Transport of Dietary Anti-Inflammatory Peptide, γ-Glutamyl Valine (γ-EV), across the Intestinal Caco-2 Monolayer

The present study analyzed the transepithelial transport of the dietary anti-inflammatory peptide, γ-glutamyl valine (γ-EV). γ-EV is naturally found in dry edible beans. Our previous study demonstrated the anti-inflammatory potency of γ-EV against vascular inflammation at a concentration of 1mM, and that it can transport with the apparent permeability coefficient (Papp) of 1.56 × 10−6 ± 0.7 × 10−6 cm/s across the intestinal Caco-2 cells. The purpose of the current study was to explore whether the permeability of the peptide could be enhanced and to elucidate the mechanism of transport of γ-EV across Caco-2 cells. The initial results indicated that γ-EV was nontoxic to the Caco-2 cells up to 5 mM concentration and could be transported across the intestinal cells intact. During apical-to-basolateral transport, a higher peptide dose (5 mM) significantly (p < 0.01) enhanced the transport rate to 2.5 × 10−6 ± 0.6 × 10−6 cm/s. Cytochalasin-D disintegrated the tight-junction proteins of the Caco-2 monolayer and increased the Papp of γ-EV to 4.36 × 10−6 ± 0.16 × 10−6 cm/s (p < 0.001), while theaflavin 3′-gallate and Gly-Sar significantly decreased the Papp (p < 0.05), with wortmannin having no effects on the peptide transport, indicating that the transport route of γ-EV could be via both PepT1-mediated and paracellular.

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Mammalian stanniocalcin-1 activates mitochondrial antioxidant pathways: new paradigms for regulation of macrophages and endothelium

AJP Renal Physiology ◽

10.1152/ajprenal.00260.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. F248-F254 ◽

Cited By ~ 53

Author(s):

David Sheikh-Hamad

Keyword(s):

Endothelial Cells ◽

Uncoupling Protein ◽

Physiological Role ◽

Tight Junction Proteins ◽

Renal Protection ◽

Superoxide Generation ◽

Cell Mobility ◽

Anti Inflammatory ◽

Inflammatory Action ◽

Junction Proteins

The mammalian homolog of the fish calcium regulatory hormone stanniocalcin-1 (STC1) is ubiquitously expressed and likely functions in an autocrine/paracrine fashion. Mammalian STC1 does not appear to exert significant effects on serum calcium, and its physiological role remains to be determined. In macrophages, STC1 decreases intracellular calcium and cell mobility; attenuates the response to chemoattractants; and diminishes superoxide generation through induction of uncoupling protein-2 (UCP2). In cytokine-treated endothelial cells, STC1 attenuates superoxide generation and the activation of inflammatory pathways [c-Jun NH2-terminal kinase (JNK) and NF-κB]; maintains the expression of tight junction proteins, preserving the endothelial monolayer seal; and decreases transendothelial migration of leukocytes. Combined, the effects of STC1 on endothelial cells and macrophages predict potent anti-inflammatory action. Indeed, application of the anti-glomerular basement membrane (GBM) glomerulonephritis model to STC1 transgenic mice that display increased expression of STC1 transgene in endothelial cells and macrophages yields renal protection. Our data suggest that STC1 activates antioxidant pathways in endothelial cells and macrophages and displays cytoprotective and anti-inflammatory actions.

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

10.21203/rs.3.rs-64813/v1 ◽

2020 ◽

Author(s):

Chao Qin ◽

Yi Jiang ◽

Xing Chen ◽

Yingxue Bian ◽

Yaoqi Wang ◽

...

Keyword(s):

