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Andrographolide inhibits HMGB1-induced inflammatory responses in human umbilical vein endothelial cells and in murine polymicrobial sepsis W. Lee,1,2* S. Ku,3* H. Yoo,1 K. Song1 and J. Bae1 1 College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea 2 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu, Korea 3 Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan, Korea
Received 7 October 2013, revision requested 6 December 2013, revision received 21 December 2013, accepted 24 February 2014 Correspondence: J. Bae, College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702701, Korea. E-mail: [email protected]
*First two authors contributed equally to this work.
Abstract Aim: Nuclear DNA-binding protein high-mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions, such as septic shock, upregulating pro-inflammatory cytokines. Andrographolide (AG) is isolated from the plant of Andrographis paniculata and used as a folk medicine for treatment of viral infection, diarrhoea, dysentery and fever. However, the effect of AG on HMGB1-induced inflammatory response has not been studied. Methods: Firstly, we accessed this question by monitoring the effects of post-treatment AG on lipopolysaccharide (LPS) and caecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Results: Post-treatment AG was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. AG also inhibited HMGB1-mediated hyperpermeability and leucocyte migration in septic mice. In addition, AG inhibited production of tumour necrosis factor-a (TNF-a) and activation of AKT, nuclear factor-jB (NF-jB) and extracellular-regulated kinases (ERK) 1/2 by HMGB1 in HUVECs. AG also induced downregulation of CLP-induced release of HMGB1, production of interleukin (IL) 1b/6/8 and mortality. Conclusion: Collectively, these results suggest that AG may be regarded as a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signalling pathway. Keywords Andrographolide, HMGB1, sepsis, vascular inflammation.
High-mobility group box chromosomal protein 1 (HMGB1) is a highly conserved ubiquitously expressed nuclear protein involved in nucleosome stabilization and gene transcription (Lotze & Tracey 2005). HMGB1 can be actively released by immune cells (activated monocytes, macrophages, neutrophils and platelets) into the extracellular environment (Degryse et al. 2001, Bianchi & Manfredi 2004, Ito et al. 2007). Extracellular HMGB1 secretes inflammatory stimulation function as a pro-inflammatory cytokine 176
from damaged and necrotic cells and triggers proinflammatory responses from macrophages and endothelial cells (Andersson & Tracey 2011). HMGB1 binds to several transmembrane receptors, such as receptor for advanced glycation end products (RAGE) and toll-like receptors (TLR)-2 and -4, and activates NF-jB and extracellular-regulated kinase (ERK) 1 and 2 (Hori et al. 1995, Park et al. 2004). Activation of endothelial cells by HMGB-1 leads to expression of cell adhesion molecules (CAMs), such as intercellular
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adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and E-selectin, which induces upregulated inflammation through recruitment of leucocytes (Bae & Rezaie 2011). Accumulation of HMGB1 occurs during sepsis, leading to multiple organ collapse and a lethal outcome (Yang et al. 2004). Therefore, it is a therapeutic target for clinical management of lethal systemic inflammatory diseases. Herbal medicines derived from plant extracts are increasingly utilized for the treatment of a wide variety of diseases. Recently, post-treatment of herbal extract could reduce the inflammatory responses (Park et al. 2012, Persson & Persson 2012). Andrographis paniculata, a popular herb, has been widely used in China as a folk medicine and is cultivated in various Asian countries. Andrographolide (AG) has numerous reported pharmacological properties and has been used extensively in the clinical setting for the treatment of infections, inflammation, colds, fever and diarrhoea. The anti-inflammatory activities of A. paniculata have been thoroughly studied (Chiou et al. 2000, Chao et al. 2009). In addition, andrographolide (AG), extracted and purified from A. paniculata, is currently prescribed for treatment of inflammationrelated diseases, such as laryngitis, upper respiratory tract infection and rheumatoid arthritis in China (Puri et al. 1993, Amroyan et al. 1999, Shen et al. 2000, 2002, Batkhuu et al. 2002). Studies on the biological effects of AG have demonstrated that AG decreases TNF-a-induced expression of ICAM-1 and monocyte adhesion in human endothelial cells (Habtemariam 1998, Yu et al. 2010). Treatment with AG resulted in reduced TNF-a-induced expression of ICAM-1 in HUVECs via the PI3K/Akt pathway and downstream target NF-jB activation (Chen et al. 2011). However, the effects of AG in HMGB1-mediated pro-inflammatory responses in HUVECs have not yet been studied. In this study, we described for the first time the effects of AG on HMGB1 release, HMGB1-mediated proinflammatory responses and the molecular mechanisms responsible for the barrier protective effects of AG in vitro and in vivo.
