Intestinal Epithelial Cell Barrier Research Articles

Herbal cake-partitioned moxibustion improves intestinal epithelial barrier dysfunction by suppressing TNF-α-mediated apoptosis pathway of intestinal epithelium in rats with Crohn's disease

To observe the effect of herbal cake-partitioned moxibustion (Moxi) on tumor necrosis factor (TNF)-α/ TNF receptor 1 (TNFR1)-associated death domain (TRADD) / Fas-associated death domain (FADD) pathway-mediated apoptosis of intestinal epithelial cells in Crohn's disease (CD) rats, so as to explore its underlying mechanisms in the treatment of CD. Forty-eight SD male rats were randomly divided into normal, model, Moxi and medication groups (n＝12 rats in each). The CD model was established by intra-annual perfusion of 2，4，6-trinitrobenzene sulfonic acid (TNBS) solution (TNBS∶50% alcohol＝2∶1, 3 mL/kg), once every 7 days, 4 times altogether. For rats of the Moxi group, moxibustion was given to "Tianshu" (ST25) and "Qihai" (CV6), two moxa-cones every time, once daily for 10 days. For rats of the medication group, intragastric perfusion of mesalazine solution was given twice daily for 10 days. After the treatment, the colonic epithelium tissue was sampled. The epithelial cells were purified and cultured to establish an in vitro intestinal epithelial barrier, and added with TNF-α (a pro-inflammatory factor, 100 ng/mL) in the culture medium for 24 h for making an increased epithelial permeability model. The permeability of intestinal epithelial cell barrier was evaluated by detecting the fluorescence yellow transmittance of the TNF-α-incubated cell medium. Western blot was used to detect the expression levels of TNFR1, TRADD, receptor-interacting protein 1 (RIP1), FADD and zinc finger protein A20 (A20, a ubiquitination enzyme for inhibiting activation of TRADD and RIP1) of the cultured intestinal epithelium cells. The apoptosis of the TNF-α-incubated intestinal epithelial cells was detected by flow cytometry. After modeling and compared with the normal group, the fluorescence yellow transmittance of intestinal epithelia cells, apoptosis rate, and expression levels of TNFR1, TRADD, and RIP1 proteins were significantly increased (P<0.001, P<0.01), and the expression of A20 was significantly decreased (P<0.01) in the model group. In comparison with the model group, the fluorescence yellow transmittance of intestinal epithelial cells, the apoptosis rate and expression levels of TRADD, RIP1 and FADD were remarkably down-regulated (P<0.001, P<0.01), and the expression of A20 was significantly up-regulated (P<0.01) in both the Moxi and medication groups. Herbal cake-partitioned moxibustion may down-regulate the permeability of intestinal epithelial barrier and the apoptosis of intestinal epithelial cells by way of suppressing TNF-α-mediated cellular apoptosis pathway of intestinal epithelium in CD rats.

Bisphenol A inhibits mucin 2 secretion in intestinal goblet cells through mitochondrial dysfunction and oxidative stress

AimsBisphenol A (BPA) can induce intestinal epithelial cell barrier dysfunction; however, its effects on the intestinal mucus barrier remain unclear. We used LS174T cells as a model to investigate the effects of BPA on the functions of intestinal goblet cells. Main methodsThis study used CCK-8, flow cytometry, ELISA and real-time PCR to investigate the effects of BPA on mitochondrial dynamics, oxidative stress and apoptosis in goblet cells. In addition, mucin synthesis and secretion were evaluated using PAS staining and a PAS assay, respectively. Key findingsOur results indicate that BPA reduced cell viability in a time- and concentration-dependent manner. BPA induced mitochondrial dysfunction, as indicated by the depolarization of the mitochondrial membrane potential, inhibition of mitochondrial respiratory chain complex enzyme activity and reduction of ATP production. Moreover, BPA caused oxidative stress by significantly increasing the accumulation of ROS, as well as oxidative stress products, and reducing the antioxidant capacity. Furthermore, BPA induced intestinal goblet cell apoptosis, accompanied by increased DNA fragmentation, caspase-3, -8, -9,-10 gene expression and enzyme activity. Additionally, BPA inhibited the synthesis and secretion of mucin 2. SignificanceOur data suggest that BPA affected the secretory function of intestinal goblet cells by inducing mitochondrial dysfunction, oxidative stress, and apoptosis.

