Inflamm. Res. (2014) 63:197–206 DOI 10.1007/s00011-013-0689-x

Inflammation Research

ORIGINAL RESEARCH PAPER

Anti-inflammatory effects of rutin on HMGB1-induced inflammatory responses in vitro and in vivo Hayoung Yoo • Sae-Kwang Ku • Young-Doo Baek • Jong-Sup Bae

Received: 8 June 2013 / Revised: 22 October 2013 / Accepted: 20 November 2013 / Published online: 1 December 2013 Ó Springer Basel 2013

Abstract Objective and design High mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions. Rutin (RT), an active flavonoid compound, is well known to possess potent antiplatelet, antiviral and antihypertensive properties. In this study, we investigated the antiinflammatory effects of RT against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) induced by HMGB1 and the associated signaling pathways. Methods The anti-inflammatory activities of RT were determined by measuring permeability, monocytes adhesion and migration, and activation of pro-inflammatory proteins in HMGB1-activated HUVECs and mice. Results We found that RT potently inhibited HMGB1 release, down-regulated HMGB1-dependent inflammatory responses in human endothelial cells, and inhibited HMGB1mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with RT resulted in reduced cecal ligation and puncture-induced release of HMGB1 and sepsisResponsible Editor: Mauro Teixeira. H. Yoo and S.-K. Ku contributed equally to this work. H. Yoo  J.-S. Bae (&) College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Dahak-ro, Buk-gu, Daegu 702-701, Republic of Korea e-mail: [email protected] S.-K. Ku Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan 712-715, Republic of Korea Y.-D. Baek Department of Clinical Pathology, Daegu Health College, Daegu 702-722, Republic of Korea

related mortality. Further studies revealed that RT suppressed the production of tumor necrosis factor-a and interleukin 6 and the activation of nuclear factor-jB and extracellular regulated kinases 1/2 by HMGB1. Conclusion Collectively, these results indicate that RT could be a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway. Keywords Rutin  HMGB1  Endothelium  Inflammation  Barrier integrity

Introduction High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein present in the nuclei and cytoplasm of nearly all cell types [1]. In response to infection or injury, HMGB1 is actively secreted by innate immune cells and/or released passively by injured or damaged cells [2]. HMGB-1 is released into the extracellular space and binds to several transmembrane receptors, including receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLR)-2 and 4, and activates nuclear factor (NF)-jB and extracellular regulated kinases (ERK) 1 and 2 [3, 4]. In most cases of sepsis or septic shock, serum levels of HMGB-1 are elevated 1 week after diagnosis, and the degree of elevation reflects organ dysfunction [5, 6]. Overexpression of HMGB-1 occurs during the late stages of sepsis, providing a wide therapeutic window for clinical intervention. Therefore, it remains an attractive target for sepsis treatment [1, 2]. Sepsis is a clinical syndrome that complicates severe infection and is characterized by systemic inflammation and widespread tissue injury [7]. A continuum of severity from sepsis to sever sepsis and septic shock exits [7].

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The inflammatory response is an important component in the pathogenesis of vascular injury and endothelial dysfunction is related especially to leukocyte recruitment during formation of the vascular inflammatory lesion [8, 9]. During vascular inflammatory processes, two important mediators are tumor necrosis factor-a (TNF-a) and NF-jB. NF-jB is a well known proinflammatory transcriptional factor [10, 11] that is activated in response to proinflammatory cytokines such as TNF-a and interleukin 1b (IL-1b) [10, 11]. There is considerable evidence suggesting that suppression of NF-jB signaling pathway confers significant vascular protective effects [11, 12] which delays or prevents vascular diseases in animal models of disease [13, 14]. Therefore, preventing the production of TNF-a and NF-jB activation in vascular endothelial cells are considered to be promising therapeutic targets for vascular inflammatory diseases. Many studies have shown that polyphenolic compounds including flavonoids and phenolic acids have diverse functions including antioxidant, antihyperglycemic and antihypertensive properties [15]. Recent studies have also focused on the prophylaxis effect in cardiovascular inflammatory disease since flavonoid intake was proven to be effective in reducing the risk of chronic disease [16]. This prevailing accelerates the elucidation of their underlying mechanism as well as the survey of phytomedicinal plants that play a pharmacological role in regulating vascular tone. Rutin (RT), one of the major flavonoids, is also known as vitamin P and has antiplatelet, antiviral and antihypertensive properties. It also strengthens capillaries due to its high radical scavenging activity and antioxidant capacity [17]. Milde et al. [18] found that RT inhibits low-density lipoprotein oxidation and reduces the risk of atherosclerosis. However, the effect of RT on HMGB1-mediated inflammatory responses and the underlying mechanisms of its effect in vascular endothelial cells and in vivo have not yet been elucidated.

