Intestinal Cell Barrier Research Articles

Protective effects of galacto-oligosaccharide and polydextrose on Caco-2 intestinal cell barrier injury model

To explore the protective effect of different ratios of galactose oligosaccharide(GOS) and polydextrose(PDX) on intestinal cell barrier damage model of Caco-2. The same batch of Caco-2 cells were cultured to form a cell barrier model and randomly divided into damaged model group without calcium, calcium-containing blank control group(1.8 mmol/L Ca~(2+)), low-ratio/low-dose group(1.8 mmol/L Ca~(2+)+2 mg/mL GOS+2 mg/mL PDX) and low-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+4 mg/mL GOS+4 mg/mL PDX), low-ratio/high-dose group(1.8 mmol/L Ca~(2+)+8 mg/mL GOS+8 mg/mL PDX) and high-ratio/low-dose group(1.8 mmol/L Ca~(2+)+0.8 mg/mL GOS+3.2mg/mL PDX), high-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+1.6 mg/mL GOS+6.4 mg/mL PDX), high-ratio/high-dose group(1.8 mmol/L Ca~(2+)+3.2mg/mL GOS+12.8 mg/mL PDX), a total of 8 groups, three parallel groups were performed in each group. The Trans Epithelial Electrical Resistance value and apparent permeability coefficient value of each group were determined after 4 d culture, and the morphology of tight junction proteins ZO-1, Occludin and Claudin-1 were observed by immunofluorescence method, and the expression levels of inflammatory related factors in each group were determined by protein microarray method. Compared with damaged model group, TEER ratio in calcium-containing blank control group was significantly increased(P<0.05), while Papp value was significantly decreased(P<0.05);Compared with calcium-containing blank control group, TEER ratio in low-ratio/medium-dose group and high-ratio/high-dose group was significantly increased(P<0.05) while Papp value was significantly decreased(P<0.05), and they could significantly down-regulate some inflammatory response related cytokines. The cell barrier was intact in all groups except for the compact junction protein structure in the model group. Compared with Ca~(2+) alone, the combination of two prebiotics can enhance the density of Caco-2 cell barrier and reduced the permeability of cell bypass. And it can significantly reduce the expression level of some inflammatory cytokines and effectively protect the intestinal cell barrier.

A New Caco-2 Cell Model of in Vitro Intestinal Barrier: Application for the Evaluation of Magnesium Salts Absorption.

Experimental data concerning the bioavailability of the different Mg-salts in human organism is inconsistent. Mg-absorption reported by clinical studies largely varies depending on the method used for evaluation. The aim of this study was to evaluate the bioavailability and accessibility of magnesium bound in different Mg-salt compounds, using an in vitro model of intestinal cell barrier. The study included a variety of inorganic (oxide, sulphate, chloride, carbonate) and organic salts (lactate, citrate, pidolate). Caco-2 cells were cultivated in a complete culture medium with different magnesium salts treatments in ascending concentrations. The viability and quantity of cells was analysed by FACS. Mg-absorption was analysed by a direct colorimetric assay, measured by spectrometry. T-test identified a significant decrease in cell count treatment with mg-lactate compared with citrate. Mg-pidolate showed a significantly higher cell viability compared with Mg-citrate, Mg-lactate and Mg-chloride. Even though the difference was not significant, we showed that an increase in Mg2+ salt concentration progressively decreased the cell count and the viability and the effect was universal for all the used Mg-salt treatments. Mg-citrate, chloride, and sulphate showed a significantly lower absorption compared to Mg-carbonate, pidolate and oxide. Our in vitro monolayer model of human intestinal transport showed that viability and quantity of cell decreased with increasing Mg-concentration. We admit that our experiment model may have some limitations in accurately describing an in vivo Mg2+ absorption. Moreover, it is also necessary to assess the relevance of our data in vivo and especially in clinical practice.

