| BackgroundThe recently discovered advanced oxidation protein products (AOPPs) are dityrosine-containing and cross-linking protein products formed during oxidative stress that are formed mainly by the reaction of plasma proteins with chlorinated compounds. The total AOPPs level in plasma contains two distinct molecular weight 670Kda and 67Kda. AOPPs is characterized by a specific absorption peak in 340 nm at the acidic conditions. AOPPs could also formed in vitro by incubating serum albumin with hypochlorous acid (HOCl), the biological effects of AOPPs prepared by this method are similar with those extracted from patients. In vivo, serum concentration of AOPPs closely correlated with levels of dityrosine (a hallmark of oxidized protein) and pentosidine (a marker of protein glycoxidation) that is tightly associated with oxidative stress. Thus, AOPPs have been considered as novel markers of oxidative stress-mediated protein injury.Increased plasma AOPPs formation has been reported in patients with chronic kidney disease (CKD), diabetes, and chronic hepatitis C. As a novel protein marker of oxidant-mediated protein damage, AOPPs participate in these pathophysiologic conditions. AOPPs accelerated proteinuria, glomerulosclerosis and interstitium fibrosis in CKD. They are capable of inducing vascular endothelial dysfunction via a receptor of advanced glycation endproducts (RAGE)-mediated signaling pathway.AOPPs have also been reported to induce overproduction of extracellular matrix(ECM) and the fibrogenic factor transforming growth factor (TGF)-?1. Furthermore,Zhou et al reported that AOPPs accumulation promotes podocyte apoptosis and depletion through RAGE. Our recent study demonstrated that AOPPs inhibit the proliferation and differentiation of rat osteoblast-like cells via ROS generation and nuclear factor (NF)-?B signaling.Crohn's disease (CD) is a chronic or recurrent relapsing gastrointestinal inflammation, mainly affecting the gastrointestinal tract with extraintestinal manifestations and associated immune disorders, and frequently presents with fever,abdominal pain, diarrhoea, abdominal mass, fistulization and bowel obstruction. CD etiology and pathophysiological changesis that environmental factors triggered susceptibility loci, under the participation of diminished diversity of commensal microbiota,resulted in a disturbed innate and adaptive immune responseie,disturbed intestinal barrier, cell death, goblet and paneth cell dysfunction, endoplasmic reticulum stress, defective unfolded protein response and autophagy, impaired recognition of microbes by pattern recognition receptors, such as nucleotide binding domain and Toll like receptors on dendritic cells and macrophages, imbalance of eff ector and regulatory T cells and cytokines, migration and retention of leukocytes. A peroxidation/antioxidation imbalance has been demonstrated in CD development, and this results in excessive levels of reactive oxygen species (ROS) generation and oxidative stress. Such changes are able to induce the oxidative modification of proteins, thus causing structural and functional changes. As the result of oxidative stress-induced protein damage, plasma AOPPs accumulated in CD patients. However,the role of AOPPs on intestine remains unknown.IEC are organized as a single cell layer that forms a contiguous lining and functional barrier that maintains gut structural integrity to separate the bowel wall from microbes and toxins. Tight junctions (TJs) are the closely associated areas of two epithelial cells whose membranes join together forming a virtually impermeable barrier to fluid. Although more proteins formed the TJs, the major types are the claudins, the occludins and peripheral membrane proteins such as ZO-1. The structural failure and dysfunction of IEC and TJs is the main physiopathologic changes of CD: (1) IEC proliferation and cell death must be tightly regulated to maintain the structural integrity of the intestinal mucosal epithelium, and changing this balance can have pathological consequences. There is a growing body of literature showing that excessive cell death is associated with chronic inflammation,as seen in patients with CD, and this could contribute to CD pathophysiology. (2)Mutation of MUCI?MUC19?PTGER4 deceased mucus secretion and resulted epithelial barrier dysfunction in IEC. (3) Recent reports have shown that barrier dysfunction in CD is linked to the down-regulation of the tight junction (TJ) scaff olding protein zonula occludens 1 (ZO-1) and to the degradation of the TJ structural protein occludin by the proteasome in the colonic mucosa.Although previous studies have