Rat Intestinal Epithelial Cell Line Research Articles

Metabolomics and Lipidomics Reveal the Metabolic Disorders Induced by Single and Combined Exposure of Fusarium Mycotoxins in IEC-6 Cells.

Deoxynivalenol (DON), fumonisin B1 (FB1), and zearalenone (ZEN) are typical fusarium mycotoxins that occur worldwide in foodstuffs, posing significant health hazards to humans and animals. Single and combined exposure of DON, FB1, and ZEN leads to intestinal toxicity but the toxicology mechanism research is still limited. In this study, we explored the cytotoxicity effects of DON, FB1, ZEN, and their combination in rat intestinal epithelial cell line 6 (IEC-6) cells. Cell viability results showed that the cytotoxicity potency ranking was DON > ZEN > FB1. Furthermore, both DON + FB1 and DON + ZEN presented synergism to antagonism effects based on a combination index (CI)-isobologram equation model. Integrated metabolomics and lipidomics was adopted to explore cell metabolism disorders induced by fusarium mycotoxin exposure. A total of 2011 metabolites and 670 lipids were identified. An overlap of 37 and 62 differential compounds was confirmed after single and combined mycotoxin exposure by multivariate analysis, respectively. Some of the differential compounds were endocellular antioxidants and were significantly downregulated in mycotoxin exposure groups, indicating metabolic disorders as well as antioxidant capacity damage in cells. Pathway enrichment analysis annotated ethanol metabolism production of ROS by CYP2E1 was mainly involved in the disturbance of DON, FB1, and ZEN. The results obtained in this study help to define the toxicity effects of DON, FB1, and ZEN singly and in co-existence, providing an important scientific basis for combined risk recognition of mycotoxin contamination.

Orally Administrated Glutamate Restored EAAT1 and 3 Expression Levels Suppressed in 5-Fluorouracil-damaged Intestinal Epithelial Cells.

5-Fluorouracil (5-FU) treatment induces intestinal mucositis, with diarrhea as the primary symptom. Mucositis significantly reduces patients' quality of life (QOL). Amino acids such as glutamate are beneficial for treating gastrointestinal disorders; however, the underlying mechanism remains unclear. Therefore, this study aimed to clarify the role of excitatory amino acid transporters (EAATs) in 5-FU-induced intestinal injury. The rat intestinal epithelial cell line (IEC-6) was used to evaluate whether the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) affects 5-FU-induced cytotoxicity. Mice with 5-FU-induced mucositis were used to determine the effects of glutamate on EAATs expression levels. Treatment with L-trans-PDC suppressed IEC-6 cell growth. It also exacerbated the 5-FU-induced cell growth suppression and increased inflammatory cytokine expression. In addition, mice treated with 5-FU+Glutamate showed higher EAAT1,3 expression than 5-FU only-treated mice. Decreased EAAT levels worsen intestinal cell damage caused by 5-FU, suppress cell growth, and induce inflammation. This study contributes to the understanding EAAT and its relationship with intestinal mucositis, which can aid in the development of novel preventive strategies for cancer chemotherapy.

PSXIV-8 Comparison of the Effects of Hydroxy-Selenomethionine and Selenium-Enriched Yeast Against Hydrogen Peroxide-Induced Oxidative Stress in Rat Intestinal Epithelial Cells

