Porcine Jejunal Epithelial Cell Research Articles

Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2.

The microbiological environment and their corresponding secreted metabolite spectrum are an essential modulator of the enterocyte function, effecting the whole organism. Intestinal porcine jejunal epithelial cell line (IPEC-J2) is an established in vitro model for differentiation of enterocytes in different cell culture models. An improved oxygen supply seems to be the main reason for differentiation in an air-liquid-interface culture, but this has not yet been conclusively clarified. In this context, the nutrition of the cell and its influence on the metabolism is also of crucial importance. The interest in short-chain fatty acids (SCFAs) has grown steadily in recent years due to their clinical relevance in certain diseases such as multiple sclerosis and other inflammatory diseases, but not much is known of FFAR2 and FFAR3 (free fatty acid receptor 2 and 3) in pigs. We want to address the questions: 1. about the distribution of FFAR2 and FFAR3 in vivo and in vitro in sus scrofa 2. whether there is an influence of propionic acid, glucose content and cultivation on metabolism of enterocytes? The morphological analysis of FFAR2 and FFAR3 in vivo was investigated through immunostaining of frozen sections of the porcine gut segments jejunum, ileum and colon. Both receptors are expressed along the gut and were found in the smooth muscle cells of the tunica muscularis and lamina muscularis mucosae. Furthermore, a high expression of FFAR2 and a low expression of FFAR3 in the enteric nerve system was also observed in jejunum, ileum and colon of sus scrofa. In addition, FFAR2 and FFAR3 within the vessels was investigated. FFAR3 showed a strong expression on endothelial cells of veins and lymphatic vessels but was not detectable on arteries. Furthermore, we demonstrate for the first time, FFAR2 and FFAR3 in IPEC-J2 cells on RNA- and protein level, as well as with confocal microscopy. In addition, ENO1 and NDUFA4 were investigated on RNA-level in IPEC-J2 cells as 2 important genes, which play an essential role in metabolism. Here, NDUFA4 is detected in the model animal sus scrofa as well as in the porcine cell line IPEC-J2. A potential impact of propionic acid and/or glucose and/or cultivation method on the metabolism of the cells was tested with the Seahorse analyzer. Here, a significant higher ECAR was observed in the SMC than in the OCR. In summary, we were able to show that the cultivation system appears to have a greater influence than the medium composition or nutrition of the cells. However, this can be modulated by incubation time or combination of different SCFAs.

CMPK2 is a host restriction factor that inhibits infection of multiple coronaviruses in a cell-intrinsic manner.

Coronaviruses (CoVs) comprise a group of important human and animal pathogens. Despite extensive research in the past 3 years, the host innate immune defense mechanisms against CoVs remain incompletely understood, limiting the development of effective antivirals and non-antibody-based therapeutics. Here, we performed an integrated transcriptomic analysis of porcine jejunal epithelial cells infected with porcine epidemic diarrhea virus (PEDV) and identified cytidine/uridine monophosphate kinase 2 (CMPK2) as a potential host restriction factor. CMPK2 exhibited modest antiviral activity against PEDV infection in multiple cell types. CMPK2 transcription was regulated by interferon-dependent and interferon regulatory factor 1 (IRF1)-dependent pathways post-PEDV infection. We demonstrated that 3'-deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) catalysis by Viperin, another interferon-stimulated protein, was essential for CMPK2's antiviral activity. Both the classical catalytic domain and the newly identified antiviral key domain of CMPK2 played crucial roles in this process. Together, CMPK2, viperin, and ddhCTP suppressed the replication of several other CoVs of different genera through inhibition of the RNA-dependent RNA polymerase activities. Our results revealed a previously unknown function of CMPK2 as a restriction factor for CoVs, implying that CMPK2 might be an alternative target of interfering with the viral polymerase activity.

Biogenic selenium nanoparticles alleviate intestinal epithelial barrier injury by regulating mitochondria-lysosome crosstalk.