The impact of tomato fruits containing multi-walled carbon nanotube residues on human intestinal epithelial cell barrier function and intestinal microbiome composition.

Carbon nanomaterials (CNMs) can positively regulate seed germination and enhance plant growth. However, clarification of the impact of plant organs containing absorbed CNMs on animal and human health is a critical step of risk assessment for new nano-agro-technology. In this study, we have taken a comprehensive approach to studying the effect tomato fruits derived from plants exposed to multi-walled carbon nanotubes (CNTs) have on gastrointestinal epithelial barrier integrity and their impact on the human commensal intestinal microbiota using an in vitro cell culture and batch human fecal suspension models. The effects of CNTs on selected pure cultures of Salmonella enterica Typhimurium and Lactobacillus acidophilus were also evaluated. This study demonstrated that CNT-containing fruits or the corresponding residual level of pure CNTs (0.001 μg ml-1) was not sufficient to initiate a significant change in transepithelial resistance and on gene expression of the model T-84 human intestinal epithelial cells. However, at 10 μg ml-1 concentration CNTs were able to penetrate the cell membrane and change the gene expression profile of exposed cells. Moreover, extracts from CNT-containing fruits had minimal to no effect on human intestinal microbiota as revealed by culture-based analysis and 16S rRNA sequencing.

Development of mutlifunctional nanoparticles self-assembled from trimethyl chitosan and fucoidan for enhanced oral delivery of insulin

Oral administration is a highly attractive approach for the delivery of protein drugs. However, oral protein therapeutics typically exhibit extremely poor bioavailability due to the harsh gastrointestinal (GI) environments and low permeability of protein across the intestinal barrier. Trimethyl chitosan (TMC) shows excellent mucoadhesive and absorption-enhancing properties while fucoidan (FD) has hypoglycemic effects and can prevent diabetes-related complications. Here we report, for the first time, that TMC combined with FD can be developed to a mutlifunctional nanoplatform for enhancing the transepithelial permeation of insulin through the intestinal epithelial cell barrier and inhibiting the α-glucosidase activity. TMC and FD self-assembled into spherical nanoparticles (NPs) for insulin encapsulation. TMC/FD NPs protected insulin against degradation by releasing insulin in a pH-dependent manner in the gastrointestinal tract fluids. The NPs were able to modulate the barrier function of the Caco-2 intestinal epithelial cell monolayer, and enhance paracellular transport of insulin across the intestinal barrier. TMC/FD NPs also showed α-glucosidase inhibitory activity, with an inhibition ratio of 33.2% at 2 mg/mL. The superior transepithelial absorption enhancing property of the TMC/FD NPs is expected to combine in the future with the functions of fucoidan against diabetes-related complications for development of advanced mutlifunctional therapeutic platforms for diabetes.

Neutrophils Promote Amphiregulin Production in Intestinal Epithelial Cells through TGF-β and Contribute to Intestinal Homeostasis.

Neutrophils are the first responders to sites of inflammation when the intestinal epithelial barrier is breached and the gut microbiota invade. Despite current efforts in understanding the role of neutrophils in intestinal homeostasis, the complex interactions between neutrophils and intestinal epithelial cells (IECs) is still not well characterized. In this study, we demonstrated that neutrophils enhanced production of amphiregulin (AREG), a member of the EGFR ligand family, by IECs, which promoted IEC barrier function and tissue repair. Depletion of neutrophils resulted in more severe colitis in mice because of decreased AREG production by IECs upon dextran sodium sulfate (DSS) insult. Administration of AREG restored epithelial barrier function and ameliorated colitis. Furthermore, neutrophil-derived TGF-β promoted AREG production by IECs. Mechanistically, TGF-β activated MEK1/2 signaling, and inhibition of MEK1/2 abrogated TGF-β-induced AREG production by IECs. Collectively, these findings reveal that neutrophils play an important role in the maintenance of IEC barrier function and homeostasis.

Probiotic Bacillus enhance the intestinal epithelial cell barrier and immune function of piglets.

