Zingerone reduces HMGB1-mediated septic responses and improves survival in septic mice

Toxicology and Applied Pharmacology 329 (2017) 202–211

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Zingerone reduces HMGB1-mediated septic responses and improves survival in septic mice Wonhwa Lee a,1, Sae-Kwang Ku b,1, Jong-Sup Bae a,⁎ a

College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics based Creative Drug Research Team, Kyungpook National University, Daegu 41566, Republic of Korea Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan 38610, Republic of Korea

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Article history: Received 18 March 2017 Revised 28 May 2017 Accepted 9 June 2017 Available online 10 June 2017 Keywords: Zingerone Endothelium HMGB1 Sepsis

a b s t r a c t High mobility group box 1 (HMGB1) is considered a late mediator of sepsis and the inhibition of HMGB1-mediated severe inflammatory responses and restoration of endothelial integrity have emerged as attractive therapeutic strategies for the management of sepsis. Zingerone (ZGR), a phenolic alkanone isolated from ginger, has been reported to possess various pharmacological activities. We examined the effects of ZGR on HMGB1-mediated septic responses and survival rate in a mouse model of sepsis. ZGR was administered after HMGB1 challenge. The antiseptic activity of ZGR was determined from the measurements of permeability, leukocyte adhesion and migration, activation of pro-inflammatory proteins, and the production of tissue injury markers in HMGB1-activated HUVECs and mice. ZGR significantly reduced HMGB1 release in LPS-activated HUVECs via the SIRT1-mediated deacetylation of HMGB1. And, ZGR suppressed the production of TNF-α and IL-6 and the activation of NF-κB and ERK 1/2 by HMGB1. ZGR also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with ZGR reduced the CLP-induced release of HMGB1, sepsis-related mortality, and tissue injury in vivo. Our results indicated that ZGR might be useful in the treatment of sepsis by targeting HMGB1. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Sepsis is defined as a systematic inflammatory response syndrome caused by infection and is a common cause of morbidity and mortality, despite recent advances in antibiotic therapy and intensive care (Russell, 2006). Previous reports have demonstrated that the persistent increase in plasma levels of the cytokine high mobility group box 1 (HMGB1) in septic patients was correlated with the degree of organ dysfunction and eventual patient outcome (Wang et al., 2001; Sunden-Cullberg et al., 2005; Gibot et al., 2007). In contrast to early inflammatory mediators such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, HMGB1 is a late mediator of sepsis and is associated with patient prognosis (Abraham et al., 2000). In response to infection or injury, HMGB1 is actively secreted by innate immune cells and/or passively released by injured or damaged cells (Ulloa and Tracey, 2005; Bae, 2012). Secreted HMGB1 can trigger a lethal inflammatory process by significantly increasing the release of inflammatory cytokines, as well as enhancing the expression of cell adhesion molecules (CAMs), such as vascular cell-adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and E-selectin, which promote inflammation via the ⁎ Corresponding author at: College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea. E-mail address: [email protected] (J.-S. Bae). 1 First two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.taap.2017.06.006 0041-008X/© 2017 Elsevier Inc. All rights reserved.

recruitment of leukocytes (Andersson et al., 2000; Bae and Rezaie, 2011). As a late inflammatory mediator of sepsis, HMGB-1 provides a wide therapeutic window for clinical intervention and therefore remains an attractive target for sepsis treatment (Bae, 2012). The herbal plant Zingiber officinale is a natural dietary spice with potent anti-inflammatory, antioxidative, and anticancer properties (Park et al., 1998). Zingerone (ZGR) [4-(4-hydroxy-3-methoxyphenyl) butan-2-one] is a stable active component of dry ginger rhizome (Sies and Masumoto, 1997) that has been reported to exhibit various pharmacological activities such as anti-inflammatory and anti-apoptotic effects and to confer protection from myocardial infarction and irritable bowel disorder (Kim et al., 2010; Rao et al., 2011; Banji et al., 2014; Hemalatha and Prince, 2015). To the best of our knowledge, only a few studies are available on the in vivo protective effect of ZGR against HMGB1-induced septic responses. Herein, we report the in vivo and in vitro antiseptic effects of ZGR on HMGB1-mediated responses.

2. Materials and methods 2.1. Reagents ZGR, bacterial lipopolysaccharide (LPS, serotype: 0111:B4, L5293), Evans blue, crystal violet, 2-mercaptoethanol, and antibiotics (penicillin G and streptomycin) were purchased from Sigma (St. Louis, MO).

W. Lee et al. / Toxicology and Applied Pharmacology 329 (2017) 202–211

Human recombinant HMGB1 was purchased from Abnova (Taipei City, Taiwan) and fetal bovine serum and Vybrant DiD were purchased from Invitrogen (Carlsbad, CA).

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stopped by the addition of 50 μL 8 N H2SO4 and the absorbance was measured at 490 nm. 2.6. Preparation of cytoplasmic and nuclear extracts