The intestinal epithelial barrier plays a fundamental role in human and animal health. Mitochondrial dysfunction can lead to intestinal epithelial barrier damage. The interaction between mitochondria and lysosomes has been proved to regulate each other's dynamics. Our previous studies have demonstrated that biogenic selenium nanoparticles (SeNPs) can alleviate intestinal epithelial barrier injury through regulating mitochondrial autophagy. In this study, we hypothesize that the protective effects of SeNPs against intestinal epithelial barrier dysfunction are associated with mitochondrial-lysosomal crosstalk. The results showed that lipopolysaccharide (LPS) and TBC1D15 siRNA transfection both caused the increase of intestinal epithelial permeability, activation of mitophagy, and mitochondrial and lysosomal dysfunction in porcine jejunal epithelial cells (IPEC-J2). SeNP pretreatment significantly up-regulated the expression levels of TBC1D15 and Fis1, down-regulated Rab7, caspase-3, MCOLN2 and cathepsin B expression levels, reduced cytoplasmic Ca2+ concentration, effectively alleviated mitochondrial and lysosomal dysfunction, and maintained the integrity of the intestinal epithelial barrier in IPEC-J2 cells exposed to LPS. Furthermore, SeNPs obviously reduced cytoplasmic Ca2+ concentration and activated the TBC1D15/Fis/Rab7-mediated signaling pathway, shortened the contact time between mitochondria and lysosomes, inhibited mitophagy, maintained mitochondrial and lysosomal homeostasis, and effectively attenuated intestinal epithelial barrier injury in IPEC-J2 cells transfected with TBC1D15 siRNA. These results indicated that the protective effect of SeNPs on intestinal epithelial barrier injury is closely associated with the TBC1D15/Rab7-mediated mitochondria-lysosome crosstalk signaling pathway.

Zearalenone Exposure Affects the Keap1-Nrf2 Signaling Pathway and Glucose Nutrient Absorption Related Genes of Porcine Jejunal Epithelial Cells.

This study aims to examine the impact of zearalenone (ZEA) on glucose nutrient absorption and the role of the Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in zearalenone-induced oxidative stress of porcine jejunal epithelial cells (IPEC-J2). For 24 and 36 h, the IPEC-J2 cells were exposed to ZEA at concentrations of 0, 10, 20, and 40 (Control, ZEA10, ZEA20, ZEA40) mol/L. With the increase of ZEA concentration and prolongation of the action time, the apoptosis rate and malondialdehyde level and relative expression of sodium-dependent glucose co-transporter 1 (Sglt1), glucose transporter 2 (Glut2), Nrf2, quinone oxidoreductase 1 (Nqo1), and hemeoxygenase 1 (Ho1) at mRNA and protein level, fluorescence intensity of Nrf2 and reactive oxygen species increased significantly (p < 0.05), total superoxide dismutase and glutathione peroxidase activities and relative expression of Keap1 at mRNA and protein level, fluorescence intensity of Sglt1 around the cytoplasm and the cell membrane of IPEC-J2 reduced significantly (p < 0.05). In conclusion, ZEA can impact glucose absorption by affecting the expression of Sglt1 and Glut2, and ZEA can activate the Keap1-Nrf2 signaling pathway by enhancing Nrf2, Nqo1, and Ho1 expression of IPEC-J2.

MitomiR-504 alleviates the copper-induced mitochondria-mediated apoptosis by suppressing Bak1 expression in porcine jejunal epithelial cells

Copper (Cu), an environmental heavy metal pollutant, has been widely researched in its toxicology. Recently, an increasing number of mitochondrial microRNAs (mitomiRs) have been shown to involve in the metabolic regulation. However, the underlying mechanisms of mitomiRs on regulating apoptosis under Cu exposure are still unclear. Here, we proved that Cu induced mitochondria-mediated apoptosis in porcine jejunal epithelial cells, concomitant with distinct reduction of mitomiR-504 in vivo and in vitro. The miR-504 mimic notably enhanced the mRNA and protein expressions of Bak1, Bax, Cleaved-caspase3 and Caspase-9, and significantly decreased the apoptosis rate and Bcl-2 mRNA and protein levels, indicating that overexpression of mitomiR-504 attenuated the Cu-induced mitochondria-mediated apoptosis. Besides, Bak1 was confirmed as a direct target of mitomiR-504 by the bioinformatics analysis and dual-luciferase reporter assay. Subsequently, transfection of siRNA targeting Bak1 significantly enhanced the alleviating effect of miR-504 mimic on the Cu-induced mitochondria-mediated apoptosis. Overall, these suggested that overexpression of mitomiR-504 alleviated the Cu-induced mitochondria-mediated apoptosis in jejunal epithelial cells by suppressing Bak1 expression. These findings are conducive to elucidating the mechanism of Cu-induced jejunal epithelial pathologies, providing a new research idea for the Cu toxicology.

