Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata).

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.

Frontline Science: Two flavonoid compounds attenuate allergic asthma by regulating epithelial barrier via G protein-coupled estrogen receptor: Probing a possible target for allergic inflammation.

Allergic asthma is a common chronic lung inflammatory disease and seriously influences public health. We aim to investigate the effects of formononetin (FMN) and calycosin (CAL), 2 flavonoids in Radix Astragali, on allergic asthma and elucidate possible therapeutic targets. A house dust mite (HDM)-induced allergic asthma mouse model and TNF-α and Poly(I:C) co-stimulated human bronchial epithelial cell line (16HBE) were performed respectively in vivo and in vitro. The role of G protein-coupled estrogen receptor (GPER) was explored by its agonist, antagonist, or GPER small interfering RNA (siGPER). E-cadherin, occludin, and GPER were detected by western blotting, immunohistochemistry, or immunofluorescence. The epithelial barrier integrity was assessed by trans-epithelial electric resistance (TEER). Cytokines were examined by enzyme-linked immunosorbent assay (ELISA). The results showed that flavonoids attenuated pulmonary inflammation and hyperresponsiveness in asthmatic mice. These flavonoids significantly inhibited thymic stromal lymphopoietin (TSLP), increased occludin and restored E-cadherin in vivo and in vitro. The effects of flavonoids on occludin and TSLP were not interfered by ICI182780 (estrogen receptor antagonist), while blocked by G15 (GPER antagonist). Furthermore, compared with PPT (ERα agonist) and DPN (ERß agonist), G1 (GPER agonist) significantly inhibited TSLP, up-regulated occludin, and restored E-cadherin. siGPER and TEER assays suggested that GPER was pivotal for the flavonoids on the epithelial barrier integrity. Finally, G1 attenuated allergic lung inflammation, which could be abolished by G15. Our data demonstrated that 2 flavonoids in Radix Astragali could alleviate allergic asthma by protecting epithelial integrity via regulating GPER, and activating GPER might be a possible therapeutic strategy against allergic inflammation.

Longan pulp polysaccharides relieve intestinal injury in vivo and in vitro by promoting tight junction expression.

The integrity of the intestinal mucosal barrier is important for the health of the host. In this study, longan pulp polysaccharides (LP) prevented the intestinal mucosal injury by increasing the expression of mucin 2, tight junction proteins zonulae occludens-1 (ZO-1), claudin-1, claudin-4, and adherens junction E-cadherin in cyclophosphamide-treated mice. To further identify the principle bioactive component of LP, four acidic polysaccharides (LPIa, LPIIa, LPIIIa, and LPIVa) were purified, and their intestinal protection activity in vitro was compared. LPIa, LPIIa, and LPIIIa displayed an ability to increase mRNA expression of ZO-1, claudin-1, occludin, and E-cadherin in differentiated Caco-2 cells, especially LPIa. LPIa has specific structure characteristics: porous surface structure, a high molecular weight (1.47 × 105 Da), and two specific glycosidic linkages of α-Araf-(1→ and →5)-α-Araf-(1→. These structure characteristics might primarily contribute to greater intestinal barrier protective effect of LPIa.

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression.

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that ß-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit the expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of ß-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases.

Experimental Colitis Is Attenuated by Cardioprotective Diet Supplementation That Reduces Oxidative Stress, Inflammation, and Mucosal Damage.

Inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD) are multifactorial, relapsing disorders of the gastrointestinal tract. However, the etiology is still poorly understood but involves altered immune responses, epithelial dysfunction, environmental factors, and nutrition. Recently, we have shown that the diet supplement corabion has cardioprotective effects due to reduction of oxidative stress and inflammation. Since oxidative stress and inflammation are also prominent risk factors in IBD, we speculated that corabion also has beneficial effects on experimental colitis. Colitis was induced in male mice by administration of 3.5% (w/v) dextran sulfate sodium (DSS) in drinking water for a period of 3 or 7 days with or without daily gavage feeding of corabion consisting of vitamin C, vitamin E, L-arginine, and eicosapentaenoic and docosahexaenoic acid. We found that corabion administration attenuated DSS-induced colon shortening, tissue damage, and disease activity index during the onset of colitis. Mechanistically, these effects could be explained by reduced neutrophil recruitment, oxidative stress, production of proinflammatory cytokines, and internalization of the junctional proteins ZO-1 and E-cadherin leading to less edema formation. Thus, corabion may be a useful diet supplement for the management of chronic inflammatory intestinal disorders such as IBD.