2.2. Cell culture Primary human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Bio Science (Charles City, IA) and maintained using a previously described method (Ku and Bae, 2015; Ku et al., 2015; Yoon et al., 2015). HUVECs were used at cell culture passages 3– 5. Human neutrophils were freshly isolated from whole blood (15 mL) obtained by venipuncture from five healthy volunteers and maintained as previously described (Jung et al., 2016a; Ku et al., 2016; Min et al., 2016). 2.3. Animals and husbandry Male C57BL/6 mice (6–7 weeks old, 27 g) purchased from Orient Bio Co. (Sungnam, Republic of Korea) were used in this study after an acclimatization period of 12 days. Five animals were housed per polycarbonate cage, which was kept under controlled temperature (20–25 °C), humidity (40–45% RH), and a 12:12 h light/dark cycle. The animals received a normal rodent pellet diet and were given ad libitum access to water during acclimatization. All animals were treated in accordance with the “Guidelines for the Care and Use of Laboratory Animals” issued by Kyungpook National University (IRB No. KNU 2016-54). 2.4. Cecal ligation and puncture (CLP) To induce sepsis, male mice were anesthetized with Zoletil (tiletamine and zolazepam, 1:1 mixture, 30 mg/kg) and Rompum (xylazine, 10 mg/kg). The CLP-induced sepsis model was prepared as previously described (Wang et al., 2004; Bae et al., 2014). In brief, a 2cm 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 to induce high grade sepsis (Rittirsch et al., 2009). The cecum was then gently squeezed to extrude a small amount of feces from the perforation site and returned to the peritoneal cavity. The laparotomy site was then sutured with 4.0 silk. 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 (IRB No. KNU 2016-54). 2.5. Competitive enzyme-linked immunosorbent assay (ELISA) for HMGB1 A competitive ELISA was performed to determine the HMGB1 concentrations in cell culture media or mice serum, as previously described (Jung et al., 2016a). HUVEC monolayers were treated first with LPS (100 ng/mL) for 16 h and then with ZGR for 6 h. After treatment, the cell culture media was collected for the determination of HMGB1. To perform the ELISA, 96-well flat plastic microtiter plates (Corning, NY) were coated with HMGB1 protein in 20 mM carbonate-bicarbonate buffer (pH 9.6) containing 0.02% sodium azide and allowed to dry overnight at 4 °C. The plates were then rinsed three times in PBS-0.05% Tween 20 (PBS-T) and stored at 4 °C. Lyophilized culture media was pre-incubated with anti-HMGB1 antibody (Abnova, diluted 1:1000 in PBS-T) in 96-well plastic round microtiter plates for 90 min at 37 °C, transferred to the pre-coated plates, and incubated for 30 min at room temperature. The plates were then rinsed three times in PBS-T, incubated for 90 min at room temperature with peroxidase-conjugated antirabbit IgG antibodies (Amersham Pharmacia Biotech, diluted 1:2000 in PBS-T), rinsed three times with PBS-T, and incubated for 60 min at room temperature in the dark with 200 μL of substrate solution (100 μg/mL o-phenylenediamine and 0.003% H2O2). The reaction was

The cells were harvested rapidly by sedimentation and nuclear and cytoplasmic extracts were prepared on ice, as previously described (Mackman et al., 1991). Briefly, the cells were harvested and were washed with 1 mL of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 19 mM KCl) for 5 min at 600 × g. Subsequently, the cells were resuspended in buffer A, centrifuged at 600 × g for 3 min, resuspended in 30 μl of buffer B (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA), rotated for 30 min at 4 °C, and centrifuged at 13,000 ×g for 20 min. The supernatant was used as the nuclear extract. The nuclear and cytosolic extracts were analyzed for protein content by using the Bradford assay. 2.7. Immunoprecipitation and western blotting Cells were lysed in 200 μL of Pierce immunoprecipitation (IP) Lysis Buffer (Thermo Fisher Scientific) and centrifuged for 15 min at 15,000 ×g at 4 °C. After protein extraction, 200–300 μg of total cellular protein was precleared with Protein G Sepharose 4 Fast Flow (GE Healthcare Life Sciences, Buckinghamshire, UK) for 1 h at 4 °C and then briefly centrifuged. The precleared cellular lysate was incubated with anti-HMGB1 (Santa Cruz Biotechnology) overnight at 4 °C with constant rotation and then incubated for 4 h with Protein G Agarose. After centrifugation, the Sepharose beads were washed with PBS and prepared for analysis via western blotting. For western blotting, the cells were first rinsed with ice-cold phosphate-buffered saline (PBS) and treated with lysis buffer composed of 0.5% SDS, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease inhibitors. The protein concentration of each sample was determined using the Bradford assay and the absorbance of the mixture at 595 nm was measured using an ELISA plate reader. To detect the presence of HMGB1 in the supernatants, samples of the culture medium were briefly centrifuged to remove cellular debris. Equal sample volumes were mixed with 2× loading dye and boiled at 95 °C for 5 min. Samples of culture medium and whole cell lysate were separated by electrophoresis in polyacrylamide gels of different percentages, depending on the size of the protein of interest. The gels were transferred to polyvinylidene difluoride (PVDF) membranes via semidry electrophoretic transfer at 20 mA for 2 h. The PVDF membranes were blocked for 2 h at room temperature in 5% bovine serum albumin (BSA), incubated with a 1:500 dilution of primary antibody in Tris-buffered saline/Tween 20 (TBS-T) containing 5% BSA overnight at 4 °C, and then incubated with a 1:5000 dilution of secondary antibody in TBS-T containing 1% BSA at room temperature for 1 h. After incubation, the membranes were washed three times with TBS-T solution, incubated with western blot enhanced chemiluminescence (ECL) detection reagents (Amersham, Piscataway, NJ), and exposed to Xomat AR films (Eastman Kodak, Rochester, NY). Scanning densitometry was performed using an Image Master® VDS (Pharmacia Biotech Inc., San Francisco, CA). For western blotting of NF-kB and ERK 1/2, anti-phospho-ERK1/2, anti-total ERK1/2, anti-NF-κB p65, and anti-lamin A/C, or β-actin antibodies (Santa Cruz) were used. β-actin or lamin A/C was used as a loading control for cytoplasmic or nuclear extracts, respectively. 2.8. Cell viability assay MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) was used as an indicator of cell viability. The cells were grown in 96-well plates at a density of 5 × 103 cells/well. After 24 h, the cells were washed with fresh medium and incubated for 48 h with ZGR. After the incubation period, the cells were washed and 100 μL MTT