Abstract Superior bioavailability of organic selenium (Se) has been extensively studied. However, limited research has been conducted to evaluate the effect of organic forms on intestinal redox potential to support gut health and animal well-being. Two experiments (Exp) were conducted to compare the effect of either hydroxy-selenomethionine (OH-SeMet) or Se-enriched yeast (Se-yeast) on ameliorating hydrogen peroxide (H2O2)-induced oxidative stress in a rat intestinal epithelial cell line (IEC-6). A cell viability test was initially performed to validate the Se level (0.15, 0.3, 0.6 ug/mL) used for the reactive oxygen species (ROS) assay. In both studies, OH-SeMet or Se-yeast promoted cell viability and attenuated cell apoptosis indicating no negative impact on cell growth. However, the Se-yeast sources showed considerably greater effectiveness than OH-SeMet. To quantify redox potential, confluent cells were treated with DCFDA, an indicator of ROS production. Briefly, IEC-6 cells were seeded into 96-well plates (4 ×104 cells per well) with 3 replicates per treatment (Trt) and incubated with a fresh solution of DCFDA. The ROS production, in response to 30µM H2O2, was quantified by fluorescence emission at 485 nm. Data were analyzed with GLIMMIX procedure of SAS. In both Exp, Se inclusion in all Se-Trts was 0.3 ppm. In Exp 1, Trts included: Non treated (cells only), H2O2, H2O2 + 2mM Trolox (Vit. E analog), H2O2 + sodium selenite, H2O2 + OH-SeMet, H2O2 + Se-yeast product A, H2O2 + Se-yeast product B. Cells treated with H2O2 had 4.6-fold increase (P < 0.05) in ROS production relative to non-H2O2 treated cells. Trolox reduced ROS 7.6-fold (P < 0.05) relative to H2O2 only cells, OH-SeMet and sodium selenite did not ameliorate (P > 0.35) the negative impact of H2O2 on ROS production. However, cells treated with H2O2 + Se-yeast product A or B reduced ROS production by 15% (P = 0.07) and 32% (P < 0.01), respectively, compared with cells treated with H2O2 only. To validate the redox potential of OH-SeMet, Exp 2 included a pure form of selenomethionine. Treatments in Exp 2. were similar to Exp 1 with the inclusion of H2O2 + seleno-L-methionine. Selenomethionine, either as a pure source or the hydroxy analog, did not prevent (P > 0.40) H2O2-induced oxidative stress. However, Exp 2 confirmed the effectiveness of Se-Yeast products on reducing ROS production. More specifically, Se-Yeast product B (52% reduction, P = 0.04) was superior to product A (32% reduction, P = 0.17) in comparison to cells treated with H2O2 only. These findings suggest that the type of organic Se source differs in their ability to regulate intestinal redox potential, with Se-yeast sources being more effective than hydroxy analogs. Selenomethionine content in Se-yeast does not appear to be the main driver in reducing ROS in intestinal cells, suggesting that livestock fed Se-yeast can support better intestinal health during oxidative stress.

Complete subunit structure of serotype C and D botulinum progenitor toxin complex induces vacuolation in the specific epithelial cell line

Clostridium botulinum produces seven botulinum neurotoxin (BoNT) serotypes. In nature, BoNT exists as a part of the progenitor toxin complex (PTC) through associations with neurotoxin associated proteins (NAPs), including nontoxic nonhemagglutinin and hemagglutinin (HA) complex, consists of HA-70, HA-17 and HA-33. Because PTC displays higher oral toxicity than pure BoNTs, NAPs play a critical role in food poisoning. In a previous study, we demonstrated that the NAP complex in mature large-sized PTC (L-PTC) from serotypes C and D concomitantly induced cell death and cytoplasmic vacuolation in the rat intestinal epithelial cell line IEC-6. Here, we found that the serotype D NAP complex induces only cytoplasmic vacuolation in the normal rat kidney cell line NRK-52E without reducing cell viability. NAP complexes from serotype A and B L-PTCs did not affect cell viability or cytoplasmic vacuolation in IEC-6 and NRK-52E cells. Furthermore, we assessed the effect of immature L-PTCs with fewer HA-33/HA-17 trimers (two HA-33 and one HA-17) than mature L-PTCs on cell viability and cytoplasmic vacuolation in IEC-6 and NRK-52E cells. As a result, mature L-PTCs with the maximum number of HA-33/HA-17 trimers displayed the greatest potency. Consequently, the reduction in cell viability and vacuolation induction are related to the number of HA-33/HA-17 trimers in PTC. The discovery of an epithelial cell model where botulinum PTC specifically induces vacuolization may help clarify the unknown cytotoxicity of PTC, which plays an important role in the trans-epithelial transport of the toxin.

The biological activity and signaling profile of EGF/EGFR were affected under heat stress conditions in IEC6 cells

Epidermal growth factor (EGF) is an effective cytoprotective peptide. It is the main nutritional factor involved in the development of the intestinal tract. It has many important biological effects on the intestinal mucosa. After binding to epidermal growth factor receptor (EGFR), it initiates a signal transduction cascade to jointly promote the migration, proliferation, and differentiation of various cell types. Heat stress severely affects the intestinal health of livestock and is becoming increasingly prevalent due to the yearly increase in ambient temperature and intestinal diseases. However, the effect of heat stress on the activity and signaling of EGF/EGFR in intestinal cells is still unclear. Therefore, rat intestinal crypt epithelial cell line (IEC6) was used as a model to explore this issue, and the results showed that EGF/EGFR is internalized into IEC6 cells in a time-dependent manner under physiological conditions. However, the activity of EGF/EGFR was altered under heat stress. Furthermore, we explored the effect of heat stress on EGF/EGFR-activated signaling transduction in IEC6 cells, and the results showed that levels of factors involved in EGFR-mediated intracellular signaling (such as EGFR, signal transducers and activators of transcription 3/protein kinase B, and extracellular regulatory kinase 1/2) were downregulated under heat stress. In summary, this study shows that heat stress could damage the biological activity and intracellular signaling of EGF/EGFR. These findings have scientific importance in the field of animal husbandry; and lay the foundation for the further study of the biological activities of EGF/EGFR in the intestine.

