Transepithelial Electrical Resistance Values Research Articles

Construction of Human Three-Dimensional Lung Model Using Layer-by-Layer Method.

The respiratory tract is one of the frontline barriers for biological defense. Lung epithelial intercellular adhesions provide protection from bacterial and viral infections and prevent invasion into deep tissues by pathogens. Dysfunction of lung epithelial intercellular adhesion caused by pathogens is associated with development of several diseases, such as acute respiratory distress syndrome, pneumonia, and asthma. To elucidate the pathological mechanism of respiratory infections, two-dimensional cell cultures and animal models are commonly used, although are not useful for evaluating host specificity or human biological response. With the rapid progression and worldwide spread of severe acute respiratory syndrome coronavirus-2, there is increasing interest in the development of a three-dimensional (3D) in vitro lung model for analyzing interactions between pathogens and hosts. However, some models possess unclear epithelial polarity or insufficient barrier functions and need the use of complex technologies, have high cost, and long cultivation terms. We previously reported about the fabrication of 3D cellular multilayers using a layer-by-layer (LbL) cell coating technique with extracellular matrix protein, fibronectin (FN), and gelatin (G). In the present study, such a LbL cell coating technique was utilized to construct a human 3D lung model in which a monolayer of the human lower airway epithelial adenocarcinoma cell line Calu-3 cells was placed on 3D-cellular multilayers composed of FN-G-coated human primary pulmonary fibroblast cells. The 3D lung model thus constructed demonstrated an epithelial-fibroblast layer that maintained uniform thickness until 7 days of incubation. Moreover, expressions of E-cadherin, ZO-1, and mucin in the epithelial layer were observed by immunohistochemical staining. Epithelial barrier integrity was evaluated using transepithelial electrical resistance values. The results indicate that the present constructed human 3D lung model is similar to human lung tissues and also features epithelial polarity and a barrier function, thus is considered useful for evaluating infection and pathological mechanisms related to pneumonia and several pathogens. Impact statement A novel in vitro model of lung tissue was established. Using a layer-by-layer cell coating technique, a three-dimensional cultured lung model was constructed. The present novel model was shown to have epithelial polarity and chemical barrier functions. This model may be useful for investigating interaction pathogens and human biology.

Beneficial Effects of Phytogenic Feed Additives on Epithelial Barrier Integrity in an In Vitro Co-Culture Model of the Piglet Gut.

Industrial farming of livestock is increasingly focused on high productivity and performance. As a result, concerns are growing regarding the safety of food and feed, and the sustainability involved in their production. Therefore, research in areas such as animal health, welfare, and the effects of feed additives on animals is of significant importance. In this study, an in vitro co-culture model of the piglet gut was used to investigate the effects of two phytogenic feed additives (PFA) with similar compositions. Intestinal porcine epithelial cells (IPEC-J2) were co-cultivated with peripheral blood mononuclear cells (PBMC) to model the complex porcine gut environment in vitro. The effects of treatments on epithelial barrier integrity were assessed by means of transepithelial electrical resistance (TEER) in the presence of an inflammatory challenge. Protective effects of PFA administration were observed, depending on treatment duration and the model compartment. After 48 h, TEER values were significantly increased by 12-13% when extracts of the PFA were applied to the basolateral compartment (p < 0.05; n = 4), while no significant effects on cell viability were observed. No significant differences in the activity of a PFA based mainly on pure chemical compounds versus a PFA based mainly on complex, natural essential oils, and extracts were found. Overall, the co-culture model was used successfully to investigate and demonstrate beneficial effects of PFAs on intestinal epithelial barrier function during an inflammatory challenge in vitro. In addition, it demonstrates that the two PFAs are equivalent in effect. This study provides useful insights for further research on porcine gut health status even without invasive in vivo trials.

Protective effects of Bifidobacterium bifidum FL-228.1 on dextran sulfate sodium-induced intestinal damage in mice.