Materials and methods Reagents RT, bacterial lipopolysaccharide (LPS; serotype 0111:B4, L5293, used at 100 ng/ml), Evans blue, and crystal violet were obtained from Sigma (St. Louis, MO, USA). Vybrant DiD (used at 5 lM) was obtained from Invitrogen (Carlsbad, CA, USA). HMGB1 was purchased from Abnova (Taipei City, Taiwan). Emodin-6-O-b-D-glucoside (EG) was prepared as described previously [19]. Animals and husbandry Male C57BL/6 mice (6–7 weeks old, weighing 18–20 g), purchased from Orient Bio Inc. (Seongnam, Republic of

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Korea), were used after a 12-day acclimatization period. Animals were housed five per polycarbonate cage under controlled temperature (20–25 °C) and humidity (40–45 %) and a 12:12 h light:dark cycle. Animals received a normal rodent pellet diet and water ad libitum during acclimatization. All animals were treated in accordance with the ‘Guidelines for the care and use of laboratory animals’ issued by Kyungpook National University. Cell culture Primary human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Bio Science (Charles City, IA, USA) and maintained as described previously [20]. Briefly, the cells were cultured to confluency at 37 °C and 5 % CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science). A human monocyte cell line, THP-1, was maintained at a density of 2 9 105 to 1 9 106 cells/ml in RPMI 1640 with L-glutamine and 10 % heat-inactivated FBS supplemented with 2-mercaptoethanol (55 lM) and antibiotics (penicillin G and streptomycin). Cecal ligation and puncture (CLP) For induction of sepsis, male mice were anesthetized with zoletil 50 and rompun. The CLP-induced sepsis model was prepared as previously described [21]. In brief, a 2-cm midline incision was made to expose the cecum and adjoining intestine. The cecum was then tightly ligated with a 3.0 silk suture at 5.0 mm from the cecal tip and punctured once using a 22-gauge needle, followed by gentle squeezing to extrude a small amount of feces from the perforation site, and then returned to the peritoneal cavity. The laparotomy site was then sutured with 4.0 silk suture. In sham control animals, the cecum was exposed but not ligated or punctured and then returned to the abdominal cavity. This protocol was approved by the animal care committee at Kyungpook National University prior to conducting the study. Cell viability assay MTT was used as an indicator of cell viability. Cells were grown in 96-well plates at a density of 5 9 103 cells/well. After 24 h, cells were washed with fresh medium, followed by treatment with RT with or without LPS (100 ng/ml) for 16 h. After a 48 h incubation period, cells were washed, and 100 ll of MTT (1 mg/ml) was added, followed by incubation for 4 h. Finally, DMSO (150 ll) was added to solubilize the formazan salt formed and the amount of formazan salt was determined by measuring the optical

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density at 540 nm using a microplate reader (Tecan Austria GmbH, Austria). Data were expressed as mean ± SD from at least three independent experiments. Enzyme-linked immunosorbent assay (ELISA) for HMGB1, RAGE, TLR2, TLR4 or phosphorylated p38 mitogen-activated protein kinase (MAPK) A competitive ELISA was performed as described previously to determine HMGB1 concentrations in cell culture medium and serum [22]. Whole-cell ELISA was performed as described previously to determine the expression levels of RAGE, TLR2 and TLR4 in HUVECs [23]. Phosphorylated p38 MAPK expression was quantified according to the manufacturer’s instructions using a commercially available ELISA kit (Cell Signaling Technology, Danvers, MA, USA). RNA interference The expression of TLR4 and RAGE by endothelial cells in response to LPS (100 ng/ml for 3 h) or HMGB1 (1 lg/ml for 16 h) was evaluated following the knockdown of TLR4 and RAGE expression by pools of target-specific 20–25 nucleotide siRNAs obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) according to the manufacturer’s instruction and as described [24]. A nontargeting 20–25 nucleotide siRNA obtained from the same company was used as a negative control.

