Tight Junction Protein Abundance Research Articles

Poria cocos polysaccharides alleviate dextran sulphate sodium-induced ulcerative colitis in mice by modulating intestinal inflammatory responses and microbial dysbiosis

Poria cocos polysaccharide (PCP), one of the main active components of P. cocos, is extensively used worldwide and exhibits strong pharmacological effects. However, whether PCP can attenuate inflammatory bowel disease remains unclear. In this study, we assessed the effects of PCP supplementation on dextran sulphate sodium (DSS)-induced ulcerative colitis (UC) in mice. We found that PCP supplementation mitigated UC symptoms in DSS-treated mice, as evidenced by reductions in body weight loss, colon length shortening and disease activity index score. Importantly, PCP supplementation enhanced colonic barrier integrity by increasing tight junction protein abundance and exerted anti-inflammatory effects by suppressing nuclear factor-κB (NF-κB) activation in DSS-treated mice. Furthermore, PCP supplementation reversed DSS-induced dysbiosis in colonic microbiota by increasing the colonic abundance of beneficial bacteria (e.g. Akkermansiaceae) and decreasing the colonic abundance of harmful bacteria (e.g. Erysipelotrichaceae) in DSS-treated mice. Although PCP supplementation failed to ameliorate DSS-induced UC in antibiotic-treated mice, faecal microbiota transplantation from PCP-administered mice ameliorated DSS-induced UC in antibiotic-treated mice. In summary, PCP alleviates UC in mice by attenuating intestinal inflammation via the inhibition of NF-κB activation and modulating the intestinal microbiota.

Loss of filamentous actin, tight junction protein expression, and paracellular barrier integrity in frataxin-deficient human brain microvascular endothelial cells-implications for blood-brain barrier physiology in Friedreich's ataxia.

Introduction: Friedreich's Ataxia (FRDA) is the most prevalent inherited ataxia. FRDA results from loss of Frataxin (FXN), an essential mitochondrial iron trafficking protein. FRDA starts with an early burst of neurodegeneration of the dorsal root ganglion and cerebellar dentate nuclei, followed by progressive brain iron accumulation in the latter. End stage disease includes cardiac fibrosis that contributes to hypertrophic cardiomyopathy. The microvasculature plays an essential barrier role in both brain and heart homeostasis, thus an investigation of this tissue system in FRDA is essential to the delineation of the cellular dysfunction in this genetic disorder. Previous reports have identified cytoskeletal alterations in non-barrier forming FRDA cell models, but physiological consequences are limited. Methods: We investigated brain microvascular endothelial cell integrity in FRDA in a model of the blood-brain barrier (BBB). We have knocked down FXN in immortalized human brain microvascular endothelial cells (hBMVEC), which compose the microcapillaries of the BBB, by using shRNA. We confirmed known cellular pathophysiologies of FXN-knockdown including decreased energy metabolism, markers of oxidative stress, and increased cell size. Results: We investigated cytoskeletal architecture, identifying decreased filamentous actin and Occludin and Claudin-5 tight junction protein expression in shFXN hBMVECs. This was consistent with decreased transendothelial electrical resistance (TEER) and increased paracellular tracer flux during early barrier formation. shFXN hBMVEC start with only 67% barrier integrity of the controls, and flux a paracellular tracer at 800% of physiological levels. Discussion: We identified that insufficient FXN levels in the hBMVEC BBB model causes changes in cytoskeletal architecture and tight junction protein abundance, co-incident with increased barrier permeability. Changes in the integrity of the BBB may be related to patient brain iron accumulation, neuroinflammation, neurodegeneration, and stroke. Furthermore, our findings implicate other barrier cells, e.g., the cardiac microvasculature, loci of disease pathology in FRDA.

Dietary supplementation with dihydroartemisinin improves intestinal barrier function in weaned piglets with intrauterine growth retardation by modulating the gut microbiota.

