IPEC-J2 Cell Monolayers Research Articles

Transport of glycinin, the major soybean allergen, across intestinal epithelial IPEC-J2 cell monolayers.

Soybean allergen entering the body is the initial step to trigger intestinal allergic response. However, it remains unclear how glycinin, the major soybean allergen, is transported through the intestinal mucosal barrier. The objective of this study was to elucidate the pathway and mechanism of glycinin hydrolysatetransport through the intestinal epithelial barrier using IPEC-J2 cell model. Purified glycinin was digested by in vitro static digestion model. The pathway and mechanism of glycinin hydrolysates transport through intestinal epithelial cells were investigated by cellular transcytosis assay, cellular uptake assay, immunoelectron microscopy and endocytosis inhibition assay. The glycinin hydrolysates were transported across IPEC-J2 cell monolayers in a time/dose-dependent manner following the Michaelis equation. Immunoelectron microscopy showed a number of glycinin hydrolysates appeared in the cytoplasm, but no glycinin hydrolysates were observed in the intercellular space of IPEC-J2 cells. The inhibitors, colchicine, chlorpromazine and methyl-β-cyclodextrin,significantly inhibited the cellular uptake of glycinin hydrolysates. The glycinin hydrolysates crossed IPEC-J2 cell monolayers through the transcellular pathway. Both clathrin- and caveolae-dependent endocytosis were involved in the epithelial uptake of the hydrolysates. These findings provided potential targets for the prevention and treatment of soybean allergy.

Allicin Improves Intestinal Epithelial Barrier Function and Prevents LPS-Induced Barrier Damages of Intestinal Epithelial Cell Monolayers.

Gut barrier disruption is the initial pathogenesis of various diseases. We previously reported that dietary allicin improves tight junction proteins in the endoplasmic reticulum stressed jejunum. However, whether the allicin benefits the gut barrier within mycotoxin or endotoxin exposure is unknown. In the present study, IPEC-J2 cell monolayers within or without deoxynivalenol (DON) or lipopolysaccharide (LPS) challenges were employed to investigate the effects of allicin on intestinal barrier function and explore the potential mechanisms. Results clarified that allicin at 2 μg/mL increased the viability, whereas the allicin higher than 10 μg/mL lowered the viability of IPEC-J2 cells via inhibiting cell proliferation. Besides, allicin increased trans-epithelial electric resistance (TEER), decreased paracellular permeability, and enhanced ZO-1 integrity of the IPEC-J2 cell monolayers. Finally, allicin supplementation prevented the LPS-induced barrier damages via activating Nrf2/HO-1 pathway-dependent antioxidant system. In conclusion, the present study strongly confirmed allicin as an effective nutrient to improve intestinal barrier function and prevent bacterial endotoxin-induced barrier damages.

Effects of 1,2-Dimethylhydrazine on Barrier Properties of Rat Large Intestine and IPEC-J2 Cells.

Colon cancer is accompanied by a decrease of epithelial barrier properties, which are determined by tight junction (TJ) proteins between adjacent epithelial cells. The aim of the current study was to analyze the expression of TJ proteins in a rat model of 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, as well as the barrier properties and TJ protein expression of IPEC-J2 cell monolayers after incubation with DMH. Transepithelial electrical resistance and paracellular permeability for sodium fluorescein of IPEC-J2 were examined by an epithelial volt/ohm meter and spectrophotometry. The expression and localization of TJ proteins were analyzed by immunoblotting and immunohistochemistry. In the colonic tumors of rats with DMH-induced carcinogenesis, the expression of claudin-3 and -4 was significantly increased compared to controls. The transepithelial electrical resistance of IPEC-J2 cells increased, while paracellular permeability for sodium fluorescein decreased, accompanied by an increased expression of claudin-4. The increase of claudin-4 in rat colon after chronic DMH exposure was consistent with the acute effect of DMH on IPEC-J2 cells, which may indicate an essential role of this protein in colorectal cancer development.

4-Phenylbutyric acid accelerates rehabilitation of barrier function in IPEC-J2 cell monolayer model.