Low Dietary Manganese Levels Exacerbate Experimental Colitis in Mice

Abstract Objectives Inflammatory Bowel Disease (IBD), including Crohn's disease and ulcerative colitis, is a family of chronic inflammatory disorders of the gastrointestinal tract that affects over 3 million Americans. Diet and micronutrient intake plays a critical role in the pathogenesis of IBD. A recent epidemiological study showing an inverse relationship between nutritional manganese (Mn) status and IBD patients. Mn is an essential micronutrient required for normal growth and physiological processes. To date, the roles of Mn in the maintenance of intestinal health remain unknown, and the contribution of dietary Mn to IBD has yet to be explored. The goal of the present study is to determine the effects of dietary Mn on induced colitis in mice. Methods To investigate whether dietary Mn alters IBD progression in a dose-dependent manner, mice were fed purified diets containing either deficient, very low, low, or sufficient concentrations of Mn (0, 3, 15, or 35 ppm, respectively). Colitis was induced by providing mice drinking water containing 3% dextran sulfate sodium (DSS) for 6 days (inflammatory phase) followed by providing regular drinking water for 7 days (recovery phase). Results Upon treatment with DSS, the mice fed deficient, very low, and low dietary Mn showed increased morbidity, weight loss, and colon injury in a dose-dependent manner compared to the mice fed sufficient dietary Mn. Inflammation and oxidative damage was exacerbated in the deficient, very low, and low dietary Mn groups, which was displayed through greater spleen weights and elevated inflammatory cytokine levels. In contrast, mice fed sufficient dietary Mn showed greater tolerance to DSS-induced experimental colitis. Strikingly, even in the absence of DSS-induced colitis, mice with deficient intakes of dietary Mn displayed increases in gut permeability and losses in tight junction protein function while mice fed sufficient intakes of dietary Mn maintained intestinal integrity. Conclusions This study provides the first evidence that deficient, very low, and low levels of dietary Mn may exacerbate intestinal injury and inflammation dose-dependently in an experimental colitis model. These findings suggest that sufficient dietary Mn plays a critical role in intestinal cell barrier function, which may act as a protective mechanism against DDS-induced acute colitis in mice. Funding Sources NIH.

Evolution of cranberry juice compounds during in vitro digestion and identification of the organic acid responsible for the disruption of in vitro intestinal cell barrier integrity.

Cranberry juice is increasingly consumed for its richness in polyphenols having a positive impact on human health. Unfortunately, when regularly consumed, its high concentration in organic acids may cause some intestinal discomforts. In the present study, its organic acid content was reduced of 41% by electrodialysis with bipolar membrane (EDBM), and the resulted deacidified juice was divided in five different juices readjusted or not with different concentrations of citric and/or malic acid(s) corresponding to the concentration of this/these acid(s) recovered during EDBM or at the titratable acidity (TA) of the non-deacidified cranberry juice. The evolution of the cranberry juice main interesting compounds (organic acids and polyphenols), according to the concentration and nature of the organic acids present, was studied for the first time at each specific stages of the digestion. After digestion, Caco-2 cells were exposed to all digested juices to identify the organic acid(s) responsible for the loss of integrity of the epithelial barrier. It appeared that organic acid contents did not change during the different steps of the digestion while polyphenolic compounds decreased starting from the gastric phase. Whatever the organic acid concentration or nature, the concentration of PACs significantly decreased between the salivary and the gastric steps but was different according to their structure when the concentration of most of anthocyanins significantly decreased at the gastric step. Also, to the best of our knowledge, it was the first time that citric acid was demonstrated as the organic acid responsible for the loss of integrity of Caco-2 cell monolayers.

Chitosan-modified lipid nanodrug delivery system for the targeted and responsive treatment of ulcerative colitis

Targeted and sensitive drug release at the colitis site is critical for the effective therapy of ulcerative colitis and reduction of side effects from the drug. Herein, we used 3,3′-dithiodipropionic acid (DTPA) to covalently link quercetin (Qu) and glyceryl caprylate-caprate (Gcc) via ester bonds to prepare Qu-SS-Gcc lipid nanoparticles (Qu-SS-Gcc LNPs). Dexamethasone (Dex) was used as a model drug, and chitosan (CSO) was modified on the surface of Qu-SS-Gcc LNPs to obtain CSO-modified Dex-loaded Qu-SS-Gcc LNPs (CSO/Dex/LNPs). The encapsulation efficiency and drug loading of CSO/Dex/LNPs were 93.1 % and 8.1 %, respectively. The in vitro release results showed that CSO/Dex/LNPs had esterase-responsive characteristics and could release the drug rapidly in esterase-containing artificial intestinal fluid. A human colorectal adenocarcinoma cell (Caco-2) monolayer was used as the intestinal cell barrier model. Transmembrane resistance measurements and permeation experiments showed that CSO/Dex/LNPs had a protective effect on the lipopolysaccharide (LPS)-stimulated Caco-2 cell monolayer and increased the expression of E-cadherin in LPS-stimulated Caco-2 cells. Moreover, CSO/Dex/LNPs could significantly reduce the expression of the inflammatory factors TNF-α, IL-6 and NO in LPS-stimulated RAW 264.7 cells. The ulcerative colitis mouse model was constructed by using C57BL/6 mice. The in vivo distribution results showed that CSO/Dex/LNPs had colon-targeting effects and strong retention ability in the colons of mice with colitis. The results also showed that CSO/Dex/LNPs had better anti-inflammatory effects than free Dex, which could reduce colonic atrophy, reduce histomorphological changes and increase the expression of E-cadherin in the colon. Furthermore, the expression levels of TNF-α, IL-6 and NO in the CSO/Dex/LNP-treated group were 37.4 %, 35.5 % and 33.2 % of those in mice with colitis, respectively.