An Active Fraction of Trillium tschonoskii Promotes the Regeneration of Intestinal Epithelial Cells After Irradiation.

Despite significant scientific advances toward the development of safe and effective radiation countermeasures, no drug has been approved for use in the clinic for prevention or treatment of radiation-induced acute gastrointestinal syndrome (AGS). Thus, there is an urgent need to develop potential drugs to accelerate the repair of injured intestinal tissue. In this study, we investigated that whether some fractions of Traditional Chinese Medicine (TCM) have the ability to regulate intestinal crypt cell proliferation and promotes crypt regeneration after radiation. By screening the different supplements from a TCM library, we found that an active fraction of the rhizomes of Trillium tschonoskii Maxim (TT), TT-2, strongly increased the colony-forming ability of irradiated rat intestinal epithelial cell line 6 (IEC-6) cells. TT-2 significantly promoted the proliferation and inhibited the apoptosis of irradiated IEC-6 cells. Furthermore, in a small intestinal organoid radiation model, TT-2 promoted irradiated intestinal organoid growth and increased Lgr5+ intestinal stem cell (ICS) numbers. More importantly, the oral administration of TT-2 remarkably enhanced intestinal crypt cell proliferation and promoted the repair of the intestinal epithelium of mice after abdominal irradiation (ABI). Mechanistically, TT-2 remarkably activated the expression of ICS-associated and proliferation-promoting genes and inhibited apoptosis-related gene expression. Our data indicate that active fraction of TT can be developed into a potential oral drug for improving the regeneration and repair of intestinal epithelia that have intestinal radiation damage.

Knockdown of TRIM9 attenuates irinotecan‑induced intestinal mucositis in IEC‑6 cells by regulating DUSP6 expression via the P38 pathway.

Intestinal mucositis is a common side effect of cancer chemotherapy and it limits the dose of chemotherapy given to a patient. Tripartite motif family (TRIM) proteins have been reported to be implicated in the regulation of cancer chemotherapy. The present study aimed to investigate the effect of TRIM9 on irinotecan-induced intestinal mucositis in the rat intestinal epithelial cell line IEC-6. The expression of several TRIMs, such as TRIM1, TRIM9, TRIM18, TRIM36, TRIM46 and TRIM67, was examined. After TRIM9 knockdown or overexpression by lentivirus infection, cell proliferation and apoptosis, epithelial barrier tight-junction proteins, inflammatory cytokines, transepithelial electrical resistance (TEER) and FITC dextran were measured. Treatment with irinotecan significantly inhibited cell proliferation and induced cell apoptosis, TRIM9 expression, intestinal mucosal barrier impairment, the levels of inflammatory cytokines and P38 phosphorylation in IEC-6 cells, while the expression levels of epithelial barrier tight-junction protein ZO-1 and Claudin-4 were decreased. Knockdown of TRIM9 partly counteracted the effect of irinotecan treatment, and inhibition of P38 potently reversed the effect of TRIM9 overexpression in IEC-6 cells. Moreover, co-immunoprecipitation showed an interaction between TRIM9 and DUSP6 in IEC-6 cells, and overexpression of DUSP6 notably counteracted the effect of TRIM9 overexpression. The results demonstrated that TRIM9 knockdown may benefit patients with intestinal mucositis by inhibiting inflammatory cytokine expression and repairing intestinal barrier functions, which was probably due to inhibition of the activation of the P38 pathway via targeting DUSP6.

Glycation sites and bioactivity of lactose-glycated caseinate hydrolysate in lipopolysaccharide-injured IEC-6 cells