As the first line of defence against pathogens and endotoxins crossing the intestine-blood barrier, the intestinal epithelial barrier plays a determinant role in pigs' health and growth. 4-Phenylbutyric acid (4-PBA), an aromatic fatty acid, was reported to benefit homeostasis of endoplasmic reticulum and protein synthesis. However, whether 4-PBA affects intestinal epithelial barrier function in pigs is unknown. This study aimed to explore the effects of 4-PBA on the intestinal barrier function, using in vitro models of well-differentiated intestinal porcine epithelial cell (IPEC-J2) monolayers in the transwell plates. Cell monolayers with or without 4-PBA (1.0 mmol/L) treatment were challenged with physical scratch, deoxynivalenol (DON, 2.0 μg/mL, 48 h), and lipopolysaccharide (LPS, 5.0 μg/mL, 48 h), respectively. Transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FD-4) permeability were measured to indicate barrier integrity and permeability. Real-time PCR and Western blot were conducted to determine relative gene and protein expressions of tight junction proteins. As expected, physical scratch, DON, and LPS challenges decreased TEER and increased FD-4 permeability. 4-PBA treatment accelerated cell mitigation and rehabilitation of the physical scratch-damaged intestinal epithelial barrier but did not alleviate DON or LPS induced barrier damage. However, once 48-h DON and LPS challenges were removed, rehabilitation of the epithelial barrier function of IPEC-J2 monolayer was accelerated by the 4-PBA treatment. Also, the relative gene and protein expressions of zonula occludens-1 (ZO-1), occludin, and claudin-1 were further upregulated by the 4-PBA treatment during the barrier rehabilitation. Taken together, 4-PBA accelerated the IPEC-J2 cell monolayer barrier recovering from physical scratch, DON-, and LPS-induced damage, via enhancing cell mitigation and expressions of tight junction proteins.

The Protective Effects of Orthosiphon stamineus Extract Against Intestinal Barrier Injury in High-Fat Diet-Induced Mouse and Oxidative Stress Cell Models

Orthosiphon stamineus Benth. (Lamiaceae) is commonly used for the treatment of kidney diseases, but its role in intestinal barrier function remains unknown. The present study investigated the protective effects of O. stamineus extract (OE) against oxidative stress-induced injury to the small intestinal epithelium and the possible mechanism. High-performance liquid chromatography fingerprinting was used to analyze OE. Oxidative stress was induced by hydrogen peroxide (1 mM for 1 hour) in an IPEC-J2 cell monolayer model and a high-fat diet in C57BL/6 mice (8 weeks). The malondialdehyde (MDA) content was tested in both models. To evaluate permeability, transepithelial electrical resistance (TEER) was tested in a cell model. Serum diamine oxidase (DAO) and endotoxin contents were determined in a mouse model, and histological sections were analyzed. The messenger ribonucleic acid expression of tight junction proteins was measured by quantitative real-time polymerase chain reaction. Pretreatment with OE (50 µg/mL) increased the IPEC-J2 cell monolayer TEER (12.4%) and decreased MDA (from 6.1 to 4.7 mmol/mg prot). Oral administration of OE (100 mg/kg) decreased serum DAO (34.2%), endotoxin (13.4%), and MDA (from 21.3 to 11.0 mmol/mL) in mice. OE upregulated ZO-1 (42.8% in the cell model and 125.0% in mice) and occluding (127.0% in the cell model and 120.3% in mice) gene expression. These results confirmed the protective effect of OE on the intestinal barrier, which was associated with the antioxidant effect of OE; thus, OE is suitable for the prevention and treatment of intestinal barrier injury.