The aim of this study was to investigate whether dietary dihydroartemisinin (DHA) supplementation could improve intestinal barrier function and microbiota composition in intrauterine growth restriction (IUGR) weaned piglets. Twelve normal birth weight (NBW) piglets and 24 IUGR piglets at 21 d of age were divided into three groups, which were fed a basal diet (NBW-CON and IUCR-CON groups) and an 80mg/kg DHA diet (IUGR-DHA group). At 49 d of age, eight piglets of each group with similar body weights within groups were slaughtered, and serum and small intestine samples were collected. The results showed that IUGR piglets reduced growth performance, impaired the markers of intestinal permeability, induced intestinal inflammation, decreased intestinal immunity, and disturbed the intestinal microflora. Dietary DHA supplementation increased average daily gain, average daily feed intake, and body weight at 49 d of age in IUGR-weaned piglets (P < 0.05). DHA treatment decreased serum diamine oxidase activity and increased the numbers of intestinal goblet cells and intraepithelial lymphocytes, concentrations of jejunal mucin-2 and ileal trefoil factor 3, and intestinal secretory immunoglobin A and immunoglobin G (IgG) concentrations of IUGR piglets (P < 0.05). Diet supplemented with DHA also upregulated mRNA abundances of jejunal IgG, the cluster of differentiation 8 (CD8), major histocompatibility complex-I (MHC-I), and interleukin 6 (IL-6) and ileal IgG, Fc receptor for IgG (FcRn), cluster of differentiation 8 (CD4), CD8, MHC-I, IL-6 and tumor necrosis factor α (TNF-α), and enhanced mRNA abundance and protein expression of intestinal occludin and ileal claudin-1 in IUGR piglets (P < 0.05). In addition, DHA supplementation in the diet improved the microbial diversity of the small intestine of IUGR piglets and significantly increased the relative abundance of Actinobacteriota, Streptococcus, Blautia and Streptococcus in the jejunum, and Clostridium sensu_ stricto_in the ileum (P < 0.05). The intestinal microbiota was correlated with the mRNA abundance of tight junction proteins and inflammatory response-related genes. These data suggested that DHA could improve the markers of intestinal barrier function in IUGR-weaned piglets by modulating gut microbiota. DHA may be a novel nutritional candidate for preventing intestinal dysfunction in IUGR pigs.

Acute and persistent effects of oral glutamine supplementation on growth, cellular proliferation, and tight junction protein transcript abundance in jejunal tissue of low and normal birthweight pre-weaning piglets.

Breeding for higher fertility has resulted in a higher number of low birthweight (LBW) piglets. It has been shown that LBW piglets grow slower than normal birthweight (NBW) littermates. Differences in growth performance have been associated with impaired small intestinal development. In suckling and weaning piglets, glutamine (Gln) supplementation has been associated with improved growth and intestinal development. This study was designed to examine the effects of oral Gln supplementation on growth and small intestinal parameters in LBW and NBW suckling piglets. At birth (day 0), a total of 72 LBW (1.10 ± 0.06 kg) and 72 NBW (1.51 ± 0.06) male piglets were selected. At day 1, litters were standardized to 12 piglets, and experimental piglets supplemented daily with either Gln (1 g/kg BW) or isonitrogenous amounts of Alanine (Ala) as control (1.22 g/kg BW) until day 12. Creep feed was offered from day 14 onward. Subgroups of piglets were euthanized at days 5, 12, and 26 for the analyses of jejunal morphometry, cellular proliferation, glutathione concentration and transcript abundance of tight junction proteins. From age day 11 to 21, Gln supplemented LBW (LBW-Gln) piglets were heavier than Ala supplemented LBW (LBW-Ala) littermates (P = 0.034), while NBW piglets were heavier until age day 26 compared to LBW littermates. Villus height was higher in LBW-Gln compared to LBW-Ala on age day 12 (P = 0.031). Sporadic differences among supplementation and birthweight groups were detected for jejunal cellular proliferation, cellular population and glutathione concentration, whereas age was the most dominant factor. These results show that Gln supplementation improved the growth of LBW piglets compared to LBW-Ala beyond the termination of Gln supplementation, but this was not associated with consistent effects on selected parameters of jejunal development.

Essential oils improve nursery pigs' performance and appetite via modulation of intestinal health and microbiota

Optimal intestinal health and functionality are essential for animal health and performance, and simultaneously intestinal nutrient transporters and intestinal peptides are also involved in appetite and feed intake control mechanisms. Given the potential of essential oil (EO) in improving animal performance and improving feed palatability, we hypothesized that dietary supplementation of cinnamaldehyde and carvacrol could improve performance and appetite of nursery pigs by modulating intestinal health and microbiota. Cinnamaldehyde (100 mg/kg), carvacrol (100 mg/kg), and their mixtures (including 50 mg/kg cinnamaldehyde and 50 mg/kg carvacrol) were supplemented into the diets of 240 nursery pigs for 42 d, and data related to performance were measured. Thereafter, the influence of EO on intestinal health, appetite and gut microbiota and their correlations were explored. EO supplementation increased (P < 0.05) the body weight, average daily gain (ADG) and average daily feed intake (ADFI) of piglets, and reduced (P < 0.05) diarrhea rates in nursery pigs. Furthermore, EO increased (P < 0.05) the intestinal absorption area and the abundance of tight junction proteins, and decreased (P < 0.05) intestinal permeability and local inflammation. In terms of intestinal development and the mucus barrier, EO promoted intestinal development and increased (P < 0.05) the number of goblet cells. Additionally, we found that piglets in the EO-supplemented group had upregulated (P < 0.05) levels of transporters and digestive enzymes in the intestine, which were significantly associated with daily gain and feed utilization. In addition, EO supplementation somewhat improved appetite in nursery pigs, increased the diversity of the gut microbiome and the abundance of beneficial bacteria, and there was a correlation between altered bacterial structure and appetite-related hormones. These findings indicate that EO is effective in promoting growth performance and nutrient absorption as well as in regulating appetite by improving intestinal health and bacterial structure.