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non-migrating cells on top of the filter were removed with a cotton swab. Monocytes on the lower side of the filter were fixed with 8 % glutaraldehyde and stained with 0.25 % crystal violet in 20 % methanol (w/v). Each experiment was repeated in duplicate wells and within each well. Nine randomly selected high power microscopic fields (HPFs, 2009) were counted and expressed as a migration index. In vivo permeability and leukocyte migration assay Mice were pretreated intravenously (i.v.) with RT (30 or 60 lg/mouse) or EG (9 lg/mouse) and 6 h later injected with 1 % Evans blue dye solution in normal saline followed immediately by HMGB1 (2 lg/mouse i.v.). Thirty minutes later, mice were killed and peritoneal exudates were collected by washing cavities with 5 ml of normal saline and centrifuging at 2009g for 10 min. Supernatant absorbance at 650 nm was read. Vascular permeabilities are expressed as micrograms of dye/mouse that leaked into the peritoneal cavity, and concentrations determined using a standard curve, as described previously [2]. For assessment of leukocyte migration, mice were pretreated i.v. with RT (30 or 60 lg/mouse) or EG (9 lg/ mouse), and 1 h later HMGB1 (2 lg/mouse, i.v.) in normal saline was administered. Mice were killed after 6 h and peritoneal cavities were washed with 5 ml of normal saline. Samples (20 ll) of peritoneal fluids obtained were mixed with 0.38 ml of Turk’s solution (0.01 % crystal violet in 3 % acetic acid) and numbers of leukocytes were counted under a light microscope.

Permeability assay in vitro Expression of cell adhesion molecules Permeability was quantitated by spectrophotometric measurement of the flux of Evans blue-bound albumin across functional HUVEC monolayers using a modified twocompartment chamber model as previously described [20]. Briefly, HUVECs were plated (5 9 104/well) in transwells of 3 lm pore size and 12 mm diameter for 3 days. The confluent monolayers were incubated with indicated RT or EG for 6 h followed by HMGB1 (1 lg/ml, for 16 h). Migration assay in vitro Migration assays were performed in transwell plates of 6.5 mm diameter, with 8 lm pore size filters. HUVECs (6 9 104) were cultured for 3 days to obtain confluent endothelial monolayers. Before adding monocytes to the upper compartment, cell monolayers were treated with increasing concentrations of RT or EG for 6 h prior to addition of THP-1 cells to the upper compartment. Transwell plates were incubated at 37 °C, 5 % CO2 for 2 h. Cells in the upper chamber of the filter were aspirated and

The expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin on HUVECs were determined by a wholecell ELISA as described [25, 26]. Briefly, expression levels of VCAM-1, ICAM-1, and E-selectin were determined by whole-cell ELISA, as described previously [25, 26]. Briefly, HUVEC monolayers were treated for 6 h with RT or EG at the indicated concentrations, followed by treatment with 1 lg/ml HMGB1 for 16 h. Cell–cell adhesion assay Adherence of monocytes to endothelial cells was evaluated by fluorescent labeling of monocytes as described [26–28]. Briefly, THP-1 cells (1.5 9 106/ml, 200 ll/well) were labeled with Vybrant DiD dye and then added to washed and stimulated HUVECs. HUVEC monolayers were treated with RT or EG for 6 h, followed by treatment with HMGB1 (1 lg/ml) for 16 h.

