Eui-Baek Byun

Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

Apple Procyanidins Induced Vascular Relaxation in Isolated Rat Aorta through NO/cGMP Pathway in Combination with Hyperpolarization by Multiple K+ Channel Activations

Apple (Malus pumila) procyanidins led to a potent vasorelaxation effect in 1.0 μM phenylephrine-contractive rat thoracic aorta. Relaxation was greatly reduced by 70 mM KCl as well as by removal of the endothelium, suggesting that it was associated with endothelium-dependent hyperpolarization. Neither cAMP synthesis inhibition nor NAD(P)H oxidase inhibition abolished the effect. In contrast, complete abolition by a soluble guanylyl cyclase inhibitor revealed that apple procyanidins were mainly involved in the cGMP production pathways. In the presence of N G-monoethyl-L-arginine or tetraethylammonium chloride, the effect was still observed at higher concentrations (>25 μg/ml), while their combination completely diminished the effect. Vasorelaxation was to some extent affected by paxillin, apamin and glybenclamide, and was greatly affected by 4-aminopyridine and by BaCl2. These results indicate that procyanidin-induced vasorelaxation is associated with NO-cGMP pathway in combination with hyperpolarization due to multiple activation of Ca2+-dependent and -independent K+ channels.

Gamma-irradiated resveratrol negatively regulates LPS-induced MAPK and NF-κB signaling through TLR4 in macrophages

Resveratrol was irradiated at various doses of 15, 30, 50, and 70kGy for the development of physiological functionalities through modification of the structural properties. Gamma irradiation induced a decrease in the resveratrol peak, and the appearance of several new peaks by gamma irradiation was gradually increased up to 70kGy. Gamma-irradiated resveratrol did not exert cytotoxicity to macrophages in dose ranges from 15 to 70kGy; therefore, 70kGy gamma-irradiated resveratrol was used as the maximum dose throughout subsequent experiments. Treatment of LPS-stimulated macrophages with 70kGy gamma-irradiated resveratrol resulted in a dose-dependent decrease in iNOS-mediated NO, PGE2, and pro-inflammatory cytokine level, such as TNF-α, IL-6 and IL-1β. 70kGy gamma-irradiated resveratrol significantly inhibited cyclooxygenase-2 levels, as well as the expression of cell surface molecules, such as CD80 and CD86, in LPS-induced macrophages. Furthermore, the inhibitory action of these pro-inflammatory mediators occurred through an inhibition of MAPKs (ERK1/2, p38 and JNK) and NF-κB signaling pathways based on a toll-like receptor 4 in macrophages, which may be closely mediated with the radiolysis products of resveratrol transformed by gamma-irradiation. From these findings, it seems likely that gamma irradiation can be an effective tool for a reduction of the toxicity and play a potent role in the treatment of inflammatory disease.

Enhancement of anti-tumor activity of gamma-irradiated silk fibroin via immunomodulatory effects.

Silk fibers have proven to be effective in many clinical applications as well as for clothing. In addition to the substantial effect of silk fibers, the present study was conducted to explore its importance in a new dimension to reinforce the effects of its physiological function regarding anti-tumor activity and immune response with gamma-irradiated silk fibroin (GISF). The cytotoxicity results showed that pre-treatment of GISF in the mouse peritoneal macrophages (MPM) indicated a higher proliferative effect than that of non-irradiated silk fibroin (NISF) in a concentration-dependent manner. Based on the cytotoxicity result of MPM, GISF (50 and 150 kGy) was selected for an ex vivo study in an animal (C57BL6) system and evaluated about whether the non-specific immune response was also related to GISF. GISF (50 and 150 kGy) augmented immune responsiveness via activation of NK cells, T lymphocytes proliferation, NO production, and cytokine level, such as IL-6, IL-2, IL-12, IFN-gamma, TNF-alpha, as compared with NISF, which strongly suggested that GISF significantly augmented an important element of all aspects of the innate and adaptive immune system. Therefore, from these results, it seems likely that the GISF will play a potent role in eliciting the effect of the non-specific immune response and anti-tumor activity as a value-added product in the medical industry.