Aflatoxin B1 disrupts the intestinal barrier integrity by reducing junction protein and promoting apoptosis in pigs and mice

With the growing diversity and complexity of diet, animals and humans are at risk of exposure to aflatoxin B1 (AFB1), which is a well-known contaminant in the food chain that causes various toxicological effects. The intestine acts as the first barrier against external contaminants, but the effect of AFB1 on intestinal barrier has not been determined. This study aimed to evaluate AFB1 on the intestinal barrier function in vitro and in vivo. In vitro, porcine jejunal epithelial cells (IPEC-J2) were treated with increasing concentrations of AFB1 (10–60 mg/L). In vivo, Kunming (KM) mice were used as controls or gavaged with 1% dimethyl sulfoxide (110 mg/kg b.w.) and AFB1 (0.3 mg/kg b.w.) for 28 days. In IPEC-J2 cells, the cell viability decreased with increasing mycotoxin concentrations, and the viability of IPEC-J2 cells decreased significantly (P < 0.05) when the AFB1 concentrations were greater than 30 mg/L. In addition, quantitative real-time PCR, Western blot analysis, and immunofluorescence results show that AFB1 can downregulate the tight junction proteins and increase the expression levels of Caspase-3 and the ratio of Bax/Bcl-2, suggesting that AFB1 was cytotoxic to IPEC-J2. In vivo, the ratio of villus height to crypt depth, the intestinal wall thickness, the number of intestinal villus per 1000 µm in the jejunum, the expression levels of ZO-1, Claudin-3, Occludin, MUC2, and Caspase-3, and the ratio of Bax/Bcl-2 were significantly affected in mice exposed to AFB1. In vitro and in vivo results showed that the effects of exposure to AFB1 on the intestinal function in the jejunum of KM mice and in the IPEC-J2 was similar, suggesting that AFB1 may adversely affect animal intestine.

Biogenic Selenium Nanoparticles Alleviate Intestinal Epithelial Barrier Damage through Regulating Endoplasmic Reticulum Stress-Mediated Mitophagy

The intestinal barrier plays a fundamental role in body health. Intracellular redox imbalance can trigger endoplasmic reticulum stress (ERS) and mitophagy, leading to intestinal barrier damage. Our previous studies demonstrated that mitophagy is closely associated with the protective effects of biogenic selenium nanoparticles (SeNPs) on intestinal epithelial barrier function. Thus, we hypothesize that ERS and mitophagy are likely involved in the regulatory effects of SeNPs on oxidative stress-induced intestinal epithelial barrier dysfunction. The results showed that oxidative stress or ERS caused the increase of intestinal epithelial permeability. SeNPs effectively alleviated hydrogen peroxide (H2O2-)-induced structural damage of endoplasmic reticulum (ER) and mitochondria of porcine jejunal epithelial cells (IPEC-J2). SeNPs significantly decreased intracellular inositol triphosphate (IP3) and Ca2+ concentration, down-regulated inositol trisphosphate receptor (IP3R) expression level, and up-regulated ER-resident selenoproteins mRNA levels in IPEC-J2 cells exposed to H2O2. In addition, SeNPs pretreatment significantly decreased the intracellular Ca2+, IP3, IP3R, and reactive oxygen species (ROS) levels; protected the structure and function of ER and mitochondria; and effectively alleviated the increase of intestinal epithelial permeability of IPEC-J2 cells exposed to tunicamycin (TM). Moreover, SeNPs significantly inhibited the colocalization of mitochondria and lysosomes. Furthermore, compared with TM model group, SeNPs significantly inhibited the activation of PERK/eIF2α/ATF4 and AMPK/mTOR/PINK1 signaling pathway. The PERK agonist (CCT020312) and the AMPK agonist (AICAR) could reverse the protective effects of SeNPs on IPEC-J2 cells. The PERK inhibitor (GSK2656157) and the AMPK inhibitor (compound C) had a similar effect on IPEC-J2 cells as that of SeNPs. In summary, the protective effects of SeNPs on intestinal barrier dysfunction are closely associated with ERS-related PERK and mitophagy-related AMPK signaling pathway.

Comparison of structural and antioxidant activity of polysaccharide extracted from truffles.