Inclusion of Quillaja Saponin Clarity Q Manages Growth Performance, Immune Response, and Nutrient Transport of Broilers during Subclinical Necrotic Enteritis.

Necrotic enteritis (NE) is an intestinal disease that results in poor performance, inefficient nutrient absorption, and has a devastating economic impact on poultry production. This study evaluated the effects of a saponin-based product (Clarity Q, CQ) during an NE challenge. A total of 1200 male chicks were randomly assigned to four dietary treatments (10 pens/treatment; 30 birds/pen): treatment 1 (NC), a non-medicated corn-soybean basal diet; treatment 2 (PC), NC + 50 g/metric ton (MT) of bacitracin methylene disalicylate (BMD); and treatments 3 (CQ15) and 4 (CQ30), NC + 15 and 30 g/MT, respectively. On the day (d) of placement, birds were challenged by a coccidia vaccine to induce NE. On d 8, 14, 28, and 42, performance parameters were measured. On d 8, three birds/pen were necropsied for NE lesions. On d 8 and d 14, jejunum samples from one bird/pen were collected for mRNA abundance of tight junction proteins and nutrient transporter genes. Data were analyzed in JMP (JMP Pro, 16), and significance (p ≤ 0.05) between treatments was identified by Fisher's least significant difference (LSD) test. Compared to PC and NC, CQ15 had higher average daily gain (ADG), while CQ30 had lower average daily feed intake (ADFI) and feed conversion ratio (FCR). NE lesions in the duodenum were lower in CQ15 compared to all other treatments. On d 8, mRNA abundance of CLDN1, CLDN5, AMPK, PepT2, GLUT2, and EAAT3 were significantly greater in CQ30 (p < 0.05) compared to both PC and NC. On d 14, mRNA abundance of ZO2 and PepT2 was significantly lower in PC when compared to all treatments, while that of ANXA1, JAM3, and GLUT5 was comparable to CQ15. In summary, adding Clarity Q to broiler diets has the potential to alleviate adverse effects caused by this enteric disease by improving performance, reducing intestinal lesions, and positively modulating the mRNA abundance of various tight junction proteins and key nutrient transporters during peak NE infection.

Organotypic sinonasal airway culture systems are predictive of the mucociliary phenotype produced by bronchial airway epithelial cells

Differentiated air–liquid interface models are the current standard to assess the mucociliary phenotype using clinically-derived samples in a controlled environment. However, obtaining basal progenitor airway epithelial cells (AEC) from the lungs is invasive and resource-intensive. Hence, we applied a tissue engineering approach to generate organotypic sinonasal AEC (nAEC) epithelia to determine whether they are predictive of bronchial AEC (bAEC) models. Basal progenitor AEC were isolated from healthy participants using a cytological brushing method and differentiated into epithelia on transwells until the mucociliary phenotype was observed. Tissue architecture was assessed using H&E and alcian blue/Verhoeff–Van Gieson staining, immunofluorescence (for cilia via acetylated α-tubulin labelling) and scanning electron microscopy. Differentiation and the formation of tight-junctions were monitored over the culture period (day 1–32) by quantifying trans-epithelial electrical resistance. End point (day 32) tight junction protein expression was assessed using Western blot analysis of ZO-1, Occludin-1 and Claudin-1. Reverse transcription qPCR-array was used to assess immunomodulatory and autophagy-specific transcript profiles. All outcome measures were assessed using R-statistical software. Mucociliary architecture was comparable for nAEC and bAEC-derived cultures, e.g. cell density P = 0.55, epithelial height P = 0.88 and cilia abundance P = 0.41. Trans-epithelial electrical resistance measures were distinct from day 1–14, converged over days 16–32, and were statistically similar over the entire culture period (global P < 0.001). This agreed with end-point (day 32) measures of tight junction protein abundance which were non-significant for each analyte (P > 0.05). Transcript analysis for inflammatory markers demonstrated significant variation between nAEC and bAEC epithelial cultures, and favoured increased abundance in the nAEC model (e.g. TGFβ and IL-1β; P < 0.05). Conversely, the abundance of autophagy-related transcripts were comparable and the range of outcome measures for either model exhibited a considerably more confined uncertainty distribution than those observed for the inflammatory markers. Organotypic air–liquid interface models of nAEC are predictive of outcomes related to barrier function, mucociliary architecture and autophagy gene activity in corresponding bAEC models. However, inflammatory markers exhibited wide variation which may be explained by the sentinel immunological surveillance role of the sinonasal epithelium.