Bacillus is widely used in the livestock industry. This study was designed to evaluate the effects of probiotic Bacillus amyloliquefaciens SC06 (Ba), originally isolated from soil, in piglets diet as an alternative to antibiotics (aureomycin), mainly on intestinal epithelial barrier and immune function. Ninety piglets were divided into three groups: G1 (containing 150 mg/kg aureomycin in the diet); G2 (containing 75 mg/kg aureomycin and 1×108 cfu/kg Ba in the diet); G3 (containing 2×108 cfu/kg Ba in the diet without any antibiotics). The results showed that, compared with the antibiotic group (G1), villus length, crypt depth and villus length/crypt depth ratio of intestine significantly increased in the G2 and G3 groups. In addition, intestinal villi morphology, goblet-cell number, mitochondria structure and tight junction proteins of intestinal epithelial cells in G2 and G3 were better than in G1. The relative gene expression of intestinal mucosal defensin-1, claudin3, claudin4, and human mucin-1 in G3 was significantly lower, while the expression of villin was significantly higher than in the antibiotic group. Probiotic Ba could significantly decrease serum interferon (IFN)-α, IFN-γ, interleukin (IL)-1β, and IL-4 levels, whereas increase tumour necrosis factor (TNF)-α and IL-6 secretion. Ba could also significantly decrease cytokines TNF-α, IFN-γ, IL-1β, and IL-4 level in liver, whereas it significantly increased IFN-α. Furthermore, replacing antibiotics with Ba also significantly down-regulated gene expression of TNF and IL-1α in intestinal mucosa, but up-regulated IL-6 and IL-8 transcription. Dietary addition of Ba could significantly reduce the gene expression of nuclear factor kappa beta (NFκB)-p50 and Toll-like receptor (TLR)6, while there was no significant difference for that of myeloid differentiation primary response 88, TNF receptor-associated factor-6, nucleotide-binding oligomerisation domain-containing protein 1, TLR2, TLR4, and TLR9. Taken together, our findings demonstrated that probiotic Ba could increase the intestinal epithelial cell barrier and immune function by improving intestinal mucosa structure, tight junctions and by activating the TLRs signalling pathway.

Total polysaccharides of the Sijunzi decoction attenuate tumor necrosis factor-α-induced damage to the barrier function of a Caco-2 cell monolayer via the nuclear factor-κB-myosin light chain kinase-myosin light chain pathway.

AIMTo explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction (TPSJ) on the epithelial barriers in vitro.METHODSCaco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance (TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respectively used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. Western blotting was also used to evaluate the cellular expression of myosin light chain (MLC), phosphorylated MLC (pMLC), MLC kinase (MLCK), and nuclear factor (NF)-κB p65.RESULTSTPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cytokines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor (TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65.CONCLUSIONOur results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.

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

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

Hypoxanthine as a Checkpoint Metabolite in Epithelial Energy Modulation and Barrier Function

A primary function of intestinal epithelial cells (IECs) is to provide a selectively permeable barrier to protect the host from luminal microbiota and antigens, and to mediate the flux of nutrients, fluid, and waste across that barrier. Loss of intestinal barrier function is a hallmark of inflammatory bowel disease (IBD). Dysregulation of IEC barrier in IBD coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically‐vulnerable state of physiologic hypoxia. The precise mechanism(s) by which metabolic pathways contribute to and control inflammation have yet to be elucidated. To delineate barrier‐related energy flux, an HPLC‐based profiling method was developed to track changes in energy and adenylate metabolites during intestinal epithelial barrier development and restitution. Cultured epithelia demonstrated a remarkable capacity to maintain ATP and phosphocreatine during barrier development. Further epithelial cell profiling identified a significant capacity for cultured human IECs to salvage hypoxanthine (Hpx) and shift their metabolic equilibrium toward the production of ATP and phosphocreatine. In a consistent manner, we have demonstrated that Hpx‐mediated shifts in metabolism result in enhanced epithelial barrier formation, increased epithelial restitution, and improved wound healing. Modulation of cellular energetics by Hpx were recapitulated with in vivo murine models, where orally‐delivered Hpx increased colonic ATP levels. Guided by an unbiased microarray analysis of Hpx treated human IECs, we identified a number of target genes important in mucosal differentiation, energy metabolism, and reactive oxygen species mediation. As the ATP consumed by IECs under energetic stress is shuttled to and pooled as Hpx, it appears not only as a central energy metabolite, but also as a regulator of gene expression. Taken together, our ongoing studies provide strong evidence that Hpx functions as a central, checkpoint energy metabolite in IEC function.Support or Funding InformationNIH/NIDDKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P115 THE IBD CANDIDATE GENE, PTPN2, RESTRICTS INTESTINAL BARRIER DEFECTS AND CLAUDIN-2 EXPRESSION VIA STAT INHIBITION