During the thermal processing of milk, Maillard reactions occur between proteins and lactose to generate glycated proteins. In this study, a lactose-glycated caseinate was hydrolyzed by trypsin. The obtained glycated caseinate (GCN) hydrolysate had a lactose content of 10.8 g/kg of protein. We identified its glycation sites and then assessed it for its protective effect against lipopolysaccharide-induced barrier injury using a rat intestinal epithelial cell line (IEC-6 cells) as a cell model and unglycated caseinate (CN) hydrolysate as a reference. Results from our liquid chromatography-mass spectrometry analysis of the GCN hydrolysate verified that lactose glycation occurred at the Lys residues in 3 casein components (αS1-casein, β-casein, and κ-casein), and this resulted in the formation of 5 peptides with the following amino acid sequences: EMPFPKYPKYPVEPF, HIQKEDVPSE, GSENSEKTTMPL, NQDKTEIPT, and EGIHAQQKEPM. The results from cell experiments showed that the 2 hydrolysates could promote cell growth and decrease lactate dehydrogenase release in the lipopolysaccharide-injured cells; more importantly, they could partially protect the damaged barrier function of the cells by increasing trans-epithelial electrical resistance, decreasing epithelial permeability, and upregulating the expression of the 3 tight junction proteins zonula occludens-1, occludin, and claudin-1. However, compared with CN hydrolysate, GCN hydrolysate showed lower efficacy in protecting against cellular barrier dysfunction. We propose that the different chemical characteristics of the CN hydrolysate and the GCN hydrolysate (i.e., amino acid loss and lactose conjugation) contributed to the lower barrier-protective efficacy of the GCN hydrolysate. During dairy processing, protein glycation of the Maillard type might have a non-negligible, unfavorable effect on dairy proteins, in view of the resulting protein glycation we found and the critical function of proteins for maintaining the integrity of the intestinal barrier.

Nanoemulsion structure and food matrix determine the gastrointestinal fate and in vivo bioavailability of coenzyme Q10

In this work, we sought to incorporate coenzyme Q10-loaded nanoemulsions into a food system and to understand the impact of food digestion on the in vivo bioavailability of this bioactive compound. We selected octenyl succinic anhydride modified starch as emulsifier to prepare the nanoemulsions (with approximately 200 nm droplet diameter) after comparing with two other food-grade surfactants (whey protein isolate and lecithin) in terms of their colloidal stability during simulated gastrointestinal digestion. The change in ζ-potential revealed that the initial emulsifier might be partially replaced by bile salts under intestinal conditions, and the mixed micelles formed after digestion showed an apparent permeability coefficient of 4.79 × 10−6 cm/s in a rat intestinal epithelial cell line, without compromising the trans-epithelial electrical resistance. In a second step, a high protein beverage that incorporated the coenzyme Q10-loaded nanoemulsion was developed in a food pilot plant. The beverage had a particle size of D4,3 = 18 μm and D3,2 = 2.5 μm, corresponding to its different components. The changes in particle morphology and size distribution were analysed to understand the behaviour of this beverage during simulated gastrointestinal digestion. When coenzyme Q10 was encapsulated into the nanoemulsions and the beverage, its bioavailability in vivo increased 1.8- and 2.8-fold respectively, compared with coenzyme Q10 dissolved in oil. The higher coenzyme Q10 bioavailability in the beverage was probably because of a significantly higher level of lipolytic activity found for beverage than for nanoemulsions during gastrointestinal digestion. These results show the potential of using natural food materials to generate formulations to improve the bioavailability of bioactive compounds. More importantly, we highlight the influence of food digestion (i.e. lipolysis) on the absorption of hydrophobic bioactive components and suggest that food systems can be utilised as a dosage form to further enhance oral bioavailability.

Activity of the peptic-tryptic caseinate digest with caseinate oligochitosan-glycation in rat intestinal epithelial (IEC-6) cells via the Wnt/β-catenin signaling pathway

The caseinate and glycated caseinate generated from the transglutaminase-catalyzed reaction of caseinate and oligochitosan were digested using pepsin and trypsin, and the activity of the resultant digests was measured in rat intestinal epithelial cell line (IEC-6) using several biological responses as indicators. Compared with the caseinate digest, the glycated caseinate digest had similar contents in 17 amino acids but less reactable –NH2 contents, and 6.57 g glucosamine per kg protein; moreover, it showed higher activity in the cells (P < 0.05) to promote cell growth, accumulate the cell-cycle progression at the S-phase, and prevent the camptothecin-induced cell apoptosis. The glycated caseinate digest also showed higher differentiation activity in the cells than the caseinate digest, resulting in enhanced activities of the three brush-border membrane enzymes (P < 0.05) and increased microvilli on the cell surfaces. The real-time reverse transcription-polymerase chain reaction, Western-blot assay, and Dickkopf-1 (a receptor inhibitor of the Wnt/β-catenin signaling pathway) were used to determine both gene and protein expression changes in the cells. A Wnt/β-catenin signaling pathway responsible for these enhanced effects was proposed because the five genes (glycogen synthase kinase 3β, Wnt3a, β-catenin, c-Myc, and cyclin D1) and three proteins (nuclear and cytosolic β-catenin, cyclin D1, and c-Myc) as part of this signaling pathway were regulated in the treated cells. The oligochitosan glycation of caseinate induced by transglutaminase is thus suggested endowing the peptic-tryptic caseinate digest with higher activity in the cells through its effects on the Wnt/β-catenin signaling pathway.