Materials and methods Reagents Andrographolide, 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, USA). Human recombinant HMGB1 was purchased from Abnova (Taipei City, Taiwan). Foetal bovine serum (FBS) and Vybrant DiD were purchased from Invitrogen (Carlsbad, CA, USA). Anti-HMGB1
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neutralizing antibody was purchased from BioLegend (Tokyo, Japan).
Cell culture Primary human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Bio Science (Charles City, IA, USA) and maintained as described previously (Bae & Rezaie 2008, Lee et al. 2012b). Briefly, cells were cultured to confluency at 37 °C and 5% CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science). THP-1 cells, a monocyte cell line, were maintained as previously described (Kim & Bae 2010).
Animals and husbandry Male C57BL/6 mice (6–7 week old, weighting 18–20 g) purchased from Orient Bio (Sungnam, KyungKiDo, Korea) were used in this study 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 hour light/dark cycle. During acclimatization, animals were supplied a normal rodent pellet diet and water ad libitum. All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University.
Cell viability assay Microculture Tetrazolium Test (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 AG. After a 48 h incubation period, cells were washed, and 100 lL of MTT (1 mg mL 1) 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 OD at 540 nm using a microplate reader (Tecan Austria GmbH, Gr€ odig/Salzburg, Austria).
Competitive ELISA (enzyme-linked immunosorbent assay) for HMGB1 Concentration of HMGB1 was determined using the competitive ELISA method as previously described (Lee et al. 2012a). Briefly, 96-well flat plastic microtitre plates (Corning, NY, USA) were coated with HMGB1 protein in 20 mM carbonate–bicarbonate buffer (pH 9.6) containing 0.02% sodium azide, overnight at 4 °C. Plates were then rinsed three times in PBS-0.05% Tween 20 (PBS–T) and kept at 4 °C. Lyophilized culture media were pre-incubated with anti-HMGB1
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antibodies (Abnova, diluted 1 : 1000 in PBS-T) in 96well plastic round microtitre plates for 90 min at 37 °C, and these pre-incubated samples were then transferred to pre-coated plates and incubated for 30 min at room temperature. Plates were then rinsed three times in PBS-T, incubated for 90 min at room temperature with peroxidase-conjugated anti-rabbit IgG antibodies (diluted 1 : 2000 in PBS-T, Amersham Pharmacia Biotech, Uppsala, Sweden), re-rinsed three times with PBS-T and incubated for 60 min at room temperature in the dark with 200 lL substrate solution (100 lg mL 1 o-phenylenediamine and 0.003% H2O2). After stopping the reaction with 50 lL of 8N H2SO4, absorbance was read at 490 nm.
Expression of CAMs and receptors Expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin on HUVECs was determined by wholecell ELISA as previously described (Che et al. 2002, Kim et al. 2012). Briefly, confluent monolayers of HUVECs were treated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG for 6 h. The medium was removed, and cells were washed with PBS and fixed with 50 lL of 1% paraformaldehyde for 15 minutes at room temperature. After washing, 100 lL of mouse antihuman monoclonal antibodies (VCAM-1, ICAM-1, E-selectin, Temecula, CA, USA, 1 : 50 each) was added. After 1 h (37 °C, 5% CO2), the cells were washed three times, followed by addition of 100 lL of 1 : 2000 peroxidase-conjugated anti-mouse IgG antibodies (Sigma) for 1 h. The cells were washed again three times and developed using o-phenylenediamine substrate (Sigma). Colorimetric analysis was performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells. The same experimental procedures were used for monitoring the cell surface expression of TLR2, TLR4 and RAGE receptors using specific antibodies (A-9, H-80 and A-9, respectively) obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
ELISA for phosphorylated p38 mitogen-activated protein kinase (MAPK) Expression of phosphorylated p38 MAPK was quantified according to the manufacturer’s instructions using a commercially available ELISA kit (Cell Signaling Technology, Danvers, MA, USA).