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(1 mg/mL) was added to each well and incubated for 4 h. Finally, dimethyl sulfoxide (DMSO, 150 μL) was added to solubilize the formed formazan salt. The amount of formazan salt was determined by measuring the OD at 540 nm using a microplate reader (Tecan Austria GmbH, Austria).

previously described (Jung et al., 2016a, 2016b). To assess leukocyte migration, mice were euthanized after 6 h, and the peritoneal cavities were washed with 5 mL normal saline. The obtained samples of peritoneal fluids (20 μL) were mixed with 0.38 mL Turk's solution (0.01% crystal violet in 3% acetic acid) and the number of leukocytes was counted under a light microscope.

2.9. In vitro permeability assay For the spectrophotometric quantification of endothelial cell permeability in response to increasing concentrations of each compound, the flux of Evans blue-bound albumin across functional cell monolayers was measured using a modified 2-compartment chamber model, as previously described (Bae and Rezaie, 2011; Jung et al., 2016b). HUVECs were plated (5 × 104 cells/well) in transwells (pore size, 3 μm; diameter, 12 mm) for 3 days. Confluent monolayers of HUVECs were first treated with LPS (100 ng/mL) for 4 h or HMGB1 (1 μg/mL) for 16 h, which was followed by subsequent treatment with ZGR. Transwell inserts were then washed with PBS (pH 7.4) and growth medium containing 0.5 mL Evans blue (0.67 mg/mL) and 4% BSA (Evans blue/BSA) was added. Fresh growth medium was then added to the lower chamber and the medium in the upper chamber was replaced with Evans blue/ BSA. After ten minutes, the optical density in the lower chamber was measured at 650 nm. 2.10. Cell-cell adhesion assay Purified human neutrophils (1.5 × 106 cells/mL, 200 μL/well) were labeled with Vybrant DiD dye and then added to washed and stimulated HUVECs. HUVEC monolayers were first treated with HMGB1 (1 μg/mL) for 16 h and then with ZGR for 6 h. The neutrophils were allowed to adhere and non-adherent neutrophils were removed by washing. The percentage of adherent neutrophils was calculated using the formula: % adherence = (adherent signal/total signal) × 100. 2.11. In vitro migration assay Migration assays were performed in transwell plates (diameter, 6.5 mm) that contained filters with a pore size of 8 μm. HUVECs (6 × 104) were cultured for 3 days to obtain confluent endothelial monolayers. Prior to the addition of neutrophils to the upper compartment, cell monolayers were treated with HMGB1 (1 μg/mL) for 16 h, followed by treatment with ZGR for 6 h. Transwell plates were then incubated at 37 °C in 5% CO2 for 2 h. The cells in the upper chamber were aspirated and non-migrating cells on top of the filter were removed using a cotton swab. Neutrophils on the lower side of the filter were fixed in 8% glutaraldehyde and stained with 0.25% crystal violet in 20% methanol (w/v). The experiments were repeated twice per well in duplicate wells, and nine randomly selected high power microscopic fields (HPF, 200 ×) were counted. The results were presented as migration indices. 2.12. In vivo permeability and the leukocyte migration assay For the in vivo study, male mice were anesthetized with 2% isoflurane (Forane, JW Pharmaceutical, South Korea) in oxygen delivered via a small rodent gas anesthesia machine (RC2, Vetequip, Pleasanton, CA), first in a breathing chamber and then via facemask. Mice were allowed to breath spontaneously during the procedure. The mice were first treated with HMGB1 (2 μg/mouse, i.v.) for 16 h and then treated with ZGR (0.36 or 0.72 mg/kg, i.v.). For the in vivo permeability assay, after 6 h, 1% Evans blue dye solution in normal saline was injected intravenously into each mouse. After 30 min, the mice were euthanized and the peritoneal exudates were collected by washing the cavities with normal saline (5 mL) and centrifugation of the wash solution (200 ×g, 10 min). The absorbance of the supernatants was measured at 650 nm. Vascular permeability was expressed as μg of dye in the peritoneal cavity per mouse, and determined using a standard curve as