Increased expression of EGR1 and KLF4 by polysulfide via activation of the ERK1/2 and ERK5 pathways in cultured intestinal epithelial cells.

Sodium trisulfide (Na2S3) releases hydrogen polysulfide (H2Sn) and is useful for the investigation of the effects of H2Sn on the cell functions. In the present study, we first examined the effects of Na2S3 on the gene expression of IEC-6 cells, a rat intestinal epithelial cell line. Microarray analysis and reverse transcription-polymerase chain reaction analysis revealed that Na2S3 increased the gene expression of early growth response 1 (EGR1) and Kruppel-like transcription factor 4 (KLF4). It was interesting that U0126, an inhibitor of the activation of extracellular signal-regulated kinase 1 (ERK1), ERK2, and ERK5, inhibited the Na2S3-induced gene expression of EGR1 and KLF4. Na2S3 activated ERK1 and ERK2 (ERK1/2) within 15 min. In addition to ERK1/2, Na2S3 activated ERK5. We noticed that the electrophoretic mobility of ERK5 was decreased after Na2S3 treatment. Phos-tag analysis and in vitro dephosphorylation of the cell extracts indicated that the gel-shift of ERK5 was due to its phosphorylation. The gel-shift of ERK5 was inhibited completely by both U0126 and ERK5-IN-1, a specific inhibitor of ERK5. From these results, we concluded that the gel-shift of ERK5 was induced through autophosphorylation by activated ERK5 after Na2S3 treatment. The present study suggested that H2Sn affected various functions of intestinal epithelial cells through the activation of the ERK1/2 and ERK5 pathways.

Role of Clostridium perfringens Enterotoxin on YAP Activation in Colonic Sessile Serrated Adenoma/ Polyps with Dysplasia.

Sessile serrated adenoma/polyp with dysplasia (SSA/P-D) is an SSA/P with cellular dysplasia and has a higher risk of progressing to colon carcinogenesis. Previously, we reported that tight junction impairment by Clostridium perfringens enterotoxin (CPE) leads to activation of the transcriptional co-activator yes-associated protein (YAP) in oral squamous cell carcinoma. Here, we investigated whether CPE activates YAP to promote the malignant progression of SSA/P. E-cadherin expression was lower in the 12 cases with SSA/P-D examined than that in normal mucosa, SSA/P, or tubular adenoma (TA). Furthermore, intracellular translocation of claudin-4 (CLDN4) and nuclear translocation of YAP were observed. The CPE gene was detected in DNA extracted from SSA/P-D lesions, but not in SSA/P or TA. Treatment of the rat intestinal epithelial cell line IEC6 with low-dose CPE resulted in intracellular translocation of CLDN4 to the cytoplasmic membrane. Cytoplasmic CLDN4 showed co-precipitation with transcriptional co-activator with PDZ-binding motif, zonula occludens (ZO)-1, large tumor suppressor, and mammalian Ste20-like. Additionally, YAP co-precipitated with ZO-2 under CPE treatment led to decreased YAP phosphorylation and nuclear translocation. YAP activation promoted increase in nuclear TEA domain family member level, expression of cyclin D1, snail, vimentin, CD44, NS and decrease in E-cadherin levels, thereby inducing stemness and epithelial-mesenchymal-transition (EMT). The Hippo complex with the incorporation of CLDN4 increased stability. Upon low-dose CPE treatment, HT29 cells with BRAFV600E gene mutation showed increased growth, enhanced invasive potential, stemness, and induced EMT phenotype, whereas HCT116 cells, which carry KRASG13D gene mutation, did not show such changes. In an examination of 10 colorectal cancers, an increase in EMT and stemness was observed in CPE (+) and BRAF mutation (+) cases. These findings suggest that C. perfringens might enhance the malignant transformation of SSA/P-D via YAP activation. Our findings further highlight the importance of controlling intestinal flora using probiotics or antibiotics.

