Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation

The deuterohemin-peptide conjugate, DhHP-6 (Dh-β-AHTVEK-NH₂), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H₂O₂); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-β-AH(Me)T(Me)V(Me)EK-NH₂ derived from this study may be developed as a promising candidate for oral administration.     

Mixed gastric- and intestinal-type metaplasia is formed by cells with dual intestinal and gastric differentiation.

We have proposed to divide intestinal metaplasia (IM) into two categories, i.e., a mixed gastric and intestinal (GI) type, and a solely intestinal (I) type, based on the residual gastric phenotype cells. The GI-mixed-type IM can be identified by the presence of both cells with either gastric or intestinal phenotypes in a single gland. This study is conducted to elucidate whether cells in the GI-mixed-type IM glands can simultaneously present both gastric and intestinal phenotypes. MUC5AC, MUC2, CD10 and villin expressions were investigated in 20 samples from five gastric cancer cases, directly using either AlexaFluor 488- or 568-labeled specific monoclonal antibodies and observed by fluorescent microscopy and confocal laser-scanning microscopy. GI-mixed IM glands comprise a population expressing MUC5AC and MUC2, MUC5AC and villin, and MUC5AC and CD10. MUC2 and villin expressions were reciprocally increased with decreasing MUC5AC expression, while CD10 expression was limited to cells with only a residual MUC5AC expression or no expression. These results suggest that a heterogeneous cell population with both gastric and intestinal phenotypes would develop into a single intestinal phenotype, as reflected in the progression of intestinal metaplasia from GI-mixed-type- to I-type IM-type glands.     

Synthesis and Characterization of a New Peptide Prodrug of Glucosamine with Enhanced Gut Permeability

The aim of this study was to synthesize a peptide prodrug of glucosamine (GlcN) with increased gut permeability through the gut peptide transporter 1 (PepT1). Glycine-Valine ester derivative of GlcN (GVG) was synthesised using solid phase synthesis followed by characterization and evaluation of its physicochemical and intestinal stability. In addition, GVG was evaluated for its ability to be biotransformed to GlcN in the liver homogenate. In vitro absorption of the new prodrug through everted rat gut was also assessed. GVG demonstrated significant and meaningful increased gut permeability as compared with GlcN. It showed favorable stability in the gut and a quick cleavage to GlcN after exposure to the liver homogenate. In conclusion, a novel prodrug of glucosamine with superior gut permeability compared to GlcN was developed and successfully tested in vitro.     

Caco-2 cell permeability and stability of two d-glucopyranuronamide conjugates of thyrotropin-releasing hormone

Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.     

In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.     

IL-1beta causes an increase in intestinal epithelial tight junction permeability

IL-1beta is a prototypical proinflammatory cytokine that plays a central role in the intestinal inflammation amplification cascade. Recent studies have indicated that a TNF-alpha- and IFN-gamma-induced increase in intestinal epithelial paracellular permeability may be an important mechanism contributing to intestinal inflammation. Despite its central role in promoting intestinal inflammation, the role of IL-1beta on intestinal epithelial tight junction (TJ) barrier function remains unclear. The major aims of this study were to determine the effect of IL-1beta on intestinal epithelial TJ permeability and to elucidate the mechanisms involved in this process, using a well-established in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. IL-1beta (0-100 ng/ml) produced a concentration- and time-dependent decrease in Caco-2 transepithelial resistance. Conversely, IL-1beta caused a progressive time-dependent increase in transepithelial permeability to paracellular marker inulin. IL-1beta-induced increase in Caco-2 TJ permeability was accompanied by a rapid activation of NF-kappaB. NF-kappaB inhibitors, pyrrolidine dithiocarbamate and curcumin, prevented the IL-1beta-induced increase in Caco-2 TJ permeability. To further confirm the role of NF-kappaB in the IL-1beta-induced increase in Caco-2 TJ permeability, NF-kappaB p65 expression was silenced by small interfering RNA transfection. NF-kappaB p65 depletion completely inhibited the IL-1beta-induced increase in Caco-2 TJ permeability. IL-1beta did not induce apoptosis in the Caco-2 cell. In conclusion, our findings show for the first time that IL-1beta at physiologically relevant concentrations causes an increase in intestinal epithelial TJ permeability. The IL-1beta-induced increase in Caco-2 TJ permeability was mediated in part by the activation of NF-kappaB pathways but not apoptosis.     