2.13. Expression of cell adhesion molecules (CAMs) and HMGB1 receptors The expressions of VCAM-1, ICAM-1, and E-selectin were determined by whole-cell ELISA, as previously described (Jung et al., 2016a, 2016b). Briefly, confluent monolayers of HUVECs were treated with HMGB1 (1 μg/mL) for 16 h (VCAM-1 and ICAM-1) or 22 h (E-Selectin), treated with ZGR, and fixed in 1% paraformaldehyde. After three washes, mouse anti-human monoclonal antibodies (VCAM-1, ICAM-1, and E-selectin; Temecula, CA; 1:50 dilution) were added and the samples were incubated for 1 h (37 °C, 5% CO2). The cells were then washed, treated with peroxidase-conjugated anti-mouse IgG antibody (Sigma, St. Louis, MO) for 1 h, washed three times, and treated with ophenylenediamine substrate (Sigma, St. Louis, MO). The same experimental procedures were used to monitor the cell surface expression of the TLR2, TLR4, and RAGE receptors, using specific antibodies (A-9, H80, and A-9, respectively) obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 2.14. ELISA for phosphorylated p38 mitogen-activated protein kinase (MAPK), NF-κB, TNF-α, extracellular regulated kinases (ERK) 1/2, and IL-6 The activity of phosphorylated p38 MAPK was quantified in accordance with the manufacturer's instructions using a commercially available ELISA kit (Cell Signaling Technology, Danvers, MA). The activities of total and phosphorylated p65 NF-κB (#7174, #7173, Cell Signaling Technology, Danvers, MA) or total and phosphorylated ERK 1/2 (R&D Systems, Minneapolis, MN) in nuclear lysates were also determined using ELISA kits. The concentrations of IL-6 and TNF-α in cell culture supernatants were determined using ELISA kits (R&D Systems, Minneapolis, MN). For all assays, the values were measured using an ELISA plate reader (Tecan, Austria GmbH, Austria). 2.15. Hematoxylin & eosin staining and histopathological examination Male C57BL/6 mice were subjected to CLP and were administered ZGR (0.36 or 0.72 mg/kg, i.v.) at 12 h and 50 h after CLP (n = 5). At 96 h after CLP, the mice were euthanized. To analyze the phenotypic change in the lung, kidney and liver, samples were removed from each mouse, washed three times in PBS (pH 7.4) to remove the remaining blood, and fixed in 4% formaldehyde solution (Junsei, Tokyo, Japan) in PBS for 20 h at 4 °C. After fixation, the samples were dehydrated using an ethanol series, embedded in paraffin, sectioned into 4-μm slices, and placed on a slide. The slides were deparaffinized in a 60 °C oven, rehydrated, and stained with hematoxylin (Sigma). To remove overstaining, the slides were quickly dipped three times in 0.3% acid alcohol and counterstained with eosin (Sigma). Over-staining was then removed by washes in an ethanol series and xylene, and the samples were placed under a coverslip. Light microscopic analysis of the lung specimens was performed by a blinded observer who evaluated pulmonary architecture, tissue edema, and infiltration of the inflammatory cells by a previously defined method (Ozdulger et al., 2003). The results were classified into four grades: Grade 1 represented normal histopathology; Grade 2 indicated minimal neutrophil leukocyte infiltration; Grade 3 represented moderate neutrophil leukocyte infiltration, perivascular edema formation, and the partial destruction of pulmonary architecture; and Grade 4 included dense neutrophil leukocyte infiltration, abscess formation, and the complete destruction of pulmonary architecture.

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2.16. Immunofluorescence staining HUVECs were grown to confluence on glass coverslips coated with 0.05% Poly-L-Lysine in complete media supplemented with 10% FBS and then maintained for 48 h. The cells were stimulated with HMGB1 (1 μg/mL) for 16 h, with or without 6-h ZGR treatment (25 or 50 μM). For cytoskeletal staining, the cells were fixed in 4% formaldehyde in PBS for 15 min at room temperature, permeabilized in 0.05% Triton X100 in PBS for 15 min, and blocked in blocking buffer (5% BSA in PBS) overnight at 4 °C. Then, the cells were incubated with F-actin labeled fluorescein phalloidin (F 432; Molecular Probes, Invitrogen) or primary rabbit monoclonal NF-κB p65 antibody and anti-rabbit Alexa 488 overnight at 4 °C. Nuclei were counterstained with 4,6-diamidino-2phenylindole dihydrochloride (DAPI) and visualized by confocal microscopy at 630 × magnification (TCS-Sp5, Leica microsystem, Germany).

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using commercial assay kits (T3 and T4: Calbiotech, Spring Valley, CA) or (TSH: LSBio, Seattle, WA). 2.18. Statistical analysis All experiments were independently performed a minimum of three times. Values were expressed as the mean ± standard deviation (SD). The statistical significance of differences between test groups was evaluated by SPSS for Windows, version 16.0 (SPSS, Chicago, IL). Statistical relevance was determined by one-way analysis of variance (ANOVA) and Tukey's post-test. Values of p b 0.05 were considered to indicate statistical significance. The survival of CLP-induced sepsis outcomes was assessed using Kaplan-Meier analysis. 3. Results and discussion 3.1. Effects of ZGR on LPS and CLP-induced secretion of HMGB1

2.17. Measurement of organ injury markers and thyroid hormones The plasma levels of aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine, lactate dehydrogenase (LDH) were measured using commercial assay kits (Pointe Scientific, Lincoln Park, MI). The levels of triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) were also measured

It is well established that HMGB1 is actively secreted by activated macrophages and necrotic cells, and functions as a “danger signal” to augment severe inflammatory responses (Mullins et al., 2004; El Gazzar, 2007; van Beijnum et al., 2008; Bae and Rezaie, 2011). The levels of HMGB1 slowly increase after 8 h and are correlated with the progression of sepsis (Czura et al., 2003). Thus, we investigated the effect of ZGR on the LPS-induced secretion of HMGB1 by HUVECs. Treatment with