47 Burn-induced Ileal Extracellular Matrix Upregulation Associated with Epithelial to Mesenchymal Transition

Abstract Introduction Severe burns have long been associated with systemic inflammation and intestinal dysfunction. Recent evidence suggests that intestinal fibrosis may be responsible for intestinal dysfunction after burns. In response to inflammatory stimuli, intestinal epithelial cells may undergo epithelial-mesenchymal transition (EMT). EMT is a process by which epithelial cells acquire a mesenchymal-like phenotype, thereby compromising epithelial barrier function. EMT has been implicated in the pathogenesis of intestinal fibrosis. In the present study, we examined the cellular mechanism of burn-induced intestinal dysfunction. Methods Male BALA/c mice (8–12 weeks) received 30% total body surface area full-thickness scald burns or sham procedure. Ileal tissue was collected 5 days after burn for immunofluorescence (IF) and Western blot. Rat intestinal epithelial cell line IEC-6 was treated with cytokines and EMT marker proteins were analyzed by Western blot. Results IF data demonstrated that burn significantly increased extracellular matrix (ECM) protein laminin in ileal tissues, suggesting that burn injury induces ileal fibrogenesis. In IEC-6 cells treated with Tumor necrosis factor (TNF) α, IL-1β dose-dependently upregulated α-smooth muscle actin (α-SMA), a well known marker for myofibroblasts. ECM proteins fibronectin and laminin were found to be significantly increased after TNFα treatment. Tight junction protein E-cadherin was decreased after TNFα treatment. Futhermore, TNF receptor signaling antagonist R-7050 and SM7368 blocked TNFα-induced α-SMA upregulation. Conclusions Burn-induced mouse ileum ECM upregulation may be through TNFα-mediated EMT. Applicability of Research to Practice TNFα receptor antagonism could represent a potential pathway for drug development for treatment of burn-induced intestinal dysfunction.

Novobiocin, a Newly Found TRPV1 Inhibitor, Attenuates the Expression of TRPV1 in Rat Intestine and Intestinal Epithelial Cell Line IEC-6.

Background and Purpose: Novobiocin (NOVO), an ABC transporter inhibitor, decreases intestinal wall permeability of capsaicin (CAP), an ABC transporter substrate. However, the mechanism of this effect is not consistent with the action of NOVO as an ABC transporter inhibitor. We previously found that CAP can also be transported via TRPV1, which was site-specific in the permeability of CAP across the intestine. We explored the regulation by NOVO of TRPV1 in the present study.Methods: Rats and transfected IEC-6 cells were used as the models to assess intestinal permeability and expression of TRPV1. Ussing chamber and intracellular accumulation were used to evaluate the influence of NOVO on the transport of CAP in vitro. The expression of TRPV1 was detected after administration of NOVO by qRT-PCR, western blot and immunofluorescent imaging. In addition, MTT and lactate dehydrogenase (LDH) were used to evaluate the cytotoxicity of NOVO in both rat and cell models. Finally, the effect of NOVO on the absorption of CAP in vivo was studied by LC-MS/MS.Results: In vitro data showed that there existed a dose-dependent relationship in the range of concentration between 5 and 50 μM, and even 5 μM NOVO could decrease intestinal permeability of CAP across the intestine. Meanwhile, cytosolic accumulation of CAP decreased when NOVO was used simultaneously or 24 h in advance. NOVO exhibited an inhibition level similar to that of ruthenium red (RR) or SB-705498, a TRPV1-specific inhibitor. NOVO down-regulated TRPV1 expression in the intestine and in transfected cells in a concentration-dependent fashion, hinting that its inhibition of the permeability of CAP is due to its inhibition of TRPV1 expression. Immunofluorescent imaging data showed that the fluorescence intensity of TRPV1 was reduced after pre-treatment with NOVO and SB-705498. In vivo data further demonstrated that oral co-administration of NOVO decreased Cmax and AUC of CAP in dosage-dependent ways, consistent with its role as a TRPV1 inhibitor.Conclusion: NOVO could be a potential TRPV1 inhibitor by attenuating the expression of TRPV1 and may be used to attenuate permeability of TRPV1 substrates.

Salmonella enterica Serovar Typhimurium Increases Functional PD-L1 Synergistically with Gamma Interferon in Intestinal Epithelial Cells via Salmonella Pathogenicity Island 2.

Nontyphoidal serovars of Salmonella enterica are pathogenic bacteria that are common causes of food poisoning. Whereas Salmonella mechanisms of host cell invasion, inflammation, and pathogenesis are mostly well established, a new possible mechanism of immune evasion is being uncovered. Programmed death ligand 1 (PD-L1) is an immunosuppressive membrane protein that binds to activated T cells via their PD-1 receptor and thereby halts their activation. PD-L1 expression plays an essential role in the immunological tolerance of self-antigens but is also exploited for immune evasion by pathogen-infected cells and cancer cells. Here, we show for the first time that Salmonella infection of intestinal epithelial cells causes the induction of PD-L1. The increased expression of PD-L1 through Salmonella infection was seen in both human and rat intestinal epithelial cell lines. We determined that cellular invasion by the bacteria is necessary for PD-L1 induction, potentially indicating that Salmonella strains are delivering mediators from inside the host cell that trigger the increased PD-L1 expression. Using knockout mutants, we determined that this effect largely originates from the Salmonella pathogenicity island 2. We also show for the first time in any cell type that Salmonella combined with gamma interferon (IFN-γ) causes a synergistic induction of PD-L1. Finally, we show that Salmonella plus IFN-γ induction of PD-L1 decreased the cytokine production of activated T cells. Understanding Salmonella immune evasion strategies could generate new therapeutic targets and help to manipulate PD-L1 expression in other diseases.

