Vascular barrier protective effects of phlorotannins on HMGB1-mediated proinflammatory responses in vitro and in vivo

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Abstract

The phlorotannins (phloroglucinol, eckol, and dieckol) are active compounds found in Eisenia bicyclis, and have been widely investigated for their antioxidant, anti-tumor, and anti-cancer activities. In this study, we investigated the protective effects of these phlorotannins against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and in mice treated by high mobility group box 1 protein (HMGB1), and the signaling pathways involved. The protective activities of the phlorotannins were determined by measuring permeability, leukocyte adhesion and migration, and the activations of pro-inflammatory proteins in HMGB1-activated HUVECs. We found that the phlorotannins inhibited; lipopolysaccharide (LPS)-induced HMGB1 release, HMGB1-mediated barrier disruption, the expressions of cell adhesion molecules (CAMs), and the adhesion/transendothelial migration of leukocytes to human endothelial cells. The phlorotannins also suppressed acetic acid induced-hyperpermeability and carboxymethylcellulose-induced leukocytes migration in vivo. Further studies revealed that the hydroxyl groups on dieckol positively regulated these vascular barrier protective effects. Collectively, these results suggest that phloroglucinol, eckol, and dieckol protect vascular barrier integrity by inhibiting hyperpermeability, the expressions of CAMs, and the adhesion and migration of leukocytes, which confirms their potential usefulnesses for the treatment of vascular inflammatory diseases.

Highlights

HMGB1 is pro-inflammatory cytokine as a mediator of vascular inflammation. ► Phlorotannins inhibited LPS induced HMGB1 release. ► Phlorotannins inhibited HMGB1-mediated hyperpermeability in vitro and in vivo. ► Rutin inhibited HMGB1-mediated inflammatory responses. ► Hydroxyl groups on dieckol positively regulate these vascular barrier protective effects.

Introduction

High-mobility group proteins are small DNA-binding proteins that serve an important role in transcriptional regulation (Bustin et al., 1990). One of these proteins, HMGB1, has been identified to be a late acting mediator of LPS-induced (Wang et al., 1999) or sepsis-induced lethality in mice (Yang et al., 2004). Extracellular HMGB1 can stimulate the secretion of proinflammatory cytokines from endothelial cells, monocytes, and macrophages, which leads to inflammatory responses in target tissues (Andersson et al., 2000, Bae and Rezaie, 2011, Fiuza et al., 2003). Furthermore, it triggers the activations of endothelium and leukocytes by binding to at least three pathogen-associated cell surface pattern recognition receptors, such as, toll-like receptors (TLR) 2 and 4 and the receptor for advanced glycation end products (RAGE), and by so doing induces TNF-α expression and NF-κB activation in target cells (Fiuza et al., 2003, Hori et al., 1995, Park et al., 2004). A high plasma concentration of HMGB1 in patients with inflammatory diseases is known to be related to poor prognosis and high mortality, and the pharmacological inhibition of HMGB1 is known to improve survival in animal models of acute inflammation in response to endotoxin challenge (Sama et al., 2004). Therefore, the preventions of HMGB1 production and of HMGB1-mediated proinflammatory responses are considered promising therapeutic strategies for the treatment of vascular inflammatory diseases.

Previous studies have focused on disease preventing roles of dietary factors, such as, phenolic compounds and polyphenols, in the contexts of serious diseases, such as, cancer, coronary heart disease, and inflammatory diseases (Kohyama et al., 1997, Koshihara et al., 1984, Yang et al., 1999). Accordingly, the search for anticancer drugs and anti-inflammatory agents in natural products represents an area of great interest (Aggarwal et al., 2006). Eisenia bicyclis (Kijellman) Setchell is a common perennial brown alga of the family Laminariaceae that inhabits the middle Pacific coast around Korea and Japan (Okada et al., 2004), and the phlorotannins, which are oligomers and polymers of phloroglucinol, are found exclusively in brown algae (Koivikko et al., 2007). E. bicyclis have been reported to have several biological activities, which include antioxidant, anti-tumor, anti-cancer, and bactericidal activities, and to Alzheimer’s disease (Jung et al., 2010, Nagayama et al., 2002, Okada et al., 2004). However, anti-inflammatory effects of the phlorotannins of E. bicyclis on HMGB1-mediated proinflammatory responses in endothelial cells and in mice have not been previously studied. In the present study, we evaluated the effects of three phlorotannins from E. bicyclis, namely, phloroglucinol, eckol, and dieckol, on HMGB1-activated human endothelial cells and in mice.

Section snippets

Reagents

Phloroglucinol (Fig. 1), bacterial lipopolysaccharide (LPS, #4391, used at 100 ng/ml), Evans blue, crystal violet, and MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) were obtained from Sigma (St. Louis, MO, USA). Human recombinant HMGB1 was purchased from Abnova (Taipei City, Taiwan).

Plant materials, extraction, and the isolations of eckol and dieckol

Fresh E. bicyclis was washed three times with water to remove salt, lyophilized, and ground into a powder. Dried E. bicyclis powder (1.0 kg) was extracted with MeOH (10 L × 3) at room temperature, and the

Effects of phloroglucinol, eckol, and dieckol on LPS-mediated HMGB1 release

Previous studies have demonstrated that LPS stimulates HMGB1 release in murine macrophages and human endothelial cells (Bae and Rezaie, 2011, El Gazzar, 2007, Mullins et al., 2004). Following HMGB1 release to intravascular spaces, it interacts with specific cell surface receptors to amplify inflammatory responses by inducing the expressions of proinflammatory cytokines (Fiuza et al., 2003). In agreement with previous results, LPS (100 ng/ml) was found to stimulate HMGB1 release by HUVECs (Fig. 2

Discussion

Phlorotannins are commonly found in brown algae and have been reported to exert various biological effects, including on antioxidant, anti-tumor, anti-cancer, and bactericidal effects and Alzheimer’s disease (Jung et al., 2010, Nagayama et al., 2002, Okada et al., 2004). In the present study, the vascular protective effects of purified phlorotannins (phloroglucinol, eckol, and dieckol) from E. bicyclis and of synthesized phlorotannin (Me-dieckol) were examined for the first time on the

Conflict of Interest

The authors have no conflict of interest to declare.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) funded by the Korea government [MEST] (Grant No. 2011-0026695, 2011-0030124).

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