Nontoxic-dose deoxynivalenol aggravates lipopolysaccharides-induced inflammation and tight junction disorder in IPEC-J2 cells through activation of NF-κB and LC3B

Lipopolysaccharide (LPS) is the key factor in various intestinal inflammation which could disrupt the epithelial barrier function. Deoxynivalenol (DON), a well-known mycotoxin, can induce intestinal injury. However, the combined enterotoxicity of LPS and DON has rarely been studied. In this study, IPEC-J2 cell monolayers were exposed to LPS and nontoxic-dose DON for 12 and 24 h to investigate the effects of DON on LPS-induced inflammatory response and tight junction variation, and specific inhibitor and CRISPR-Cas9 were used to explore the underlying mechanisms. Our results showed that nontoxic-dose DON aggravated LPS-induced cellular inflammatory response, reflecting on more significant changes of inflammatory cytokines mRNA expression, higher protein expression of NOD-like receptor protein 3 (NLRP3) and procaspase-1. Moreover, nontoxic-dose DON aggravated LPS-induced mRNA and protein expression decreased, and distribution confused of tight junction proteins. We found that DON further enhanced LPS-induced phosphorylation and nucleus translocation of p65, and expression of LC3B-Ⅱ. NF-κB inhibitor and CRISPR-Cas9-mediated knockout of LC3B attenuated the effects of combination which indicated nontoxic-dose DON aggravated LPS-induced intestinal inflammation and tight junction disorder through activating NF-κB signaling pathway and autophagy-related protein LC3B. It further warns that ingesting low doses of mycotoxins may exacerbate the effects of intestinal pathogens on the body.

Cecropin A Modulates Tight Junction-Related Protein Expression and Enhances the Barrier Function of Porcine Intestinal Epithelial Cells by Suppressing the MEK/ERK Pathway.

Inflammatory bowel disease (IBD) in humans and animals is associated with bacterial infection and intestinal barrier dysfunction. Cecropin A, an antimicrobial peptide, has antibacterial activity against pathogenic bacteria. However, the effect of cecropin A on intestinal barrier function and its related mechanisms is still unclear. Here, we used porcine jejunum epithelial cells (IPEC-J2) as a model to investigate the effect and mechanism of cecropin A on intestinal barrier function. We found that cecropin A reduced Escherichia coli (E. coli) adherence to IPEC-J2 cells and downregulated mRNA expression of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). Furthermore, cecropin A elevated the transepithelial electrical resistance (TER) value while reducing the paracellular permeability of the IPEC-J2 cell monolayer barrier. Finally, by using Western blotting, immunofluorescence and pathway-specific antagonists, we demonstrated that cecropin A increased ZO-1, claudin-1 and occludin protein expression and regulated membrane distribution and F-actin polymerization by increasing CDX2 expression. We conclude that cecropin A enhances porcine intestinal epithelial cell barrier function by downregulating the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We suggest that cecropin A has the potential to replace antibiotics in the treatment of IBD due to its antibacterial activity on gram-negative bacteria and its enhancement effect on intestinal barrier function.

OTA induces intestinal epithelial barrier dysfunction and tight junction disruption in IPEC-J2 cells through ROS/Ca2+-mediated MLCK activation

Ochratoxin A (OTA) is a frequent contaminant of feed and food worldwide. The toxicity of OTA on intestinal barrier was investigated in porcine intestinal epithelial cells (IPEC-J2). We observed that OTA induced intestinal barrier dysfunction as indicated by the reduction in transepithelial electrical resistance (TEER) and elevation in paracellular permeability to 4 kDa dextran. The barrier dysfunction was accompanied with tight junction disruption including a down-regulation in ZO-1 expression and redistribution of Occludin and ZO-1. Moreover, OTA exposure increased reactive oxygen species (ROS) generation, elevated the intracellular calcium level ([Ca2+]c) and activated myosin light chain kinase (MLCK). Simultaneously, NAC, a ROS scavenger, blocked OTA-induced ROS generation, [Ca2+]c elevation, barrier dysfunction and tight junction disruption, suggesting that OTA-induced ROS generation may act as a trigger. Next, we found that OTA-induced MLCK activation was inhibited by BAPTA-AM, the cytosolic Ca2+ chelator, demonstrating that OTA-induced MLCK activation is dependent on [Ca2+]c elevation. Furthermore, inhibition of MLCK with ML-7 or inhibition of [Ca2+]c elevation with BAPTA-AM markedly prevented OTA-induced barrier dysfunction and tight junction disruption. Taken together, our results indicated that OTA induces ROS generation, and then elevates the [Ca2+]c and MLCK activity in turn, which finally induces barrier dysfunction and disrupts tight junction in IPEC-J2 cell monolayers.