revealed that oxidative stress results in plasma accumulation of AOPPs in CD,the effects of AOPPs on IECs remain unclear. It is unknown whether AOPPs affect IEC proliferation and death or intestinal tissue injury.Moreover, there is no information regarding the possible deposition of AOPPs in the intestinal tissue of patients with IBD. Thus, in the present study, (1) we determined the effects of AOPPs on IEC death both in vitro and in vivo. (2) investigated the role of AOPPs on intesinal TJs. (3) investigated the cellular pathway underlying the effect of AOPPs.Methods1. AOPP-RSA preparation and determinationAOPP-RSA was prepared in vitro by incubation of RSA (Sigma, St. Louis, MO,USA) with hypochlorous acid (HOCl, Fluke, Buchs, Switzerland) as described previously. Prepared samples were dialyzed against phosphate-buffered saline (PBS)for 24 h to remove free HOCl and passed through a Detoxi-Gel column (Pierce,Rockford, IL, USA) to remove contaminated endotoxin. Endotoxin levels in AOPP-RSA were measured with a Limulus Amoebocyte Lysate kit (BioWhittaker,Walkersville, MD, USA) and were found to be below 0.05 ng/mg protein. AOPPs contents in the preparations were determined with an OxiSelectTM AOPP Assay Kit(Cell Biolabs, San Diego, CA, USA); AOPPs contents in the AOPP-RSA and unmodified RSA were 50.10±3.92 and 0.22±0.06 ?mol/g protein, respectively.2. Cell cultureAn immortalized rat IEC line (IEC-6, The Committee on Type Culture Collection, Chinese Academy of Sciences, Beijing, China) and Caco-2 were cultured in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% fetal bovine serum, 100 mg/mL penicillin, and 100 IU/mL streptomycinin a 5% carbon dioxide atmosphere at 37?. Experiments were performed using passages 10 to 20.3. Proliferation of IEC-6 cells.Cells were seededon 96-wells plate at the density of 4×103 cells per well. 24 hours after cell seeding, RSA, different concentration of AOPP-RSA were added into each well. 0, 24h, 48h and 72h after the treatment, cell proliferation was assessed according to the instruction of CCK-8 kit (BI Yuntian Co, China), respectively. In brief, at the end of each time point, change the medium with 100?L fresh DMEM contained 10?L WST-8, and the plates were incubated for an 1 hours at 37?. The absorbance was measured at 450 nm with spectrophotometer.4. Apoptosis assays in IEC-6 culturesFlow cytometry was performed to analyze cell apoptosis.Assessment of fluorescein isothiocyanate (FITC) annexin V-labeled apoptotic cells was performed according to the protocol provided by the manufacturer (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were seeded on six-well plates andtreated with or without AOPP-RSA for the indicated time; cells (1×106) were suspended in buffer containing FITC annexin V and propidium iodide (PI). The samples were analyzed with a FACS Calibur flow cytometer (Becton Dickinson). A total of 10,000 cells were analyzed per determination. Cells were considered apoptotic if they were undergoing either early (Annexin-V-positive, PI-negative) or late apoptosis (Annexin-V-positive,PI-positive).DAPI stainingIEC-6 cells were inoculated into 24-wells plateat the density of 2×1 04cells per well, The cells were stained with DAPIafter AOPPs treatment,the apoptotic morphology was observed under OLYMPUS XB-51 fluorescence inverted microscope (Olympus, Tokyo, Japan).Apoptosis assays of intestinal tissuesApoptotic cells in the intestinal tissue sections were assessed with TUNEL assays (In situ cell death detection kit,Roche,Mannheim,Germany). Briefly, tissue sections were incubated with proteinase K for 20 min at room temperature and then washed with PBS. After inactivating endogenous peroxidase, sections were incubated in TdT buffer containing FITC-conjugated dUTP at 37? for 60 min. Morphological nuclear changes were observed by counterstaining with DAPI (Beyotime). The sections were analyzed under a confocal microscope (Carl Zeiss, Inc., Oberkochen,Germany). The apoptotic cells were counted in five random high-power fields (HPF,each 300 cells), and a total of 1500 epithelial cells were counted. The positive cells were scored for apoptosis. Data were expressed as numbers of apoptotic cells/HPF.5. Activation of NADPH oxidasep47phox phosphorylationp47phox phosphorylation in IEC-6 cultures was measured by immunoprecipitation as described previously. Briefly, cell lysates were incubated with protein A/G agarose beads (Santa Cruz Biotechnology),and a polyclonal rabbit anti-phosphoserine Ab(Abcam,). The precipitated immunocomplexes were resolved by SDS-PAGE,transferred onto PVDF membranes (Millipore), incubated with an HRP-conjugated rabbit