The procyanidin trimer C1 inhibits LPS-induced MAPK and NF-κB signaling through TLR4 in macrophages

Natural products and dietary components rich in polyphenols have been shown to reduce inflammation; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. This research was carried out to clarify the potential role of procyanidin trimer C1 in the anti-inflammatory effect of polyphenols. Procyanidin C1 inhibited inducible nitric oxide synthase-mediated nitric oxide production and the release of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α) in lipopolysaccharide (LPS)-induced macrophages. Treatment with procyanidin C1 resulted in a significant decrease in prostaglandin E2 and cyclooxygenase-2 levels, as well as the expression of cell surface molecules (CD80, CD86, and MHC class II), which was induced by LPS. Furthermore, our data demonstrated that the anti-inflammatory effect of procyanidin C1 occurs through inhibition of mitogen-activated protein kinase (p38 and c-Jun N-terminal kinase) and nuclear factor-κB signaling pathways. These 2 factors play a major role in controlling inflammation, through toll-like receptor 4, suggesting that procyanidin C1 plays a potent role in promoting anti-inflammatory activity in macrophages. These results represent a novel and effective therapeutic intervention for the treatment of inflammatory disease.

Quercetin negatively regulates TLR4 signaling induced by lipopolysaccharide through Tollip expression

Polyphenolic compounds have been regarded as one of the most promising dietary agents for the prevention and treatment of inflammation-related chronic diseases; however, the anti-inflammatory activities of flavonoids, such as quercetin, are not completely characterized, and many features remain to be elucidated. In this study, we showed the molecular basis for the downregulation of TLR4 signal transduction by quercetin. Quercetin markedly elevated the expression of the Toll-interacting protein, a negative regulator of TLR signaling. Lipopolysaccharide-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6, and IL-12p70) were inhibited by quercetin, and this action was prevented by Toll-interacting protein silencing. In addition, quercetin-treated macrophages inhibited lipopolysaccharide-induced activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase, and the translocation of nuclear factor-κB and p65 through Toll-interacting protein. Treatment with quercetin resulted in a significant decrease in prostaglandin E2 and cyclooxygenase-2 levels as well as inducible nitric oxide synthase-mediated nitric oxide production induced by lipopolysaccharide. Taken together, these findings represent new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and effective therapeutic intervention for the treatment of inflammatory disease.

Effect of gamma irradiation on mistletoe (Viscum album) lectin-mediated toxicity and immunomodulatory activity

This study evaluated the effect of gamma irradiation on the reduction of the toxicity of mistletoe lectin using both in vitro and in vivo models. To extract the lectin from mistletoe, an (NH4)2SO4 precipitation method was employed and the precipitant purified using a Sepharose 4B column to obtain the pure lectin fraction. Purified lectin was then gamma‐irradiated at doses of 0, 5, 10, 15, and 20 kGy, or heated at 100 °C for 30 min. Toxic effects of non‐irradiated, irradiated, and heat‐treated lectins were tested using hemagglutination assays, cytotoxicity assays, hepatotoxicity, and a mouse survival test and immunological response was tested using cytokine production activity. Hemagglutination of lectin was remarkably decreased (P < 0.05) by irradiation at doses exceeding 10 kGy and with heat treatment. However, lectin irradiated with 5 kGy maintained its hemagglutination activity. The cytotoxicity of lectin was decreased by irradiation at doses over 5 kGy and with heat treatment. In experiments using mouse model, glutamate oxaloacetate transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels were decreased in the group treated with the 5 kGy irradiated and heat‐treated lectins as compared to the intact lectin, and it was also shown that 5 kGy irradiated and heat‐treated lectins did not cause damage in liver tissue or mortality. In the result of immunological response, tumor necrosis factor (TNF‐α) and interleukin (IL‐6) levels were significantly (P < 0.05) increased in the 5 kGy gamma‐irradiated lectin treated group. These results indicate that 5 kGy irradiated lectin still maintained the immunological response with reduction of toxicity. Therefore, gamma‐irradiation may be an effective method for reducing the toxicity of lectin maintaining the immune response.

Epigallocatechin-3-gallate-mediated Tollip induction through the 67-kDa laminin receptor negatively regulating TLR4 signaling in endothelial cells.

