Fluorescein Isothiocyanate-labeled Dextran Research Articles

Deeper Insights into Mixed Crowding through Enzyme Activity, Dynamics, and Crowder Diffusion.

Given the fact that the cellular interior is crowded by many different kinds of macromolecules, it is important that in vitro studies be carried out in the presence of mixed crowder systems. In this regard, we have used binary crowders formed by the combination of some of the commonly used crowding agents, namely, Ficoll 70, Dextran 70, Dextran 40, and PEG 8000 (PEG 8), to study how these affect enzyme activity, dynamics, and crowder diffusion. The enzyme chosen is AK3L1, an isoform of adenylate kinase. To investigate its dynamics, we have carried out three single point mutations (A74C, A132C, and A209C) with the cysteine residues being labeled with a coumarin-based solvatochromic probe [CPM: (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin)]. Both enzyme activity and dynamics decreased in the binary mixtures as compared with the sum of the individual crowders, suggesting a reduction in excluded volume (in the mixture). To gain deeper insights into the binary mixtures, fluorescence correlation spectroscopy studies were carried out using fluorescein isothiocyanate-labeled Dextran 70 and tetramethylrhodamine-labeled AK3L1 as the diffusion probes. Diffusion in binary mixtures was observed to be much more constrained (relative to the sum of the individual crowders) for the labeled enzyme as compared to the labeled crowder showing different environments being faced by the two species. This was further confirmed during imaging of the phase-separated droplets formed in the binary mixtures having PEG as one of the crowding agents. The interior of these droplets was found to be rich in crowders and densely packed, as shown by confocal and digital holographic microscopy images, with the enzymes predominantly residing outside these droplets, that is, in the relatively less crowded regions. Taken together, our data provide important insights into various aspects of the simplest form of mixed crowding, that is, composed of just two components, and also hint at the enhanced complexity that the cellular interior presents toward having a detailed and comprehensive understanding of the same.

Electromagnetic pulse induced blood-brain barrier breakdown through tight junction opening in rats.

The blood-brain barrier (BBB) is the main obstacle to hydrophilic and large molecules to enter the brain, maintaining the stability of the central nervous system (CNS). But many environmental factors may affect the permeability and structure of the BBB. Electromagnetic pulses (EMP) irradiation has been proven to enhance the permeability of the BBB, but the specific mechanism is still unclear. To explore the potential mechanism of EMP-induced BBB opening, this study investigated the permeability, fine structure and the proteins expression of the tight junction (TJ) of the BBB in the rats exposed to EMP. Using the leakage of fluorescein isothiocyanate-labeled dextran with different molecular mass under different field intensity of EMP exposure, we found that the tracer passing through the BBB is size-dependent in the rat exposed to EMP as field intensity increased. Transmission electron microscopy showed TJ of the endothelial cells in the EMP-exposed group was open, compared with the sham-irradiated group. But the levels of TJ proteins including ZO-1, claudin-5, or occludin were not changed as indicated by western blot. These data suggest that EMP induce BBB opening in a field intensity-dependent manner and probably through dysfunction of TJ proteins instead of their expression. Our findings increase the understanding of the mechanism for EMP working on the brain and are helpful for CNS protection against EMP.

Quercetin Ameliorates Deoxynivalenol-Induced Intestinal Injury and Barrier Dysfunction Associated with Inhibiting Necroptosis Signaling Pathway in Weaned Pigs.

Quercetin (Que) is a flavonol compound found in plants, which has a variety of biological activities. Necroptosis, a special form of programmed cell death, plays a vital role in the development of many gastrointestinal diseases. This study aimed to explore whether Que could attenuate the intestinal injury and barrier dysfunction of piglets after deoxynivalenol (DON) exposure through modulating the necroptosis signaling pathway. Firstly, twenty-four weaned piglets were used in a 2 × 2 factorial design and the main factors, including Que (basal diet or diet supplemented with 100 mg/kg Que) and DON exposure (control feed or feed contaminated with 4 mg/kg DON). After feeding for 21 d, piglets were killed for samples. Next, the intestinal porcine epithelial cell line (IPEC-1) was pretreated with or without Que (10 μmol/mL) in the presence or absence of a DON challenge (0.5 μg/mL). Dietary Que increased the body weight, average daily gain, and average daily feed intake (p < 0.05) through the trial. Que supplementation improved the villus height, and enhanced the intestinal barrier function (p < 0.05) indicated by the higher protein expression of occludin and claudin-1 (p < 0.05) in the jejunum of the weaned piglets after DON exposure. Dietary Que also down-regulated the protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated RIP1 (p-RIP1), p-RIP3, total mixed lineage kinase domain-like protein (t-MLKL), and p-MLKL (p < 0.05) in piglets after DON exposure. Moreover, Que pretreatment increased the cell viability and decreased the lactate dehydrogenase (LDH) activity (p < 0.05) in the supernatant of IPEC-1 cells after DON challenge. Que treatment also improved the epithelial barrier function indicated by a higher transepithelial electrical resistance (TEER) (p < 0.001), lower fluorescein isothiocyanate-labeled dextran (FD4) flux (p < 0.001), and better distribution of occludin and claudin-1 (p < 0.05) after DON challenge. Additionally, pretreatment with Que also inhibited the protein abundance of t-RIP1, p-RIP1, t-RIP3, p-RIP3, t-MLKL, and p-MLKL (p < 0.05) in IPEC-1 cells after DON challenge. In general, our data suggest that Que can ameliorate DON-induced intestinal injury and barrier dysfunction associated with suppressing the necroptosis signaling pathway.