anti-p47phox antibody (Sigma),and subjected to chemiluminescence detection as described above.p47phox translocation: detected using immunofluorescence staining.p47phox, p22phox, gp91phox expressions: detected using western blotting.6. Determination of ROS generationIntracellular ROS generation was measured with a flow cytometer (Becton Dickinson) with the probe DCFH-DA (2',7'-dichlorofluorescein-diacetate), which is a cell-permeable, nonfluorescent dye that can be oxidized to the fluorescent 2',7'-dichlorofluorescein (DCF) by ROS inside cells. Briefly, IEC-6 cultures were incubated with 10?M DCFH-DA for 30 min at 37? followed by AOPPs treatment as described above.7. Western blottingCultured cells or frozen rat intestinal tissue sampleswere lysed in radio-immunoprecipitation assay (RIPA) buffer, and protein was collected after centrifugationand mixed with5× sodium dodecyl sulfate (SDS) sample buffer. The samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 8-12% acrylamide gels and then transferred to polyvinylidene fluoride (PVDF)membranes (Millipore, Billerica, MA, USA). After incubation with primary and secondary antibodies, the protein bandswere detected with chemiluminescence detection reagents.8. Nuclear/Cytosolic FractionationSubfractionation was performed using a Nuclear/Cytosolic Fractionation Kit(Beyotime, Wuhan, China). IEC-6 cultures were washed with ice-cold PBS, scraped from the plates, and collected. After centrifugation, the supernatant was discarded,and the cells were suspended with Cytosol Extraction Buffer containing DTT/protease inhibitors, incubated on ice for 10 min, and Cell Lysis Reagent was added.The nuclei fraction was fractioned at 800×g for 10min. The supernatant was further centrifuged at 12,000×g for 10min, and the final supernatant was collected for cytoplasmic fraction. The nuclei pellet was washed and resuspended with nuclear extraction buffer containing DTT/protease inhibitors.9. Immunofluorescence stainingp47phox translocation from the cytoplasm to the membrane, AIF migration and TJ proteins were detected using immunofluorescence staining. Cells were fixed with paraformaldehyde, washed, and permeabilized with 0.1% Triton X-100 for 20 min.After blocking with nonfat milk for 1 h, the cells were incubated with primary antibodies overnight at 4?. The cells were then incubated with Alex 555-conjugated donkey anti-goat IgG (Invitrogen, Carlsbad, CA, USA) or rhodamine-conjugated chicken anti-rabbit IgG-R, stained with DAPI (4',6-diamidino-2-phenylindole), and observed under an OLYMPUS XB-51 fluorescence inverted microscope (Olympus,Tokyo, Japan).10. Permeability and transepithelial electrical resistance(TEER) measurementCaco-2 cells were seeded on the transwell inserts with 0.4?m pore sizeplaced in 24 well plates (Corning,USA) with 300 ?L cell culture mediumadded into the apical chamber and 500?L into the basolateral chamber. Periodically,TEER values were read using an electrical resistance system (EVOM; World Precision Instruments,Berlin, Germany). Experiments were carried out until the TEER had risen teadily above 300?/cm2. AOPPs wereadded to the apical chamber. TEER measurements were performed prior to the change of medium at 0-48h treatment with AOPPs.Paracellular permeability across cell monolayers was determined by measuring the flux of Fitc?daxtran.11. Animal studiesThe protocols of this study were approved by the Laboratory Animal Care and Use Committee of Southern Medical University. Male Sprague Dawley rats (initial weight, 160-200g,Southern Medical University Animal Experiment Center,Guangzhou, China) were housed in a pathogen-free environment and allowed free access to water and diet. The rats were randomly divided into four groups containing six animals per group and received daily intraperitoneal injections of vehicle (PBS,pH 7.4), unmodified RSA (50mg/kg per day), AOPP-RSA (50mg/kg per day), or AOPP-RSA (50 mg/kg per day) with or without separate intragastric administration of NADPH oxidase inhibitor apocynin (Sigma, 50 mg/kg per day). AOPP-RSA dosages were based on our preliminary experiment indicating that by this procedure,plasma AOPPs concentrations in the AOPP-RSA-treated group increased approximately 0.5-fold compared to the vehicle group (the level that has been found in IBD patients). At the end of 12 weeks, rats were anesthetized with sevoflurane and exsanguinated. The duodenum, jejunum,and ileum were collected, flushed with ice-cold PBS, and stored for further analyses.12. H&E staining, PAS staining, and immunohistochemistryDuodenum, jejunum, and ileum tissues were separately removed and fixed in neutral-buffered