Numerous studies have found that probiotics benefit the intestinal barrier. However, the prophylactic effects of probiotics on the intestinal barrier, i.e., if probiotics exert protective effects in healthy individuals to defend them against harmful elements, have seldomly been reported. The present study aimed to investigate the possible mechanisms of potential strains with the function of preventing intestinal barrier damage. This study investigated nine potential probiotic strains using in vitro and in vivo models on their intestinal barrier-protecting properties. Transcriptomic was then employed to decipher the underlying mechanisms of action of the strains. The results showed that the strains, to varying degrees, regulated the ratio of interleukin (IL)-10 and IL-12 in peripheral blood mononuclear cells (PBMCs), increased the transepithelial electrical resistance (TEER) values, and decreased Caco-2 cell monolayers permeability. Correspondingly, the strains showed different prophylactic efficacies in protecting mice from dextran sulfate sodium (DSS)-induced intestinal barrier damage. Remarkably, Bifidobacterium bifidum FL-228.1 (FL-228.1) showed the best prophylactic efficacies in protecting mice from DSS-induced intestinal barrier damage. Further research suggested that FL-228.1 exerted its prophylactic effects by enhancing mucin 2 (Muc2) production and Claudin (Cldn)-4 in the colon. Furthermore, the transcriptomic and protein-protein interactions (PPI) analyses indicated that the inhibition of NLRP3 and the activation of PPARγ and TLR2 could be involved in protecting the intestinal barrier by FL-228.1. Bifidobacterium bifidum FL-228.1 may be developed as a promising probiotic for the prevention of intestinal barrier damage via PPARγ/NLRP3/ TLR2 pathways by enhancing Muc2 and Cldn-4.

Influence of permeability enhancers on the paracellular permeability of metformin hydrochloride and furosemide across Caco-2 cells.

Permeability enhancers can affect absorption of paracellularly transported drugs. This study aims to evaluate effects of permeability enhancers (chitosan, methyl-β -cyclodextrin, sodium caprate, sodium lauryl sulfate, etc.) on the permeability of paracellularly absorbed furosemide and metformin hydrochloride. Methyl thiazole tetrazolium bromide test was carried out to determine the drug concentrations in permeability study. Trans-epithelial electrical resistance (TEER) values determined to assess the integrity of tight junctions. Permeability enhancers were applied at different concentrations alone, in dual/triple combinations. Permeability was determined using human colorectal adenocarcinoma (Caco-2) cells (TEER>400Ω·cm2). Permeability enhancers have no significant effect (<2-fold; p>0.05) on the permeability of furosemide (1.80×10-5±4.55×10-7cm/s); however, metformin permeability (1.36×10-5±1.25×10-6cm/s) increased significantly (p<0.05) with 0.3% and 0.5% (w/v) chitosan (2.0- and 2.7-fold, respectively), 1% methyl-β -cyclodextrin (w/v) (3.5-fold), 10 and 20µmol/L sodium caprate (2.2- and 2.8-fold, respectively), and 0.012% sodium lauryl sulfate (w/v) (1.9-fold). Furosemide permeability increased significantly (p<0.05) with chitosan-sodium lauryl sulfate combination (1.7-fold), and all triple combinations (1.4- to 1.9-fold). Chitosan containing dual/triple combinations resulted in significant increase (p<0.05) in metformin permeability (1.7 to 2.8-fold). All results indicated that absorption of furosemide and metformin can be improved by the combination of permeability enhancers. Therefore, it can be evaluated for the formulation of development strategies containing furosemide and metformin by the pharmaceutical industry.

Tannic acid attenuates intestinal oxidative damage by improving antioxidant capacity and intestinal barrier in weaned piglets and IPEC-J2 cells.

Tannic acid (TA) has received widespread attention for its beneficial biological function with antioxidant capacity. This study investigated the protective role of TA on the intestinal antioxidant capacity and intestinal barrier in weaned piglets and porcine intestinal epithelial cells (IPEC-J2). A total of 18 weaned piglets were randomly allocated into two groups (n = 9) and fed with a basal diet (control, CON) and a basal diet containing 1,000 mg/kg TA for two weeks. The in vivo results showed that treatment with TA increased both glutathione peroxidase (GSH-PX) activity and the protein expression of ZO-1 in the jejunum of weaned piglets, and reduced the level of malondialdehyde (MDA) in the serum and the mRNA and protein expression of Keap1 in the jejunum of weaned piglets. Furthermore, in vitro results indicated that TA treatment effectively alleviated tert-butyl hydroperoxide (TBH)-induced oxidative stress in IPEC-J2 cells, improved the antioxidant capacity by elevating the cell redox state and activating the Nrf2 pathway, and improved the intestinal barrier by upregulating the mRNA and protein expression of intestinal tight junction proteins and increasing the transepithelial electrical resistance (TEER) value. In conclusion, these results confirmed that TA relieves oxidative injury and improves intestinal barrier function and intestinal antioxidant capacity by activating the Nrf2 signaling pathway. These findings suggest that TA has the potential application in alleviating oxidative stress in the intestine of weaned piglets.