Loss-of-function mutations in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene locus increase IBD susceptibility. We reported that PTPN2 knockdown (PTPN2-KD) in intestinal epithelial cells (IECs): (i) increased IFN-g activation of STAT signaling; (ii) reduced IEC barrier function; (iii) increased expression of the tight junction (TJ) protein, claudin-2, by IFN-g; and that the claudin-2 promoter contains a STAT binding sequence. Increased claudin-2 expression occurs in IBD and increases permeability to cations thus reducing transepithelial electrical resistance (TER). To confirm PTPN2 regulation of barrier function in vivo and identify the mechanisms of STAT regulation of claudin-2 expression. In Ussing chamber studies, TER was significantly reduced in cecum from Ptpn2 knockout (KO) and heterozygous (Het) mice vs. wild-type (WT) littermates indicating increased electrolyte permeability. Immunostaining and western blotting showed progressive increases in claudin-2 expression and STAT-1 phosphorylation (Y701) in Het and KO mice vs. wild-type intestine. Claudin-2 was also elevated in intestinal enteroids from Ptpn2-deficient mice. STAT-1 siRNA significantly decreased the elevated claudin-2 expression in PTPN2-KD IECs. Chromatin immunoprecipitation (ChIP) analysis showed increased STAT-1 binding to the claudin-2 promoter in PTPN2-KD vs. control cells. Claudin-2 promoter-luciferase reporter construct activity was significantly elevated in PTPN2-KD vs. Con-shRNA IEC and was further increased by IFN-g (1000 U/ml). This effect was lost by two different mutations of the STAT binding site thus confirming the requirement of STAT binding for increased claudin-2 transcription in PTPN2-KD IECs. Loss of functional PTPN2 increases susceptibility to inflammation-induced tight junction reorganization, providing an explanation for the association of PTPN2 mutants with UC and CD.

The Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis in Intestinal Epithelial Cells Regulates Intestinal Barrier Function During Intestinal Epithelial Cells–CD4+T-Cell Interactions

Background/Aims: Epithelial cells line the intestinal mucosa and form an important barrier for maintaining host health. This study aimed to explore the mechanism of the Sphingosine-1-phosphate (S1P)/Sphingosine-1-phosphate receptor 2 (S1PR2) pathway in intestinal epithelial cells (IECs) that participate in the intestinal barrier function. Methods: In this study, we constructed a knockout of the S1PR2 gene in mice, and Dextra sulfate sodium (DSS) was used to induce colitis. We isolated IECs from wild type (WT) and S1PR2–/– mice, and the endogenous expression of S1PR2 and Zonula occludens 1 (ZO-1) in IEC were detected by Western blot. Next, the major histocompatibility complex II (MHC-II) expression was analyzed by reverse transcription quantitative real-time (RT-qPCR) and flow cytometry. The in vivo and in vitro intestinal permeability were evaluated by serum fluorescein isothiocyanate (FITC) concentration. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) levels in cell suspension were analyzed by enzyme-linked immuno sorbent assay (ELISA). A carboxyfluorescein diacetate succinimidyl ester (CFSE) assay was used to detect the T-cell proliferation in a co-culture system. Results: The intestinal mucosal barrier damage in S1PR2–/– mice was more severe than in the WT mice, and there were more CD4+T-cells in the colon tissue of DSS-treated S1PR2–/– mice. Either the mouse colon carcinoma cell line (CT26. WT) or the IECs upregulated MHC-II expression, which then promoted CD4+T-cell proliferation. The S1P/S1PR2 pathway controlled MHC-II expression to regulate CD4+T-cell proliferation via the extracellular signal-regulated kinase (ERK) pathway. In addition, the IFN-γ that was secreted by CD4+T-cells increased DSS-induced damage of intestinal epithelial cell barrier function. ZO-1 expression was increased by S1P in CT26.WT cells, while S1PR2 antagonist JTE-013 expression was downregulated. However, in CT26.WTsi-S1PR2 cells, S1P had no effect on ZO-1 expression. Conclusions: The S1P/S1PR2 axis in IECs mediated CD4+T-cell activation via the ERK pathway and MHC-II expression to regulate intestinal barrier function.