Dietary Supplementation with Epicatechin Improves Intestinal Barrier Integrity in Mice

Epicatechin (EPI) is a dietary flavonoid that is present in many foods and possesses various bioactivities. We assessed the effects of EPI supplementation on intestinal barrier integrity in mice. Thirty-six mice were assigned to three groups and fed a standard diet or a standard diet supplemented with 50 or 100 mg EPI/kg (n = 12 per group). After 21 days of rearing, blood and intestinal samples were collected from eight randomly selected mice. Supplementation with 50 and 100 mg/kg EPI decreased (p < 0.05) the serum diamine oxidase activity and D-lactic acid concentration and increased (p < 0.05) the duodenal, jejunal, and ileal abundance of tight junction proteins, such as occludin. Moreover, it lowered (p < 0.05) the duodenal, jejunal, and ileal tumor necrosis factor-α contents and enhanced (p < 0.05) the duodenal and jejunal catalase activities and ileal superoxide dismutase activity. Supplementation with a lower dose (50 mg/kg) decreased (p < 0.05) the ileal interleukin-1β content, whereas supplementation with a higher dose (100 mg/kg) increased (p < 0.05) the duodenal and jejunal glutathione peroxidase activities. Furthermore, supplementation with 50 and 100 mg/kg EPI decreased (p < 0.05) cell apoptosis, cleaved cysteinyl aspartate-specific proteinase-3 (caspase-3), and cleaved caspase-9 contents in the duodenum, jejunum, and ileum. In conclusion, EPI could improve intestinal barrier integrity in mice, thereby suppressing intestinal inflammation and oxidative stress and reducing cell apoptosis.

Raptor knockdown concurrently increases the electrical resistance and paracellular permeability of Caco-2 cell monolayers

AimsAs a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. Intestinal barrier integrity ensures tissue homeostasis by closely regulating the permeability of the epithelium to lumenal contents. The role of mTORC1 in the regulation of intestinal barrier function and permeability remains to be fully elucidated. Materials and methodsIn this study, we employed lentivirus-mediated knockdown of mTORC1 signaling-associated proteins Raptor (regulatory-associated protein of mTOR) and TSC2 (tuberin) to ascertain the effects of constitutive activation or repression of mTORC1 activity on barrier function in Caco-2 cell monolayers. Key findingsResults showed that the loss of Raptor concomitantly raised the transepithelial electrical resistance (TEER) and para/transcellular permeability leading to a cell monolayer that is leaky for dextran yet electrically resistant to the movement of ions. Paracellular permeability was linked to the downregulation of tight junction protein expression and enhanced autophagy. Raptor-depleted cells had the highest abundance of myosin binding subunit MYPT1 concomitantly with the lowest abundance of p-MYPT1 (Thr696) and phosphorylated myosin light chain (p-MLC, Ser19) implying that MLC phosphatase activity was increased resulting in MLC relaxation. Although rapamycin suppressed mTORC1 activity and decreased the abundance of tight junction proteins in control cells, rapamycin caused a modest increase of TEER compared to Raptor knockdown. SignificanceThe study showed that epithelium paracellular permeability of small molecular weight dextran is dissociated from TEER.