Anthocyanins protect the gastrointestinal tract from high fat diet-induced alterations in redox signaling, barrier integrity and dysbiosis

The gastrointestinal (GI) tract can play a critical role in the development of pathologies associated with overeating, overweight and obesity. We previously observed that supplementation with anthocyanins (AC) (particularly glycosides of cyanidin and delphinidin) mitigated high fat diet (HFD)-induced development of obesity, dyslipidemia, insulin resistance and steatosis in C57BL/6J mice. This paper investigated whether these beneficial effects could be related to AC capacity to sustain intestinal monolayer integrity, prevent endotoxemia, and HFD-associated dysbiosis. The involvement of redox-related mechanisms were further investigated in Caco-2 cell monolayers. Consumption of a HFD for 14 weeks caused intestinal permeabilization and endotoxemia, which were associated with a decreased ileum expression of tight junction (TJ) proteins (occludin, ZO-1 and claudin-1), increased expression of NADPH oxidase (NOX1 and NOX4) and NOS2 and oxidative stress, and activation of redox sensitive signals (NF-κB and ERK1/2) that regulate TJ dynamics. AC supplementation mitigated all these events and increased GLP-2 levels, the intestinal hormone that upregulates TJ protein expression. AC also prevented, in vitro, tumor necrosis factor alpha-induced Caco-2 monolayer permeabilization, NOX1/4 upregulation, oxidative stress, and NF-κB and ERK activation. HFD-induced obesity in mice caused dysbiosis and affected the levels and secretion of MUC2, a mucin that participates in intestinal cell barrier protection and immune response. AC supplementation restored microbiota composition and MUC2 levels and distribution in HFD-fed mice. Thus, AC, particularly delphinidin and cyanidin, can preserve GI physiology in HFD-induced obesity in part through redox-regulated mechanisms. This can in part explain AC capacity to mitigate pathologies, i.e. insulin resistance and steatosis, associated with HFD-associated obesity.

Multifactorial Modulation of Food-Induced Anaphylaxis.

Prevalence of food-induced anaphylaxis increases progressively and occurs in an unpredictable manner, seriously affecting the quality of life of patients. Intrinsic factors including age, physiological, and genetic features of the patient as well as extrinsic factors such as the intake of drugs and exposure to environmental agents modulate this disorder. It has been proven that diseases, such as mastocytosis, defects in HLA, or filaggrin genes, increase the risk of severe allergic episodes. Certain allergen families such as storage proteins, lipid transfer proteins, or parvalbumins have also been linked to anaphylaxis. Environmental factors such as inhaled allergens or sensitization through the skin can exacerbate or trigger acute anaphylaxis. Moreover, the effect of dietary habits such as the early introduction of certain foods in the diet, and the advantage of the breastfeeding remain as yet unresolved. Interaction of allergens with the intestinal cell barrier together with a set of effector cells represents the primary pathways of food-induced anaphylaxis. After an antigen cross-links the IgEs on the membrane of effector cells, a complex intracellular signaling cascade is initiated, which leads cells to release preformed mediators stored in their granules that are responsible for the acute symptoms of anaphylaxis. Afterward, they can also rapidly synthesize lipid compounds such as prostaglandins or leukotrienes. Cytokines or chemokines are also released, leading to the recruitment and activation of immune cells in the inflammatory microenvironment. Multiple factors that affect food-induced anaphylaxis are discussed in this review, paying special attention to dietary habits and environmental and genetic conditions.

Deacidification of cranberry juice protects against disruption of in-vitro intestinal cell barrier integrity

Cranberry juice is a well-known functional juice that has many beneficial effects on human health. However, it also has a high concentration of organic acids which may cause gastrointestinal discomfort. Hence, the organic acid content in cranberry juice was reduced to different levels of deacidification (0%, 19%, 37%, 50%, and 77%) by electrodialysis to study the impact of the deacidification rate on intestinal cell integrity. Before in vitro tests on Caco-2 cells, all samples underwent three steps of in vitro digestion: oral, gastric and intestinal. Digested and deacidified cranberry juices were applied to Caco-2 cells and the transepithelial electrical resistance (TEER) was measured after 24 hours of contact to evaluate the resulting cell integrity. In the presence of deacidified cranberry juice, the integrity of caco-2 cell monolayers measured by the ΔTEER was increased by 56% in comparison with raw cranberry juice, but a minimal deacidification rate of 37% was necessary to reach this level of protection.