Tight Junction ◽

Intestinal Permeability ◽

Light Chain ◽

Myosin Light Chain ◽

Burn Injury ◽

Intestinal Barrier ◽

Signalling Pathway ◽

Tight Junction Proteins ◽

Anti Inflammatory ◽

Junction Proteins

Abstract Background Dexmedetomidine, a potent α2-adrenoceptor agonist with analgesic, sedative, anti-inflammatory, and anti-apoptotic effects, is commonly used in patients with critical illness in intensive care units. Accumulating evidence indicates that dexmedetomidine can protect against intestinal dysfunction. However, the specific mechanisms of its protective effects against burn-induced intestinal barrier injury remain unclear. Here, we aimed to explore the possible positive effects of dexmedetomidine on burn-induced intestinal barrier injury and the role of the myosin light chain kinase (MLCK)/phosphorylated myosin light chain (p-MLC) signalling pathway in an experimental model of burn injury.Methods In this study, the intestinal permeability of burn-induced intestinal barrier damage was assessed by estimating the plasma concentration of 4.4 kDa fluorescein isothiocyanate-labelled dextran (FITC-dextran). 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 determined by enzyme-linked immunosorbent assay (ELISA).Results Our findings demonstrated 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.Conclusions In 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, suggesting that it may be an effective drug in the treatment of burn-induced intestinal injury.Trial registration Not appliance.

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Benzotropolone moiety in theaflavins is responsible for inhibiting peptide-transport and activating AMP-activated protein kinase in Caco-2 cells

Functional Foods in Health and Disease ◽

10.31989/ffhd.v3i5.60 ◽

2013 ◽

Vol 3 (5) ◽

pp. 111 ◽

Cited By ~ 4

Author(s):

Ha-Young Park ◽

Yuri Kunitake ◽

Toshiro Matsui

Keyword(s):

Protein Kinase ◽

Ussing Chamber ◽

Fixed Time ◽

Peptide Transport ◽

Peptide Transporter ◽

Apparent Permeability ◽

Structural Requirement ◽

Compound C ◽

Apparent Permeability Coefficient ◽

Amp Activated Protein Kinase

Objective: In the small intestine, peptide transporter 1 (PEPT1) plays a role in the transport of di- and tri-peptides. Recently, we found that theaflavins (TFs), dimeric catechins, inhibited the transport of di-peptides across Caco-2 monolayers by suppressing the expression of PEPT1 through AMP-activated protein kinase (AMPK) activation. In this study, we investigated the structural requirement of theaflavins for the effect, and the mechanism(s) underling theaflavin-induced AMPK activation.Methods: Theaflavin-3’-O-gallate (TF3’G) was used for this study, since it possessed the most potent inhibition power for peptide-transport among theaflavins. Absorption ability was measured with Caco-2 cell monolayers treated with or without 20 μM sample (TF3’G or its related compounds) in an Ussing Chamber. The amount of Gly-Sar (a model of PEPT1-transporing peptide) transport at fixed time-points to 60 min was determined by fluorescent naphthalene-2,3-dicarboxaldehyde-derivatized assay (Ex/Em: 405 nm/460 nm). The apparent permeability coefficient (Papp) was used to evaluate the permeability. Expression of PEPT1 protein in Caco-2 cells treated with or without 20 μM TF3’G in the presence or absence of inhibitor (10 μM compound C as AMPK inhibitor or 25 μM STO-609 as CaMKK inhibitor) was evaluated by Western blot.Results: The Papp value of Gly-Sar significantly (P < 0.05) decreased in 20 μM purprogallin-treated Caco-2 cells as well as in TF3’G-treated cells, together with the reduction of PEPT1 expression, while their monomeric catechins did not show any Papp reduction. In TF3'G-treated Caco-2 cells, the recovery of the reduced PEPT1 expression was found by 10 μM compound C, but not STO-609.Conclusion: The study demonstrated that the benzotropolone moiety in theaflavins was a crucial structural requirement for exerting the inhibition of intestinal peptide-transport, and the suppression of PEPT1 expression by theaflavins would be caused by activating LKB1/AMPK pathway, but not CaMKK/AMPK pathway.Keywords: Theaflavin-3’-Ο-gallate, Peptide transport, PEPT1, Benzotropolone, AMP-activated protein kinase, Calmodulin-dependent protein kinase kinase

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Transepithelial Transport Characteristics of the Cholesterol- Lowing Soybean Peptide, WGAPSL, in Caco-2 Cell Monolayers

Molecules ◽

10.3390/molecules24152843 ◽

2019 ◽

Vol 24 (15) ◽

pp. 2843

Author(s):

Huijuan Zhang ◽

Yawen Duan ◽

Yulin Feng ◽

Jing Wang

Keyword(s):