Protective Effect of Sweetpotato (Ipomoea batatas L.) Leaf Phenolic Acids Extract on IL-1β-Induced Barrier Injury of Caco-2 Monolayers

The status quo of a high-fat diet can impair the integrity of the intestinal barrier and promote inflammatory bowel diseases. The exploration of natural products as sources of new lead compounds that control such diseases becomes essential. Sweetpotato leaves (SPLs) have strong biological activity, and their phenolic acids were recently reported to be able to impart antioxidative, anti-inflammatory, and neuroprotection. The aim of this study was to investigate the effects and mechanisms of sweetpotato leaf phenolic acids (SPLPAs) extract on interleukin 1 beta (IL-1β)-induced barrier injury of Caco-2 monolayers. The safety from 0.02 to 0.2 mg/mL SPLPA extracts were demonstrated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method and Trypan blue staining. The 0.2 mg/mL SPLPA extract significantly decreased the release of Nitric oxide (NO) generation and its contribution to the expression of inflammation-related nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and nuclear factor kappa B (NF-κB) were evaluated. Further investigation suggested that the SPLPA extract inhibited the IL-1β-induced decrease in the transepithelial electrical resistance (TEER) value and could upregulate the IL-1β induced decline in tight junction protein levels. The results suggested that the SPLPA extract could enhance the integrity of Caco-2 monolayers. These results clarified the beneficial effects of SPLPA extract on inflammatory Caco-2 monolayers, indicating that the application of SPLPA extract in medicine and functional food has great potential for exploration.

Attenuation of Palmitic Acid-Induced Intestinal Epithelial Barrier Dysfunction by 6-Shogaol in Caco-2 Cells: The Role of MiR-216a-5p/TLR4/NF-κB Axis.

Palmitic acid (PA) can lead to intestinal epithelial barrier dysfunction. In this study, the protective effects and working mechanisms of 6-shogaol against PA-induced intestinal barrier dysfunction were investigated in human intestinal epithelial Caco-2 cells. Transepithelial electrical resistance (TEER), paracellular flux, qRT-PCR, immunofluorescence, and Western blot experiments showed that the 24-h treatment with 400 μM PA damaged intestinal barrier integrity, as evidenced by a reduction of 48% in the TEER value, a 4.1-fold increase in the flux of fluorescein isothiocyanate-dextran 4000 (FD-4), and decreases in the mRNA and protein expression of tight junction (TJ)-associated proteins (claudin-1, occludin, and ZO-1), compared with the control. The PA treatment significantly (p &lt; 0.05) increased the levels of pro-inflammatory cytokines (interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α)) in Caco-2 cells due to the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylated nuclear factor kappa-B (NF-κB) proteins, and downregulation of miR-216a-5p (which directly targeted TLR4). Co-treatment with PA and 6-shogaol (2.5 μM) significantly (p &lt; 0.05) attenuated PA-induced changes through regulation of TJs via the miR-216a-5p/TLR4/NF-κB signaling pathway. This study provides insights into the functions and working mechanisms of 6-shogaol as a promising food-derived agent against PA-induced intestinal epithelial barrier dysfunction.

Preparation and Characterization of Aprepitant Solid Dispersion with HPMCAS-LF.