Low dose of arsenic exacerbates toxicity to mice and IPEC-J2 cells exposed with deoxynivalenol: Aryl hydrocarbon receptor and autophagy might be novel therapeutic targets

Deoxynivalenol (DON) and arsenic (As) are widespread environmental contaminants, which are frequently found in human and animal food products. The intestine is a common target of As and DON when they are digested. Numerous studies mainly evaluate the individual effects whereas their combined toxicity has rarely been elucidated. Hence, this study was to assess the effect of low dose of NaAsO2 on DON-induced intestinal damage and explore the underling mechanism in mice and IPEC-J2 cells. The results showed that low dose of NaAsO2 exacerbated DON-induced intestinal impairment by increasing intestinal permeability and decreasing the abundance of tight junction proteins (ZO-1, Occludin, Claudin-1). Further, low dose of NaAsO2 enhanced the AhR signaling pathway and autophagy-related mRNA/protein expressions induced by DON. Interestingly, FICZ, an AhR activator, instead of CH223191, an AhR inhibitor, could alleviate toxicity of the low dose of NaAsO2 in the mice and IPEC-J2 cells. Compared to the WT IPEC-J2 cells, the intestinal barrier damage was more serious in LC3B−/− IPEC-J2 cells induced by low dose of NaAsO2 combination with DON. Collectively, our study demonstrated that low dose of NaAsO2 exacerbated DON-induced intestinal barrier impairment in vivo and in vitro. The present study also demonstrated that activation of AhR-mediated autophagy might be a self-protection mechanism. Hence, AhR and autophagy might be novel therapeutic targets to prevent or alleviate NaAsO2 combined with DON-induced intestinal barrier impairment.

Capsaicin Attenuates Lipopolysaccharide-Induced Inflammation and Barrier Dysfunction in Intestinal Porcine Epithelial Cell Line-J2.

Capsaicin is a spicy, highly pungent, colorless, vanilloid compound found in chili peppers with anti-inflammatory, antioxidant, anti-cancer, and analgesic properties. However, the protective effects of capsaicin on the pig intestine during inflammation are yet to be explored. This study investigated the effects of capsaicin on the gut inflammatory response, intestinal epithelial integrity, and gene expression level of nutrient transporters in a model of lipopolysaccharide (LPS)-induced inflammation in non-differentiated intestinal porcine epithelial cell line-J2 (IPEC-J2). The results showed that the pre-treatment of cells with capsaicin (100 μM) significantly decreased the gene expression and secretion of proinflammatory cytokines induced by LPS through Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. In addition, pre-treatment of cells with capsaicin also increased both gene and protein abundance of tight junction proteins. Furthermore, pre-treatment cells with capsaicin significantly increased trans-epithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-dextran (FD4) from the apical side to the basolateral side compared with the control (P < 0.05). Additionally, pre-treatment of cells with capsaicin upregulated the mRNA abundance of nutrients transporters such as Na+/glucose cotransporter 1 (SGLT1). These results suggested that capsaicin could attenuate LPS-induced inflammation response through TLR4/NF-κB pathway and improve barrier integrity and glucose absorption.

Inactivation of the Wnt/β-catenin signaling contributes to the epithelial barrier dysfunction induced by sodium oxalate in canine renal epithelial cells.

High oxalate consumption has been recognized as a risk factor for renal calcium oxalate stones in companion animals (dogs and cats). However, the cellular signaling involved in oxalate-induced dysfunction in renal tubular epithelial cells remains not fully elucidated. In this study, Mardin-Darby canine kidney (MDCK) cells, an epithelial cell line derived from canine kidney tubule, were tested for cell proliferation activity and barrier function after being exposed to sodium oxalate (NaOx). Further, the involvement of Wnt/β-catenin in NaOx-induced renal epithelial barrier dysfunction was evaluated. MDCK cells treated with NaOx exhibited reduction in cell proliferation and migration. Besides, NaOx exposure led to a decrease in transepithelial electrical resistance and an increase in paracellular permeability. The deleterious effects of NaOx on epithelial barrier function were related to the suppressed abundance of tight junction proteins including zonula occludens, occludin, and claudin-1. Of note, protein levels of β-catenin and phosphorylated (p)-β-catenin (Ser552) in MDCK cells were repressed by NaOx, indicating inhibitory effects on Wnt/β-catenin signaling. An inhibition of glycogen synthase kinase-3β (GSK-3β) by SB216763 enhanced the abundance of β-catenin and p-β-catenin (Ser552), and protected against epithelial barrier dysfunction in NaOx-treated MDCK cells. The results revealed a critical role of Wnt/β-catenin signaling in the epithelial barrier function of MDCK cells. Activation of Wnt/β-catenin signaling might be a potential therapeutic target for the treatment of oxalate-linked renal stones.

Glycine represses endoplasmic reticulum stress-related apoptosis and improves intestinal barrier by activating mammalian target of rapamycin complex 1 signaling.