Tight Junctions ◽

Soybean Protein ◽

Blood Stream ◽

Transepithelial Transport ◽

Apparent Permeability ◽

Cholesterol Lowering ◽

Phase Ii Metabolism ◽

Cell Monolayers ◽

Apparent Permeability Coefficient ◽

Lowering Activity

Recent studies have shown that soybean protein and its peptides have cholesterol-lowering activities. However, it is not clear whether these peptides could overcome physiological barriers, such as phase II metabolism in gastrointestinal tract and poor permeability, to reach the blood stream in its intact form. Therefore, the transepithelial transport characteristics of soybean peptide Trp-Gly-Ala-Pro-Ser-Leu (WGAPSL) with cholesterol- lowering activity were investigated in Caco-2 cells. In this study; the transepithelial absorption of WGAPSL was studied using human intestinal Caco-2 cell monolayers. The results showed that WGAPSL had good stability (83.9% ±1.98%) after simulated gastric and intestinal digestion. During the apical (AP) side to basolateral (BL) side transport, WGAPSL was absorbed intact through Caco-2 cell monolayers with apparent permeability coefficient (Papp) values of 4.4 × 10−8 to 1.2 × 10−8 cm/s. Cytochalasin D loosened the tight junctions of Caco-2 cell monolayers and significantly (p < 0.05) improved the transport process. Sodium azide, wortmannin, and Gly-Pro had minimal effects on transport, demonstrating that the major transport route of WGAPVL was paracellular via tight junctions. Finally, LC-MS analysis showed that Gly-Ala-Pro (GAP) was the important part for the intact absorption of WGAPVL and Trp (W) was the most unstable amino acid residue.

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The blood-brain barrier studied in vitro across species

PLoS ONE ◽

10.1371/journal.pone.0236770 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0236770

Author(s):

Maj Schneider Thomsen ◽

Nanna Humle ◽

Eva Hede ◽

Torben Moos ◽

Annette Burkhart ◽

...

Keyword(s):

Tight Junction ◽

Cell Size ◽

Blood Brain Barrier ◽

Transferrin Receptor ◽

Brain Barrier ◽

Tight Junction Proteins ◽

Apparent Permeability ◽

Blood Brain ◽

Junction Proteins

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis and protects the brain against toxic substances circulating in the blood, meaning that only a few drugs can pass the BBB. Thus, for drug screening, understanding cell interactions, and pathology, in vitro BBB models have been developed using BECs from various animal sources. When comparing models of different species, differences exist especially in regards to the transendothelial electrical resistance (TEER). Thus, we compared primary mice, rat, and porcine BECs (mBECs, rBECs, and pBECs) cultured in mono- and co-culture with astrocytes, to identify species-dependent differences that could explain the variations in TEER and aid to the selection of models for future BBB studies. The BBB models based on primary mBECs, rBECs, and pBECs were evaluated and compared in regards to major BBB characteristics. The barrier integrity was evaluated by the expression of tight junction proteins and measurements of TEER and apparent permeability (Papp). Additionally, the cell size, the functionality of the P-glycoprotein (P-gp) efflux transporter, and the expression of the transferrin receptor were evaluated and compared. Expression and organization of tight junction proteins were in all three species influenced by co-culturing, supporting the findings, that TEER increases after co-culturing with astrocytes. All models had functional polarised P-gp efflux transporters and expressed the transferrin receptor. The most interesting discovery was that even though the pBECs had higher TEER than rBECs and mBECs, the Papp did not show the same variation between species, which could be explained by a significantly larger cell size of pBECs. In conclusion, our results imply that the choice of species for a given BBB study should be defined from its purpose, instead of aiming to reach the highest TEER, as the models studied here revealed similar BBB properties.

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Eleutheroside B increase tight junction proteins and anti‐inflammatory cytokines expression in intestinal porcine jejunum epithelial cells (IPEC‐J2)

Journal of Animal Physiology and Animal Nutrition ◽

10.1111/jpn.13087 ◽

2019 ◽

Author(s):

Dongsheng Che ◽

Bao Zhao ◽

Yueli Fan ◽

Rui Han ◽

Chun Zhang ◽

...

Keyword(s):

Epithelial Cells ◽

Tight Junction ◽

Inflammatory Cytokines ◽

Tight Junction Proteins ◽

Eleutheroside B ◽

Anti Inflammatory ◽

Junction Proteins

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Resveratrol defends blood-brain barrier integrity in experimental autoimmune encephalomyelitis mice

Journal of Neurophysiology ◽

10.1152/jn.00510.2016 ◽

2016 ◽

Vol 116 (5) ◽

pp. 2173-2179 ◽

Cited By ~ 30

Author(s):

Dong Wang ◽

Shi-Ping Li ◽

Jin-Sheng Fu ◽

Sheng Zhang ◽

Lin Bai ◽

...