formalin. Formalin-fixed specimens were embedded in paraffin, cut into 3-4-?m-thick transverse sections, and stained with hematoxylin and eosin (H&E)to assess epithelial morphology and eosinophilic infiltration. PAS staining was performed according to standard protocol using PAS Staining System reagents from Sigma.For immunohistochemistry studies, after antigen retrieval, endogenous peroxidase activity, and normal serum blocking, the sections were incubated with primary antibody overnight followed bybiotinylated secondary antibodies(Zhongshanjinqiao, Beijing, China). Proteins were visualized as brown pigments via standard diaminobenzidine (DAB) protocol. The slides were lightly counterstained with hematoxylin.13. Patients and specimensA total of 23 formalin-fixed, paraffin-embedded intestinal resection specimens from CD patients who underwent segmental small bowel resection were obtained from the Nanfang hospital of Southern Medical University (Guangzhou, China) from 2010-2012. The diagnosis of CD was based on established clinical and histologic criteria. Patients with malignant tumor, cardiovascular disease, severe infection, or infliximab use were excluded. Normal intestinal tissue adjacent to diseased tissue was used as normal control. This study was approved by the Medical Ethical Committee of Nanfang hospital, and specimens were treated anonymously according to ethical and legal standards.14. Statistical analysisAll experiments were repeated at least three times. Continuous variables are expressed as mean±standard deviation (SD). For multiple comparisons within a dataset, one-way analysis of variance (ANOVA) with least significant difference(LSD) or Dunnett's T3 test was performed. Spearman's rank correlation was used toevaluate the association of AOPPs and cell apoptosis in CD patients. A two-tailed P-value of less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS 13.0 software (SPSS Inc., Chicago, IL, USA).Results1. Increased extracellular AOPPs inhibited the proliferation of IEC-6 cellsIEC-6 cells were treated with increasing concentrations of AOPP-RSA for 24h,CCK-8 determination showed AOPPs inhibited the proliferation of IEC-6 monolayers in a dose-dependent way.2. Increased extracellular AOPPs triggered IEC apoptosis in vitroTo determine whether AOPPs accumulation induces IEC apoptosis, we subjected conditionally immortalized IEC-6 cultures to increasing concentrations of AOPP-RSA for 48 h or 200?g/mL of AOPP-RSA for increasing times. Healthy IEC-6 cultures contained intactnuclei, but AOPP-RSA-treated cells exhibited nuclear condensation followed by fragmentation. Quantitative fluorescence-activated cell sorting (FACS) analysis offluorescein isothiocyanate (FITC)-annexin-V/propidium iodide (PI) staining showed that AOPP-RSA caused IEC-6 apoptosis in a concentration- and time-dependent manner compared with cells cultured in control medium and treated with unmodified RSA.3. AOPPs activated NADPH oxidaseWe evaluated NADPH oxidase activity in IEC-6 cultures stimulated with AOPP-RSA. Treatment with AOPPs led to of p47phox phosphorylation and membrane translocation, as well asincreased expression levels of NADPH oxidase key components p22phox, p47phox, and gp91phox.4. AOPPs-triggered apoptosis was mediated by NADPH oxidase-dependent ROS productionPrevious studies demonstrated that intracellular ROS mediate AOPP-induced podocyte and mesangial cell apoptosis. Therefore, we examined intracellular ROS levels in AOPPs-treated IEC-6 cultures; DCF fluorescence in the FITC/FL-1 channel was used to assess ROS generation. Incubation of IEC-6 cultures with AOPP-RSA induced time- and dose-dependent increases in ROS production. To evaluate whether NADPH oxidases were responsible for intracellular ROS generation, the experiment was repeated with the NADPH oxidase inhibitors diphenylene iodinium (DPI) and apocynin. AOPPs-induced ROS generation was significantly decreased in IEC-6 cultures that were pretreated with superoxide dismutase (SOD), DPI, and apocynin.These results suggested that AOPP-triggered ROS production was dependent on cellular NADPH oxidase activation in IEC-6 cultures.Next, we sought .to elucidate the role of ROS and NADPH oxidase in AOPPs-induced apoptosis. In IEC-6 cultures treated with 200?g/mL AOPPs in the presence of the general ROS scavenger SOD, AOPPs-triggered apoptosis was largely abolished. Similarly, inhibition of NADPH oxidase with apocynin and DPI significantly reduced EEC-6 apoptosis induced by AOPPs. Taken together, these findings imply that AOPPs are sufficient to induce IEC-6 apoptosis by increasing ROS synthesis, which is mediated through cellular NADPH