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Results and discussion RT is found in many plants, especially the buckwheat plant Fagopyrum tataricum Gaertn, family Polygonaceae. In this study the effects of RT (Fig. 1) on the release of HMGB1 and the HMGB1-mediated vascular barrier disruptive response were determined in vitro and in vivo. EG was used as a positive control [19]. Effects of RT on LPS and CLP-mediated HMGB1 release

Fig. 1 Chemical structure of RT

ELISA for NF-jB, TNF-a, ERK1/2, and IL-6 Total and phosphorylated p65 NF-jB (#7174, #7173, Cell Signaling Technology) or total and phosphorylated ERKs 1/2 (R&D Systems, Minneapolis, MN, USA) activities in nuclear lysates were determined using ELISA kits. The concentrations of IL-6 and TNF-a in cell culture supernatants were determined using ELISA kits (R&D Systems). Values were measured using an ELISA plate reader (Tecan). Immunofluorescence staining HUVECs were grown to confluence on glass cover slips coated with 0.05 % poly-L-lysine in complete media containing 10 % FBS and maintained for 48 h. Cells were pretreated with RT (50 lM, for 6 h) and then stimulated with 1 lg/ml HMGB1 for 16 h. For cytoskeletal staining, cells were fixed in 4 % formaldehyde in PBS (v/v) for 15 min at room temperature. After fixation, cells were blocked in blocking buffer (5 % BSA in PBS) overnight at 4 °C. Cells were then incubated with primary rabbit monoclonal NF-jB p65 antibody (Cell Signaling), and anti-rabbit alexa 488 (green). Nuclei were counterstained with 4,6-diamidino-2-phenylindole dihydroChloride (DAPI), blue (Roche, USA). And cells were visualized by confocal microscopy at a 639 magnification (TCS-SP5, Leica Microsystems, Germany). Statistical analysis Results are expressed as mean ± standard error of mean (SEM) of at least three independent experiments. Statistical significance was determined using analysis of variance (ANOVA; SPSS, version 14.0, SPSS Science, Chicago, IL, USA) and p values \0.05 were considered significant.

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Previous studies have demonstrated stimulation of HMGB1 release by LPS from murine macrophages and human endothelial cells [29–31], and Chen et al. [32] reported that 100 ng/ml LPS is sufficient to induce release of HMGB1. Similarly, in the current study 100 ng/ml LPS stimulated release of HMGB1 by HUVECs (Fig. 2a). To investigate the effects of RT on LPS-mediated release of HMGB1, HUVECs were pretreated with increasing concentrations of RT for 6 h, followed by stimulation with 100 ng/ml LPS for 16 h. As shown in Fig. 2a, RT inhibited release of HMGB1 in HUVECs, with a maximal effective concentration [25 lM. However, in the absence of LPS pretreatment, RT did not affect HMGB1 release (Fig. 2a). To confirm these effects in vivo, CLP-induced septic mice were used, because this model more closely resembles human sepsis than LPS-induced endotoxemia [33]. As shown in Fig. 2b, treatment with RT resulted in marked inhibition of CLP-induced release of HMGB1. Assuming that the average weight of a mouse was 20 g, and the average blood volume was 2 ml, the amount of RT injected (30 or 60 lg per mouse) was equivalent to 25 or 50 lM in peripheral blood. We then investigated the effects of RT on expression of the HMGB1 receptors, TLR2, TLR4, and RAGE in HUVECs. As shown in Fig. 2c, d, LPS or HMGB1 increased TLR2, TLR4, RAGE expression threefold in HUVECs, and treatment with RT resulted in significant inhibition of RAGE and TLR4 but not TLR2 expressions. Therefore, the inhibitory effects of RT on HMGB1 release were mediated by the suppression of the LPS or HMGB1-mediated TLR4 and RAGE expressions. To confirm this result, we determined the effects of RT on LPS-induced HMGB1 release after TLR4 or/and RAGE receptors blocked by siRNA. As shown in Fig. 2e, blocking TLR4 (LPS receptor) alone sufficiently inhibited LPSinduced HMGB1 which is consistent with a previous report [34]. However, blocking RAGE alone partially inhibited LPS-induced HMGB1 release. After TLR4 was blocked, RT did not affect LPS-induced HMGB1 release because TLR4 was already blocked by TLR4 siRNA. However, after RAGE was blocked, the effects of RT on LPSinduced HMGB1 release were better than those of RAGE