Bovine colostrum increases the viability and the healing process of IPEC-J2 intestinal porcine epithelial cells while modulating the barrier function and Escherichia coli-mediated inflammatory responses.

Abstract Piglet gut response to weaning results in impaired intestinal barrier function, increased inflammatory response and susceptibility to enteric diseases. As the use of antibiotics as growth promoters will be restricted in the swine industry in Canada, novel feeding strategies to improve gut health are needed. This study aims to determine the potential of defatted bovine colostrum (BC) and its fractions, the serocolostrum (SC) and caseins (CAS), to promote viability and the healing process and to modulate barrier integrity and the inflammatory response induced by enterotoxigenic E. coli (ETEC) in IPEC-J2 intestinal epithelial porcine cells. To evaluate cell viability and wound healing, XTT cell proliferation and migration scratch assays were performed on IPEC-J2 cells incubated with BC, SC, or CAS. Monolayer permeability of ETEC infected IPEC-J2 cells incubated with BC, SC or CAS was monitored by transepithelial electrical resistance measurements. Inflammatory and oxidative responses were assessed by gene expression analysis of several cytokines and chemokines by qPCR. Incubation with BC derivatives led to 8 to 14% increases IPEC-J2 cell viability, in comparison to untreated cells (P ≤ 0.05). IPEC-J2 cell migration was increased by 42% after SC addition, as opposed to non-treated cells (P ≤ 0.05). BC and SC treatments significantly reduced ETEC-mediated increases in IL-8 and CCL20 expression (P ≤ 0.05). BC treatment also significantly reduced IPEC-J2 cell monolayer permeability induction after ETEC infection. These results show that BC and SC fractions improve intestinal epithelial cell integrity and decrease ETEC-mediated inflammatory responses. Therefore, BC should be considered as a potential alternative to antibiotics.

In Vitro Investigation of Six Antioxidants for Pig Diets.

Oxidative stress in the small intestinal epithelium can lead to barrier malfunction. In this study, the effect of rosmarinic acid (RA), quercetin (Que), gallic acid (GA), lipoic acid (LA), ethoxyquin (ETQ) and Se-methionine (SeMet) pre-treatments using 2 mM Trolox as a control on the viability and the generation of intracellular reactive oxygen species (iROS) of oxidatively (H2O2) stressed intestinal porcine epithelial cells (IPEC-J2) was investigated. A neutral red assay showed that RA (50–400 µM), Que (12.5–200 µM), GA (50–400 µM), ETQ (6.25–100 µM), and SeMet (125–1000 µM) pre-treatments but not LA significantly increased the viability of H2O2-stressed IPEC-J2 cells (p < 0.05). A 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) fluorescent probe showed that RA (100–600 µM), Que (25–800 µM), ETQ (3.125–100 µM) and SeMet (500–2000 µM) pre-treatments significantly reduced iROS in IPEC-J2 monolayers (p < 0.05). Moreover, RA and Que were most effective in reducing iROS. Therefore, the effects of RA and Que on barrier functioning in vitro were examined. RA and Que pre-treatments significantly decreased fluorescein isothiocyanate (FITC)-conjugated dextran-4 (4 kDa) permeability and transepithelial electrical resistance (TEER) of an IPEC-J2 cell monolayer (p < 0.05). These in vitro results of RA and Que hold promise for their use as antioxidants in pig feed.