This study focused on improving the physicochemical characteristics of aprepitant with poor water solubility by preparing solid dispersion (SD). To prepare the SD with HPMCAS-LF, the solvent evaporation method was applied. Based on dissolution analysis, the dissolution rate of SD increased by five times compared with aprepitant. In addition, scanning electron microscopy (SEM), X-ray powder diffraction (XRPD), and differential scanning calorimetry (DSC) results suggested the presence of amorphous-form aprepitant inside SD. According to Fourier transform infrared (FTIR) spectroscopy, intermolecular hydrogen bonds were detected between polymer and aprepitant. The Caco-2 cell experiment proved that SD did not lower the transepithelial electrical resistance (TEER) values but improved the permeation amount of aprepitant. Additionally, the SD of aprepitant displayed excellent stability.

Investigation into the anti-inflammatory mechanism of coffee leaf extract in LPS-induced Caco-2/U937 co-culture model through cytokines and NMR-based untargeted metabolomics analyses

In this study, lipopolysaccharide (LPS)-induced Caco-2/U937 co-culture was used to investigate the anti-inflammatory mechanism of coffee leaf extract (CLE). HPLC analysis found that 5-caffeoylquinic acid (5-CQA) and epicatechin were degraded by 96.66 % and 85.35 %, respectively after 24 h incubation, while rutin and trigonelline remained unchanged. The absorption efficiency of 4,5-dicaffeoylquinic acid (4,5-diCQA) and mangiferin were higher than the other phytochemicals, reaching 46.90 % and 37.65 %, respectively. CLE significantly inhibited TNF-α, IL-1β, and IL-8 produced by LPS-induced U937 cells in the basolateral side as well as IL-8 produced by apical Caco-2 cells, thereby inhibiting the intestinal monolayer leakage evidenced by the increase of transepithelial electrical resistance (TEER) values. CLE ameliorated some of the LPS-induced impaired cellular immunometabolism, including amino acid and energy metabolisms. Our study indicated that CLE inhibited the pro-inflammatory cytokines and regulated the metabolites in the co-culture system, thus recovering the disrupted intestinal monolayer caused by inflammation.

Gypenosides counteract hepatic steatosis and intestinal barrier injury in rats with metabolic associated fatty liver disease by modulating the adenosine monophosphate activated protein kinase and Toll-like receptor 4/nuclear factor kappa B pathways

Context Non-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease, can develop into metabolic associated fatty liver disease (MAFLD). Gypenosides (GP), the main phytochemical component of Gynostemma pentaphylla (Thunb.) Makino (Cucurbitaceae), have been applied for treatment of metabolic diseases. Objective We investigate how GP modulate MAFLD-related hepatic steatosis and intestinal barrier injury. Materials and methods In cell experiments, Caco-2 cells were treated with GP (150 or 200 μmol/L, 24 h), following lipopolysaccharide (LPS) exposure (10 μg/mL, 24 h) to mimic MAFLD in vitro. In in vivo experiments, control, model and model + GP groups were set. High fructose diet/high fat (HFD/HF)-fed (12 weeks) MAFLD rats received GP treatment (300 mg/kg, 6 weeks), followed by intra-peritoneal glucose tolerance test and histopathological examination of rat liver and intestinal mucosa using haematoxylin–eosin staining. Results GP at 200 μM significantly reversed LPS-induced decreases in transepithelial electrical resistance (TER) value (25%), protein expression of occludin (two fold) and ZO-1 (four fold), and the ratio of p-AMPK to AMPK (five fold), while partially repressing LPS-induced leakage of FD4 (50%) and LPS-induced increases in the Toll-like receptor 4 (TLR4) level (50%) and the ratio of p-p65 to p65 (55%). Compared with the model rats, rats with GP treatment presented a reduction in gain of weight and glucose tolerance. In addition, GP alleviated HFD/HF-induced histopathological abnormalities in rat liver and intestinal mucosa. Conclusions GP attenuates hepatic steatosis and intestinal barrier injury in MAFLD rats via the AMPK and TLR4/nuclear factor kappa B (NF-κB) pathways, providing a potential treatment for MAFLD patients.