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Fig. 2 Effects of RT on release of HMGB1 and expressions of TLR2, TLR4 and RAGE. a HUVECs were pretreated with the indicated concentrations of RT or EG (10 lM) for 6 h, stimulated with 100 ng/ml LPS for 16 h, and HMGB1 release measured by ELISA. b Male C57BL/6 mice who underwent CLP received i.v. administration of RT or EG 12 h later (n = 5). Mice were killed 24 h after CLP and serum HMGB1 levels were measured by ELISA. Confluent HUVECs were pretreated with or without RT for 6 h, followed by incubation with c 100 ng/ml LPS for 3 h or d 1 lg/ml HMGB1 for 16 h. As a positive control, HUVECs were pretreated with 10 lM EG for 6 h. Expression of TLR2 (white bar), TLR4 (gray bar) and RAGE (black bar) on HUVECs was determined by cell-based ELISA. e The same as a except that HUVECs were pretreated with siRNA against TLR4 or/ and RAGE. f, g HUVECs were pretreated with RT or EG for 6 h followed by treating without f or with g LPS (100 ng/ml, for 16 h) and then cellular viability was measured by MTT assay. Results are expressed as the mean ± SEM of three independent experiments. *p \ 0.05 and **p \ 0.01 versus LPS alone (a, c, e, g), CLP alone (b), or HMGB1 alone (d)

alone blocking because presumably RT could block TLR4 receptor (Fig. 2e). To assess the cytotoxicity of RT, cell viability assays were performed in HUVECs treated with RT for 24 h. At concentrations up to 100 lM, RT did not affect cell viability (Fig. 2f). Noting that HMGB1 is released upon cell death and released HMGB1 could induce cell death [35, 36], we monitored the effects of RT on cell viability after LPS administration. As shown in Fig. 2g, RT was able to reduce cell death induced by LPS or/and LPS-induced HMGB1. High plasma concentrations of HMGB1 in patients with inflammatory diseases are known to be related to a poor

prognosis and high mortality. In addition, pharmacological inhibition of HMGB1 is known to improve survival in animal models of acute inflammation in response to endotoxin challenge [37]. Therefore, prevention of LPS- or CLP-induced HMGB1 release by RT suggests the potential of RT in the treatment of vascular inflammatory diseases. Effect of RT on HMGB1-mediated vascular barrier disruption A permeability assay was performed to determine the effects of RT on the barrier integrity of HUVECs. Treatment with 50 lM RT alone did not alter barrier integrity

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Fig. 3 Effects of RT on HMGB1-mediated permeability in vitro and in vivo. a The effects of pretreatment with different concentrations of RT or EG (10 lM) for 6 h on barrier disruptions caused by 1 lg/ml HMGB1 for 16 h were monitored by measuring the flux of Evans blue dye-bound albumin across HUVECs. b The effects of RT (i.v. injection) on HMGB1-induced (2 lg/mouse, i.v.) vascular permeability in mice were determined by measuring the levels of Evans blue

dye in peritoneal washings (expressed lg/mouse, n = 5). c HUVECs were treated with RT for 6 h, followed by activation with HMGB1. The effects RT on HMGB1-mediated expression of phosphorylated p38 (p-p38) were determined by ELISA. Results are expressed as the mean ± SEM of at least three independent experiments. *p \ 0.05 and **p \ 0.01 versus HMGB1 alone

(Fig. 3a). In contrast, HMGB1 is known to cause cleavage and disruption of endothelial barrier integrity [38, 39]. Thus, HUVECs were pretreated with various concentrations of RT for 6 h prior to addition of 1 lg/ml HMGB1. As shown in Fig. 3a, treatment with RT resulted in a dosedependent decrease in HMGB1-mediated endothelial barrier disruption. To confirm this vascular barrier protective effect in vivo, HMGB1-mediated vascular permeability in mice was assessed. As shown in Fig. 3b, treatment with RT resulted in markedly inhibited peritoneal leakage of dye induced by HMGB1. HMGB1 is known to induce pro-inflammatory responses by promoting phosphorylation of p38 MAPK [40, 41]. To determine whether RT inhibits HMGB1-induced activation of p38 MAPK in HUVECs, HUVECs were pre-incubated with RT and then activated with HMGB1, followed by determination of phosphorylated p38 MAPK levels by ELISA. As shown in Fig. 3c, HMGB1 induced up-regulated expression of phosphorylated p38, which was inhibited significantly by treatment with RT. These findings demonstrate inhibition of HMGB1-mediated endothelial disruption and maintenance of human endothelial cell barrier integrity by RT in mice treated with HMGB1.