Endoplasmic reticulum (ER) stress has been associated with the dysfunction of intestinal barrier in humans and animals. We have previously shown that oral administration of glycine to suckling-piglets improves ER stress-related intestinal mucosal barrier impairment and jejunal epithelial apoptosis. However, the underlying mechanism remains unknown. In this study, the protective effect and the mechanism of glycine on apoptosis and dysfunction in intestinal barrier induced by brefeldin A (BFA), an ER stress inducer, was explored in porcine intestinal epithelial cells (IPEC-1). The results showed that BFA treatment led to enhanced apoptosis and upregulation of proteins involved in ER stress signaling, including inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6α (ATF6α), c-Jun N-terminal kinase (JNK), and C/EBP-homologous protein (CHOP). In addition, BFA induced a dysfunction in intestinal epithelial barrier, as evidenced by the increased paracellular permeability, decreased transepithelial electrical resistance (TEER), and reduced abundance of tight junction proteins (occludin, claudin-1, zonula occludens [ZO]-1, and ZO-2). These alterations triggered by BFA were significantly abolished by glycine treatment (P < 0.05), indicating a protective effect of glycine on barrier function impaired by ER stress. Importantly, we found that the regulatory effect of glycine on intestinal permeability, proteins implicated in ER stress and apoptosis, as well as the morphological alterations of the ER were reversed by rapamycin. In summary, our results indicated that glycine alleviates ER stress-induced apoptosis and intestinal barrier dysfunction in IPEC-1 cells in a mammalian target of rapamycin complex 1 (mTORC1)-dependent manner. The data provides in vitro evidence and a mechanism for the protective effect of glycine against the disruption of intestinal barrier integrity induced by ER stress.

Quercetin Alleviates Oxidative Damage by Activating Nuclear Factor Erythroid 2-Related Factor 2 Signaling in Porcine Enterocytes.

Oxidative stress has been implicated in the etiology of multiple gastrointestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease. This study was conducted to evaluate effects of natural product quercetin on diquat-induced oxidative stress in porcine enterocytes and underlying mechanisms. Intestinal porcine epithelial cell line 1 (IPEC-1) cells pretreated with or without quercetin (5 μM, 24 h) were incubated with vehicle or diquat (100 μM) for 6 h. The results showed that diquat treatment induced apoptosis in a caspase-3-dependent manner, as accompanied by elevated reactive oxygen species (ROS) production, increased mitochondrial depolarization, and reduced the abundance of tight junction proteins. These adverse effects of diquat were remarkably abrogated by quercetin administration. Further study indicated that the protective effect of quercetin was associated with elevated protein abundance of nuclear factor erythroid 2-related factor 2 (Nrf2) and increased intracellular glutathione (GSH) content. Interestingly, the beneficial effects of quercetin on diquat-induced oxidative damage were abolished by all-trans-retinoic acid (Atra), a specific inhibitor of Nrf2, indicating a Nrf2-dependent regulation manner. The results show that quercetin attenuates diquat-induced cell injury by promoting protein abundance of Nrf2 and regulating GSH-related redox homeostasis in enterocytes. These findings provide new insights into a function role of quercetin in maintaining intestinal homeostasis.

Mechanism of endocytic regulation of intestinal tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells.

Intestinal epithelial cells are tightly bound by tight junction proteins (TJP) which are dynamic and sensitive to environmental stress. However, the role of the endocytic pathway in the regulation of TJP abundance and tight junction integrity during nutrient stress is poorly understood. Therefore, this study was conducted to investigate the regulation of TJP abundance during nutrient starvation and the role of the endocytic mechanism in this process. IPEC-J2 cells were subjected to nutrient starvation in Krebs-Ringer bicarbonate buffer (KRB) and abundance of TJP, an indication of tight junction remodeling, was characterized with RT-PCR, western blotting and immunofluorescence. Abundance of TJP was dynamically regulated by nutrient starvation. The protein levels of claudin-1, 3, and 4 were initially downregulated within the first 6 hours of starvation, and then, increased thereafter (P<.01). However, there was no change in occludin and ZO-1. Lysosome and proteasome inhibitors were used to determine the contribution of these protein degradation pathways to the TJP remodeling. Short-term starvation-induced degradation of claudin-1, 3, and 4 was found to be lysosome dependent. Specifically, the downregulation of claudin-3 and 4 was via a dynamin-dependent, but clathrin and caveolae independent, endocytic pathway and this downregulation was partly reversed by amino acids supplementation. Interestingly, the re-synthesis of TJP with prolonged starvation partly depended on proteasome function. Collectively, this study, for the first time, elucidated a major role for dynamin-dependent endocytosis of claudin-3 and 4 during nutrient stress in intestinal epithelial cells. Therefore, transient endocytosis inhibition may be a potential mechanism for preserving tight junction integrity and function in metabolic or pathological states such as inflammatory bowel disease that involves destruction of intestinal epithelial TJP.