As the main component of truffles, polysaccharides have a variety of biological activities such as anti-oxidation, anti-tumor, and hypoglycemic activity, and these activities are closely related to its structure. In this study, Tuber Aestivum crude polysaccharide (TACP) and Tuber Melanosporum crude polysaccharide (TMCP) were obtained from Tuber Aestivum and Tuber Melanosporum by using microwave-assisted hot water, and then the Sephadex G-200 column was utilized to further separate and purify Tuber Aestivum polysaccharide (TAP) and Tuber Melanosporum polysaccharide (TMP) from TACP and TMCP. The structural characterization results showed that the molecular weight of TAP was 2.18 × 104 kDa, while TMP was 8.79 × 103 kDa. Although the two polysaccharide components were mainly composed of mannose (Man) and glucose (Glc), the molar ratio of Man and Glc in TAP was 14.76: 12.31, with a molar ratio of 5.43:10.94 in TMP. Furthermore, the antioxidant activity of two polysaccharide components was evaluated. TAP and TMP could protect porcine jejunal epithelial (IPEC-J2) cells from oxidative damage by H2 O2 , but TAP exhibited stronger antioxidant effects. It was mainly reflected that TAP could increase the secretion level of intracellular antioxidant enzymes (superoxide dismutase and catalase) in IPEC-J2 cells, and had a significant effect on the total antioxidant capacity of cells. The reactive oxygen species and malondialdehyde had better scavenging ability at the concentration of 20µg/ml.Thedifference between TAP and TMP may be due to the dissimilar structure. Its structure-activity relationship needs further study. PRACTICAL APPLICATION: The structure of TAP and TMP were different, and TAP had higher molecular weight. Besides, TAP and TMP can protect IPEC-J2 cells from oxidative stress, providing a theoretical basis for developing potential antioxidant drugs of practical significance.

Cellular Iron Imbalance Alters Metabolite Profile in Gut Epithelial Cells

ObjectiveThe study aims to investigate the metabolic profile and proliferative activity of small intestinal enterocytes in response to dietary iron deficiency and excess. We hypothesize that iron imbalance affects cellular iron, energy and fatty acid metabolism and alters cell proliferation.MethodsA porcine jejunal epithelial cell line (IPEC‐J2) was used as an in vitro model and cultured in DMEM/F12 containing 10% fetal bovine serum and 1% antibiotics. Cells were treated with the complete growth media for as control (CON), or the growth media supplemented with 500 µM Deferiprone (DFP) to induce iron deficiency (ID), 10 µM ferric ammonium citrate (FAC) to induce iron overload, or 500 µM DFP followed by 10 µM FAC in treatment for iron deficiency and replenishment (IDIR). An XTT assay determined doses of 500 µM of DFP and 10 µM of FAC as non‐cytotoxic. The relative expression of genes encoding iron regulatory proteins and zinc transporters were determined at 24, 48, 72, and 96 hours of treatment using RT‐qPCR. A BrdU assay was used to assess the proliferative activity of cells cultured after 48 hours of IE, ID or control treatments. Cell lysate was extracted after 48 hours of treatment media and analyzed for untargeted metabolomics using GC coupled to time‐of‐flight MS. Data were subjected to two‐way ANOVA mixed‐effect analysis with Geisser‐Greenhouse correction and statistical significance was determined at p&lt;0.05.ResultsThe relative expression of transferrin receptor 1 was significantly higher in IE compared to ID (P &lt; 0.05) regardless of the time of exposure. Iron treatment did not affect the expression of cytochrome B reductase (CYBRD1), divalent metal transporter 1 (DMT1), Solute Carrier Family 39 (ZIP14), or ferritin light chain (FTL). FPN expression increased significantly at 96 hour post DFP treatment compared to 24 hours. (P &lt;0.05). Iron deficiency gradually increased the expression of CYBRD1, DMT1 and FTL with the increase of exposure time (Ptime &lt; 0.05). The results suggested a compensatory induction of genes associated with iron uptake in IPEC‐J2 cells in response to extended exposure to iron deficiency. BrdU assay revealed that ID significantly reduced the rate of DNA replication compared to IE (P &lt; 0.05), which did not alter cell proliferation rate in comparison with IR. Metabolome analysis revealed that iron treatment altered 71 metabolites (FDR &lt; 0.1), among which 19 were mapped in the user database. In comparison with CON, the most drastic changes in metabolite profile were observed in ID‐treated cells and 33 altered metabolites changed to an opposite direction in IE‐ and ID‐treated cells. Iron replenishment dampened or reversed changes of metabolites affected by ID. The profile of altered metabolites highlighted reduction of cytidine‐5‐monophosphate and changes in glucose metabolism (e.g. glucuronic acid and glucose‐1‐phosphate), the Kreb’s cycle (e.g. citric acid and aconitic acid) suggesting a significant impact of ID on cellular energy metabolism.ConclusionIntracellular iron status can have a significant impact on the cellular metabolism in enterocytes. Given the absorptive function of the enterocyte, whether and how ID and IE affect nutrient absorption in the intestinal epithelium and proliferative activity of crypt‐based intestinal stem cells warrants future study.