The effects of sodium propionate on intestinal barrier function of genetically improved farmed tilapia in a high-lipid formulation

Sodium propionate has long been used as a feed additive in fish diets for its remarkable anti-mold properties. In this study, two-month old genetically improved farmed (GIF) tilapia (initial weight 0.55 ± 0.0004 g/fish) were fed with five isonitrogenous diets, including a low-fat diet (LFD), high-fat diet (HFD), 0.25% sodium propionate-supplemented HFD (HFSP0.25), 0.5% sodium propionate-supplemented HFD (HFSP0.5) and 1.0% sodium propionate-supplemented HFD (HFSP1.0). This study investigated the effects of sodium propionate on fish growth performance, intestinal epithelium integrity and permeability, intestinal “physiological hypoxia” and gut microbiota composition of GIF tilapia fed with the HFD. After a four-week feeding trial, sodium propionate supplementation (0.25%, 0.5% and 1.0%) had no effects on survival rate, body weight gain and feed conversion ratio of GIF tilapia. However, feeding the HFSP0.5 and HFSP1.0 impaired tight junctions and lamina propria compared to feeding the HFD (P < 0.05). Serum fluorescence intensities of fish fed the HFSP0.5 and HFSP1.0 were higher than that fed the HFD after an oral gavage of fluorescein isothiocyanate-labeled carboxymethylated dextran (P < 0.05). Besides, the level of serum endotoxin was higher in fish fed the HFSP1.0 (P < 0.05). The expressions of genes encoding tight junction-related proteins including occludin and claudin-1 were remarkably decreased by feeding the HFSP0.5 and HFSP1.0 (P < 0.05). Moreover, intestinal pimonidazole retention was significantly reduced by HFSP1.0 feeding (P < 0.05). Furthermore, feeding the HFSP0.5 significantly increased the relative abundance of Cetobacterium somerae (P < 0.05) while feeding the HFSP1.0 significantly increased the relative abundance of Plesiomonas shigelloides (P < 0.05) in the gut. Consistently, the presence of Cetobacterium somerae was observed in the liver and muscle of fish fed the HFSP0.5 as well as in the muscle of fish fed the HFSP1.0. Presence of Plesiomonas shigelloides was observed in the liver of fish fed the HFSP1.0. In conclusion, higher levels of sodium propionate (0.5% and 1.0%) disrupt intestinal epithelium integrity and increase intestinal permeability of GIF tilapia when fed a HFD. Besides, sodium propionate at 1.0% additive amount abolishes intestinal “physiological hypoxia”. Sodium propionate supplementation alters the species diversity and composition of gut microbial community.

Advanced Binary Guanosine and Guanosine 5'-Monophosphate Cell-Laden Hydrogels for Soft Tissue Reconstruction by 3D Bioprinting.

Soft tissue defects or pathologies frequently necessitate the use of biomaterials that provide the volume required for subsequent vascularization and tissue formation as autrografts are not always a feasible alternative. Supramolecular hydrogels represent promising candidates because of their 3D structure, which resembles the native extracellular matrix, and their capacity to entrap and sustain living cells. Guanosine-based hydrogels have emerged as prime candidates in recent years since the nucleoside self-assembles into well-ordered structures like G-quadruplexes by coordinating K+ ions and π-π stacking, ultimately forming an extensive nanofibrillar network. However, such compositions were frequently inappropriate for 3D printing due to material spreading and low shape stability over time. Thus, the present work aimed to develop a binary cell-laden hydrogel capable of ensuring cell survival while providing enough stability to ensure scaffold biointegration during soft tissue reconstruction. For that purpose, a binary hydrogel made of guanosine and guanosine 5'-monophosphate was optimized, rat mesenchymal stem cells were entrapped, and the composition was bioprinted. To further increase stability, the printed structure was coated with hyperbranched polyethylenimine. Scanning electron microscopic studies demonstrated an extensive nanofibrillar network, indicating excellent G-quadruplex formation, and rheological analysis confirmed good printing and thixotropic qualities. Additionally, diffusion tests using fluorescein isothiocyanate labeled-dextran (70, 500, and 2000 kDa) showed that nutrients of various molecular weights may diffuse through the hydrogel scaffold. Finally, cells were evenly distributed throughout the printed scaffold, cell survival was 85% after 21 days, and lipid droplet formation was observed after 7 days under adipogenic conditions, indicating successful differentiation and proper cell functioning. To conclude, such hydrogels may enable the 3D bioprinting of customized scaffolds perfectly matching the respective soft tissue defect, thereby potentially improving the outcome of the tissue reconstruction intervention.