Madin-Darby Canine Kidney (MDCK) Cell line permeability of Curcumin loaded Phycocyanin nanosponges - In-Vitro study

Blood Brain barrier (BBB) is a natural protective wall in the brain to restrict the invasion of xenobiotics or toxic chemicals. This, in turn, becomes a major obstacle for researchers and industry people in formulating new drugs to treat brain disorders like brain tumors, Alzheimer's disease, multiple sclerosis, meningitis, and so on. The purpose of this research is to study the in-vitro cytotoxicity &amp; BBB permeation of curcumin-loaded phycocyanin nanosponges (Cur-PC NS) using Madin-Darby Canine Kidney (MDCK) cell lines. Cell viability of Cur-PC NS was performed using 3-(4,5-dimethylthiazol-2-yl)-2.5- diphenyltetrazolium bromide (MTT) assay, the transepithelial electrical resistance (TEER) values, and permeability coefficient were measured to test the integrity of monolayer of MDCK cell line. Results of the current study showed that Cur-PC NS at 50µM, 85% of MDCK cells are more viable and there was a significant (p&lt;0.01) reduction in TEER values up to 48 hours when compared to the curcumin. The permeability coefficient of nanosponges produced a 2.5-fold increase in enhancement ratio with a Papp value of 1.94±0.11×10-6 cm/s and 4.86±0.04×10-6cm/s for curcumin and Cur-PC NS respectively. Results of the study can be concluded that phycocyanin nanosponges can be used as a carrier for curcumin to permeate the BBB which may play a major role in the treatment of various brain disorders. Future studies are needed to substantiate the exact mechanism of permeability with clarification of efflux transporters presented in BBB.

RNA-seq reveals a novel porcine lncRNA MPHOSPH9-OT1 induces CXCL8/IL-8 expression in ETEC infected IPEC-J2 cells.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of bacterial diarrhea in piglets, leading to economic losses in the pig industry. In past decades, long non-coding RNAs (lncRNAs) have shown to be widely involved in the regulation of host immunity in porcine infection diseases. In this study, we explored the lncRNAs associated with ETEC F41 infection in IPEC-J2 cells by high-throughput sequencing and bioinformatic analysis. A total of 10150 novel porcine lncRNAs were identified. There were 161 differentially expressed (DE) lncRNAs associated with ETEC F41 infection, of which 65 DE lncRNAs were up-regulated and 96 DE lncRNAs were down-regulated. Functional and KEGG enrichment analysis of predicted target genes of DE lncRNAs indicated they are enriched in cell growth and inflammation-related pathways, such as endocytosis, focal adhesion, TGF-β signaling pathway, and adherens junctions. We revealed a novel candidate lncRNA MPHOSPH9-OT1 that was up-regulated after ETEC infection. The qRT-PCR validation and ELISA assessment showed the knockdown and overexpression of MPHOSPH9-OT1 resulted in significantly down- and up-regulation of cellular mRNA levels and secreted cytokine levels of CXCL8/IL-8, respectively. Meanwhile, MPHOSPH9-OT1 equilibrium is important to maintain the transepithelial electric resistance value and tight junction protein expression of IPEC-J2 cells. This study provides insights into the functionality of novel porcine lncRNAs in host immune responses to ETEC infection.

Comparison of the Modulating Effect of Anthocyanin-Rich Sour Cherry Extract on Occludin and ZO-1 on Caco-2 and HUVEC Cultures.

Increased permeability of the epithelial and endothelial cell layers results in the onset of pathogenic mechanisms. In both cell types, cell–cell connections play a regulatory role in altering membrane permeability. The aim of this study was to investigate the modulating effect of anthocyanin-rich extract (AC) on TJ proteins in inflammatory Caco-2 and HUVEC monolayers. Distribution of Occludin and zonula occludens-1 (ZO-1) were investigated by immunohistochemical staining and the protein levels were measured by flow cytometry. The mRNA expression was determined by quantitative real-time PCR. The transepithelial electrical resistance (TEER) values were measured during a permeability assay on HUVEC cell culture. As a result of inflammatory induction by TNF-α, redistribution of proteins was observed in Caco-2 cell culture, which was reduced by AC treatment. In HUVEC cell culture, the decrease in protein and mRNA expression was more dominant during inflammatory induction, which was compensated for by the AC treatment. Overall, AC positively affected the expression of the examined cell-binding structures forming the membrane on both cell types.