Fig. 1. Effects of ZGR on HMGB1 release and expression of HMGB1 receptors. (A) After stimulation with LPS (100 ng/mL, 16 h), HUVECs were treated with the indicated concentrations of ZGR for 6 h and HMGB1 release was measured by ELISA. (B) Twelve hours after CLP, male C57BL/6 mice (n = 5) were intravenously administered the indicated amount of ZGR, and euthanized 24 h after CLP. Serum HMGB1 levels were measured by ELISA. (C, D) Cells were treated with ZGR (50 μM) for 0, 2, 4, 6, 8, 10, and 12 h. After incubation for the indicated time, the cells were lysed and analyzed via western blotting to measure the expression levels of SIRT1 (C). Effect of ZGR on the acetylation of HMGB1 and the SIRT1 expression in HUVECs. Cells were treated with LPS (100 ng/mL) with or without ZGR (50 μM). Alternatively, cells were treated with the SIRT1 inhibitor (sirtinol, Srtnl, 10 mM) for 1 h prior to ZGR treatment. After incubation for 6 h, the cells were lysed for immunoprecipitation. Cell lysates were subjected to immunoprecipitation with anti-HMGB1 antibody and HMGB1 acetylation and total HMGB1 protein level were measured by immunoblot analysis using anti-acetyl-lysine (K) or anti-HMGB1, respectively (D, rows 1 and 2). After incubation for 16 h, equal volumes of media were collected and released HMGB1 was detected by western blot (D, row 3). (E) Confluent HUVECs were activated with HMGB1 (1 μg/mL, 16 h) and then incubated with ZGR for 6 h. Expression of TLR2 (white bar), TLR4 (gray bar), or RAGE (black bar) was determined by cell-based ELISA. (F) The effect of ZGR on cellular viability was measured by MTT assay. The results shown are the mean ± SD from three separate experiments conducted in triplicate on different days. D = 0.2% DMSO is the vehicle control. *p b 0.05 versus LPS alone (A), CLP alone (B), or HMGB1 alone (E).

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ZGR inhibited the secretion of HMBG1 in a dose-dependent manner, with a minimal effective concentration of 10 μM (Fig. 1A). To confirm these activities in vivo, we evaluated the ability of ZGR to inhibit HMGB1 release using a mouse model of CLP-induced sepsis. Treatment with ZGR also resulted in a significant reduction in HMGB1 secretion (Fig. 1B). As the average circulating blood volume for mice is 72 mL/kg (Diehl et al., 2001) and the average weight of mouse used was 27 g, the average blood volume was 2 mL. Hence, the amount of injected ZGR (0.36 or 0.72 mg/kg) yielded a maximum concentration of 25 or 50 μM in the peripheral blood. Previous studies indicated that the hyperacetylation of HMGB1 affected its ability to bind DNA and redirected it toward the cytoplasm (Bonaldi et al., 2003) and that the hyperacetylation on the serine residues of HMGB1 blocked its nuclear import and promoted cytoplasmic secretion (Youn and Shin, 2006). In addition, the activation of Sirtuin 1 (SIRT1) plays a pivotal role in the deacetylation of HMGB1; HMGB1 is a novel deacetylation target of SIRT1 (Rabadi et al., 2015). Therefore, to determine the effects of ZGR on the induction of SIRT1 expression, we performed a western blot analysis. As shown in Fig. 1C, the protein expression of SIRT1 was apparent after 4 h of incubation, peaked after 6 h, was maintained until 8 h, and disappeared at 12 h. In addition, to define the molecular mechanism by which ZGR suppressed the secretion of HMGB1 by LPS, we determined the effects of ZGR on the deacetylation of HMGB1 and the induction of SIRT1 expression. As shown in Fig. 1D, stimulation with LPS increased the acetylation of HMGB1; this change was mostly reduced by the addition of ZGR. To

confirm that SIRT1 activity was responsible for the inhibition of HMGB1 release via the deacetylation of HMGB1 in LPS-activated HUVECs, we investigated the effect of sirtinol, a SIRT1 inhibitor. We observed that treatment with sirtinol clearly reversed the effect of ZGR (Fig. 1D) and significantly increased both the acetylation and secretion of HMGB1. Therefore, these results suggested that ZGR significantly reduced HMGB1 release in LPS-activated HUVECs via the SIRT1-mediated deacetylation of HMGB1 based on the following data: 1) the protein expression of SIRT1 was significantly increased by ZGR; 2) the secretion of HMGB1 was significantly reduced by ZGR via HMGB1 deacetylation; and 3) the ZGR-mediated inhibition of HMGB1 release and acetylation were significantly antagonized by sirtinol, an inhibitor of SIRT1. Next, we determined whether ZGR inhibited the expression of HMGB1 receptors such as TLR2, TLR4, and RAGE, in HUVECs. The data showed that HMGB1 increased the expression of each receptor and ZGR diminished this increased expression (Fig. 1E). Additionally, cell viability assays were performed to probe the toxicity of ZGR in HUVECs after 48 h. At the tested concentrations (up to 100 μM), ZGR did not affect cell viability (Fig. 1F). Collectively, these results indicated that ZGR may be a viable early intervention to prevent the release of HMGB1 and subsequent progression to severe sepsis and septic shock. 3.2. Effect of ZGR on HMGB1-mediated vascular barrier disruption As HMGB1 and LPS are known to disrupt vascular barrier integrity (Lee et al., 2014b), a vascular permeability assay was applied to evaluate

Fig. 2. Effects of ZGR on HMGB1-mediated permeability in vitro and in vivo. (A, B) Effects of treatment with different concentrations of ZGR for 6 h on barrier disruption caused by LPS (100 ng/mL, 4 h; A) or HMGB1 (1 μg/mL, 16 h; B) were monitored by the measurement of the flux of Evans blue-bound albumin across the HUVECs. (C) The effects of ZGR on HMGB1-induced (2 μg/mouse, i.v.) vascular permeability in mice were examined through the measurement of Evans blue dye in peritoneal washings (expressed as μg/mouse, n = 5). (D) HUVECs were activated with HMGB1 (1 μg/mL, 16 h), followed by treatment with different concentrations of ZGR for 6 h. Effects of ZGR on the HMGB1-mediated expression of phospho-p38 were determined by ELISA. (E) Staining for F-actin. HUVEC monolayers grown on glass coverslips were stimulated with HMGB1 for 1 h, treated with ZGR (25 or 50 μM) for 6 h, and stained for F-actin. Arrows indicate intercellular gaps. The images are representative of three separate experiments conducted on different days with similar results. Results are expressed as the mean ± SD of three separate experiments on different days. D = 0.2% DMSO is the vehicle control. *p b 0.05 versus LPS (A) or HMGB1 (B, C, D).