The tight junction protein occludin modulates blood–brain barrier integrity and neurological function after ischemic stroke in mice

Blood–brain barrier (BBB) disruption contributes to brain injury and neurological impairment. Tight junctions (TJs) and cell–cell adhesion complexes develop between endothelial cells in the brain to establish and maintain the BBB. Occludin, the first transmembrane protein identified in TJs, has received intense research interest because numerous in vitro studies have suggested its importance in maintaining BBB integrity. However, its role in maintaining BBB integrity after ischemic stroke is less clear owing to the lack of in vivo evidence. This study aimed to investigate the dynamics and function of occludin across the acute and chronic phases after stroke using occludin-deficient mice. By photochemically induced thrombosis model, the expression of occludin was decreased in brain endothelial cells from ischemic lesions. The neurological function of occludin-deficient mice was continuously impaired compared to that of wild-type mice. BBB integrity evaluated by Evans blue and 0.5-kDa fluorescein in the acute phase and by 10-kDa fluorescein isothiocyanate-labeled dextran in the chronic phase was decreased to a greater extent after stroke in occludin-deficient mice. Furthermore, occludin-deficient mice showed decreased claudin-5 and neovascularization after stroke. Our study reveals that occludin plays an important role from the acute to the chronic phase after ischemic stroke in vivo.

Protocatechuic acid and quercetin attenuate ETEC-caused IPEC-1 cell inflammation and injury associated with inhibition of necroptosis and pyroptosis signaling pathways

BackgroundNecroptosis and pyroptosis are newly identified forms of programmed cell death, which play a vital role in development of many gastrointestinal disorders. Although plant polyphenols have been reported to protect intestinal health, it is still unclear whether there is a beneficial role of plant polyphenols in modulating necroptosis and pyroptosis in intestinal porcine epithelial cell line (IPEC-1) infected with enterotoxigenic Escherichia coli (ETEC) K88. This research was conducted to explore whether plant polyphenols including protocatechuic acid (PCA) and quercetin (Que), attenuated inflammation and injury of IPEC-1 caused by ETEC K88 through regulating necroptosis and pyroptosis signaling pathways.MethodsIPEC-1 cells were treated with PCA (40 μmol/L) or Que (10 μmol/L) in the presence or absence of ETEC K88.ResultsPCA and Que decreased ETEC K88 adhesion and endotoxin level (P < 0.05) in cell supernatant. PCA and Que increased cell number (P < 0.001) and decreased lactate dehydrogenases (LDH) activity (P < 0.05) in cell supernatant after ETEC infection. PCA and Que improved transepithelial electrical resistance (TEER) (P < 0.001) and reduced fluorescein isothiocyanate-labeled dextran (FD4) flux (P < 0.001), and enhanced membrane protein abundance of occludin, claudin-1 and ZO-1 (P < 0.05), and rescued distribution of these tight junction proteins (P < 0.05) after ETEC infection. PCA and Que also declined cell necrosis ratio (P < 0.05). PCA and Que reduced mRNA abundance and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8 (P < 0.001), and down-regulated gene expression of toll-like receptors 4 (TLR4) and its downstream signals (P < 0.001) after ETEC infection. PCA and Que down-regulated protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated-RIP1 (p-RIP1), p-RIP1/t-RIP1, t-RIP3, p-RIP3, mixed lineage kinase domain-like protein (MLKL), p-MLKL, dynamin- related protein 1 (DRP1), phosphoglycerate mutase 5 (PGAM5) and high mobility group box 1 (HMGB1) (P < 0.05) after ETEC infection. Moreover, PCA and Que reduced protein abundance of nod-like receptor protein 3 (NLRP3), nod-like receptors family CARD domain-containing protein 4 (NLRC4), apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D (GSDMD) and caspase-1 (P < 0.05) after ETEC infection.ConclusionsIn general, our data suggest that PCA and Que are capable of attenuating ETEC-caused intestinal inflammation and damage via inhibiting necroptosis and pyroptosis signaling pathways.