Citral alleviates peptidoglycan‐induced inflammation and disruption of barrier functions in porcine intestinal epithelial cells

Peptidoglycan (PGN) is a major polymer in bacterial cell walls and may constrain gut functionality and lower intestinal efficiencies in livestock. Citral has been reported to exhibit antibacterial and anti-inflammatory biological activities, improving the gastrointestinal function of swine. However, the protective effect of citral against PGN-elicited cellular responses and possible underlying mechanisms are unknown. In this study, the porcine jejunal epithelial cell line (IPEC-J2) was challenged with PGN from Staphylococcus aureus (S. aureus) or Bacillus subtilis (B. subtilis) to explore PGN-induced inflammatory responses. Our data showed that the inflammatory response stimulated by PGN from harmful bacteria (S. aureus) was more potent than that from commensal bacteria (B. subtilis) in IPEC-J2 cells. Based on the inflammatory model by PGN from S. aureus, it was demonstrated that PGN could significantly induce inflammatory cytokine production and influence nutrient absorption and barrier function in a dose-dependent manner. However, the PGN-mediated immune responses were remarkably suppressed by citral. In addition, citral significantly attenuated the effect of PGN on the intestine nutrient absorption and barrier function. The expression of TLR2 was strongly induced by PGN stimulation, which was suppressed by citral. All data nominated that citral downregulated PGN-induced inflammation via TLR2-mediated activation of the NF-κB signaling pathway in IPEC-J2 cells. Furthermore, the results also indicate that the PGN degradation through the inclusion of enzymes (e.g., muramidase) as well as the inclusion of citral for attenuating inflammation may improve pig gut health and functionality.

Nano chitosan-zinc complex improves the growth performance and antioxidant capacity of the small intestine in weaned piglets.

The present study was conducted to test the hypothesis that dietary supplementation with a nano chitosan-zinc complex (CP-Zn, 100 mg/kg Zn) could alleviate weaning stress in piglets challenged with enterotoxigenic Escherichia coli K88 by improving growth performance and intestinal antioxidant capacity. The in vivo effects of CP-Zn on growth performance variables (including gastrointestinal digestion and absorption functions and the levels of key proteins related to muscle growth) and the antioxidant capacity of the small intestine (SI) were evaluated in seventy-two weaned piglets. The porcine jejunal epithelial cell line IPEC-J2 was used to further investigate the antioxidant mechanism of CP-Zn in vitro. The results showed that CP-Zn supplementation increased the jejunal villus height and decreased the diarrhoea rate in weaned piglets. CP-Zn supplementation also improved growth performance (average daily gain and average daily feed intake), increased the activity of carbohydrate digestion-related enzymes (amylase, maltase, sucrase and lactase) and the mRNA expression levels of nutrient transporters (Na+-dependent glucose transporter 1, glucose transporter type 2, peptide transporter 1 and excitatory amino acid carrier 1) in the jejunum and up-regulated the expression levels of mammalian target of rapamycin (mTOR) pathway-related proteins (insulin receptor substrate 1, phospho-mTOR and phospho-p70S6K) in muscle. In addition, CP-Zn supplementation increased glutathione content, enhanced total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-px) activity, and reduced malondialdehyde (MDA) content in the jejunum. Furthermore, CP-Zn decreased the content of MDA and reactive oxygen species, enhanced the activity of T-SOD and GSH-px and up-regulated the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway-related proteins (Nrf2, NAD(P)H:quinone oxidoreductase 1 and haeme oxygenase 1) in lipopolysaccharide-stimulated IPEC-J2 cells. Collectively, these findings indicate that CP-Zn supplementation can improve growth performance and the antioxidant capacity of the SI in piglets, thus alleviating weaning stress.

Selenium deficiency induced necroptosis, Th1/Th2 imbalance, and inflammatory responses in swine ileum.

