisms involved in disruption of intestinal epithelium mechanical barrier induced by PAF.Intestinal trefoil factor(ITF)is a member of trefoil peptide family,which is important in maintenance and reparation of the intestinal mucosal barrier.It can not only stimulate cell migrating and proliferating,promote epithelial cell reparation,but also interact with the mucus,stabilize the mucus gel by perhaps interacting with intestinal mucin and increasing the viscosity.So it is important in the self-protection mechanism of intestine.Many researches in vivo also demonstrated ITF was important in the maintenance of intestinal epithelium barrier integrity and in the rescovery of mucosal normal permeability.Recent in vitro experiment find ITF can regulate the expression of TJ proteins of Claudin-1 and Claudin-2,then to affect TEER.But whether PAF can affect the expression of ITF? Administer of rITF can protect the intestinal mucosal barrier? What is the mechanisms? To answer these questions and to provide theory base for clinical application of rITF were the basic consideration of this study.Materials and methods1.Cell cultures Caco-2 cells were grown in a culture medium composed of RPMI 1640 with 4.5 mg/ml glucose,50 U/ml penicillin,50 U/ml streptomycin,and 15%FBS in an atmosphere of 5%CO₂ and 90%relative humidity maintained at 37℃.Parental cells were split at a ratio of 1:3 on reaching confluency,and set up on Transwell filters,6 well plates or T-50 flasks for propagation and experiments.2.PAF and ITF treatmentCell monolayers were exposed in RPMI 1640 without FBS for 24hs then PAF of different concentrations(0,50,100,200ng/L)was administered,incubating for different time(0,2,4,8,12,24,48h);ITF(0.3g/L)was administered 30mins before or after PAF(100ng/L)was given and incubated for 24hs in 6 well plates,T-50 flasks or transwell Cell Culture Inserts to undertake different experiments.3.Experimental methods and analysis index(1)Establishing intestinal epithelial barrier model in vitroCaco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by TEER and unidirectional flux of lucifer yellow measurements to ensure the form of intestinal epithelial barrier.(2)Cell proliferation and apoptosisMTT was used to detect cell vigor;Annexin V-EGFP and Hoechst were used to detect cell apoptosis through immunofluorescence and FCM.(3)Determination of epithelial monolayer resistance and paracellular permeabilityThe transepithelial electrical resistance(TEER)of the filter-grown Caco-2 intestinal monolayers was measured by using an epithelial voltohm-meter(EVOM); the unidirectional flux of paracellular marker lucifer yellow was measured to determination the Caco-2 monolayer paracellular permeability using fluorescence spectrophotometer.(4)Transmission electron microscopyCells were treated with PAF or ITF and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate.Ultrastructure of junctional complexes was observated by transmission electron microscopy.(5)F-actin rearrangment and assaysWhen treated by PAF and ITF with different dose and different time,TRITC-phalloidin staining was performed.Direct immunofluorescent staining observe cytoskeleton F-actin rearrangement,Flow cytometry was used to assays F-actin contents.(6)Assessment of ITF protein localization and expressionCells were treated with PAF and the localization of ITF protein were detected by immunohistochemistry and ITFmRNA expression was measured useing SYBR Green I based real-time quantitative PCR.(7)Assessment of Occludin,Claudin-1,ZO-1 protein localization and expressionCells were treated with PAF or ITF for different time,the localization and expression of occluding,Claudin-1,ZO-1 protein were detected by immunofluorescence and Western blot analysis.SYBR Green I based real-time quantitative PCR was used to measure the expression of mRNA.4.StatisticsData was expressed as Mean±SD.SPSS version 13.0 was used to perform statistical analysis.Results were analysed with one-way ANOVA,using LSD test for inter-group comparison,p＜0.05 was regarded as significant difference.Results1.Establishing intestinal epithelial barrier model using Caco-2 cellCultured Caco-2 cell confluencd as monolayer with time passed.When visualized with an inverted microscope,like polygon,irregular polygon or cobble cells presented, there were tight junctions amomg cells.When cultured about 21days,TEER reached 300-400Ω·cm² and there was little flux of lucifer yellow,transmission electron microscopy also found cells differentiated better,had well-arranged villi and polarity alined as monolayer,which was the marker of intestinal epithelial barrier model in vitro.2.PAF not affects the Cell proliferation and apoptosisThere was no effect on the Caco-2's vigor and proliferation by PAF even very high dose(P＞0.05)and no positive results of apoptosis.3.PAF increases intestinal epithelial paracellular permeability and abolished by ITFPAF treatment of Caco-2 monolayers produced a concentration and time-dependent drop in TEER and increase of paracellular