Analysis of absorption-enhancing mechanisms for combinatorial use of spermine with sodium taurocholate in Caco-2 cells

Previously, we reported that the combined use of spermine (SPM) and sodium taurocholate (STC) (SPM–STC) significantly improves the oral absorption of rebamipide (BCS class IV) and pulmonary absorption of interferon-α without any harmful histopathological changes in the gastrointestinal tract and lungs, respectively. In the present study, we examined the effect of SPM–STC on the transport of fluorescein isothiocyanate-labeled dextrans (FDs) across Caco-2 cell monolayers and attempted to clarify the mechanisms underlying the transport enhancement caused by SPM–STC. SPM–STC were found to significantly enhance the transport of FDs, while the treatment with SPM–STC was not harmful, and the decrease in transepithelial electrical resistance was transient and reversible. The voltage-clamp study clearly indicated that the opening of the paracellular route could be mainly responsible for the enhanced transport of FD-4. As for the mechanisms, it was found that SPM–STC caused a significant increase in membrane fluidity, which would lead to the enhanced transport of small-molecule drugs such as rebamipide. Since SPM–STC increased intracellular Ca2+ via Ca2+ uptake through Ca2+ channels and Ca2+ release from the endoplasmic reticulum stimulated by the IP3 pathway, the subsequent possible activation of the MLCK signaling pathway would have led to the contraction of the actin–myosin ring. The rearrangement of tight junction-constituting proteins induced through the MAPK pathway has also been suggested as a possible mechanism for opening tight junctions. Claudin-4, a key protein constituting the tight junction, merged with F-actin along with the plasma membrane, was significantly decreased, which would be at least partial structural evidence for the tight-junction opening.

Improving endothelial cell junction integrity by diphenylmethanone derivatives at oxidative stress: A dual-action directly targeting caveolar caveolin-1

Directly targeting caveolar caveolin-1 is a potential mechanism to regulate endothelial permeability, especially during oxidative stress, but little evidence on the topic limits therapeutics discoveries. In this study, we investigated the pharmacological effect of an antioxidant LM49 (5,2′-dibromo-2,4′,5′-trihydroxydiphenylmethanoe) and its five diphenylmethanone derivatives on endothelial permeability and establish two distinct mechanisms of action. Multiplex molecular assays with theoretical modeling indicate that diphenylmethanone molecules, including LM49, directly bind the caveolin-1 steric pocket of ASN53/ARG54, ILE49/ASP50, ILE18, LEU59, ASN60, GLU48 and ARG19 residues. They also indicated dynamic binding-affinity for diphenylmethanone derivatives. First, this molecular interaction at caveolin-1 pocket inhibits its phosphorylation at TYR14 residue in H2O2-injured endothelial cell. A positive correlation was established between diphenylmethanone derivative binding-affinity and caveolin-1 phosphorylation inhibition. Inhibition of caveolin-1 phosphorylation, however, was independent of the LM49-mediated variation of protein tyrosine kinase activity, suggesting a direct blockage of adenosine triphosphate substrate diffusion into cavelion-1 structure. Second, LM49 increases the expression of cellular adhesive and tight junction proteins, VE-cadherin and occludin, in H2O2-injured cell, in a dose dependent manner. A leakage assay of fluorescein isothiocyanate-labeled dextran 40 across cell monolayer suggested improvement in endothelial barrier integrity with diphenylmethanone treatments. Our results demonstrate a direct targeting effect of caveolin-1 on endothelial permeability, and should guide the diphenylmethanone therapy against oxidative stress-induced junction dysfunction, especially at caveolar membrane invagination.

The Pyruvate Dehydrogenase Complex Mitigates LPS-Induced Endothelial Barrier Dysfunction by Metabolic Regulation.

Sepsis is a fatal health issue induced by an aberrant host response to infection, and it correlates with organ damage and a high mortality rate. Endothelial barrier dysfunction and subsequent capillary leakage play major roles in sepsis-induced multiorgan dysfunction. Anaerobic glycolysis is the primary metabolic mode in sepsis and the pyruvate dehydrogenase complex (PDHC) serves as a critical hub in energy regulation. Therefore, it is important to understand the role of PDHC in metabolic regulation during the development of sepsis-induced endothelial barrier dysfunction.In present study, human umbilical vein endothelial cells (HUVECs) and C57 BL/6 mice were treated with lipopolysaccharide (LPS) as models of endotoxemia. LPS increased basal glycolysis, compensatory glycolysis, and lactate secretion, indicating increased glycolysis level in endothelial cells (ECs). Activation of PDHC with dichloroacetate (DCA) reversed LPS-induced glycolysis, allowing PDHC to remain in the active dephosphorylated state, thereby preventing lactic acid production and HUVECs monolayers barrier dysfunction, as assessed by transendothelial electrical resistance and Fluorescein Isothiocyanate-labeled dextran. The in vivo study also showed that the lactate level and vascular permeability were increased in LPS-treated mice, but pretreatment with DCA attenuated these increases. The LPS-treated HUVEC model showed that DCA reversed LPS-induced phosphorylation of pyruvate dehydrogenase E1α Ser293 and Ser300 to restore PDHC activity. Immunoprecipitation results showed that LPS treatment increased the acetylation level of PDH E1α in HUVECs.Our study suggested that activation of PDHC may represent a therapeutic target for treatment of LPS-induced endothelial barrier dysfunction.