Early tranexamic acid administration: A protective effect on gut barrier function following ischemia/reperfusion injury.

The mucus barrier is a critical component of the gut barrier and may be disrupted by pancreatic enzymes following trauma/hemorrhagic shock (T/HS). Luminal strategies against pancreatic enzyme activation or contact with the intestine are protective of the mucus layer and gut barrier integrity following T/HS. There is increasing evidence the use of tranexamic acid (TA) attenuates inflammatory responses in cardiac surgery and is readily absorbed from the gut. We therefore postulated that systemic administration of TA would attenuate mucus degradation and gut barrier failure following T/HS. This was studied in an in vitro model. Confluent monolayers of HT29-MTX (mucus-producing clone) and Caco-2 cocultures were exposed to 90 minutes of hypoxia followed by reoxygenation (H/R), luminal trypsin (5 μM), or both treatment groups. In a subset of experiments, TA (40 μM or 150 μM) was added to the basal chamber (systemic side) of intestinal cell cultures immediately following the hypoxic period. Mucus barrier function was indexed by rheologic measurement of both mucus thickness and viscosity (G', dyne/cm) and oxidant stress. Intestinal cell barrier integrity was indexed by transepithelial electrical resistance, permeability to fluorescein isothiocyanate-dextran, and apoptosis by flow cytometry. Exposure to both trypsin and H/R of Caco-2/HT29-MTX cocultures led to the most severe effect on mucus barrier function. Administration of TA immediately following hypoxia abrogated the effects noted on mucus barrier function. The epithelial barrier was also most severely impacted by both trypsin and H/R. Addition of TA after the hypoxic event was shown to be protective. Intestinal mucus physiochemical properties and intestinal barrier function were most severely impacted by exposure to both trypsin (concentration related) and H/R. The "systemic" administration of TA immediately after the hypoxic period was protective and suggests an additional role for early administration of TA in trauma patients in shock.

Correction: Intestinal Cell Barrier Function In Vitro Is Severely Compromised by Keratin 8 and 18 Mutations Identified in Patients with Inflammatory Bowel Disease

Correction: Intestinal Cell Barrier Function In Vitro Is Severely Compromised by Keratin 8 and 18 Mutations Identified in Patients with Inflammatory Bowel Disease

Intestinal cell barrier function in vitro is severely compromised by keratin 8 and 18 mutations identified in patients with inflammatory bowel disease.

Keratin 8 and 18 (K8/K18) mutations have been implicated in the aetiology of certain pathogenic processes of the liver and pancreas. While some K8 mutations (K8 G62C, K8 K464N) are also presumed susceptibility factors for inflammatory bowel disease (IBD), the only K18 mutation (K18 S230T) discovered so far in an IBD patient is thought to be a polymorphism. The aim of our study was to demonstrate that these mutations might also directly affect intestinal cell barrier function. Cell monolayers of genetically engineered human colonocytes expressing these mutations were tested for permeability, growth rate and resistance to heat-stress. We also calculated the change in dissociation constant (Kd, measure of affinity) each of these mutations introduces into the keratin protein, and present the first model of a keratin dimer L12 region with in silico clues to how the K18 S230T mutation may affect keratin function. Physiologically, these mutations cause up to 30% increase in paracellular permeability in vitro. Heat-stress induces little keratin clumping but instead cell monolayers peel off the surface suggesting a problem with cell junctions. K18 S230T has pronounced pathological effects in vitro marked by high Kd, low growth rate and increased permeability. The latter may be due to the altered distribution of tight junction components claudin-4 and ZO-1. This is the first time intestinal cells have been suggested also functionally impaired by K8/K18 mutations. Although an in vitro colonocyte model system does not completely mimic the epithelial lining of the intestine, nevertheless the data suggest that K8/K18 mutations may be also able to produce a phenotype in vivo.