Selenium (Se) deficiency has a significant impact on the swine breeding industry by inducing digestive system damage and diarrhea. However, the molecular mechanism remains unclear. Our objectives were to investigate if different amounts of necroptosis, inflammatory responses, and T helper cell 1/T helper cell 2 (Th1/Th2) imbalances were induced by Se deficiency in intestinal porcine jejunal epithelial cells (IPEC-J2) and swine ileum tissue. Therefore, Se-deficient models were successfully established both in vitro and in vivo. In the current study, the cell morphological observation results showed that Se deficiency seriously affected the growth and differentiation of IPEC-J2 cells. Moreover, the necroptosis staining and histomorphology observation results showed that the number of necroptotic cells increased significantly, and the ileal tissue exhibited abnormal structures, including necroptotic features and inflammatory cell infiltration, in the Se-deficient group. Furthermore, Se deficiency resulted in accelerated cell necroptosis by increasing (p < .05) the expression of genes related to the tumor necrosis factor-α pathway at both the protein and messenger RNA (mRNA) levels compared to the control group. Moreover, the relative mRNA and protein expression of the inflammatory genes and their responses to dietary Se deficiency were consistent with the resultant Th1/Th2 imbalances in vitro and in vivo. Taken together, the results suggested that Se deficiency caused necroptosis, inflammatory responses, and abnormal expression of cytokines in swine ileum tissue. These findings might help us to explain the damage induced by Se deficiency to the digestive system of swine.

Lactobacillus casei ATCC 393 alleviates Enterotoxigenic Escherichia coli K88-induced intestinal barrier dysfunction via TLRs/mast cells pathway

AimsMast cells play a crucial role in gastrointestinal physiology and pathophysiology. This study was conducted to investigate the role of mast cells (MCs) in the protective effect of Lactobacillus casei ATCC 393 (L. casei ATCC 393) on intestinal barrier function. Main methodsThe regulatory effect of L. casei ATCC 393 on intestinal barrier dysfunction and MCs activation induced by Enterotoxigenic Escherichia coli K88 (ETEC K88) were evaluated by porcine mucosal mast cells (PMMCs)-porcine jejunal epithelial cells (IPEC-J2)-L. casei ATCC 393 co-culture experiments in vitro and MCs stabilizer drug experiment in vivo. Key findingsResults showed that L. casei ATCC 393 pretreatment effectively alleviated the reduction of cell viability and increase of permeability in ETEC K88-infected IPEC-J2 cells. L. casei ATCC 393 pretreatment inhibited the increase of proinflammatory cytokines and some other MCs mediators, and decrease of anti-inflammatory cytokines in ETEC K88-infected PMMCs. Cromolyn sodium or L. casei ATCC 393 prevented ETEC K88-induced increase of intestinal epithelial cell permeability in IPEC-J2 cells when co-cultivation with PMMCs. Furthermore, cromolyn sodium or L. casei ATCC 393 pretreatment attenuated ETEC K88-induced increase of MCs mediators, mast cell proteases (MCPs) and carboxypeptidase A3 (CPA3) mRNA levels, and down-regulation of tight junction proteins, Toll-like receptor 2 and 4 (TLR2 and TLR4) expression levels in mice challenged by ETEC K88. SignificanceThese results indicated that intestinal barrier dysfunction caused by ETEC K88 was mediated by intestinal mast cell activation which can be prevented by L. casei ATCC 393 via TLRs signaling pathway.

The Role of Nrf2 Signaling Pathway in Eucommia ulmoides Flavones Regulating Oxidative Stress in the Intestine of Piglets.

Eucommia ulmoides flavones (EUF) have been demonstrated to alleviate oxidative stress and intestinal damage in piglets, but their effect target is still poorly understood. NF-E2-related factor 2 (Nrf2) pathway plays a very important role in the defense mechanism. This study was designed to investigate the regulation of EUF on the Nrf2 pathway and inhibition of Nrf2 on oxidative stress in the intestine of piglets. An in vivo study was conducted in weaned piglets treated with basal diet, basal diet+diquat, and 100 mg/kg EUF diet+diquat for 14 d to determine Nrf2 and Keap1 protein expressions, as well as downstream antioxidant gene mRNA expression. An in vitro study was performed in a porcine jejunal epithelial cell line to investigate the effect of inhibiting Nrf2 on cell growth and intracellular oxidative stress parameters. The results showed that the supplementation of EUF decreased the oxidized glutathione (GSSG) concentration and the ratio of GSSG to glutathione (GSH) but increased the protein expressions of nuclear Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) as well as mRNA expression of heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO-1), and glutamate cysteine ligase catalytic subunit (GCLC) in the small intestinal mucosa of diquat-challenged piglets. When Nrf2 was inhibited by using ML385, cell viability, cellular antioxidant activities, expressions of nuclear Nrf2 and Keap1 protein, and downstream antioxidant enzyme (HO-1, NQO-1, and GCLC) mRNA were decreased in paraquat-treated enterocytes. These results showed that the Nrf2 signaling pathway played an important role in EUF-regulating oxidative stress in the intestine of piglets.