EPA and DHA inhibit endocytosis of claudin-4 and protect against deoxynivalenol-induced intestinal barrier dysfunction through PPARγ dependent and independent pathways in jejunal IPEC-J2 cells

Peroxisome proliferator activated receptor gamma (PPARγ) activation has been shown to protect against intestinal injury induced by different stimuli. PPARγ is known to regulate tight junction proteins (TJP) in epithelial cells. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are natural PPARγ agonists, but the implication of PPARγ in their physiological effects on the gut is poorly understood. Therefore, this study was conducted to investigate the mechanism of EPA and DHA effects on intestinal epithelial cell barrier function in IPEC-J2 cells exposed to deoxynivalenol (DON), a known food mycotoxin that is toxic to both humans and animals. Exposure of cells to EPA and DHA significantly increased mRNA expression of a PPARγ target gene, AP2, and concomitantly increased the protein expression of claudin-4. Treatment with EPA and DHA also reversed the endocytosis and degradation of claudin-4 caused by DON exposure. EPA and DHA also restored the membrane presence of claudin-4 and ZO-1 that was disrupted by DON. However, the protective effects of EPA and DHA against DON exposure was abolished by a specific PPARγ antagonist (T0070907), confirming the importance of PPARγ in regulating TJP expression by the fatty acids. Effect of PPARγ activation by EPA and DHA also included the restoration of transepithelial electrical resistance (TEER) and reduction of fluorescein isothiocyanate-labeled dextran (FITC-dextran) permeability that have been perturbed by DON. However, the effectiveness of EPA and DHA in opposing DON-induced decrease in TEER and the increase in FITC-dextran permeability was not affected by PPARγ inhibition, potentially suggesting the involvement of other PPARγ-independent mechanisms in the observed benefits from EPA and DHA. Collectively, this study shows that the protective effects of EPA and DHA against DON-induced intestinal barrier disruption are through both PPARγ-dependent and-independent pathways. Therefore, EPA and DHA containing ingredients could be used to prevent the DON-induced gut barrier dysfunction in humans and animals.

Barrier properties of ex vivo porcine intestinal mucus are highly independent of isolation and storage conditions

Porcine intestinal mucus (PIM) is often utilized as an ex vivo mucus model in mucus interaction studies. However, numerous isolation procedures and storage conditions for PIM are reported, yet their potential impact on preserving the critical properties of PIM remains unknown. This study investigated the effect of isolation procedures (rinsing and anatomical site of mucus isolation) and storage conditions (−20 °C, −80 °C, snap frozen in li-quid nitrogen prior to storage at −80 °C, or freeze-dried followed by storage at room temperature and reconstitution) of PIM in regard to the permeation of fluorescein-isothiocyanate-labelled dextran (FD) macromolecules of 4, 40 and 150 kDa, rheological properties as well as pH, osmolality, protein and water content. Rinsing intestines with tap water or phosphate-buffered saline as well as isolating PIM from different regions of the first five meters of the proximal jejunum did not affect the pH or osmolality of isolated PIM. The permeation of FD4, FD40 and FD150 through stored PIM was similar to permeation through fresh PIM. The rheological properties of stored PIM were similar to properties of fresh PIM. Osmolality, protein and water content were similar in stored and fresh PIM whereas pH decreased with 0.3 unit for all stored PIM. Overall, PIM samples stored at −20 °C, −80 °C, snap frozen or freeze-dried were found to have similar properties to freshly isolated PIM and can all be consi-dered good alternatives to fresh PIM for mucus studies.

Long-chain PUFA ameliorate enterotoxigenic Escherichia coli-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in porcine intestinal epithelial cells.