Lactobacillus plantarum Enhanced IL-22 Production in Natural Killer (NK) Cells That Protect the Integrity of Intestinal Epithelial Cell Barrier Damaged by Enterotoxigenic Escherichia coli.

Interleukin (IL)-22-producing Natural Killer (NK) cells protect the gut epithelial cell barrier from pathogens. A strain of probiotics, Lactobacillus plantarum (L. plantarum, LP), was previously found by our laboratory to significantly improve the mucosal barrier integrity and function of the small intestine in pigs. However, it was unclear whether LP benefited the intestinal mucosal barrier via interactions with the intestinal NK cells. The present study, therefore, was focused on the therapeutic effect of NK cells that were stimulated by LP on attenuating enterotoxigenic Escherichia coli (ETEC)-induced the damage to the integrity of the epithelial cell barrier. The results showed that LP can efficiently increase protein levels of the natural cytotoxicity receptor (NCR) family, and the expression levels of IL-22 mRNA and protein in NK cells. Transfer of NK cells stimulated by LP conferred protection against ETEC K88-induced intestinal epithelial barrier damage in NCM460 cells. We found that NK cells stimulated by LP could partially offset the reduction in NCM460 cell monolayers transepithelial electrical resistance (TEER) caused by ETEC K88, and increase ZO-1 and occludin mRNA and protein expressions by ETEC K88-infected NCM460 cells. Furthermore, adding NK cells that were stimulated by LP to ETEC K88-infected NCM460cells, IL-22R1, p-Stat3, and p-Tyk2 expression by NCM460 cells was increased. Mechanistic experiment showed that NK cells stimulated by LP lost the function of maintaining TEER of NCM460 cells challenged with ETEC K88, when polyclonal anti-IL-22 antibody was used to block IL-22 production. Collectively, our results suggested that LP stimulation of NK could enhance IL-22 production, which might be able to provide defense against ETEC-induced damage to the integrity of intestinal epithelial barrier.

Microbial-Derived Butyrate Promotes Epithelial Barrier Function through IL-10 Receptor-Dependent Repression of Claudin-2.

Commensal interactions between the enteric microbiota and distal intestine play important roles in regulating human health. Short-chain fatty acids (SCFAs), such as butyrate, produced through anaerobic microbial metabolism represent a major energy source for the host colonic epithelium and enhance epithelial barrier function through unclear mechanisms. Separate studies revealed that the epithelial anti-inflammatory IL-10 receptor α subunit (IL-10RA) is also important for barrier formation. Based on these findings, we examined if SCFAs promote epithelial barrier through IL-10RA-dependent mechanisms. Using human intestinal epithelial cells (IECs), we discovered that SCFAs, particularly butyrate, enhanced IEC barrier formation, induced IL-10RA mRNA, IL-10RA protein, and transactivation through activated Stat3 and HDAC inhibition. Loss and gain of IL-10RA expression directly correlates with IEC barrier formation and butyrate represses permeability-promoting claudin-2 tight-junction protein expression through an IL-10RA-dependent mechanism. Our findings provide a novel mechanism by which microbial-derived butyrate promotes barrier through IL-10RA-dependent repression of claudin-2.

The Pseudomonas aeruginosa dnaK gene is involved in bacterial translocation across the intestinal epithelial cell barrier.

Pseudomonas aeruginosa can penetrate through polarized epithelial cell monolayers produced by the human adenocarcinoma cell line Caco-2. We previously identified genes associated with bacterial translocation through Caco-2 cell monolayers by analysing transposon insertion mutants with dramatically reduced penetration activity relative to that of the wild-type P. aeruginosa PAO1 strain. In this study, we focused on the dnaK mutant because the association between this gene and penetration activity is unknown. Inactivation of dnaK caused significant repression of bacterial penetration through Caco-2 cell monolayers, with decreased swimming, swarming and twitching motilities; bacterial adherence; and fly mortality rate; as well as dramatic repression of type III effector secretion and production of elastase and exotoxin A. However, type IV pilus protein PilA expression was not affected. These results suggest that dnaK is associated with bacterial motility and adherence, which are mediated by flagella and pili, and with toxin secretion, which plays a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Inactivation of P. aeruginosa dnaK function may interfere with bacterial translocation and prevent septicaemia caused by P. aeruginosa.