Polyunsaturated Fatty-acids Modulate Inflammatory Response in in Vitro Models of Enteric Disease (P19-006-19)

ObjectivesDietary intervention is a potential therapy for enteric disease. The objective of this study was to investigate an in vitro model of enteric disease how long-chain polyunsaturated fatty acids (LC-PUFA) could modulate intestinal inflammation. Using neonatal pig intestinal cells, IPEC-J2, in a challenge model the inflammatory response of the enterocytes was measured when challenged with bacterial flagellin. The goal of the experiment was to determine modulation of inflammation by LC-PUFA. MethodsNeonatal pig intestinal cells, IPEC-J2, were cultured in DMEM/F12 media supplemented with 5% FBS, ITS, EGF and antibiotics. Cells were plated at a cell density of 3 × 105 cells/well and grown to 100% confluence. Porcine jejunal epithelial cells were supplemented with 30 μM PUFA for 96 h. Cells were then stimulated with 10 ng/ml flagellin and cell RNA and media were harvested. TNFa mRNA and protein were analyzed via real-time PCR and ELISA, respectively. Data were analyzed using SAS using an ANOVA and reporting means ± SE. ResultsFollowing supplementation arachidonic acid (ARA) and eicosapentanoic acid (EPA) incorporation increased from 0.96 to 5.47 and 0.03 to 1.23% of FA, respectively (P < 0.05). Flagellin treatment increased TNFα mRNA expression over a time course of 0–24 h by ∼2-fold numerically, while TNFa protein secretion reached peak levels at 2 h followed by a 50% decrease at 24 h. ARA showed a trend in reduction of TNFa protein secretion compared to unenriched and EPA enriched enterocytes following flagellin stimulation (P = 0.08). ConclusionsThese data demonstrate increased supplementation of LC-PUFAs potentially modulate proinflammatory immune response in epithelial cells. Funding SourcesOhio Agriculture Research and Development SEEDS grant.

Caprylic acid and nonanoic acid upregulate endogenous host defense peptides to enhance intestinal epithelial immunological barrier function via histone deacetylase inhibition

The intestinal epithelial barrier plays a critical role in the etiopathogenesis of ulcerative colitis. This study aims to explore the potential effects and underlying mechanisms of medium chain fatty acids (caprylic acid and nonanoic acid) on intestinal epithelial barrier function. Using the porcine jejunal epithelial cell line IPEC-J2, a well-established model, challenged with Escherichia coli ATCC 43889 (O157:H7), we found that treatment with caprylic acid (C8) and nonanoic acid (C9) significantly reduced bacterial translocation, enhanced antibacterial activity, and remarkably increased the secretion of porcine β-defensins 1 (pBD-1) and pBD-2. Mechanistically, like TSA (a histone deacetylase inhibitor), C8 and C9 attenuated the activity of the classical histone deacetylase pathway to facilitate the acetylation of histone 3 lysine 9 (H3K9) at the promoters pBD-1 and pBD-2, and consequently augmented the gene expression of pBD-1 and pBD-2. In conclusion, with their combined antibacterial and defense peptide-induced roles, the use of C8 and C9 may provide a novel method to protect the intestinal barrier of animals and humans from bacterial infection.

Selenised yeast sources differ in their capacity to protect porcine jejunal epithelial cells from cadmium-induced toxicity and oxidised DNA damage.

In recent years there has been increasing interest in the use of selenised yeast (Se-Y) as an antioxidant feed supplement. Here, three selenised yeast products are differentiated in terms of bioefficiency and the ameliorative effect on Cadmium (Cd) toxicity in porcine epithelial cells. A porcine digestion in vitro model was chosen to more accurately simulate the bioavailability of different Se-Y preparations, allowing a comprehensive understanding of the bio efficiency of each Se-Y compound in the porcine model. To elucidate a possible mechanism of action of selenium a number of bioassays were applied. Levels of Se dependent antioxidant enzymes (glutathione peroxidase and thioredoxin reductase) were evaluated to analyze the ROS neutralizing capacity of each Se-Y compound. The effects of Se-Y sources on Cd-induced DNA damage and apoptosis-associated DNA fragmentation was assessed using comet and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, respectively. Lesion-specific DNA damage analysis and in vitro DNA repair assay determined the DNA repair capacity of each Se-Y source. The results presented in this study confirm that the ability of different commercially available Se-Y preparations to enhance a range of cellular mechanisms that protect porcine gut epithelial cells from Cd-induced damage is concentration-dependent and illustrates the difference in bioefficiency of different Se-Y compounds.