Keyword(s):

Tight Junction ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Tight Junction Proteins ◽

Autoimmune Encephalomyelitis ◽

Arginase 1 ◽

Blood Brain ◽

Anti Inflammatory ◽

The Brain ◽

Junction Proteins

The mouse autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis (MS), is primarily characterized as dysfunction of the blood-brain barrier (BBB). Resveratrol exhibits anti-inflammatory, antioxidative, and neuroprotective activities. We investigated the beneficial effects of resveratrol in protecting the integrity of the BBB in EAE mice and observed improved clinical outcome in the EAE mice after resveratrol treatment. Evans blue (EB) extravasation was used to detect the disruption of BBB. Western blot were used to detected the tight junction proteins and adhesion molecules zonula occludens-1 (ZO-1), occludin, ICAM-1, and VCAM-1. Inflammatory factors inducible nitric oxide synthase (iNOS), IL-1β, and arginase 1 were evaluated by quantitative RT-PCR (qPCR) and IL-10 by ELISA. NADPH oxidase (NOX) levels were evaluated by qPCR, and its activity was analyzed by lucigenin-derived chemiluminescence. Resveratrol at doses of 25 and 50 mg/kg produced a dose-dependent decrease in EAE paralysis and EB leakage, ameliorated EAE-induced loss of tight junction proteins ZO-1, occludin, and claudin-5, as well as repressed the EAE-induced increase in adhesion proteins ICAM-1 and VCAM-1. In addition, resveratrol suppressed the EAE-induced overexpression of proinflammatory transcripts iNOS and IL-1β and upregulated the expression of anti-inflammatory transcripts arginase 1 and IL-10 cytokine in the brain. Furthermore, resveratrol downregulated the overexpressed NOX2 and NOX4 in the brain and suppressed NADPH activity. Resveratrol ameliorates the clinical severity of MS through maintaining the BBB integrity in EAE mice.

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The blood-brain barrier studied in vitro across species

10.1101/2020.07.15.204032 ◽

2020 ◽

Author(s):

Maj Schneider Thomsen ◽

Nanna Humle ◽

Eva Hede ◽

Torben Moos ◽

Annette Burkhart ◽

...

Keyword(s):

Tight Junction ◽

Cell Size ◽

Blood Brain Barrier ◽

Transferrin Receptor ◽

Brain Barrier ◽

Tight Junction Proteins ◽

Apparent Permeability ◽

Blood Brain ◽

Junction Proteins

AbstractThe blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis and protects the brain against toxic substances circulating in the blood, which consequently means that only a few drugs can pass the BBB. Thus for drug screening, understanding cell interactions, and pathology, in vitro BBB models have been developed using BECs from various animal sources. When comparing models of different species, differences exist especially in regards to the transendothelial electrical resistance (TEER). Thus, in the present study, we compared primary mice, rat, and porcine BECs (mBECs, rBECs, and pBECs) cultured in mono- and co-culture with astrocytes, to identify potential species-dependent differences that could explain the variations in TEER and aid to the selection of models for future in vitro BBB studies.The in vitro BBB models based on primary mBECs, rBECs, and pBECs were evaluated and compared in regards to major BBB characteristics. The barrier integrity was evaluated by the expression of tight junction proteins and measurements of TEER and apparent permeability (Papp). Furthermore, the cell size, the functionality of the P-glycoprotein (P-gp) efflux transporter, and the expression of the transferrin receptor were evaluated and compared.pBECs exhibit the highest TEER followed by rBECs and mBECs, but surprisingly the Papp was not that different, which might be explained by a significantly larger cell size of pBECs than rBECs and mBECs. The expression and organization of many tight junction proteins were in all three species influenced by co-culturing, which supports the findings that the TEER increases after co-culturing with astrocytes. Furthermore, all models had functional polarised P-gp efflux transporters and expressed the transferrin receptor. In conclusion, the choice of species for in vitro BBB modeling should be based on the purpose of the study.

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