BACKGROUND Green tea polyphenol epigallocatechin-3-gallate (EGCG) has the potential to impact a variety of inflammation-related diseases; however, the anti-inflammatory action of EGCG in endothelial cells has not been understood. Recently, we demonstrated that the 67-kDa laminin receptor (67LR) acts as a cell-surface EGCG receptor. AIM This research was carried out to clarify the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in lipopolysaccharide (LPS)-stimulated endothelial cells. RESULTS RNAi-mediated silencing of 67LR resulted in an abrogation of the inhibitory action of EGCG on the LPS-induced activation of downstream signaling pathways. Also, we found that EGCG induced a rapid upregulation of Toll-interacting protein (Tollip), a negative regulator of TLR signaling, through 67LR in endothelial cells. RNAi-mediated silencing of Tollip impaired the TLR4 signaling inhibitory activity of EGCG. Additionally, silencing of Tollip resulted in an abrogation of the inhibitory action of EGCG on the LPS-induced expressions of cell-associated adhesion molecules, such as ICAM-1 and VCAM-1. CONCLUSION Taken together, these novel findings provide new insights into an understanding of negative regulatory mechanisms of the TLR4 signaling pathway and effective therapeutic intervention for the treatment of inflammatory disease.

Procyanidin C1 Causes Vasorelaxation Through Activation of the Endothelial NO/cGMP Pathway in Thoracic Aortic Rings

The aim of this study was to clarify the efficacy of procyanidin C1 (Pro C1) for modulating vascular tone. Pro C1 induced a potent vasorelaxant effect on phenylephrine-constricted endothelium-intact thoracic aortic rings, but had no effect on denuded thoracic aortic rings. Moreover, Pro C1 caused a significant increase in nitric oxide (NO) production in endothelial cells. Pro C1-induced vasorelaxation and Pro C1-induced NO production were significantly decreased in the presence of a nonspecific potassium channel blocker (tetraethylammonium chloride [TEA]), an endothelial NO synthase inhibitor (N(G)-monomethyl-L-arginine [L-NMMA]), and a store-operated calcium entry inhibitor (2-aminoethyl diphenylborinate [2-APB]). Pro C1-induced vasorelaxation was also completely abolished by an inhibitor of soluble guanyl cyclase, which suggests that the Pro C1 effects observed involved cyclic guanosine monophosphate (cGMP) production. Interestingly, Pro C1 significantly enhanced basal cGMP levels. Taken together, these results indicate that Pro C1-induced vasorelaxation is associated with the activation of the calcium-dependent NO/cGMP pathway, involving potassium channel activation. Thus, Pro C1 may represent a novel and potentially therapeutically relevant compound for the treatment of cardiovascular diseases.

Gamma-irradiated black ginseng extract inhibits mast cell degranulation and suppresses atopic dermatitis-like skin lesions in mice

Gamma irradiation is able to affect various structural modification and an increase of the biological properties of biomaterials. This study was conducted to investigate the anti-allergenic effect of γ-irradiated black ginseng extract (BGE) using in vitro and in vivo experiments. IgEantigen complex-induced degranulation was measured in RBL-2H3 mast cells. In addition, an anti-atopic dermatitis (AD) test was carried out by spreading γ-irradiated BGE on the dorsal skin of 2,4-dinitrochlorobenzene (DNCB)-induced BALB/c mice. The content of arginylfructose (AF) of gamma-irradiated BGE was higher than that of BGE. In RBL-2H3 mast cells, γ-irradiated BGE treatments significantly reduced the IgE-antigen complex-induced release of β-hexosaminidase, histamine, intracellular ROS, and Ca2+ influx. A western blot analysis showed that γ-irradiated BGE had an inhibitory activity on the FcεRI-mediated signaling in mast cells. In the DNCB-induced AD model, γ-irradiated BGE significantly alleviated the ADlike skin symptoms and clinical signs. The suppression of AD by γ-irradiated BGE was accompanied by a decrease in the serum level of IgE and IL-4, as well as the number of leukocyte. Gamma-irradiated BGE also suppressed IL-4 and increased IFN-γ in splenocytes. Our data suggests that γ-irradiated BGE may be effective therapeutic agents for the treatment of AD.