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the effects of ZGR on the maintenance of barrier integrity in HUVECs. HUVECs were treated with ZGR for 6 h following the activation by LPS (Fig. 2A, 100 ng/mL) or HMGB1 (Fig. 2B, 1 μg/mL) and we observed that ZGR dose-dependently inhibited LPS- and HMGB1-mediated hyperpermeability (Figs. 2A and B). To verify these results in vivo, the in vivo effects of ZGR on vascular permeability were assessed. Fig. 2C shows that ZGR also induced a marked inhibition of the peritoneal dye leakage through the action of HMGB1. As the vascular disruptive responses caused by HMGB1 occur through various signaling pathways (the activation of ERK 1/2 and p38 MAPK downstream of TLR2/4, and the Ras/p38 pathway downstream of RAGE (Palumbo et al., 2007; Qin et al., 2009; Sun et al., 2009)), we next determined whether the activation of p38, a common signaling target of HMGB1 receptor, was affected by ZGR. To achieve this, HUVECs were treated with ZGR following activation with HMGB1. As shown in Fig. 2D, HMGB1 upregulated the expression of phosphorylated p38; this upregulation was clearly reduced by treatment with ZGR. In addition, treatment with ZGR (20 or 30 μM) inhibited the formation of HMGB1-induced paracellular gaps by the formation of dense F-actin rings (Fig. 2E). The reduction in HMGB1-induced permeability and p38 activation indicated the promising role of ZGR as an antisepsis drug. 3.3. Effects of ZGR on HMGB1-mediated expression of CAMs, adhesion, and migration of human neutrophils The upregulated levels of cellular adhesion molecules (CAMs), such as ICAM-1, VCAM-1, and E-selectin, are involved in vascular inflammatory diseases (Luo et al., 2013) by aiding the adhesion and migration of

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immune cells toward the endothelium at the site of vascular inflammation (Frenette and Wagner, 1996; Ulbrich et al., 2003). Therefore, we examined the inhibitory effects of ZGR on the expression of CAMs. As shown in Fig. 3A, ZGR reduced the HMGB1-induced expression of CAMs. In addition to reduced CAM expression, ZGR reduced the adherence of the human neutrophils to HUVECs and their subsequent migration (Figs. 3B, C, E). These results were corroborated in vivo by the inhibition of HMGB1-induced migration of leukocytes in the peritoneal space (Fig. 3D). Thus, our results suggested that ZGR inhibited the adhesion and migration of leukocytes to the inflamed endothelium. 3.4. Effects of ZGR on HMGB1-stimulated activation of NF-κB/ERK and production of IL-6/TNF-α HMGB1 is known to contribute to pathophysiological systemic inflammation by the upregulation of inflammatory cytokines, such as TNF-α and IL-6, either individually or in combination with other pro-inflammatory cytokines (Erlandsson Harris and Andersson, 2004; Lee et al., 2014a; Jung et al., 2016a; Min et al., 2016). As outlined above, HMGB1 stimulated the release of inflammatory cytokines through various signaling pathways (Erlandsson Harris and Andersson, 2004; Lee et al., 2014a; Jung et al., 2016a), including ERK 1/2, and ultimately resulted in the activation of NK-κB. Thus, in order to find the inhibitory mechanism of ZGR on HMGB1-mediated septic responses, the effects of ZGR on the HMGB1-induced production of TNF-α and IL-6, or activation of NF-κB and ERK 1/2, were evaluated. HUVECs were activated with HMGB1 for 16 h, followed by incubation with ZGR for 6 h. Our results showed that HMGB1 enhanced the production of TNF-α and IL-6 and

Fig. 3. Effects of ZGR on HMGB1-mediated pro-inflammatory responses. (A-C) HUVECs were stimulated with HMGB1 (1 μg/mL) for 16 h and then treated with ZGR for 6 h. HMGB1mediated (A) expression of VCAM-1 (white bar), ICAM-1 (gray bar), and E-selectin (black bar) in HUVECs, (B,C) adherence of human neutrophils to HUVEC monolayers, and (D) migration of neutrophils through HUVEC monolayers were analyzed. (E) The effects of treatment with ZGR on HMGB1-induced (2 μg/mouse, i.v.) leukocyte migration into the peritoneal cavities of mice were analyzed. The images are representative of three separate experiments conducted on different days with similar results. All results indicate the mean ± SD of three separate experiments on different days. D = 0.2% DMSO is the vehicle control. *p b 0.05 versus HMGB1.