This study was aimed to investigate whether EPA and arachidonic acid (ARA), the representative n-3 or n-6 PUFA, could alleviate enterotoxigenic Escherichia coli (ETEC) K88-induced inflammation and injury of intestinal porcine epithelial cells 1 (IPEC-1) by modulating pyroptosis and necroptosis signalling pathways. IPEC-1 cells were cultured with or without EPA or ARA in the presence or absence of ETEC K88. EPA and ARA reduced ETEC K88 adhesion and endotoxin content in the supernatant. EPA and ARA increased transepithelial electrical resistance, decreased permeability of fluorescein isothiocyanate-labelled dextran, increased membrane protein expression of occludin, ZO-1 and claudin-1 and relieved disturbed distribution of these proteins. EPA and ARA also reduced cell necrosis ratio. EPA or ARA reduced mRNA and concentration of TNF-α, IL-6 and IL-8 and decreased mRNA abundances of intestinal toll-like receptors 4 and its downstream signals. Moreover, EPA and ARA downregulated mRNA expression of nod-like receptor protein 3 (NLRP3), caspase 1 and IL-18 and inhibited protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D and caspase-1. Finally, EPA and ARA reduced mRNA expression of fas-associated death domain protein, caspase 8, receptor-interacting protein kinase (RIP) 1, mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase 5 (PGAM5), motility-related protein 1 (Drp1) and high mobility protein 1 (HMGB1) and inhibited protein expression of phosphorylated-RIP1, p-RIP3, p-MLKL and HMGB1. These data demonstrate that EPA and ARA prevent ETEC K88-induced cell inflammation and injury, which is partly through inhibiting pyroptosis and necroptosis signalling pathways.

Induction of gut leakage in young broiler chickens fed a diet with low rye inclusion

The aim of the present study was to assess the absence of a non-starch polysaccharide (NSP) enzyme in a broiler diet containing a low level (10%) of rye inclusion. Two experimental groups with 40 Ross broilers each, were fed a diet containing 10% rye. One group was supplemented with a NSP enzyme, and the other was not supplemented with the enzyme to increase intestinal viscosity. The birds were fed the respective diets for 14 or 28 days. Intestinal sections were submitted to morphological, morphometric and mRNA-level gene expression analyses. To assess gut leakage, 150 min before euthanasia, broilers had no access to feed and received an oral gavage with fluorescein isothiocyanate-labelled dextran (FITC-d). Serum levels of FITC-d, D-lactate, tight-junction-associated protein 1 (TJAP1), citrulline and ovotransferrin were determined. A significant increase in FITC-d levels was observed in the 14-day-old birds fed the non-supplemented rye diet, and no other serum markers were affected. These birds presented a decreased villus height/crypt depth (VH:CD) ratio and an increased degree of damage in the jejunum. The ileum VH:CD increased, and the goblet cell number decreased in 28-day-old birds fed the non-supplemented rye diet. When broilers were fed the non-supplemented rye diet, the mRNA expression of the tight-junction zona occludens 1 (ZO1) was significantly decreased in the jejunum of 14-day-old broilers, whereas a significant decrease in jejunum mRNA expression of ZO2 and mucin-2 (MUC2) was observed in the jejunum of 28-day-old broilers. In contrast, a significant increase in the mRNA expression of ZO2 was observed in the ileum from 28-day-old broilers fed the non-supplemented rye diet. In conclusion, a 10% rye diet causes intestinal stress in young broiler chickens when the feed is not supplemented with a NSP enzyme. This study may be applied as experimental model of mild gut leakage of broiler chickens.

P681 Higher in vitro mucin degradation, but no increased paracellular permeability by faecal water from Crohn’s disease patients

Abstract Background Crohn’s disease (CD) is a chronic inflammatory gastro-intestinal condition with a variable disease course. Impaired intestinal integrity and microbial dysbiosis are associated with disease onset and exacerbations. We hypothesized that a perturbed microbial activity in CD patients may contribute to the impaired barrier function. Therefore, this study aimed to examine the impact of faecal bacterial products of active CD patients, CD patients in remission, and healthy controls (HC) on mucin degradation and intestinal epithelial barrier function in vitro. Methods Six HC and twelve CD patients were included. Faecal samples were obtained within one week prior to endoscopy processed within 6 hours after collection. Disease activity was determined by the short endoscopic score for CD (SES-CD). Faecal water (FW) and bacterial membrane vesicles (MVs) were applied on mucin agar to determine mucin degradation. Further, differentiated Caco-2 cell monolayers were exposed to FW and MVs to assess transepithelial electrical resistance (TEER) and paracellular junction stability using permeation of fluorescein isothiocyanate-labelled dextran of 4 kDa. Relative abundances of faecal bacterial genera were evaluated by 16S rRNA gene amplicon sequencing. Results FW-induced mucin degradation was higher in CD samples as compared to HC (p&amp;lt;0.01), but was not linked to specific bacterial relative abundances. FW resulted in 78–87% decrease of TEER in three of the remissive (p&amp;lt;0.001) but not the active CD or HC samples. The decrease of TEER was not linked to increased paracellular permeability. MVs did not induce mucin degradation or epithelial barrier disruption. Conclusion The higher mucin degradation capacity of CD patient-derived FW might indicate contributions of microbial products to CD pathophysiology and warrants further investigation. Moreover, the altered epithelial resistance in some individuals is not due to paracellular alterations.