Enteric glial–mediated enhancement of intestinal barrier integrity is compromised by morphine

BackgroundThe opioid epidemic is a growing concern, and emerging evidence suggests that morphine use may be associated with sepsis. Enteric glial cells (EGCs) are the most numerous cell type in the enteric nervous system and regulate gastrointestinal function through the production of trophic factors, including glial-derived neurotrophic factor (GDNF). We sought to determine the effect of morphine on enteric glia and hypothesized that morphine contributes to EGC dysfunction and increased gut permeability. Materials and methodsRat intestinal epithelial cells (IECs) and EGC lines were purchased from ATCC. Immunocytochemistry was used to evaluate the impact of EGCs on IEC barrier proteins and detect the μ-opioid receptor. Co-culture assays were used to determine the effect of EGCs, GDNF, and morphine on barrier integrity. Quantitative polymerase chain reaction and western blotting were performed to determine the impact of morphine in GDNF production. Transepithelial resistance of IEC-6 cell monolayers was measured in the presence of EGC-conditioned media (EGC-CM) and morphine treated EGC-CM using electrical cell impedance sensing. ResultsEGC-CM enhanced tight junction organization in IECs. IEC barrier integrity was enhanced when co-cultured with unstimulated EGCs or with GDNF alone; this barrier protective effect was lost with morphine-treated EGCs. GDNF RNA and protein expression were decreased by morphine treatment. Transepithelial resistance was decreased in IEC confluent monolayers when exposed to morphine-treated EGC-CM compared with control. ConclusionsMorphine compromises intestinal epithelial cell barrier function through a mechanism which appears to involve GDNF. Further studies are warranted to delineate the role of enteric glial cell function in opioid signaling and sepsis.

The light subunit of mushroom Agaricus bisporus tyrosinase: Its biological characteristics and implications

The light subunit of mushroom Agaricus bisporus tyrosinase (LSMT) is a protein of unknown function that was discovered serendipitously during the elucidation of the crystal structure of the enzyme. The protein is non-immunogenic and can penetrate the intestinal epithelial cell barrier, and thus, similar to its structural homologue HA-33 from Clostridium botulinum, may be potentially absorbable by the intestine. LSMT also shares high structural homology with the ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which has been shown to display biological activity against leukemic cancer cells and dendritic cells. Therefore, we evaluated the biological activity of LSMT. An in vitro assay suggested that LSMT presentation to most of the cancer cell lines studied has a negligible effect on their proliferation. However, inhibition of cell growth and a slight stimulation of cell proliferation were observed with breast cancer and macrophage cells, respectively. LSMT appeared to be relatively resistant against proteolysis by trypsin and papain, but not bromelain. Challenges with gastric and intestinal juice suggested that the protein is resistant to gastrointestinal tract conditions. This is the first report on the biological characteristics and implication of LSMT.

Exposure to minimally processed pear and melon during shelf life could modify the pathogenic potential of Listeria monocytogenes

Survival and virulence of foodborne pathogens can be influenced by environmental factors such as the intrinsic properties of food as well as the extrinsic properties that contribute to food shelf life (e.g., temperature and gas atmosphere). The direct contribution of food matrix characteristics on the survival of L. monocytogenes during fresh-cut fruit shelf life is not very well understood. In addition, the gastrointestinal tract is the primary route of listeriosis infection and penetration of the intestinal epithelial cell barrier is the first step in the infection process. Hence, the pathogenic potential of L. monocytogenes, measured as the capability for the organism to survive a simulated gastrointestinal tract and the proportion of cells able to subsequently adhere to and invade differentiated Caco-2 cells, subjected to fresh-cut pear and melon shelf life, was investigated. Samples were inoculated, stored at 10 °C for 7 days and evaluated after inoculation and again after 2 and 7 days of storage. A decrease in L. monocytogenes’ capacity to survive a simulated gastrointestinal tract was observed with increasing storage time, regardless of the fruit matrix evaluated. Furthermore, L. monocytogenes placed on fresh-cut pear and melon was subjected to an attachment and invasion assay after crossing the simulated gastrointestinal tract. After inoculation, pathogen on fresh-cut pear showed 5-fold more capacity to adhere to Caco-2 cells than pathogen on fresh-cut melon. After 2 days of storage, L. monocytogenes grown on fresh-cut melon showed similar adhesive capacity (1.11%) than cells grown on pear (1.83%), but cells grown on melon had the higher invasive capacity (0.0093%). We can conclude that minimally processed melon could represent a more important hazard than pear under the studied shelf life.