oxidase activation.5. AOPPs-triggered apoptosis was associated with JNK activation.Intracellular mitogen-associated protein kinases (MAPKs), including extracellular signal regulating kinase 1/2 (ERK1/2 or p44/42 MAPK), c-jun N-terminal kinase (JNK), and p38 MAPK, have been shown to regulate cell growth,death, and cellular responses to stress. To determine whether the MAPK pathway is involved in AOPP-RSA-triggered cell death, we examined MAPK activity in IEC-6 cultures treated with AOPPs. JNK phosphorylation was markedly increased from 30 to 120 min after AOPPs treatment. However, AOPPs had no significant effect on phospho-p38 or phospho-ERK1/2 MAPK levels (data not shown).6. AOPPs activated PARP-1 via the NADPH oxidase-ROS-JNK pathwayIt is reported that the caspase-3 and caspase-independent (mediated by PARP-1 activation) pathways can both lead to cell death after inflammatory injury or ROS-induced injury. The former is the classic pathway marked by degradation of procaspase-3 into cleaved caspase-3. The latter is characterized by formation of polymers of ADP-ribose (PAR), decreased NAD+ levels, cytosolic apoptosis-inducing factor (AIF) nuclear translocation, nuclear condensation, and cell death. To confirm which was involved in AOPPs-induced death, we examined the activities of both pathways in IEC-6 cultures incubated with AOPPs. We verified that AOPPs stimulated robust PARP-1 activation in IEC-6 cultures from 1 h, which was accompanied by PAR formationand decreased NAD+ levels and was followed by AIF nuclear translocation from 6 h on. Interestingly, decreased procaspase-3 protein and increased cleaved caspase-3 could be detected after AOPPs treatment.To further evaluate the role of JNK-MAPK in cell apoptosis, IEC-6 cultures were incubated with a JNK inhibitor (SP600125) before AOPP-RSA treatment. The results suggested that activation of the proapoptotic JNK-MAPK pathway plays a critical role in AOPP-induced IEC-6 apoptosis.To further determine the roles of JNK, PARP-1, and caspase-3 in AOPP-induced apoptosis, IEC-6 cultures were incubated with a JNK inhibitor (SP600125), the PARP-1 inhibitor 3,4-dihydro-5- [4-( 1 -piperidiny l)butoxy] -1 (2H)-isoquinolin-one(DPQ), or the broad-spectrum caspase inhibitor Z-VAD.fmk before AOPP-RSA stimulation. SP600125 almost completely abolished the AOPP-induced increase in cell apoptosis. DPQ significantly decreased AOPP-induced cell apoptosis. However,caspase inhibitor treatment failed to statistically decrease AOPP-induced toxicity.These data indicate that AOPP-induced cell death is dependent on activation of the proapoptotic JNK-MAPK and PARP-1 pathway, not caspase-3 signaling.We also pre-treated IEC-6 cultures with DPI, apocynin SOD, and SP600125 before AOPP-RSA incubation. We found that PARP-1 activation was significantly suppressed by SOD, DPI, apocynin, and especially by SP600125. Over time, these suppressive effects became more obvious. Therefore, we concluded that AOPPs activate PARP-1 via an NADPH-dependent ROS-JNK pathway.7. IEC death was aggravated in AOPPs-treated rats and relieved by apocyninIn an attempt to examine if the effects of AOPPs on cell death observed in vitro might also occur in vivo, normal male Sprague Dawley rats were randomly assigned into four groups and received intraperitoneal injections of PBS, RSA, AOPP-RSA, or AOPP-RSA every other day with or without intragastric administration of apocynin for 12 weeks. We found that plasma AOPPs levels increased approximately 0.5-fold in AOPP-RS A-treated rats in comparison with control rats, which is equivalent to the level detected in patients with active CD.TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)staining revealed that IEC death was significantly aggravated in AOPP-treated rats when compared with that in control (vehicle- or RSA-treated rats). Inhibition of NADPH oxidase by apocynin significantly ameliorated AOPP-induced cell death.8. AOPPs decreased the number of goblet cells.Periodic acid Schiff (PAS) and Alcian blue (AC) staining showed that chronic AOPPs administration significantly decreased the number of goblet cells compared to control. We also found that goblet cell numbers were reduced in both the crypts and villi, especially the latter.9. In vivo AOPPs-triggered cell death was mediated by the NADPH oxidase-JNK-PARP-1 pathwayImmunohistochemical staining of intestine showed significant upregulations of p47phox, gp91phox, and p22phoxin AOPPs-challenged rats compared with controls.Western blotting confirmed increases in p47phox, gp91phox, and p22phox expression levels. We also performed immunohistochemistry to demonstrate increased JNK phosphorylation