Optimal branched-chain amino acid ratio improves cell proliferation and protein metabolism of porcine enterocytesin in vivo and in vitro.

The aim of this study was to investigate the effects of different branched-chain amino acid (BCAA) ratios on intestinal cell proliferation and protein metabolism. This in vivo study was conducted in growing pigs (average initial body weight = 9.85 ± 0.35 kg) exposed to different leucine, isoleucine, and valine (Leu:Ile:Val) ratios (1:1:1, 1:0.75:0.75, 1:0.51:0.63, and 1:0.25:0.25) in 17% crude protein (CP) diets for 45 d to determine intestinal morphology, cell proliferation, and amino acid transporter mRNA expression. This in vitro study was performed in porcine jejunal epithelial cell line to investigate the effects of different BCAA ratios (0, 1:0.25:0.25, 1:0.5:0.5, and 1:1:1) on cell proliferation, AA transporter expression. and protein turnover. Relative to the positive control (Leu:Ile:Val = 1:0.51:0.63, 20% CP), the 1:0.75:0.75 group (17% CP) significantly increased the jejunal villus height, the percentage of proliferating cell nuclear antigen-positive cells and decreased the ileal crypt depth (P <0.05). Moreover, most AA concentrations and AA transporter expression in the jejunal and ileal mucosa in 17% CP diets were increased to the levels similar to those in the positive control. Additionally, this in vitro study showed that the BCAA ratio of 1:0.25:0.25 significantly increased intestinal cell proliferation, mRNA expression of AA transporters, AA concentrations, and protein turnover (P <0.05). These findings suggest that optimal BCAA ratio (in vivo = 1:0.75:0.75; in vitro = 1:0.25:0.25) would improve intestinal morphology and cell proliferation, increase intestinal AA absorption through mediating expression of intestinal AA transporters, and promote intestinal protein turnover. The findings yield new insights into the use of BCAAs in humans and animals.

Zinc strengthens the jejunal barrier by reversibly tightening the paracellular route.

During the postweaning period, piglets are prone to gastrointestinal infections. The resulting impairment of intestinal barrier function may cause diarrhea associated with growth retardation or even death of piglets. Orally applied Zn is commonly used to prevent and treat diarrhea, but its mode of action still needs to be elucidated. To analyze the molecular mechanism whereby Zn acts on porcine intestinal barrier function, ex vivo studies on piglet jejunum and accompanying in vitro studies on a porcine jejunal epithelial cell line, IPEC-J2/PS, were performed with electrophysiological tools. Feeding pharmacological Zn doses exerted no significant electrophysiologically ascertainable short- and long-term effects on jejunal barrier function ex vivo. However, in IPEC-J2/PS, basolateral Zn was cytotoxic since its application caused a release of lactate dehydrogenase and an irreversible breakdown of the epithelial barrier. In contrast, apical Zn application caused an immediate increase in paracellular resistance and a decrease in permeability to the paracellular marker fluorescein, reflecting overall barrier strengthening in vitro. Apical effects were fully reversible upon washout. This indicates that Zn supplemented to feed was completely washed out during ex vivo jejunum preparation. We conclude that there is no evidence for long-term barrier effects through prophylactic Zn supplementation and that extracellular Zn acts acutely and reversibly from the apical side via tightening the paracellular route, thus counteracting leak-flux diarrhea.NEW & NOTEWORTHY Therapeutically administered Zn successfully treats diarrhea in veterinary and human medicine. Here we present data that Zn strengthens the porcine jejunal epithelial barrier by reversibly tightening the paracellular route for inorganic ions and small solutes. Acute or long-lasting Zn effects on transcellular transport (Cl- secretion) were not detected. We therefore conclude that Zn is useful for acutely treating leak-flux diarrhea rather than secretory diarrhea. Suitability as prophylactic feed supplement, however, is questionable.

Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells.

Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1mM) of butyrate had no effect on cell proliferation. However, at 5 and 10mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.