Effects of RT on HMGB1-mediated CAM expression, THP-1 adhesion, and migration

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Previous studies have demonstrated that HMGB1 mediates inflammatory responses through increased expression of CAMs, such as ICAM-1, VCAM-1, and E-selectin, on the surface of endothelial cells, thereby promoting adhesion and migration of leukocytes across the endothelium to sites of inflammation [42–45]. We found that HMGB1 induced up-regulation of the surface expression of VCAM-1, ICAM-1, and E-selectin (Fig. 4a) and that RT inhibited this effect in a concentration-dependent manner, suggesting that the inhibitory effects of RT on CAMs expression are mediated via attenuation of the HMGB1 signaling pathway by RT. In addition, elevated expression of CAMs was found to correspond well with enhanced binding of THP-1 monocytic cells to HMGB1-activated endothelial cells, followed by their migration. In addition, treatment with RT resulted in down-regulation of THP-1 cell adherence and their subsequent migration across activated endothelial cells in a concentration-dependent manner (Fig. 4b, c). RT also suppressed HMGB1-mediated neutrophil adhesion and migration toward HUVECs (data not shown). These results suggest that RT not only inhibits endotoxin-mediated

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Fig. 4 Effects of RT on HMGB1-mediated pro-inflammatory responses. a HMGB1-induced (1 lg/ml) expression of VCAM-1 (white box), ICAM-1 (gray box), and E-selectin (black box) on HUVECs was determined after treatment of cells with the indicated concentrations of RT for 6 h. b HMGB1 (1 lg/ml, for 16 h)-mediated adherence of monocytes to HUVEC monolayers was assessed after pretreatment of cells with RT for 6 h. c HMGB1 (1 lg/ml for 16 h)-

mediated migration of monocytes through HUVEC monolayers was assessed after treatment of cells with the indicated concentrations of RT for 6 h. d HMGB1 (2 lg/mouse, i.v.)-mediated migration of leukocytes into the peritoneal cavities of mice was assessed after treatment of mice with i.v. RT. Data are expressed as the mean ± SEM of three independent experiments (n = 5). **p \ 0.01 versus HMGB1 alone

release of HMGB1 in endothelial cells, but also downregulates the pro-inflammatory signaling effect caused by HMGB1 release, thereby inhibiting amplification of inflammatory pathways by nuclear cytokines. To confirm this effect in vivo, we examined HMGB1-induced migration of leukocytes in mice. HMGB1 was found to stimulate migration of leukocytes into the peritoneal cavities of mice, and treatment with RT resulted in a significant reduction of peritoneal leukocyte counts (Fig. 4d). Experiments on CAMs are widely used in vitro for studying the regulation of interactions between leukocytes and endothelial cells [46, 47]. In the current study, treatment with RT resulted in down-regulation of HMGB1-induced levels of VCAM-1, ICAM-1, and E-selectin, suggesting that RT inhibits the adhesion and migration of leukocytes to inflamed endothelium.

ELISA. IL-6 and TNF-a levels increased in HMGB1stimulated endothelial cells; these increases were significantly reduced by RT (Fig. 5a, b), indicating that RT regulates the most important signals that induce proinflammatory responses in human endothelial cells. Activation of NF-jB and ERK1/2 is required for proinflammatory responses [10, 11, 48]. HMGB1 is known to activate NF-jB and ERK 1/2 in vascular inflammatory responses [49–51]. Therefore, we evaluated the effects of RT on activation of NF-jB and ERK 1/2 by HMGB1. As shown in Fig. 5c, d, HMGB1 increased activation of NFjB and ERK 1/2 and these increases were significantly reduced by RT. Furthermore, immunofluorescence staining (Fig. 5e) shows that HMGB1 stimulation caused obvious translocation of NF-jB p65 from cytoplasm into nucleus, which was counteracted obviously by pretreatment of RT.