PSIII-36 The endocytic pathway plays a major role in the regulation of tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells

Abstract Postweaning pigs are subjected to nutrient deprivation during which intestinal epithelial cells undergo increased turnover. To preserve intestinal function, intestinal epithelial cells must activate adaptive mechanisms that allow them to cope with starvation-induced stress; most importantly, the preservation of intestinal barrier function. The objective of this study was to investigate the underlying mechanisms involved in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were subjected to total nutrient starvation in Krebs-Ringer bicarbonate (KRB) buffer for 0, 3, 6, 12 and 24 h. Abundance of tight junction proteins was determined by RT-PCR, western blotting and immunofluorescence. Compared with control group (0 h), the protein expression of claudin 1, claudin 3 and claudin 4 protein was downregulated up to 6 h of starvation and then increased thereafter (P &amp;lt; 0.01). However, there was no change in the protein level of occludin and ZO-1. To determine the contribution of the lysosome and the ubiquitin proteasome pathways to regulation of tight junction protein abundance, the lysosome (Bafilomycin A1) and the proteasome (MG132) inhibitors were used in nutrient starved cells. Results showed the degradation of claudin 1, 3 and 4 up to 6 h of starvation was through the lysosomal pathway. Surprisingly, re-synthesis of claudins 4 and claudin 3 after prolonged starvation (12 and 24 h) was prevented when cells were treated with bafilomycin A1 and MG132, respectively. The autophagy-lysosome pathway inhibitors (Wortmannin and MHY1485) and endosome-lysosome pathway inhibitors (Dynasore and Pitstop 2) were further used to determine the specific roles of these pathways. In summary, the degradation of claudin 3 and claudin 4 during short-term starvation (up to 6 h) was through the dynamin-dependent endocytic pathway. However, re-synthesis of these proteins after prolonged starvation relies on both the lysosome and proteasome pathways.

Eugenol attenuates inflammatory response and enhances barrier function during lipopolysaccharide-induced inflammation in the porcine intestinal epithelial cells.

Eugenol (4-allyl-2-methoxyphenol) is an essential oil component, possessing antimicrobial, anti-inflammatory, and antioxidative properties; however, the effect of eugenol on porcine gut inflammation has not yet been investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model in porcine intestinal epithelial cells (IPEC-J2) has been set up. Cells were pretreated with 100 μM (16.42 mg/L) eugenol for 2 h followed by 10 μg/mL LPS stimulation for 6 h. Proinflammatory cytokine secretion; reactive oxygen species; gene expression of proinflammatory cytokines, tight junction proteins, and nutrient transporters; the expression and distribution of zonula occludens-1 (ZO-1); transepithelial electrical resistance (TEER); and cell permeability were measured to investigate the effect of eugenol on inflammatory responses and gut barrier function. The results showed that eugenol pretreatment significantly suppressed the LPS-stimulated interleukin-8 level and the mRNA abundance of tumor necrosis factor-α and restored the LPS-stimulated decrease of the mRNA abundance of tight junction proteins, such as ZO-1 and occludin, and the mRNA abundance of nutrient transporters, such as B0 1 system ASC sodium-dependent neutral amino acid exchanger 2, sodium-dependent glucose transporter 1, excitatory amino acid transporter 1, and peptide transporter 1. In addition, eugenol improved the expression and even redistribution of ZO-1 and tended to increase TEER value and maintained the barrier integrity. In conclusion, a low dose of eugenol attenuated inflammatory responses and enhanced selectively permeable barrier function during LPS-induced inflammation in the IPEC-J2 cell line.

PSVIII-1 Nutrient starvation induces autophagy and alters intestinal barrier function in IPEC-J2 cells