Glutamine supplementation attenuates ethanol-induced disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier dysfunction and fatty liver in mice.

Previous in vitro studies showed that glutamine (Gln) prevents acetaldehyde-induced disruption of tight junctions and adherens junctions in Caco-2 cell monolayers and human colonic mucosa. In the present study, we evaluated the effect of Gln supplementation on ethanol-induced gut barrier dysfunction and liver injury in mice in vivo. Ethanol feeding caused a significant increase in inulin permeability in distal colon. Elevated permeability was associated with a redistribution of tight junction and adherens junction proteins and depletion of detergent-insoluble fractions of these proteins, suggesting that ethanol disrupts apical junctional complexes in colonic epithelium and increases paracellular permeability. Ethanol-induced increase in colonic mucosal permeability and disruption of junctional complexes were most severe in mice fed Gln-free diet. Gln supplementation attenuated ethanol-induced mucosal permeability and disruption of tight junctions and adherens junctions in a dose-dependent manner, indicating the potential role of Gln in nutritional intervention to alcoholic tissue injury. Gln supplementation dose-dependently elevated reduced-protein thiols in colon without affecting the level of oxidized-protein thiols. Ethanol feeding depleted reduced protein thiols and elevated oxidized protein thiols. Ethanol-induced protein thiol oxidation was most severe in mice fed with Gln-free diet and absent in mice fed with Gln-supplemented diet, suggesting that antioxidant effect is one of the likely mechanisms involved in Gln-mediated amelioration of ethanol-induced gut barrier dysfunction. Ethanol feeding elevated plasma transaminase and liver triglyceride, which was accompanied by histopathologic lesions in the liver; ethanol-induced liver damage was attenuated by Gln supplementation. These results indicate that Gln supplementation ameliorates alcohol-induced gut and liver injury.

Protective Effects of N-Acetyl Cysteine against Diesel Exhaust Particles-Induced Intracellular ROS Generates Pro-Inflammatory Cytokines to Mediate the Vascular Permeability of Capillary-Like Endothelial Tubes.

Exposure to diesel exhaust particles (DEP) is associated with pulmonary and cardiovascular diseases. Previous studies using in vitro endothelial tubes as a simplified model of capillaries have found that DEP-induced ROS increase vascular permeability with rearrangement or internalization of adherens junctional VE-cadherin away from the plasma membrane. This allows DEPs to penetrate into the cell and capillary lumen. In addition, pro-inflammatory cytokines are up-regulated and mediate vascular permeability in response to DEP. However, the mechanisms through which these DEP-induced pro-inflammatory cytokines increase vascular permeability remain unknown. Hence, we examined the ability of DEP to induce permeability of human umbilical vein endothelial cell tube cells to investigate these mechanisms. Furthermore, supplementation with NAC reduces ROS production following exposure to DEP. HUVEC tube cells contributed to a pro-inflammatory response to DEP-induced intracellular ROS generation. Endothelial oxidative stress induced the release of TNF-α and IL-6 from tube cells, subsequently stimulating the secretion of VEGF-A independent of HO-1. Our data suggests that DEP-induced intracellular ROS and release of the pro-inflammatory cytokines TNF- α and IL-6, which would contribute to VEGF-A secretion and disrupt cell-cell borders and increase vasculature permeability. Addition of NAC suppresses DEP-induced ROS efficiently and reduces subsequent damages by increasing endogenous glutathione.

Sphingomyelin and phosphatidylcholine contrarily affect the induction of apoptosis in intestinal epithelial cells.