1741 Comparison of transport characteristics of ferrous sulfate and iron glycine chelate across IPEC-J2 cell monolayers

1741 Comparison of transport characteristics of ferrous sulfate and iron glycine chelate across IPEC-J2 cell monolayers

In vitro characterization of TMPRSS2 inhibition in IPEC-J2 cells

The transmembrane serine protease, TMPRSS2 is an important target in the treatment of seasonal influenza infections and contributes to prostate carcinogenesis and metastasis. In this study, the effect of the synthetic TMPRSS2 inhibitor I-432 on jejunal IPEC-J2 cell monolayers cultured on membrane inserts was characterized. Using a fluorogenic substrate, it was found that the apical addition of I-432 could suppress trypsin-like activity in the supernatants of IPEC-J2 cells. The inhibition of TMPRSS2 did not affect physiologically produced hydrogen peroxide levels in the apical and in basolateral compartments. Loss of expression of the TMPRSS2 serine protease domain (28 kDa) was also observed when cells were pre-exposed to I-432. Partial decrease in immunofluorescent signal intensities derived from the altered distribution pattern of TMPRSS2 was detected after a 48 h long incubation of IPEC-J2 cells with the inhibitor indicating the efficacy of TMPRSS2 inhibition via I-432 administration in vitro.

Green tea polyphenol epigallocatechin‐3‐gallate improves epithelial barrier function by inducing the production of antimicrobial peptide pBD‐1 and pBD‐2 in monolayers of porcine intestinal epithelial IPEC‐J2 cells

Green tea has been known to confer numerous health benefits such as the prevention of cardiovascular disease, cancers, and obesity. Epigallocatechin-3-gallate (EGCG) is the major polyphenol present in green tea. Since EGCG is a food-derived component, intestinal epithelial cells lining the gastrointestinal tract are constantly and directly exposed to EGCG. It is anticipated that EGCG can exert beneficial effects in the intestine. The aim of this study was to explore the protective effects of EGCG on intestinal barrier functions against bacterial translocation by using a porcine jejunal epithelial cell line, IPEC-J2. EGCG reduced bacterial translocation across IPEC-J2 cell monolayers through the enhancement of the intestinal epithelial immunological barrier function by inducing secretion of antimicrobial peptides, porcine β-defensins 1 and 2 (pBD-1 and 2), which possessed higher antimicrobial activity against Escherichia coli. Further mechanistic studies demonstrated that EGCG upregulated pBD-2 but not pBD-1 via the p38 mitogen-activated protein kinase dependent pathway. Such effects were not an "artifact" of hydrogen peroxide, catechin dimers, or other auto-oxidation products generated from EGCG in cell culture media. Our results imply that EGCG may be useful for prevention of intestinal disorders or bacterial infection in animals/humans.

Reinforced Epithelial Barrier Integrity via Matriptase Induction with Sphingosine-1-Phosphate Did Not Result in Disturbances in Physiological Redox Status.

Objectives. The relationship among matriptase function, cellular redox status, and maintenance of intestinal barrier integrity has not been established yet. The aim of this study is to reveal if the crosstalk between matriptase activators and intestinal epithelial monolayers can lead to perturbations in physiological redox regulation in vitro. Methods. The effects of suramin and sphingosine-1-phosphate (S1P) were tested on viability of intestinal porcine epithelial IPEC-J2 cells using MTS assay. Measurements of transepithelial electrical resistance (TER) were performed to determine changes in barrier integrity of cell monolayers. Amplex Red assay was used to monitor extracellular hydrogen peroxide production. Occludin distribution pattern was detected prior to and after matriptase activation using immunofluorescent staining technique. Results. TER reduction was observed in suramin-treated IPEC-J2 cell monolayers, which could be attributed to cell cytotoxic properties of 48 hr 50 μM suramin administration. In contrast, S1P treatment increased TER significantly and elevated occludin accumulation in tight junctions. It was also found that extracellular hydrogen peroxide levels were maintained in IPEC-J2 cells exposed to matriptase activators. Discussion. S1P administration not accompanied by redox imbalance might be one of the key strategies in the improvement of barrier function and consequently in the therapy of intestinal inflammations.

Inhibition of Matriptase Activity Results in Decreased Intestinal Epithelial Monolayer Integrity In Vitro.