Marine toxins and the cytoskeleton

One of the most striking features of marine toxins is that many of them produce diarrhea. This effect has been attributed to an effect on the cytoskeleton, with the consequent disorganization of the adherent cells on the intestinal cell barrier leading to an excess of liquid. But this has never been fully proven, as many neurotoxins show diarrheic effects that could be explained by an effect on the innervation system rather than on the epithelial intestinal cells. There is a wide diversity of mechanisms of action for marine toxins: phosphatase inhibition (okadaic acid), phosphodiesterase activation (yessotoxin), Na ⁄K-ATPase inhibition (palytoxin), Na channel blockage (saxitoxin, ciguatoxin, brevetoxin, conotoxin), nicotinic receptor antagonism (gymnodimin), mitogen-activated protein kinase activation (azaspiracid), Ca channel opening (maitotoxin), and K blockade (gambierol, conotoxin). Despite this complex array of modes of action, only pectenotoxins are known to target specifically the actin filaments. But regardless of the mechanism of action, most of the toxin groups have profound effects on cytoskeletal dynamics. The relevance of this is difficult to clarify, as the cytoskeleton is a common transduction step for many signals, but the effect of phycotoxins on the cytoskeleton is becoming a useful tool with which to investigate the toxicity and the mechanism of action of the toxins. This minireview series is intended to bring together a comprehensive set of data on four of the best recently studied toxin groups. Okadaic acid and their analogs, the dinophysistoxins, have been known for years as phosphatase inhibitors. They are very common worldwide, and important both economically, as a major cause of loss for shellfish producers, and scientifically, as okadaic acid is often used in research as a phosphatase inhibitor. Okadaic acid has been considered to be a very selective drug, in a similar fashion as initially thought for phorbol esters and protein kinase C, and its mode of action has clear implications for the dynamics of actin reorganization, but recent evidence indicates that phosphatases may not be the only targets of these toxins. The diarrhea caused by okadaic acid and analogs is so important that in the marine toxins field, members of the group are called diarrheic shellfish poisons. Palytoxin is well known to modify the function of the Na ⁄K-ATPase, but does not compete with ouabain, and its specific binding site has not yet been identified. The complexities of this compound and its many analogs make the group unique [largest nonrepetitive (peptide or sugar) molecule in nature], and also very interesting from a food safety perspective, as this group of compounds has been recently identified in Europe as a new risk phenomenom. Azaspiracids are currently by far the most complex toxin group and, 10 years after the first document reporting their presence, the mechanism of action remains elusive. Their complex transduction pathway is tightly linked to the cytoskeleton, and deserves a review. Finally, pectenotoxins are clearly associated with actin. Their action has been reported to involve a 1 : 1 binding with actin. However, the affinity of this binding is undefined as yet, and there are many unresolved questions regarding the implications of this effect for cell structure. Of all the marine toxin groups, okadaic acid, palytoxin, azaspiracids and pectenotoxins seem to be the most intriguing and interesting with regard to their effects on the cystoskeleton, and the four minireviews in this series describe what is known to date.

BapA, a large secreted protein required for biofilm formation and host colonization of Salmonella enterica serovar Enteritidis

In environmental settings, biofilms represent the common way of life of microorganisms. Salmonella enterica serovar Enteritidis, the most frequent cause of gastroenteritis in developed countries, produces a biofilm whose matrix is mainly composed of curli fimbriae and cellulose. In contrast to other bacterial biofilms, no proteinaceous compound has been reported to participate in the formation of this matrix. Here, we report the discovery of BapA, a large cell-surface protein required for biofilm formation by S. Enteritidis. Deletion of bapA caused the loss of the capacity to form a biofilm whereas the overexpression of a chromosomal copy of bapA increased the biofilm biomass formation. We provide evidence that overproduction of curli fimbriae and not cellulose can compensate for the biofilm deficiency of a bapA mutant strain. BapA is secreted through a type I protein secretion system (BapBCD) situated downstream of the bapA gene and was found to be loosely associated with the cell surface. Experiments with mixed bacterial populations positive or negative for BapA showed that BapA minus cells are not recruited into the biofilm matrix. The expression of bapA is coordinated with that of genes encoding curli fimbriae and cellulose, through the action of csgD. Studies on the contribution of BapA to S. Enteritidis pathogenesis revealed that orally inoculated animals with a bapA-deficient strain survived longer than those inoculated with the wild-type strain. Also, a bapA mutant strain showed a significantly lower colonization rate at the intestinal cell barrier and consequently a decreased efficiency for organ invasion compared with the wild-type strain. Taken together, these data demonstrate that BapA contributes both to biofilm formation and invasion through the regular Salmonella infection route.

Removal from the small intestine of tubercle bacilli by macrophages in the guinea pig.

The present study was arranged to test the reaction of the intestinal cell barrier toward the presence of chromogenic, acid-fast bacteria. The bacilli were introduced into the alimentary tract through a modified stomach tube. It was found that these bacterial forms were transported by macrophages through the columnar epithelium, within one to two hours following the test feeding. No other cellular elements such as neutrophils or columnar epithelial cells were observed in association with this process.