SCOPE: The major alimentary sources for the plasma membrane lipid sphingomyelin (SM) are dairy products, eggs, and meat. We recently reported that the SM metabolite ceramide induces cathepsin D mediated apoptosis in murine intestinal epithelial cells (IECs) and increases inflammation in acute colitis. We investigated the impact of SM and phosphatidylcholine on apoptosis in human IECs and point out BH3-interacting death agonist (BID) as link between cathepsin D and apoptosis. METHODS AND RESULTS: HT-29 and isolated human IECs were stimulated with SM or phosphatidylcholine. SM treatment resulted in increased apoptosis. Phosphatidylcholine showed contrary effects. Western revealed higher amounts of cathepsin D and BID activation upon lipid stimulation. Western blotting revealed BID activation through SM in both an induced and a spontaneous mouse model of colitis. CONCLUSION: Dietary phospholipids may induce or abolish apoptosis in IECs and seem to play a role in the pathogenesis of inflammatory bowel diseases. This nutritional factor might be considered when evaluating the pathogenesis of inflammatory bowel diseases. Effects of SMase- and SM treatment on inflammation in dextran sulfate sodium induced animal models of colitis and in vitro experiments are discussed as controversial. Variable sources of SM, feeding techniques, and mouse strains might play a role.

Disruption of endothelial adherens junction by invasive breast cancer cells is mediated by reactive oxygen species and is attenuated by AHCC.

AIMS: The effect of antioxidants on treatment of cancer is still controversial. Previously, we demonstrated that interaction of breast cancer cells with endothelial cells leads to tyrosine phosphorylation of VE-cadherin and disruption of endothelial adherens junction (EAJ). The molecular mechanism underlying the anti-metastatic effects of mushroom-derived active hexode correlated compound (AHCC) remains elusive. MAIN METHODS: Several cellular and biochemical techniques were used to determine the contribution of oxidative stress in the disruption of EAJ and to test this hypothesis that AHCC inhibits the breast cancer cell-induced disruption of EAJ. KEY FINDINGS: Interaction of breast cancer cells (MDA-MB-231 cells) with human umbilical vein endothelial cells (HUVECs) leads to an increase in generation of reactive oxygen species (ROS). Treatment of HUVECs with H2O2 or phorbol myristate acetate (PMA) led to tyrosine phosphorylation of VE-cadherin, dissociation of ß-catenin from VE-cadherin complex and increased transendothelial migration (TEM) of MDA-MB-231 cells. Induction of VE-cadherin tyrosine phosphorylation by PMA or by interaction of MDA-MB-231 cells with HUVECs was mediated by HRas and protein kinase C-α signaling pathways. Disruption of EAJ and phosphorylation of VE-cadherin induced by interaction of MDA-MB-231 cells with HUVECs were attenuated when HUVECs were pretreated with an antioxidant, N-acetylcysteine (NAC) or AHCC. AHCC inhibited TEM of MDA-MB-231 cells and generation of ROS induced by interaction of MDA-MB-231 cells with HUVECs. SIGNIFICANCE: Our studies suggest that ROS contributes to disruption of EAJ induced by interaction of MDA-MB-231 cells with HUVECs and AHCC attenuates this alteration.

A novel approach to prevent endothelial hyperpermeability: the Crataegus extract WS® 1442 targets the cAMP/Rap1 pathway.

Endothelial hyperpermeability followed by edema formation is a hallmark of many severe disorders. Effective drugs directly targeting endothelial barrier function are widely lacking. We hypothesized that the hawthorn (Crataegus spp.) extract WS® 1442, a proven multi-component drug against moderate forms of heart failure, would prevent vascular leakage by affecting endothelial barrier-regulating systems. In vivo, WS® 1442 inhibited the histamine-evoked extravasation of FITC-dextran from mouse cremaster muscle venules. In cultured human endothelial cells, WS® 1442 blocked the thrombin-induced FITC-dextran permeability. By applying biochemical and microscopic techniques, we revealed that WS® 1442 abrogates detrimental effects of thrombin on adherens junctions (vascular endothelial-cadherin), the F-actin cytoskeleton, and the contractile apparatus (myosin light chain). Mechanistically, WS® 1442 inhibited the thrombin-induced rise of intracellular calcium (ratiometric measurement), followed by an inactivation of PKC and RhoA (pulldown assay). Moreover, WS® 1442 increased endothelial cAMP levels (ELISA), which consequently activated PKA and Rap1 (pulldown assay). Utilizing pharmacological inhibitors or siRNA, we found that PKA is not involved in barrier protection, whereas Epac1, Rap1, and Rac1 play a crucial role in the WS® 1442-induced activation of cortactin, which triggers a strong cortical actin rearrangement. In summary, WS® 1442 effectively protects against endothelial barrier dysfunction in vitro and in vivo. It specifically interacts with endothelial permeability-regulating systems by blocking the Ca(2+)/PKC/RhoA and activating the cAMP/Epac1/Rap1 pathway. As a proven safe herbal drug, WS® 1442 opens a novel pharmacological approach to treat hyperpermeability-associated diseases. This in-depth mechanistic work contributes to a better acceptance of this herbal remedy.