Disruption of intestinal barrier and endotoxemia after traumatic brain injury: Implications for post-traumatic epilepsy.

Traumatic brain injury (TBI) may lead to the disruption of the intestinal barrier (IB), and to the escape of products of commensal gut bacteria, including lipopolysaccharide (LPS), into the bloodstream. We examined whether lateral fluid percussion injury (LFPI) and post-traumatic epilepsy (PTE) are associated with the increased intestinal permeability and endotoxemia, and whether these events in turn are associated with PTE. LFPI was delivered to adult male Sprague-Dawley rats. Before, 1 week, and 7 months after LFPI, the IB permeability was examined by measuring plasma concentration of fluorescein isothiocyanate-labeled dextran (FD4) upon its enteral administration. Plasma LPS concentration was measured in the same animals, using enzyme-linked immunosorbent assay. PTE was examined 7 months after LFPI, with use of video-EEG (electroencephalography) monitoring. One week after LFPI, the IB disruption was detected in 14 of 17 and endotoxemia - in 10 of 17 rats, with a strong positive correlation between FD4 and LPS levels, and between plasma levels of each of the analytes and the severity of neuromotor deficit. Seven months after LFPI, IB disruption was detected in 13 of 15 and endotoxemia in 8 of 15 rats, with a strong positive correlation between plasma levels of the two analytes. Five of 15 LFPI rats developed PTE. Plasma levels of both FD4 and LPS were significantly higher in animals with PTE than among the animals without PTE. The analysis of seven rats, which were examined repeatedly at 1 week and at 7 months, confirmed that late IB disruption and endotoxemia were not due to lingering of impairments occurring shortly after LFPI. LFPI leads to early and remote disruption of IB and a secondary endotoxemia. Early and late perturbations may occur in different subjects. Early changes reflect the severity of acute post-traumatic motor dysfunction, whereas late changes are associated with PTE.

Dexmedetomidine protects against burn-induced intestinal barrier injury via the MLCK/p-MLC signalling pathway

BackgroundEvidence suggests that sedative dexmedetomidine can prevent intestinal dysfunction. However, the specific mechanisms of its protective effects against burn-induced intestinal barrier injury remain unclear. We aimed to explore the possible positive effects of dexmedetomidine on burn-induced intestinal barrier injury and the effects the myosin light chain kinase (MLCK)/phosphorylated myosin light chain (p-MLC) signalling pathway in an experimental model of burn injury. MethodsIn this study, the plasma concentration of fluorescein isothiocyanate-labelled dextran (FITC-dextran) was measured. Histological changes were evaluated using haematoxylin and eosin (HE) staining. Tight junction proteins were evaluated by western blot and immunofluorescence analyses to assess the structural integrity of intestinal tight junctions. The level of inflammation was detected by enzyme-linked immunosorbent assay (ELISA). ResultsThe results shows that the increase in intestinal permeability caused by burn injury is accompanied by histological damage to the intestine, decreases in the expression of the tight junction proteins Zonula Occludens-1 (ZO-1) and Occludin, increases in inflammatory cytokine levels and elevation of both MLCK protein expression and MLC phosphorylation. After dexmedetomidine treatment, the burn-induced changes were ameliorated. ConclusionsIn conclusion, dexmedetomidine exerted an anti-inflammatory effect and protected tight junction complexes against burn‑induced intestinal barrier damage by inhibiting the MLCK/p-MLC signalling pathways.

Azithromycin and ciprofloxacin inhibit interleukin-8 secretion without disrupting human sinonasal epithelial integrity in vitro.