426 Effect of L-4-thiazolylalanine on skin barrier strength

Skin barrier properties are critical for maintaining epidermal water content, protecting from environmental factors, and providing first line of defense against pathogens. In this study, the effect of a non-proteinogenic amino acid, L-4-Thiazolylalanine (L4), on this important aspect of skin health was evaluated by in vitro and in vivo biomarkers. 3D human skin models were treated with 0.3% L4. After 48h, RNA samples were collected and gene expression analyzed. Significant increase in the expression of ALOXE3 (protein eLOX-3) vs. vehicle (VEH) (+27%) was measured. eLOX-3 is a member of the lipoxygenase family required in the epidermis for the covalent linkage of ceramides to proteins of the cornified envelope, an important step in establishing skin barrier function. To further evaluate skin barrier integrity, trans-epithelial electrical resistance (TEER) was measured in the 3D tissues at basal level as well as upon barrier disruption. TEER is a quantitative method that allows barrier strength evaluation. TEER values are highly related to the tight junction barrier function and decrease upon disruption induced by calcium depletion. L4 treatment induced a significant increase in TEER vs. vehicle and protected from low calcium-induced barrier decrease. Clinically, skin treated with L4 for 4 weeks had better barrier integrity vs. vehicle control. This was shown via instrumentation and evaluation of biomarkers in skin samples collected via tape stripping. A fluorescence-based assay was used to quantify the enzymatic activity of 12R-LOX, another lipoxygenase critical for maturation of skin barrier ceramides. Significant increase in 12R-LOX activity was measured after L4 treatment vs. vehicle (+31%) with a stronger effect in subjects 40 years-old and older (+45%). In conclusion, L4 led to modulation of in vitro and in vivo biomarkers critical for skin barrier strength, making it desirable ingredient for topical treatments.

Antioxidative NAC-Loaded Silk Nanoparticles with Opening Mucosal Tight Junctions for Nasal Drug Delivery: An In Vitro and In Vivo Study.

Using nasal routes to deliver drugs to the brain using multifunctional nanoparticles (NPs) to bypass the blood–brain barrier (BBB) might enhance the delivery efficacy. Anti-oxidative N-Acetyl-L-cysteine (NAC)-loaded silk fibroin (SF/NAC) NPs are produced, characterized and studied as a potential delivery vehicle for NAC delivered to the brain via nasal for both in vitro and in vivo studies. The NPs are not cytotoxic to RPMI 2650 cells, mucosal model cells, at a concentration of 6000 μg/mL. The anti-oxidative activities of SF/NAC NPs are demonstrated by high H2O2 scavenge capacities of the NPs and shown by mitochondrial superoxide (MitoSOX) immunostaining of human mesenchymal stem cells. Tight junctions in RPMI 2650 cells are opened after 30 min of incubation with SF/NAC NPs, which are demonstrated by measuring the decrease in trans-epithelial electrical resistance (TEER) values and discreteness in ZO-1 stains. The cellular uptake of SF/NAC NPs by RPMI 2650 cells is significantly greater than that for SF NPs and increased with increasing incubation time. In an in vivo imaging study (IVIS) using rats shows that the amount of NAC that is delivered to the brain by SF/NAC NPs increased by 1.40–2.60 times and NAC is retained longer in the nasal cavity than NAC solutions in a 2-h study.

Preventive Effects of Thinned Apple Extracts on TNF-α-Induced Intestinal Tight Junction Dysfunction in Caco-2 Cells through Myosin Light Chain Kinase Suppression.

Inflammatory bowel disease (IBD) is associated with intestinal epithelial barrier dysfunction and elevation of proinflammatory cytokines such as TNF-α. Tight junctions (TJ) control the paracellular barrier of the gut. Thinned apples are an indispensable horticultural agro-waste for apple cultivation, but are disposed by most farmers. This study aimed to elucidate the preventive effect of thinned apple extracts (TAE) on the intestinal epithelial barrier dysfunction induced by TNF-α treatment in Caco-2 cells. The differentiated Caco-2 monolayers were pre-treated with mature apple extract (MAE) and TAE for 1 h and then incubated with 100 ng/mL TNF-α for 24 h. The TJ integrity was estimated by measuring the value of transepithelial electrical resistance (TEER) and the flux of fluorescein isothiocyanate-dextran through paracellular transport. TAE had a better protective effect on the intestinal epithelial barrier than MAE did. Western blot results showed that TAE pre-retreatment elevated TJ protein levels such as claudin-1, -4, and -5. Moreover, TAE inhibited the interaction between zonula occludens proteins (ZO)-1 and occludin by reducing the tyrosine phosphorylation of ZO-1. The mechanisms underlying TAE-mediated attenuation of TNF-α-induced TJ disruption included suppression of myosin light chain kinase and NF-κB p65 protein levels. Therefore, thinned apples could be a sustainable ingredient for functional foods to prevent IBD.