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the activation of NF-κB and ERK 1/2, and that these increases were significantly reduced by treatment with ZGR (Fig. 4). Furthermore, HMGB1 induced an increase in the expression of p65 NF-κB in the nucleus, whereas this was not elevated under normal conditions. Treatment with ZGR resulted in a decrease in the HMGB1-induced expression of p65 NF-κB in the nucleus (Fig. 4C, Western blotting). To confirm these data, we measured the HMGB1-induced translocation of NF-κB from the cytosol to the nucleus by using p65 NF-κB and a fluorescein isothiocyanate (FITC)-conjugated antibody. The resulting immunofluorescence staining (Fig. 4E) showed that stimulation with HMGB1 resulted in the obvious translocation of NF-κB p65 from the cytoplasm into the nucleus, which was clearly counteracted by treatment with ZGR. 3.5. Protective effect of ZGR in CLP-induced septic mice To evaluate whether the suppression of HMGB1 secretion and HMGB1-mediated septic responses by ZGR also influenced the survival rate of CLP-induced septic mice, ZGR was administered to mice after CLP surgery. A single administration of ZGR (0.36 or 0.72 mg/kg, 12 h after CLP) did not prevent CLP-induced death (data not shown). Thus, we administered two equal doses of ZGR, one at 12 h after CLP and the other at 50 h after CLP, and the results showed that ZGR increased the survival rate of septic mice, according to the Kaplan-Meier survival analysis (p b 0.00001, Fig. 5A). The marked improvement in survival rate achieved by the administration of ZGR suggested that ZGR might be used to the treatment of severe vascular inflammatory diseases, such as sepsis and septic shock. 3.6. Protective effect of ZGR in CLP-induced tissue injury We determined the effects of ZGR on CLP-induced pulmonary, renal and hepatic injury to confirm the protective activity of ZGR in CLP-induced death. CLP surgery resulted in the interstitial edema owing to massive infiltration of inflammatory cells in the interstitium and alveolar spaces and the pulmonary architecture was severely impaired

(Figs. 5B and C). These disordered morphological changes were reduced in CLP mice treated with ZGR (Figs. 5B and C). Systemic inflammation during sepsis frequently causes multiple organ failure, with the liver and kidney as the major target organs (Astiz and Rackow, 1998). CLP resulted in significant increases in the plasma level of ALT and AST (Fig. 5D), which are markers of hepatic injury, and creatinine and BUN (Fig. 5E and F), which are markers of renal injury; these increases were mitigated by ZGR. Another important marker of tissue injury, LDH, was also reduced by ZGR in CLP-induced mice (Fig. 5G). Endocrine dysfunction is common in severe sepsis and is associated with an increased risk of mortality; thyroid hormone abnormalities are very common in septic patients (Gheorghita et al., 2015). There is evidence that lower baseline thyroid hormone values, including of T3, T4, and TSH, can be substantially lower in septic patients compared with nonseptic patients with a similar severity of illness. Such abnormalities are associated with a poorer outcome in patients with sepsis (Angelousi et al., 2011). Therefore, we determined the effects of ZGR on the levels of T3, T4, and TSH in CLP-induced septic conditions. The levels of T3, T4, and TSH were lower in CLP-induced septic mice compared with the sham control; this reduction was recovered by ZGR treatment (Figs. 5H-J). Figs. 6A-B showed the renal and hepatic histological changes in the Sham and CLP with or without ZGR. Histological evaluation of the renal sections of mice in the Sham groups revealed a regular morphology of the glomeruli and tubuli (Fig. 6A). However, CLP induced tissue destruction proximal and distal tubules along with severe infiltration of polymorphonuclear leukocytes. In contrast, ZGR post-treated CLP group was found to attenuate many of the symptoms of renal injury (Fig. 6A). The histological structure of liver was observed as normal in the Sham group (Fig. 6B). CLP also induced the formation of necrotic areas in the liver, with significantly enhanced infiltration of inflammatory cells into areas surrounding the centrilobular veins of the liver (Fig. 6B). Treatment with ZGR, however, attenuated the severity of inflammation and necrosis induced by CLP (Fig. 6B). These histological data confirmed the plasma levels of renal and hepatic damage markers (Figs. 5D-F).

Fig. 4. Effects of ZGR on HMGB1-stimulated production of IL-6/TNF-α and activation of NF-κB/ERK. (A,B) HUVECs were stimulated with HMGB1 (1 μg/mL) for 16 h and then treated with ZGR for 6 h. HMGB1-mediated production of TNF-α (A) or IL-6 (B) in HUVECs was analyzed after the treatment of cells with ZGR for 6 h. (C,D) Confluent HUVECs were activated with HMGB1 (1 μg/mL, 16 h) and then incubated with ZGR for 6 h, and HMGB1-mediated activation of phospho-NF-κB p65 (white bar) or total NF-κB p65 (black bar) in (C) or phosphoERK1/2 (white box) or total ERK1/2 (black box) in HUVECs was analyzed (D). Alternatively, expression levels of NF-κB in nuclear or cytoplasmic extracts were evaluated by using western blotting. β-actin and lamin A/C were used as loading controls for cytoplasmic and nuclear extracts, respectively (C, lower panel). Phospho-ERK1/2 or total ERK1/2 in HUVECs was analyzed by Western blotting (D, lower panel). (E) Immunofluorescence (IF) microscopy of the nuclear translocation of p65 in HUVECs. HUVECs were stimulated for 1 h with 1 μg/mL HMGB1, or not stimulated, and treated with 50 μM ZGR for 6 h, or not treated. The subcellular localization of p65 was examined by IF staining. The images are representative of three separate experiments conducted on different days with similar results. Results are expressed as the mean ± SD of three separate experiments on different days. D = 0.2% DMSO is the vehicle control. *p b 0.05 versus HMGB1.

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Fig. 5. Effects of ZGR on lethality or tissue injury after CLP. (A) Male C57BL/6 mice (n = 20) were administered ZGR at 0.36 mg/kg (i.v. □) or at 0.72 mg/kg (i.v.■) at 12 h and 50 h after CLP. Animal survival was monitored every 12 h for 132 h after CLP. Control CLP mice (●) and sham-operated mice (○) were administered sterile saline (n = 20). Kaplan-Meier survival analysis was used to determine the overall survival rates versus CLP treated mice. (B) Male C57BL/6 mice were subjected to CLP, administered ZGR intravenously at 12 h and 50 h after CLP (n = 5), and euthanized at 96 h after CLP. Histopathological scores for the lung tissue were recorded as described in methods section. (C) Photomicrographs of lung tissues (H&E staining, ×200). Sham group (grade 1); CLP group (grade 3); Right, CLP + ZGR group, (grade 2). Illustrations are representative images from three independent experiments conducted on different days with similar results. (D-J) As for (B,C) except that mice were bled and euthanized to examine the plasma levels of: (D) AST, ALT; (E) creatinine; (F) BUN; (G) LDH; (H) T3; (I) T4; and (J) TSH. All results indicate the mean ± SD of three separate experiments conducted on different days with similar results. *p b 0.05 versus CLP.