Effects of RT on HMGB1-stimulated activation of NFjB/ERK, production of IL-6/TNF-a, and cytoskeletal rearrangement

Protective effect of RT in HMGB1-induced lethality

To investigate the effects of RT on production of the proinflammatory cytokines, IL-6 and TNF-a, HUVECs were pre-incubated with RT for 6 h, followed by measurement of IL-6 and TNF-a levels in culture media by

RT was administered to mice after HMGB1 injection to determine its ability to protect mice from HMGB1-induced lethality. Administration of RT in a single dose (60 lg/ mouse, 12 h after CLP) did not prevent HMGB1-induced death (data not shown). Therefore, RT was administered three times (60 lg/mouse, 12, 50, and 98 h after HMGB1

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Fig. 5 Effects of RT on HMGB1-stimulated activation of NF-jB/ERK and production of IL-6/TNF-a. HMGB1mediated (1 lg/ml, for 16 h) production of a TNF-a or b IL-6 in HUVECs was assessed after treatment of cells with the indicated concentrations of RT for 6 h. c HMGB1 (1 lg/ml)mediated activation of phosphorylated-NF-jB p65 (white box) or total NF-jB p65 (black box) in HUVECs was assessed after treatment of cells with RT. d HMGB1 (1 lg/ml)mediated activation of phosphorylated-ERK1/2 (white box) or total ERK1/2 (black box) in HUVECs was assessed after treatment of cells with RT. e Immunofluorescence microscopy analysis of the nuclear translocation of p65 in HUVECs. HUVECs, pretreated or not with 50 lM RT for 6 h, were stimulated (or not) for 16 h with 1 lg/ml HMGB1. The subcellular localization of p65 was examined by IF staining. The results are representative of three independent experiments. *p \ 0.05 and **p \ 0.01 versus HMGB1

Fig. 6 Effects of RT on HMGB1-induced lethality. a Male C57BL/6 mice (n = 10) were administered i.v. RT (unfilled square, 60 lg/ mouse) or EG (filled square, 9 lg/mouse, positive control) at 12, 50, and 98 h after HMGB1 injection (2 lg/mouse, i.v.). b The same as a except that compounds were administrated at 12 h before HMGB1

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injection. Animal survival was monitored every 6 h after HMGB1 injection for 126 h. Control CLP mice (filled circle) and shamoperated mice (unfilled circle) were administered sterile saline (n = 10). A Kaplan–Meier survival analysis was used for determination of overall survival rates versus HMGB1-injected mice

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injection). Results of a Kaplan–Meier survival analysis indicated an increase in the survival rate from 0 to 50 % (Fig. 6a). EG was used as a positive control in this experiments [19]. To determine the effects of RT pretreatment on lethality, mice were pretreated with RT (60 lg/mouse) 12 h before HMGB1 injection. Again, mice pretreated with RT survived longer after HMGB1 injection than untreated mice (Fig. 6b). This marked survival as a result of RT administration suggests suppression of HMGB1 release and of HMGB1-mediated inflammatory responses to be a therapeutic strategy for management of severe vascular inflammatory diseases. In summary, our results demonstrate that RT inhibits both LPS and CLP-mediated release of HMGB1, expression of HMGB1 receptor (TLR4 and RAGE), and HMGB1-mediated barrier disruption through increases in barrier integrity and inhibition of CAM expression. In addition, RT reduces monocyte adhesion and migration toward HUVECs. These barrier protective effects of RT were confirmed in a mouse model, in which treatment with RT resulted in reduction of HMGB1-induced mortality. Our findings indicate that RT can be regarded as a candidate for use in treatment of severe vascular inflammatory diseases, such as sepsis and septic shock.

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15. Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) funded by the Korea government [MEST] (Grant No. 2012-0009400). 16. Conflict of interest

The authors declare no conflicts of interest. 17.

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Anti-inflammatory effects of rutin on HMGB1-induced inflammatory responses in vitro and in vivo.

High mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions. Rutin (RT), an active flavonoid compound...
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