Intestinal Stem Cell Markers in the Intestinal Metaplasia of Stomach and Barrett’s Esophagus

Gastric intestinal metaplasia (IM) is a highly prevalent preneoplastic lesion; however, the molecular mechanisms regulating its development remain unclear. We have previously shown that a population of cells expressing the intestinal stem cell (ISC) marker LGR5 increases remarkably in IM. In this study, we further investigated the molecular characteristics of these LGR5⁺ cells in IM by examining the expression profile of several ISC markers. Notably, we found that ISC markers—including OLFM4 and EPHB2—are positively associated with the CDX2 expression in non-tumorous gastric tissues. This finding was confirmed in stomach lesions with or without metaplasia, which demonstrated that OLFM4 and EPHB2 expression gradually increased with metaplastic progression. Moreover, RNA in situ hybridization revealed that LGR5⁺ cells coexpress several ISC markers and remained confined to the base of metaplastic glands, reminiscent to that of normal intestinal crypts, whereas those in normal antral glands expressed none of these markers. Furthermore, a large number of ISC marker-expressing cells were diffusely distributed in gastric adenomas, suggesting that these markers may facilitate gastric tumorigenesis. In addition, Barrett’s esophagus (BE)—which is histologically similar to intestinal metaplasia—exhibited a similar distribution of ISC markers, indicating the presence of a stem cell population with intestinal differentiation potential. In conclusion, we identified that LGR5⁺ cells in gastric IM and BE coexpress ISC markers, and exhibit the same expression profile as those found in normal intestinal crypts. Taken together, these results implicate an intestinal-like stem cell population in the pathogenesis of IM, and provide an important basis for understanding the development and maintenance of this disease.     

Patterns of intestinal metaplasia in gastric biopsies. A comparison of different histochemical classifications

Summary A total of 217 cases of gastric intestinal metaplasia (IM), out of 491 cases of consecutive multiple gastric biopsies, were examined using various histochemical methods. Three classifications were adopted (1) Jass and Filipe's (1979), (2) Segura and Montero's (1983), and (3) our own distinguishing two types of small intestine IM (incomplete and complete) and two types of colonic IM (incomplete and complete). Our classification revealed (1) a group of pure IM cases (44%), (2) a group of combined IM cases (15%), either small intestinal or colonic, expressing a maturating line, and (3) a conspicuous group of mixed mosaic IM cases (41%) consisting of all possible combinations between the basic IM types. No significant correlations were found between each IM type and associated diseases (i.e. gastritis, stump gastritis, ulcer, adenoma and adenocarcinoma). However, a suggestive association between colonic IM and malignancy was observed. Our results confirmed the heterogeneity of IM and indicated two differentiation cell lines (ileal and colonic), thus providing additional evidence of impaired maturation and aberrant differentiation along various cell lines, supporting Leblond's (1976) unifying theory on the origin of intestinal epithelial cells.     

Development and validation of bioanalytical methods for Imidafenacin (KRP-197/ONO-8025) and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry

Imidafenacin (KRP-197/ONO-8025, IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a new antimuscarinic agent currently under application for the indication of treatment of overactive bladder in Japan. We developed and validated the sensitive and selective bioanalytical methods for the extremely low levels of IM and its metabolite, M-2 (Method 1), M-4 (Method 2) and M-9 (Method 3) in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In each method, plasma sample was extracted by solid phase extraction, separated on a semi-micro high performance liquid chromatography column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization or ionspray interface. Selected reaction monitoring mode was used for quantification. Each method was found to have acceptable accuracy, precision, stability, selectivity and linearity over the concentration range of 10-500 pg/mL for IM and M-2, 10-1000 pg/mL for M-4 and 50-5000 pg/mL for M-9. Using these analytical methods, concentration profiles of IM and its metabolites in human plasma were successfully determined even in the low pg/mL levels after oral administration of IM at the therapeutic dosage of 0.1 mg.     