Induction of myosin light chain kinase and CPI-17 by TGF-β accelerates contractile activity in intestinal epithelial cells.

Epithelial-mesenchymal transition (EMT) is an orchestral and functional change in epithelial cells. Many signaling pathways are involved in EMT, and transforming growth factor-beta (TGF-β) is considered to be one of the most important factors in induction of EMT. In this study, we treated the rat intestinal epithelial cell line (IEC-6) with TGF-β1 as a signaling stimulant. Gross analysis of IEC-6 cells showed typical characteristics of epithelial cells such as cuboidal morphology and cell-cell contact, whereas treatment with TGF-β1 (10 ng/ml−1) for 7 days produced robust, spindle-shaped morphology. Immunocytochemistry analysis showed distinct E-cadherin staining in IEC-6 cells, but weak and faint in EMT cells. EMT cells showed positive expression of α-SMA and tenascin-C but IEC-6 cells did not. Quantitative real-time PCR analysis showed that myosin light chain kinase and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17) mRNAs were significantly upregulated in EMT cells. Immunocytochemistry analysis also showed that EMT cells strongly expressed CPI-17 but IEC-6 cells did not. A collagen gel contraction assay revealed that EMT cells had greatly increased contraction compared with control cells. These results suggest that the increased contractile activity induced by TGF-β in EMT cells may be attributable to the upregulation of molecules responsible for myosin phosphorylation/de-phosphorylation.

Parathyroid Hormone-Related Protein (1-40) Enhances Calcium Uptake in Rat Enterocytes Through PTHR1 Receptor and Protein Kinase Cα/β Signaling

Background/Aims: Parathyroid hormone-related protein (PTHrP) is implicated in regulating calcium homeostasis in vertebrates, including sea bream, chick, and mammals. However, the molecular mechanism underlying the function of PTHrP in regulating calcium transport is still not fully investigated. This study aimed to investigate the effect of PTHrP on the calcium uptake and its underlying molecular mechanism in rat enterocytes. Methods: The rat intestinal epithelial cell line (IEC-6) was used. Calcium uptake was determined by using the fluo-4 acetoxymethyl ester fluorescence method. The expression levels of RNAs and proteins was assessed by RT-PCR and Western-blot, respectively. Results: PTHrP (1-40) induced rapid calcium uptake in enterocytes in a dose-dependent manner. PTHrP (1-40) up-regulated parathyroid hormone 1 receptor (PTHR1) protein but not 1,25D3-MARRS receptor. Pre-treatment of anti- PTHR1 antibody abolished the PTHrP (1-40)-induced calcium uptake. PTHrP (1-40) significantly up-regulated four transcellular calcium transporter proteins, potential vanilloid member 6 (TRPV6), calbindin-D9k (CaBP-D9k), sodium-calcium exchanger 1 (NCX1) and plasma membrane calcium ATPase 1 (PMCA1), in a dose- and time-dependent manner. Pre-treatment with TRPV6 or CaBP-D9k antibodies or knockout of rat TRPV6 or CaBP-D9k markedly inhibited PTHrP (1-40)-induced calcium uptake, whereas inhibition of NCX or PMCA1 by antibodies or inhibitors had no effect on PTHrP(1-40)-induced calcium uptake. Furthermore, PTHrP (1-40) treatment up-regulated protein levels of protein kinase C (PKC α/β) and protein kinase A (PKA). Pretreatment of PKC α/β inhibitor (but not PKA inhibitor) inhibited PTHrP (1-40)-induced calcium uptake. Conclusion: PTHrP (1-40) stimulates calcium uptake via PTHR1 receptor and PKC α/β signaling pathway in rat enterocytes, and calcium transporters TRPV6 and CaBP-D9k are necessary for this stimulatory effect.

Efficacy and Safety of a Multistrain Probiotic Formulation Depends from Manufacturing.