We recently developed a ciprofloxacin and azithromycin sinus stent (CASS) to target recalcitrant infections in chronic rhinosinusitis (CRS). The objective of this study was to evaluate the anti-inflammatory activity of azithromycin released from the CASS and assess the impact on the integrity and function of primary human sinonasal epithelial cells (HSNECs). Pseudomonas aeruginosa lipopolysaccharide (LPS)-stimulated HSNECs were treated with azithromycin and/or ciprofloxacin at concentrations attainable from CASS release. Interleukin-8 (IL-8) secretion was quantified by enzyme-linked immunosorbent assay (ELISA). Epithelial integrity (transepithelial resistance [TEER], paracellular permeability [fluorescein isothiocyanate-labeled dextran], lactate dehydrogenase [LDH] assays) and function (ciliary beat frequency [CBF]) were also evaluated. Azithromycin significantly reduced secreted IL-8 from P. aeruginosa LPS-stimulated HSNECs at all concentrations tested (mean ± standard deviation; control = 5.77 ± 0.39 ng/mL, azithromycin [6 μg/mL] = 4.58 ± 0.40 ng/mL, azithromycin [60 µg/mL] = 4.31 ± 0.06, azithromycin [180 µg/mL] = 4.27 ± 0.26 ng/mL, p < 0.05). Co-incubation with azithromycin (6 µg/mL) and ciprofloxacin (2.4 µg/mL) in LPS-stimulated HSNECs also displayed a significant reduction in secreted IL-8 when compared to P. aeruginosa LPS alone (co-treatment = 4.61 ± 0.29 ng/mL, P. aeruginosa LPS = 7.35 ± 0.89 ng/mL, p < 0.01). The drugs did not negatively impact TEER, paracellular permeability, LDH release, or CBF, indicating retention of cell integrity and function. Azithromycin decreased P. aeruginosa LPS IL-8 production in HSNECs at drug concentrations attainable with sustained release of azithromycin from the CASS. In addition to antibacterial activity, anti-inflammatory properties of the CASS should provide further benefit for patients with recalcitrant CRS.

EphrinA1/EphA2 Promotes Epithelial Hyperpermeability Involving in Lipopolysaccharide-induced Intestinal Barrier Dysfunction.

Background/AimsLipopolysaccharide (LPS) is the key factor inducing mucosal and systemic inflammation in various intestinal and parenteral diseases, which could initially disrupt the epithelial barrier function. EphrinA1/ephA2 is speculated to increase the epithelial permeability for its “repulsive interaction” between adjacent cells. This study aim to investigate the role of ephrinA1/ephA2 in LPS-induced epithelial hyperpermeability.MethodsIn vivo model challenged with oral LPS in C57BL/6 mice and in vitro model exposed to LPS in Caco2 monolayer were established. The barrier function was assessed including expression of tight junction proteins (occludin and claudin-1), transepithelial electrical resistance, and permeability to macromolecules (fluorescein isothiocyanate-labeled fluorescent dextran 4 kDa [FD4]). Moreover, the expression and phosphorylation of ephrinA1/ephA2 were quantified, and its roles in the process of epithelial barrier disruption were confirmed via stimulating ephA2 with ephrinA1-Fc chimera (ephrinA1-Fc) and inactivating ephA2 with ephA2-Fc chimera (ephA2-Fc), or ephA2 monoclonal antibody (ephA2-mab), as well as inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059.ResultsLPS induced significant barrier dysfunction with dismissed occludin and claudin-1 expression, reduced transepithelial electrical resistance and increased FD4 permeability, accompanied by upregulated ephrinA1/ephA2 pathway and phosphorylation of ephA2 receptor. Furthermore, ephA2-Fc, and ephA2-mab ameliorated LPS-induced epithelial hyperpermeability, which was also inhibited by PD98059. Additionally, ephrinA1-Fc led to apparent epithelial leakage in Caco2 monolayer by promoting the phosphorylation of ERK1/2, which could be obviously blocked by ephA2-mab and PD98059.ConclusionEphrinA1/ephA2 promotes epithelial hyperpermeability with an ERK1/2-dependent pathway, which involves in LPS-induced intestinal barrier dysfunction.

Efficacy of SPM-NONOate following intrapulmonary delivery in promoting absorptions of poorly absorbed macromolecules in rats and the underling mechanism.

This research aims to investigate the potential of N-[4-[1-(3-Aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3-propanediamine (SPM-NONOate) for promoting the absorption of poorly absorbed macromolecules delivered by intrapulmonary route. Influence of SPM-NONOate on the drug absorption was characterized by using a series of fluorescein isothiocyanate-labeled dextrans (FDs) as affordable models of hydrophilic macromolecules with established tools for quantitative analysis. SPM-NONOate increased concentration-dependently within 1-10mM the pulmonary absorptions of FDs in rats. Moreover, this promoting effect varied with the molecular weight of FDs, and the largest absorption enhancement effect was obtained for FD70. SPM-NONOate also showed promising enhancement potential on the absorption of some therapeutic peptides, where obvious hypoglycemic and hypocalcemic effects were observed after intrapulmonary delivery of insulin and calcitionin, respectively, with SPM-NONOate to rats. The safety of SPM-NONOate was confirmed based on measurement of some biological markers in bronchoalveolar lavage fluid (BALF) of rats. Additionally, mechanism underling the absorption enhancement action of SPM-NONOate was explored by combinatorial administration of FD4 and SPM-NONOate with various scavengers and generator to rat lungs. Results indicated that NO released from SPM-NONOate induced the enhancement in the drug absorption, and peroxynitrate, a NO metabolite, possibly participated in the absorption enhancing action of SPM-NONOate.