Alteration of tight junctions during botulinum toxin type A-inhibited salivary secretion.

Tight junctions (TJs) are involved in the regulation of salivary secretion via paracellular pathway. Botulinum toxin type A (BTXA) is widely used for the treatment of hypersecretion diseases such as sialorrhea. This study aimed to investigate the role of TJs in BTXA-inhibited secretion of the submandibular gland (SMG). BTXA was injected into the SMGs of rats, and the same amount of saline was injected as a control. Western blot, real-time PCR, and immunofluorescence staining were used to detect the expression and distribution of TJ proteins. Paracellular permeability was evaluated using the transepithelial electrical resistance (TER) measurements and fluorescent tracer detection in BTXA-stimulated SMG-C6 cells. BTXA injection into the SMGs of rats led to increased expression of claudin (Cldn) -1 and Cldn3. Immunofluorescence staining showed no significant changes in the distribution of TJ proteins. In vitro, BTXA increased the TER values and significantly reduced the permeability of fluorescent tracer, suggesting that BTXA decreased the paracellular permeability. The expression levels of Cldn1, Cldn3, and Cldn4 were upregulated after BTXA treatment. The expression of TJ proteins changed in both animal models and SMG-C6 cells after BTXA treatment, which may contribute to the inhibition of salivary secretion.

Long-Chain Polyunsaturated Fatty Acids Attenuate Enterotoxigenic Escherichia coli K88-Induced Intestinal Barrier Damage via PPARγ Regulation in IPEC-J2 Cells

ObjectivesEnterotoxigenic Escherichia coli (ETEC) impairs the intestinal barrier function and triggers inflammatory response in infants and young animals. Long-chain polyunsaturated fatty acids (LC-PUFA) have been reported to improve intestinal health and limit inflammation. The objective is to define the role of LC-PUFA on intestinal barrier integrity with ETEC induced inflammation in intestinal porcine epithelial cells (IPEC-J2). MethodsCultured IPEC-J2 cells were pretreated with 30 μM LC-PUFA and then incubated without ETEC challenge (NE) or with ETEC challenge (CE). Transepithelial electrical resistance (TEER) value, fluorescein isothiocyanate-dextran (FITC-dextran) flux, released cytosolic lactate dehydrogenase (LDH), and interleukin 8 (IL-8) measures were evaluated. Protein abundance of claudin-1, occludin, and nuclear peroxisome proliferator-activated receptor-gamma (PPARγ) were measured by Western blot analysis. ResultsTreatment of cells with docosahexaenoic acid (DHA) significantly increased TEER when compared to the bovine serum albumin (BSA; 14% NE and 25% CE, respectively) and compared to oleate control (25% NE and 27% CE, respectively) (P < 0.05). Further, DHA decreased FITC-dextran across the epithelial barrier by 56% (P < 0.05) and decreased IL-8 secretion by 26% compared to oleate, regardless of ETEC challenge (P > 0.05). In the presence of ETEC, eicosapentaenoic acid (EPA) protected against a decline in membrane claudin-1 compared to EPA non-challenged cells (P > 0.05). In addition, arachidonic acid (ARA) and DHA tended to increase membrane occludin (P < 0.1). Additionally, there was a 1.6-fold increase in nuclear PPARγ by treatments of both ARA and DHA compared to BSA, regardless of challenge (P < 0.05). Release of LDH, a measure of cell cytotoxicity, was decreased by the ARA (64%), EPA (27%), and DHA (34%) in ETEC treated cells compared to BSA ETEC treated cells (P < 0.05). ConclusionsThese data suggest that LC-PUFA protect tight junction proteins by modulating PPARγ, thereby improving epithelial integrity in ETEC-challenged IPEC-J2 cells. Funding SourcesUnited States Department of Agriculture Hatch Project.