Phosphorylation of the gastric tumor suppressor RUNX3 following H. pylori infection results in its localization to the cytoplasm

As H. pylori infection progresses, intestinal metaplasia (IM), a key event in gastric carcinogenesis, develops in the stomach. The mechanism by which H. pylori infection causes the trans-differentiation of gastric cells to intestinal-type cells remains an important question. In the current study, we found that RUNX3 is deregulated in all human IM specimens examined by either down regulation or mislocalization; Aberrant localization of a gastric tumor suppressor RUNX3 is observed in most human cases of IM with concurrent H. pylori infection, and RUNX3 is down-regulated in most cases of IM without H. pylori-infection. The cytoplasmic mislocalization of a RUNX3 was associated with H. pylori-induced c-Src activation and RUNX tyrosine phosphorylation. Moreover, gastric epithelial cells of Runx3(-/-) mice expressed the intestinal markers Muc2 and Li-Cadherin, which suggests that the deregulation of Runx3 is a key event in the intestinalization of the gastric epithelium. Collectively, the results of the current study suggest that RUNX3 deregulation is associated with H. pylori-induced pathogenesis and the development of IM.     

Correlation of in vitro and in vivo models for the oral absorption of peptide drugs

The aim of this study was to evaluate two in vitro models, Caco-2 monolayer and rat intestinal mucosa, regarding their linear correlation with in vivo bioavailability data of therapeutic peptide drugs after oral administration in rat and human. Furthermore the impact of molecular mass (Mm) of the according peptides on their permeability was evaluated. Transport experiments with commercially available water soluble peptide drugs were conducted using Caco-2 cell monolayer grown on transwell filter membranes and with freshly excised rat intestinal mucosa mounted in Using type chambers. Apparent permeability coefficients (P (app)) were calculated and compared with in vivo data derived from the literature. It was shown that, besides a few exceptions, the Mm of peptides linearly correlates with permeability across rat intestinal mucosa (R (2) = 0.86; y = -196.22x + 1354.24), with rat oral bioavailability (R (2) = 0.64; y = -401.90x + 1268.86) as well as with human oral bioavailability (R (2) = 0.91; y = -359.43x + 1103.83). Furthermore it was shown that P (app) values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between P (app) values across rat intestinal mucosa and in vivo oral bioavailability in human (R (2) = 0.98; y = 2.11x + 0.34) attests the rat in vitro model to be a very useful prediction model for human oral bioavailability of hydrophilic peptide drugs. Presented correlations encourage the use of the rat in vitro model for the prediction of human oral bioavailabilities of hydrophilic peptide drugs.     

Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-alpha-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-alpha-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-alpha-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-alpha-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-alpha increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-alpha-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-alpha-induced activation. Glucocorticoids inhibit the TNF-alpha-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-alpha-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.     

Nuclear volume in type I gastric intestinal metaplasia

OBJECTIVE: To retrospectively compare nuclear size of epithelial cells in type I intestinal metaplasia (IM) from various pathologic lesions of gastric mucosa. STUDY DESIGN: Endoscopic mucosal biopsies with type I IM from intestinal type gastric carcinoma (n = 25), chronic gastritis (n = 40) and benign ulcer (n = 32) cases were analyzed. After standard fixation, embedding, sectioning, routine hematoxylin and eosin and alcian blue--periodic acid--Schiff reaction (pH 1.0 and 2.5) staining were used for identification of IM. The mean point sampled nuclear intercept was estimated by the original test system and 100x objective at a total magnification of 1,200x. To obtain the mean nuclear volume, the cubed nuclear intercept was multiplied by pi/3. In each case 100 epithelial cell nuclei were analyzed. RESULTS: In type I IM in gastric carcinoma cases there was significantly greater nuclear volume (118.34 +/- 10.32 micron 3) than in type I IM in other pathologic states of gastric mucosa (77.72 +/- 8.58 micron 3). CONCLUSION: The karyometric findings of the present study suggest a difference between type I IM found in benign pathologic states and type I IM found in gastric mucosa surrounding carcinoma, despite morphologic and histochemical similarities. Nuclear volume may be used in early detection of precancerous states of gastric mucosa.     