markers luminal yellow, when PAF100ng/L incubated with Caco-2 monolayers for 24h,significant difference was calculated(P＜0.01).When incubated with ITF(0.3g/L),there was a significant increase of TEER values from 2h and the peak protection effect was at 24h,the variation of LY flux was paralleled to that of TEER.4.PAF disrupts the interepithelial tight junctionThe presence of electron-dense material in the space between cells near the brush border reflects the TJ.In cells without PAF,the TJ displayed an intact structure.When the cells were incubated with PAF 100ng/L for 24 hours,the TJ complex appeared reduced and contained less electron-dense material.5.Alterations in F-actin induced by PAF and ITFControl cells exhibits an intact actin network—a normal and smooth architecture of the F-actin "ring" at the areas associated with the plasma membrane(i.e.areas of cell-cell contact).PAF reduced the percentage of cells displaying normal actin.Following exposure to PAF,especially100ng/L for 24h,extensive disorganization, kinking,condensation,and beading of the actin ring could be seen.Preincubation with ITF protected the appearance of F-actin under PAF insult.Measurement of fluorescence intensity was investigated in a large number of cells (10000 cells per sample)by FACS.PAF induced a dose and time-dependent decline in fluorescent phalloidin and there were significant changes at 24h when treated with PAF of 100ng/L,at the same time,when pretreated with ITF and incubated 24h, fluorescence intensity recovered,which indicated ITF had protection effect definitely.6.Effect on ITF induced by PAFITF mRNA decreased begain with 8h and lower at 24h,recovered partly at 48h, but there was no significant difference when given PAF of 50ng/L.Group of PAF 100ng/L compared with control,ITF mRNA decreased at 4h and at a low ebb at 24h, P＜0.05,recovered partly at 48h.High dose PAF(200ng/L)has no significant difference with PAF 100ng/L.Immunohistochemistry showed that in normal Caco-2 cells,ITF positive stain sited in cytoplasm and some little part in cytomembrane.It decreased after PAF administered and reached the lowest point with PAF 100ng/L(P＜0.01). 7.Tight junction protein occludin,and ZO-1 distribution changed, mRNA and protein expression decreased caused by PAF and the protection of ITFIndirect immunofluorescent staining showed in the control Caco-2 monolayers, Occludin,Claudin-1 and ZO-1 proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders.PAF caused a progressive disturbance in the continuity of Occludin,Claudin-1 and ZO-1 localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contact,rITF can partly recover the disruption of distribution of these proteins.The three kinds of proteins were almost paralleled to each other in location and changing rule.Real time qPCR demonstrated that the mRNA expression of Occludin,Claudin-1 and ZO-1 in PAF treatment groups with different concentrations(50,100 and 200 ng/L) especilly100ng/L for 24 hours was significantly different from the corresponding time normal groups(P＜0.05),preincubated with rITF,the expression of mRNA increased,but treatment with rITF the expression of mRNA was not changed.Using Westem blot analysis found PAF produced a progressive decrease in higher molecular form,a phosphorylated form(80 kDa)of Occludin and 25 kDa of Claudin-1 protein expression(P＜0.01),whereas the low molecular form,a nonphosphorylated form (65 kDa and 20 kDa),was no significant change.ZO-1 protein was 220 kDa,when treatment with PAF of 100ng/L for 24h,there was a significant difference compared with control group(P＜0.01).Like the expression of mRNA,preincubated with rITF, protein expression also increased(P＜0.01).Conclusion1.Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.2.PAF disrupts the intestinal epithelial barrier directly,the increasing of intestinal epithelial permeability is independent of apoptosis and its target locates at paracellular pathway.3.ITF can protect intestinal epithelium by recover the normal structure of tight junction,and then decrease the intestinal epithelial permeability.4.PAF induces the structural alterations and the decrease of protein quantity of the cytoskeleton F-actin,which is one of the mechanisms for PAF increases intestinal epithelial permeability.5.PAF causes a significant decrease of ITF mRNA and protein expression, inhibiting the self-protection and reparation of the intestinal mucosal barrier.6.ITF could reverse the hyper-permeability by recover the normal structural of F-actin.It is an important evidence contributing to the function of ITF in epithelial protection.7.PAF disrupts the intestinal epithelial barrier,the mechanism of which would correlate to the decrease of Occludin,Claudin-1 and ZO-1 action proteins and mRNA expression and abnormal distribution.8.Pretreatly and therapeutically interventing of ITF may protect intestinal barrier function by recovering Occludin,Claudin-1 and ZO-1 action proteins and mRNA expression and abnormal distribution partly.