Human amniotic glycodelin actively regulates changes in ß-catenin immunoreactivity in cultured human umbilical vein endothelial cells (HUVEC).

OBJECTIVE: Assessment of the capacity of Glycodelin A (GdA) to modulate the aggregation of cultured human umbilical vein endothelial cells. METHODS: Highly purified Glycodelin A (GdA) from late first trimester amniotic fluid has been added to cultured cells and its biological activity has been observed with immunofluorescent staining of ß-catenin molecules. RESULTS: GdA induces translocation of ß-catenin molecules promoting cell-to-cell adhesion and formation of adherents junctions through cytoskeletal reorganization. CONCLUSION: These data provide further mechanistic insight into the specificity of cell-to-cell adhesion, thus corroborating the role of GdA in promoting angiogenesis.

Effect of sphingosine 1-phosphate on morphological and functional responses in endothelia and venules after scalding injury.

OBJECTIVE: The uncontrolled increase of vascular permeability is the major obstacle in treatment of severe burns. Sphingosine 1-phosphate (S1P) has emerged as an important modulator of EC barrier function. This study was designed to explore the effect of S1P on morphological alteration in cultured endothelial cells (ECs) after burned plasma stimulation, and second to investigate the hyper-permeability response in intact vessels after scalding injury. METHODS: The distribution of VE-cadherin and F-actin was observed by double staining in primary cultured human umbilical vein endothelial cells (HUVECs) with immunofluorescence and fluorescent probes; respectively. Permeability changes were measured by a fluorescence ratio technique in isolated venules from rat skin. Burned plasma was obtained from a third-degree scald covering 30% of the total body surface area. RESULTS: The intervention with burned plasma on injured rats cultured HUVECs caused a significant disruption of intercellular adherens junction labeled by VE-cadherin staining, accompanied by the formation of F-actin stress fibers in the cells. S1P prevented or reversed these burned plasma-induced morphological alterations in cultured endothelial cells. The inhibition of S1P synthesis with N,N-dimethylsphingosine (DMS) mimicked the burned plasma-evoked redistribution of VE-cadherin and reorganization of F-actin. Venules isolated from burned rats demonstrated similar endothelial cytoskeleton changes with cultured cells under the influence of S1P or DMS. Both pre- and post-burn application of S1P attenuated increased permeability in isolated and perfused skin venules after burned plasma stimulation. CONCLUSION: Our results indicate that S1P plays a role in maintaining basal vascular barrier function and could be protective in burn injury by enhancing the endothelial barrier function.

Phosphatidylethanolamine deficiency in membrane lipids inhibits keratinocyte intercellular networks formation.

Ethanolamine (Etn) is required for the growth of epithelial cells in culture. Without Etn, the amount of phosphatidylethanolamine (PE) in membrane lipids is reduced, and cell proliferation stops. When the membrane lipids are deficient of PE, some extracellular signaling processes become impaired. In this study, we examined the effect of Etn deprivation on the formation of intercellular networks in immortalized human oral keratinocytes. Keratinocytes proliferate with undifferentiated morphologies in a low-calcium medium, whereas they undergo differentiation to form intercellular networks in a high-calcium medium. The cells were first cultured with or without Etn supplement in a low-calcium (0.07 mM) medium, and then the calcium concentration was raised to 1.8 mM. The localization and organization of the following proteins were examined: (1) desmogleins and plakoglobin in desmosomes, (2) E-cadherin and beta-catenin in adherens junctions and (3) actin and keratin filaments in cytoskeletons. As expected, in the Etn-supplemented cells, the elevated level of calcium induced the junctional localization of the proteins associated with desmosomes and adherens junctions and also induced the formation of keratin and actin networks. On the contrary, in the Etn-deprived cells, the elevated level of calcium induced none of the above processes. The results suggest that having a sufficient amount of PE or proper phospholipid composition in the membranes is crucial for differentiation in epithelial cells.