This study was performed to evaluate the protective effect of ZGR against vessel barrier integrity. Under physiological conditions, the vascular endothelium plays a central role in maintaining and controlling vascular integrity in response to the extracellular environment and is therefore the primary target of sepsis induced damage (Bogatcheva and Verin, 2008). As all blood vessels are lined with endothelial cells, the presence of vascular leak and tissue edema in sepsis suggests endothelial dysfunction. The breakdown of the vascular integrity and endothelial dysfunction resulting from septic attack may result in hyperpermeability (Bogatcheva and Verin, 2008). It is known that continuous, subcutaneous, and body-cavity edema typically develop in septic patients; this is suggestive of a comprehensive increase in vascular permeability (Bogatcheva and Verin, 2008). Therefore, the restoration of vascular integrity from disruptive responses by inflammatory stimuli and the maintenance of vascular homeostasis should be the primary strategy against sepsis, which will result in spontaneous diuresis with a reduction in edema. The results of this study suggested that the antiseptic effects of ZGR occurred through the inhibition of HMGB1 release and HMGB1-mediated hyperpermeability.

The molecular mechanism of the anti-inflammatory effects of ZGR against HMGB1-mediated septic responses may be facilitated by the suppression of HMGB1 release via the SIRT1-induced deacetylation of HMGB1 (Figs. 1A-D), the expression of HMGB1 receptors such as TLR2, TLR4, and RAGE (Fig. 1E), and HMGB1-mediated hyperpermeability (Figs. 2B, C, E) via the suppression of p38 activation (Fig. 2D). Furthermore, the inhibitory mechanism of ZGR on the interaction between leukocytes and endothelial cells was produced by the inhibition of the expressions of CAMs, such as VCAM, ICAM, and E-Selectin (Fig. 3). The underlying mechanism of the anti-inflammatory effects of ZGR was the downregulated production of inflammatory cytokines TNF-α and IL-6 (Figs. 4A and B) and the activation of the inflammatory transcriptional factors NF-κB and ERK1/2 (Figs. 4C and D). ZGR also inhibited the translocation of NF-κB from the cytosol to the nucleus (Fig. 4E). With regard to the pharmacological activities of ZGR, ZGR treatment showed the beneficial effects on the modulation of age-related NF-κB activation via inhibiting MAPK pathway and maintaining redox balances (Kim et al., 2010). And, Rao et al. reported the protective effect of ZGR against radiation induced DNA damage and antiapoptotic effect in

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Fig. 6. Effects of ZGR on histopathological change in kidney and lover tissues after CLP. Male C57BL/6 mice were subjected to CLP, administered ZGR intravenously at 12 h and 50 h after CLP (n = 5), and euthanized at 96 h after CLP. Histological examination of kidney (A) or liver (B) sections from Sham control, CLP with or without ZGR treated mice (H&E staining, original magnification 200 x). The images are representative of three separate experiments conducted on different days with similar results.

human lymphocytes by scavenging of radiation induced free radicals and also by the inhibition of radiation induced oxidative stress (Rao et al., 2011). The possible usefulness of ZGR in the treatment of in irritable bowel disorder by enhancing the levels of superoxide dismutase, glutathione and decreasing the levels of corticosterone was also reported (Banji et al., 2014). In addition, ZGR offers protection to the myocardial infarction by restricting the leakage of serum creatine kinase-MB, lactate dehydrogenase (LDH)-1, and LDH-2 isoenzymes (Hemalatha and Prince, 2015). Although these reports showed various pharmacological activities such as anti-inflammatory, anti-apoptotic, and beneficial effects on myocardial infarction and irritable bowel disorder, ZGR was administered before the pathological conditions, such as aging, radiation, bowel disorder, or myocardial infarction, were established. However, in the present study, ZGR was treated after making the sepsis condition induced by LPS, HMGB1, or CLP surgery, clearly showing the beneficial efficacy of ZGR for the sepsis treatment.

Collectively, the results of this study demonstrated that ZGR reduced HMGB1 release in LPS-activated HUVECs via the SIRT1-mediated deacetylation of HMGB1, suppressed CLP-mediated release of HMGB1, expression of HMGB1 receptors, and HMGB1-mediated barrier disruption by increasing barrier integrity and inhibiting of CAM expression (Fig. 7). Furthermore, ZGR reduced leukocyte adhesion and migration toward HUVECs (Fig. 7). The barrier protective effects of ZGR were confirmed in a mouse model, in which treatment with ZGR reduced CLP-induced mortality and pulmonary injury. Our findings indicated that ZGR may be a potential candidate for use in the treatment of severe vascular inflammatory diseases, such as sepsis and septic shock. Conflict of interest statement The authors have no conflicts of interest to declare. Acknowledgements This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) (HI15C0001), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HI15C0001) and by a grant from Korea of Health & Welfare, Republic of Korea (Project No: 20-11-0-090-091-3000-3033-320). References

Fig. 7. Briefed scheme of a signal transduction pathway of ZGR in HMGB1-mediated septic responses.

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