and PARP-1 expression in AOPPs-challenged rats. PAR generation and AIF translocation were also detected after AOPPs treatment. Moreover, IECs were positive for TUNEL but negative for caspase-3 (data not shown). These data provide further evidence that AOPPs-induced cell death in vivo is mediated by activation of the NADPH oxidase-JNK-PARP-1-PAR pathway rather than by caspase-3 signaling. Treatment with apocynin significantly decreased AOPPs-induced activation of the NADPH oxidase-JNK-PARP-1-PAR pathway.10. Chronic AOPPs administration promoted inflammation and injury in rat intestinal mucosaHistological examination of the small intestine revealed that AOPPs were predominantly deposited in the crypts and lymphocytes of the lamina propria and villous epithelial cells. Systematic histological assessment of the intestinal tracts revealed significant inflammatory changes; these alterations were primarily localized to the terminal ileum and barely infiltrated the colon tissue. The lesions consisted of shortened intestinal villous; lamina propria and submucosal infiltration of lymphocytes, plasmacytes, and scattered neutrophils; lymphoid follicle hyperplasia;epithelial necrosis and exfoliation; and erosion of the intestinal mucosal layer.Apocynin treatment attenuated the degree of tissue injury.11. Intestinal mucosa AOPPs deposition was associated with cell death in CD patientsA previous study demonstrated that plasma AOPP concentrations were elevated in patients with IBD, particularly in those with active CD. To further evaluate the effects of AOPPs on IECs in patients with CD, we examined AOPP expression and cell death by immunohistochemistry and TUNEL staining, respectively, in sequential sections of intestinectomy specimens from 23 patients. The normal intestinal tissues adjacent to the diseased regions were used as normal control samples. AOPPs were predominantly deposited in IECs and inflammatory cells in the lamina propria of intestinectomy specimens, whereas AOPP staining was negative in normal intestinal tissue. Likewise, TUNEL-positive cells were detected in the diseased region but rarely in the adjacent normal region. In addition, the high immunoreactive score of AOPPs indicated increased cell death, suggesting that AOPPs accumulation is associated with cell death in patients with CD.12. AOPPs induced intestinal epithelial barrier dysfunctionAfter 3h of AOPPs stimulation, there was a significant decrease in TEER, 3-6h after AOPPs treatment increase in FITC-dextran permeability (p<0.01 compared with control group). Western blotting revealed that treatment with AOPPs significantly downregulated levels of occludin, claudin-1 and ZO-1. Immunostaining showed that in the control group, occludin and ZO-1 presented a continuous band of cells encircling the apical cellular junctions, and AOPPs treatment caused a pronounced disruption in junctional localization of occludin and ZO-1 staining, characterized by decreased intensity staining and marked discontinuity localized to the structures of intercellular junctions.13. The effect of ERK-MAPK pathway on the AOPPs-induced intestinal barrier dysfunctionAOPPs treatment increase the expression of p-ERKl/2 (p<0.05). when pretreated with 10?M U0126 1h prior to AOPPs, the increase in paracellular permeability and decease in TEER were reversed (p<0.01), the expression of tight junction proteins occludin and ZO-1 was increased compared with AOPPs group.Conclusion1. Increased extracellular AOPPs triggered IEC apoptosis in a concentration-and time-dependent manner2. The triggering effect of AOPPs was mainly mediated by a redox-dependent pathway, including NADPH oxidase-derived ROS generation, JNK phosphorylation,and PARP-1 activation.3. Chronic AOPP-RSA administration to normal rats resulted in AOPPs deposition in the villous epithelial cells and in inflammatory cells in the lamina propria. These changes were companied with IEC death, inflammatory cellular infiltration, and intestinal injury. Both cell death and intestinal injury were ameliorated by chronic treatment with apocynin.4. AOPPs deposition was also observed in IECs and inflammatory cells in the lamina propria of patients with CD. The high immunoreactive score of AOPPs showed increased apoptosis.5. AOPPs induced intestinal epithelial barrier dysfunction via activation of ERK1/2, and then resulted in intestinal epithelial barrier dysfunction.Taken together, our observations are suggestive of a novel mechanism that aggravates CD: accumulated AOPPs in CD patients have the capacity to induce ROS generation, which initiates IEC death and intestinal tissue injury. |
PDF Full Text Request