Barrier dysfunction in inflammatory bowel diseases implies enhanced paracellular flux and lowered transepithelial electrical resistance (TER) causing effective invasion of enteropathogens or altered intestinal absorption of toxins and drug compounds. To elucidate the role of matriptase-driven cell surface proteolysis in the maintenance of intestinal barrier function, the 3-amidinophenylalanine-derived matriptase inhibitor, MI-432 was used on porcine IPEC-J2 cell monolayer. Studies with two fluorescent probes revealed that short (2 h) treatment with MI-432 caused an altered distribution of oxidative species between intracellular and extracellular spaces in IPEC-J2 cells. This perturbation was partially compensated when administration of inhibitor continued for up to 48 h. Significant decrease in TER between apical and basolateral compartments of MI-432-treated IPEC-J2 cell monolayers proved that matriptase is one of the key effectors in the maintenance of barrier integrity. Changes in staining pattern of matriptase and in localization of the junctional protein occludin were observed suggesting that inhibition of matriptase by MI-432 can also exert an effect on paracellular gate opening via modulation of tight junctional protein assembly. This study confirms that non-tumorigenic IPEC-J2 cells can be used as an appropriate small intestinal model for the in vitro characterization of matriptase-related effects on intestinal epithelium. These findings demonstrate indirectly that matriptase plays a pivotal role in the development of barrier integrity; thus matriptase dysfunction can facilitate the occurence of leaky gut syndrome observed in intestinal inflammatory diseases.

Interaction exists between matriptase inhibitors and intestinal epithelial cells

The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro.

Trolox and ascorbic acid reduce direct and indirect oxidative stress in the IPEC-J2 cells, an in vitro model for the porcine gastrointestinal tract.

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H2DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pre-treatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets.

Gentamicin sulphate permeation through porcine intestinal epithelial cell monolayer.

Gentamicin is an aminoglycoside antibiotic widely used in combination with dimethyl sulphoxide (DMSO) in topical drug formulations. It is not known, however, whether DMSO can enhance the permeation of gentamicin through biological membranes, leading to oto- and nephrotoxic side effects. A simple and reliable high-performance liquid chromatographic (HPLC) method was applied for the quantitative determination of gentamicin collected from the apical and basolateral compartments of the porcine intestinal epithelial cell line IPEC-J2 cell monolayer using fluorometric derivatisation of the analyte with fluorenylmethyloxycarbonyl chloride (FMOC) prior to chromatographic run in the presence and absence of 1% DMSO. The lack of change in transepithelial electrical resistance (TER) demonstrated that gentamicin and 1% DMSO did not affect IPEC-J2 cell monolayer integrity via the disruption of cell membranes. Chromatographic data also ascertained that gentamicin penetration across the cell monolayer even in the presence of 1% DMSO was negligible at 6 h after the beginning of apical gentamicin administration. This study further indicates that the addition of this organic solvent does not increase the incidence of toxic effects related to gentamicin permeation.

Arginine and citrulline protect intestinal cell monolayer tight junctions from hypoxia-induced injury in piglets

Arginine (Arg) is deficient in the serum of the preterm neonate and is lower in those developing intestinal ischemia. We investigated whether Arg or its precursor, citrulline (Cit), protects intestinal tight junctions (TJs) from hypoxia (HX) and determined whether inducible nitric oxide (NO) plays a role. Neonatal piglet jejunal IPEC-J2 cell monolayers were treated with Arg or Cit, reversible and irreversible NO synthetase (NOS) inhibitors, and were exposed to HX. TJs were assessed by serial measurements of transepithelial electrical resistance (TEER), flux of inulin-fluorescein isothiocyanate, and immunofluorescent staining of TJ proteins. We found that Arg and Cit were protective against HX-related damage. At the final time point (14 h), the mean TEER ratio (TEER as compared with baseline) for Arg + HX and Cit + HX was significantly higher than that for HX alone. Both Arg and Cit were associated with decreased inulin flux across hypoxic monolayers and qualitatively preserved TJ proteins. Irreversible inhibition of NOS blocked this protective effect. Lipid peroxidation assay showed that our model did not produce oxidant injury. Arg and Cit, via a mechanism dependent on NO donation, protected intestinal epithelial integrity.

Transport of Surface Engineered Polyamidoamine (PAMAM) Dendrimers Across IPEC-J2 Cell Monolayers

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAMG4 dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (Papp) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.