Abstract Autophagy is a cellular process of controlled degradation of damaged organelles and cytoplasmic macromolecules during stress for maintaining homeostasis. Intestinal epithelial cells are barriers against microorganisms, toxins and food antigens. Whether autophagy plays a role in regulating intestinal barrier function is unclear. The objective of this study was to characterize the role of autophagy in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were nutrient starved in Krebs-Ringer bicarbonate (KRB) buffer for 0, 0.5, 1, 2, 3, 6, 9 and 12 h. Expression of genes implicated in autophagy regulation (AMPK, MDM2, p53 and DRAM) was determined by RT-PCR. The ratio of protein abundance of microtubule-associated protein light chain 3 (LC3-II/LC3-I) and p62, positive and negative markers of autophagy induction, respectively, was determined by western blotting. Compared with control group (0 h), the relative mRNA expression level of AMPK, MDM2, p53 and DRAM significantly decreased by an average of 52.5% (P &amp;lt; 0.01). Relative to 0 h, the mRNA expression of claudin 1 and claudin 4 significantly increased (108.0%) up to 6 h of starvation and then decreased (31.4%) thereafter (P &amp;lt; 0.01). On the contrary, abundance of claudin 1 and claudin 4 protein was downregulated (49.5%) up to 3 h of starvation and then increased (82.6%) thereafter (P &amp;lt; 0.01). Protein expression of claudin 3 was reduced (P &amp;lt; 0.01) with duration of starvation. There was no change in the protein level of occludin and ZO-1. The ratio of LC3-II/LC3-I significantly increased with duration starvation (P &amp;lt; 0.01), whereas p62 and phospho-AMPK levels decreased up to 6 h of starvation and then increased thereafter (P &amp;lt; 0.01). In summary, autophagy may be implicated in the regulation of tight junction integrity during nutrient starvation in IPEC-J2 cells.

C-type natriuretic peptide regulates the molecular components of the rainbow trout gill epithelium tight junction complex

Freshwater (FW) fish experience passive paracellular loss of ions into the surrounding environment across water-exposed epithelia such as the gill. The mitigation of paracellular ion loss is thought to be regulated by proteins of the tight junction (TJ) complex and in particular, the large superfamily of claudin (cldn) TJ proteins plays an important role. Transcript and protein levels of TJ proteins in teleosts are known to be under endocrine control of several important osmoregulatory hormones and the current study was aimed at determining whether the osmoregulatory hormone, C-type natriuretic peptide (CNP), can alter paracellular permeability and TJ protein abundance in a primary cultured gill epithelium derived from rainbow trout. Natriuretic peptide receptors were detected in the cultured trout gill epithelium. It was found that (i) developing cultured gill epithelia "grown" in the presence of 10 nM CNP, and (ii) mature cultured gill epithelia exposed to 10 nM CNP for 48 h, exhibited augmented barrier properties. This occurred in association with reduced flux rates of a paracellular permeability marker (polyethylene glycol, molecular mass 400; PEG-400) and, reduced ion efflux (i.e. ion loss) when preparations were exposed to apical FW. Exposure to CNP altered mRNA abundance of cldn-3a, -5a, -6, - 8c, -20a, -25b, -28a, -32a and cgn, but differences in the transcriptional response were observed between chronic and acute CNP exposure. In contrast, chronic and acute exposure to CNP resulted in reduced cldn-10e/Cldn-10e abundance. Data suggest that CNP may play a role in regulating the molecular physiology of the TJ complex in the fish gill epithelium and contribute to the regulation of salt and water balance by influencing the paracellular permeability properties of this tissue.

MTOR PROMOTES BBB BREAKDOWN IN A MODEL OF ALZHEIMER’S DISEASE

Cerebral amyloid angiopathy (CAA) is characterized by fibrillar amyloid β (Aβ) association with cerebrovasculature, which leads to impaired brain vascular function, and is present in 87% of people with Alzheimer’s disease (AD). We previously showed that inhibition of mTOR by rapamycin prevented BBB breakdown and reduced vascular fibrillar Aβ in 18-19 month old Tg2576 mice that model AD-associated CAA. This finding suggests that mTOR attenuation restores integrity of the blood brain barrier (BBB) and concomitantly reduces vascular Aβ accumulation in this mouse model. Objective: To determine the mechanisms by which mTOR drives BBB breakdown we measured the abundance of tight junction proteins zonula occludens 1 (ZO-1), occludin, and claudin-5. Methods: We used immunofluorescent confocal microscopy on frozen brain tissue sections of the same Tg2576 mice used in the previous study. Results: We confirm BBB breakdown in Tg2576 mouse brains and showed that some, but not all tight junction proteins measured were decreased in cerebrovasculature of Tg2576 mice. Attenuation of mTOR by rapamycin preserved BBB integrity, decreased vascular Aβ accumulation, and increased levels of tight junction protein abundance in Tg2576 mice, which also showed a reduced numbers of cerebral microhemorrhages. Conclusions: Taken together, these data suggest that mTOR promotes brain vascular Aβ deposition, BBB breakdown and vascular damage in the Tg2576 mouse model. Thus, mTOR inhibitors such as rapamycin – an FDA approved drug - may have promise in the treatment of AD and other dementias with related cerebrovascular dysfunction.