Variability in probiotics manufacturing may affect their properties, with potential implications for their efficacy and safety. This is of particular concern with probiotic products destined for use in patients with serious medical conditions, including human immunodeficiency virus (HIV) infection. The purpose of the study was to carry out a series of experiments comparing the properties of the US-made probiotic formulation originally commercialized under the brand name VSL#3®, with those of the Italian-made formulation now commercialized under the same name. The US-made formulation has previously shown beneficial effects at the intestinal and neurological levels in HIV-infected subjects as well as in patients with inflammatory bowel diseases and hepatic encephalopathy. Eleven subjects receiving combined antiretroviral therapy for HIV-1 were treated for 6 months with the US-made VSL#3 formulation. At baseline and 6 months, T-cells were analyzed for phenotype and activation markers, and fecal samples were analyzed for bifidobacteria, lactobacilli, and their metabolites. The fecal metabolome was assessed using 1H-NMR spectroscopy. Production of metabolites of interest by bacteria obtained from sachets of the two formulations was compared in vitro and their effects on a rat intestinal epithelial cell line (IEC-6) were assessed. Particular attention was paid to the metabolite 1,3-dihydroxyacetone (DHA). At 6 months, fecal samples showed a significant increase in the specific bacterial genera contained in the probiotic supplement. Immune activation was reduced as shown by a significant reduction in the percentage of CD4+CD38+HLA-DR+ T-cells at 6 months. Fecal concentrations of DHA decreased significantly. In vitro, significant differences in the production and metabolism of DHA were found between bacteria from the US-made and Italian-made formulations: the US-made formulation was able to metabolize DHA whereas the bacteria in the Italian-made formulation were producing DHA. DHA reduced the viability of Streptococcus thermophilus, reduced IEC-6 cell viability in a dose-dependent manner, and also led to a lower rate of repair to scratched IEC-6 cell monolayer. Our data, in conjunction with previously published findings, confirm that the new Italian-made formulation of VSL#3® is different from the previous US-made VSL#3 and therefore its efficacy and safety in HIV-infected subjects is still unproven.

Transforming Growth Factor β1/SMAD Signaling Pathway Activation Protects the Intestinal Epithelium from Clostridium difficile Toxin A-Induced Damage.

Clostridium difficile, the main cause of diarrhea in hospitalized patients, produces toxins A (TcdA) and B (TcdB), which affect intestinal epithelial cell survival, proliferation, and migration and induce an intense inflammatory response. Transforming growth factor β (TGF-β) is a pleiotropic cytokine affecting enterocyte and immune/inflammatory responses. However, it has been shown that exposure of intestinal epithelium to a low concentration of TcdA induces the release of TGF-β1, which has a protective effect on epithelial resistance and a TcdA/TGF-β signaling pathway interaction. The activation of this pathway in vivo has not been elucidated. The aim of this study was to investigate the role of the TGF-β1 pathway in TcdA-induced damage in a rat intestinal epithelial cell line (IEC-6) and in a mouse model of an ileal loop. TcdA increased the expression of TGF-β1 and its receptor, TβRII, in vitro and in vivo TcdA induced nuclear translocation of the transcription factors SMAD2/3, a hallmark of TGF-β1 pathway activation, both in IEC cells and in mouse ileal tissue. The addition of recombinant TGF-β1 (rTGF-β) prevented TcdA-induced apoptosis/necrosis and restored proliferation and repair activity in IEC-6 cells in the presence of TcdA. Together, these data show that TcdA induces TGF-β1 signaling pathway activation and suggest that this pathway might play a protective role against the effect of C. difficile-toxin.

12-O-tetradecanoylphorbol-13-acetate (TPA) increases murine intestinal crypt stem cell survival following radiation injury.

Radiation enteropathy is a common complication in cancer patients following radiation therapy. Thus, there is a need for agents that can protect the intestinal epithelium against radiation. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce differentiation and/or apoptosis in multiple cell lines and primary cells. In the current report, we studied the function of TPA in radiation induced enteropathy in cultured rat intestinal epithelial cell line IEC-6 after ionizing radiation (IR) and in mice after high dose total-body gamma-IR (TBI). In IEC-6 cells, there were reduced apoptosis and cell cycle arrest in TPA treated cells after IR. We detected a four-fold increase in crypt cell survival and a two-fold increase in animal survival post TBI in TPA treated mice. The beneficial effects of TPA were accompanied by upregulation of stem cells markers and higher level of proteins that are involved in PKC signaling pathway. In addition, TPA also decreased the TBI-augmented levels of the DNA damage indicators. The effects were only observed when TPA was given before irradiation. These results suggest that TPA has the ability to modulate intestinal crypt stem cells survival and this may represent a promising countermeasure against radiation induced enteropathy.