Fentanyl alleviates intestinal mucosal barrier damage in rats with severe acute pancreatitis by inhibiting the MMP-9/FasL/Fas pathway

Background Fentanyl is an analgesic used against pancreatitis-related pain, while whether it ameliorates severe acute pancreatitis (SAP) has yet to be checked. This study aims to determine fentanyl-delivered effect on SAP and the mechanism underlying this effect. Methods Rat SAP models were established, following fentanyl treatment. The serum activity of amylase (AMY), lipase (LIP), and diamine oxidase (DAO) was detected by enzyme-linked immunosorbent assay (ELISA). Histological examination was performed in the pancreatic and intestinal tissues with hematoxylin-eosin staining. After transfection with matrix metalloproteinase (MMP) 9 overexpression plasmids, Caco-2 monolayers were treated with fentanyl and subsequently exposed to lipopolysaccharide (LPS). The transepithelial electrical resistance (TEER) value was determined in rat intestinal mucosa through an Ussing chamber assisted by Analyze & Acquire, and in Caco-2 cell monolayers through a voltohmmeter. Intestinal mucosa and paracellular permeabilities were determined by fluorescein isothiocyanate (FITC)-labeled dextran assay. The expressions of ZO-1, Occludin, MMP9, Fas and Fas ligand (FasL) in rat intestinal mucosa and/or Caco-2 monolayers were analyzed by qRT-PCR or/and western blot. Results Fentanyl alleviated SAP-related histological alterations in the pancreas and intestines, reduced the elevated levels of SAP-related AMY, LIP, and DAO, but promoted the levels of ZO-1 and Occludin. In SAP rats and Caco-2 monolayers, SAP-related or LPS-induced TEER value decreases, permeability increases, and increases in the expressions of MMP9, Fas, and FasL were reversed partly by fentanyl. Notably, MMP9 overexpression could reverse the above fentanyl-delivered in vitro effects. Conclusions Fentanyl alleviates intestinal mucosal barrier damage in rats with SAP by inhibiting the MMP9/FasL/Fas pathway.

Deoxynivalenol exposure inhibits biosynthesis of milk fat and protein by impairing tight junction in bovine mammary epithelial cells

Deoxynivalenol (DON) is one of the most common feed contaminants, and it poses a serious threat to the health of dairy cows. The existing studies of biological toxicity of DON mainly focus on the proliferation, oxidative stress, and inflammation in bovine mammary epithelial cells, while its toxicity on the biosynthesis of milk components has not been well documented. Hence, we investigated the toxic effects and the underlying mechanism of DON on the bovine mammary alveolar cells (MAC-T). Our results showed that exposure to various concentrations of DON significantly inhibited cell proliferation, induced apoptosis, and altered the cell morphology which was manifested by cell distortion and shrinkage. Moreover, the transepithelial electrical resistance (TEER) values of MAC-T cells exposed to DON were gradually decreased in a time- and concentration- dependent manner, but lactate dehydrogenase (LDH) leakage was significantly increased with the maximum increase of 2.4-fold, indicating the cell membrane and tight junctions were damaged by DON. Importantly, DON significantly reduced the synthesis of β-casein and lipid droplets, along with the significantly decreases of phospho-mTOR, phospho-4EBP1, phospho-JAK2, and phospho-STAT5. Gene expression profiles showed that the expressions of several genes related to lipid synthesis and metabolism were changed, including acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid binding protein 3 (FABP3), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), and insulin-induced gene 1 (INSIG1). GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in ribosome, glutathione metabolism, and lipid biosynthetic process, which play important roles in the toxicological process induced by DON. Taken together, DON affects the proliferation and functional differentiation of MAC-T cells, which might be related to the cell junction disruption and morphological alteration. Our data provide new insights into functional differentiation and transcriptomic alterations of MAC-T cells after DON exposure, which contributes to a comprehensive understanding of DON-induced toxicity mechanism.