Fumonisin B1 affects “in vitro” intestinal epithelial cell barrier integrity and reduces claudin expression

Fumonisin B1 affects “in vitro” intestinal epithelial cell barrier integrity and reduces claudin expression

Herb-partitioned moxibustion upregulated the expression of colonic epithelial tight junction-related proteins in Crohn's disease model rats.

BackgroundHerb-partitioned moxibustion (HPM) at Tianshu (ST25) and Qihai (RN6) has been used to treat Crohn’s disease (CD). Injury to intestinal epithelial tight junctions (TJs) is the leading cause of CD onset with under expression of TJ-related proteins such as occludin, claudin-1, and zonula occludens protein-1 (ZO-1). This study aimed to investigate whether HPM can change the permeability of the intestinal epithelial barrier by affecting the expression of colonic epithelial TJ-related proteins in vitro.MethodsForty-eight male Sprague–Dawley rats were randomly divided into four groups of twelve rats: normal control (NC) group; model control (MC) group; herb-partitioned moxibustion (HPM) group; and mesalazine control (MESA) group. The rats in the latter three groups were given trinitrobenzene sulfonic acid (TNBS) enemas to establish CD models. The HPM group was treated with HPM at Tianshu (ST25) and Qihai (RN6) once daily for 14 consecutive days, while the MESA group was given mesalazine solution (at the proportion of 0.018:1) by lavage twice daily for the same period. After the treatment period, the colon tissues from all groups were partly processed for macroscopic damage assessment and histological observation, and partly purified and cultured in vitro to examine the permeability of the intestinal epithelial cell barrier by trans-epithelial electrical resistance (TEER). Western blot and fluorescence quantitative polymerase chain reaction (FQ-PCR) analyses were performed to observe the expression of occludin, claudin-1, and ZO-1 proteins and mRNAs, respectively.ResultsIn the HPM and MESA groups, the typical CD macroscopic damage, i.e., inflammatory cell infiltration in colonic mucosa and submucosa, submucosal lymphoid follicular hyperplasia, hyperemia and edema, and morphological changes were improved to different degrees in the colonic tissues (HPM, MESA vs. MC for macroscopic score of colonic damage: all P < 0.001). The decreasing tendencies were minor for colonic TEER values (HPM, MESA vs. MC: all P < 0.001), and expression of intestinal epithelial TJ-related proteins (HPM, MESA vs. MC: all P < 0.05) and mRNAs (HPM, MESA vs. MC: all P < 0.05), especially in the HPM group (HPM vs. MESA for TEER values: P < 0.001).ConclusionsHPM at Tianshu (ST25) and Qihai (RN6) upregulated the expression of occludin, claudin-1, and ZO-1 in TNBS-induced CD model rats.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0090-0) contains supplementary material, which is available to authorized users.

The impact of lemon pectin characteristics on TLR activation and T84 intestinal epithelial cell barrier function

Sufficient dietary fibre intake reduces the risk of several diseases, but the mechanisms linking fibre structure and health effects remain unclear. To analyse the influence of the amount of methyl groups esterified to the backbone, lemon pectins of different degrees of methyl esterification (30, 56, and 74DM) were studied for immune receptor activating potential and epithelial barrier protection. In reporter assays, the pectins demonstrated TLR/MyD88 dependent activation of NF-κB/AP-1, which increased with increasing pectin DM. To analyse the importance of backbone structure, the pectins were enzymatically digested into oligomers, which abrogated TLR activating potential. The 30 and 74DM pectins induced strong protection of the epithelial barrier measured by T84 transepithelial electrical resistance (TEER). These results indicate that activation of immune cells by lemon pectins is TLR dependent, and the intact polymer backbone is indispensable for activation. In addition, DM is a determining factor in activating potential and epithelial barrier protective effects.