Differential expression of laminin isoform (alpha2), integrins (alpha3beta1 and alpha6beta4) and cytokeratin 20 in H. pylori gastritis

The expression of laminin-1 chains (beta1 and gamma1), laminin-2 (merosin), integrin receptors to laminin (alpha3beta1 and alpha6beta4) and cytokeratin (CK20) were studied by immunohistochemical methods in gastric biopsies from antrum of 25 patients. H. pylori gastritis was found in 19 cases and intestinal metaplasia (IM) in four from these 19. Another 13 biopsies, all with IM were immunostained to laminin-2. Laminin-1 chains in normal and gastritis areas without IM were expressed as a strong, linear and continuous deposit in the basement membranes of the superficial and glandular epithelium. In metaplastic glands the reactivity to laminin-1 chains was decreased. Merosin was discontinuous when a moderate to accentuated H. pylori glandular colonization was present. Samples with IM were negative to laminin-2. The alpha3beta1 and alpha6beta4 integrins were negative only in IM gastric biopsies. The CK20 immunoreactivity was strong and homogeneous in the cells at the tip and the upper portion of foveolae in normal areas and in gastritis with IM the reactivity to CK 20 was heterogeneous. A differential expression of laminin isoforms is related to inflammation and subsequent IM caused by H. pylori. The alterations of alpha3beta1 and alpha6beta4 parallel both modifications in merosin and CK20 expression in H. pylori chronic gastritis.     

Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator

Mitochondrial membrane permeabilization can be a rate limiting step of apoptotic as well as necrotic cell death. Permeabilization of the outer mitochondrial membrane (OM) and/or inner membrane (IM) is, at least in part, mediated by the permeability transition pore complex (PTPC). The PTPC is formed in the IM/OM contact site and contains the two most abundant IM and OM proteins, adenine nucleotide translocator (ANT, in the IM) and voltage-dependent anion channel (VDAC, in the OM), the matrix protein cyclophilin D, which can interact with ANT, as well as apoptosis-regulatory proteins from the Bax/Bcl-2 family. Here we discuss that ANT has two opposite functions. On the one hand, ANT is a vital, specific antiporter which accounts for the exchange of ATP and ADP on IM. On the other hand, ANT can form a non-specific pore, as this has been shown by electrophysiological characterization of purified ANT reconstituted into synthetic lipid bilayers or by measuring the permeabilization of proteoliposomes containing ANT. Pore formation by ANT is induced by a variety of different agents (e.g. Ca(2+), atractyloside, thiol oxidation, the pro-apoptotic HIV-1 protein Vpr, etc.) and is enhanced by Bax and inhibited by Bcl-2, as well as by ADP. In isolated mitochondria, pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, and OM permeabilization, presumably due to physical rupture of OM. Although alternative mechanisms of mitochondrial membrane permeabilization may exist, ANT emerges as a major player in the regulation of cell death. Cell Death and Differentiation (2000) 7, 1146 - 1154     

Intestinal Alkaline Phosphatase Inhibits the Translocation of Bacteria of Gut-Origin in Mice with Peritonitis: Mechanism of Action

Exogenous intestinal alkaline phosphatase (IAP), an enzyme produced endogenously at the brush edge of the intestinal mucosa, may mitigate the increase in aberrant intestinal permeability increased during sepsis. The aim of this study was to test the efficacy of the inhibitory effect of IAP on acute intestinal inflammation and to study the molecular mechanisms underlying IAP in ameliorating intestinal permeability. We used an in vivo imaging method to evaluate disease status and the curative effect of IAP. Two Escherichia coli (E.coli) B21 strains, carrying EGFP labeled enhanced green fluorescent protein (EGFP) and RFP labeled red fluorescent protein (RFP), were constructed as tracer bacteria and were administered orally to C57/B6N mice to generate an injection peritonitis (IP) model. The IP model was established by injecting inflammatory lavage fluid. C57/B6N mice bearing the tracer bacteria were subsequently treated with (IP+IAP group), or without IAP (IP group). IAP was administered to the mice via tail vein injections. The amount of tracer bacteria in the blood, liver, and lungs at 24 h post-injection was analyzed via flow cytometry (FCM), in vivo imaging, and Western blotting. Intestinal barrier function was measured using a flux assay with the macro-molecule fluorescein isothiocyanate dextran, molecular weight 40kD, (FD40). To elucidate the molecular mechanism underlying the effects of IAP, we examined the levels of ERK phosphorylation, and the expression levels of proteins in the ERK-SP1-VEGF and ERK-Cdx-2-Claudin-2 pathways. We observed that IAP inhibited the expression of Claudin-2, a type of cation channel-forming protein, and VEGF, a cytokine that may increase intestinal permeability by reducing the levels of dephosphorylated ERK. In conclusion, exogenous IAP shows a therapeutic effect in an injection peritonitis model. This including inhibition of bacterial translocation. Moreover, we have established an imaging methodology for live-animals can effectively evaluate intestinal permeability and aberrant bacterial translocation in IP models.