Permeability assay in vitro Endothelial cell permeability in response to increasing concentrations of AG was quantified by spectrophoto178
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metric measurement of the flux of Evans blue-bound albumin across functional cell monolayers using a modified two-compartment chamber model, as previously described (Bae & Rezaie 2011). Human umbilical vein endothelial cells were plated (5 9 104/well) in 3-lm pore size, 12-mm diameter transwells for 3 days. Confluent monolayers of HUVECs were treated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG for 6 h. Transwell inserts were then washed with PBS (pH 7.4), followed by addition of 0.5 mL of Evans blue (0.67 mg mL 1) diluted in growth medium containing 4% BSA. Fresh growth medium was then added to the lower chamber, and the medium in the upper chamber was replaced with Evans blue/BSA. Ten minutes later, optical density was measured at 650 nm in the lower chamber.
Migration assay in vitro Migration assays were performed in 6.5 mm diameter transwell plates containing 8 lm pore size filters. Human umbilical vein endothelial cells (6 9 104) were cultured for 3 days to obtain confluent endothelial monolayers. Before addition of THP-1 cells to the upper compartment, cell monolayers were treated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG for 6 h. Transwell plates were then incubated at 37 °C, 5% CO2 for 2 h. Cells in the upper chamber were then aspirated, and non-migrating cells on top of the filter were removed using a cotton swab. THP-1 cells 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 twice per well, and for duplicate wells, nine randomly selected high power microscopic fields (HPF, 200 9 ) were counted and results are defined as migration indices.
In vivo permeability and leucocyte migration assays For in vivo study, male mice were anesthetized with zoletil (tiletamine and zolazepam, 1 : 1 mixture, 30 mg kg 1) and rompun (xylazine, 10 mg kg 1). CLPoperated mice or mice were pre-treated with HMGB1 (2 lg per mouse) intravenously, and after 16 h, AG (3.5 or 7.0 lg per mouse) was injected intravenously. After 6 h, 1% Evans blue dye solution in normal saline was injected intravenously into each mouse. Thirty minutes later, the mice were sacrificed and the peritoneal exudates were collected after being washed with 5 mL of normal saline and centrifuged at 200 g for 10 min. The absorbance of the supernatant was read at 650 nm. The vascular permeability was expressed in terms of dye (lg per mouse), which leaked into the peritoneal cavity according to a standard curve of Evans blue dye, as previously described (Lee et al. 2009, Bae et al. 2012).
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For assessment of leucocyte and neutrophils migration, CLP-operated mice or mice were treated with HMGB1 (2 lg per mouse, i.v.) in normal saline for 16 h followed by treatment with AG (3.5 or 7.0 lg per mouse, i.v.) for 6 h. The mice were then sacrificed, and the peritoneal cavities were washed with 5 mL of normal saline. Twenty microlitre of peritoneal fluid was mixed with 0.38 mL of Turk’s solution (0.01% crystal violet in 3% acetic acid), and the number of leucocytes was counted under a light microscope. For neutrophil migration to the peritoneal cavity, total counts were performed with a cell counter (Coulter AC T series analyzer; Coulter Corporation, Miami, Florida, USA), and differential cell counts were carried out on cytocentrifuge slides (Cytospin 3; Shandon Southern Products, Astmoore, UK) stained by the May–Gr€ umwald–Giemsa (Rosenfeld) method (Valerio et al. 2007). The results were expressed as neutrophils 9 106 per peritoneal cavity.
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Poly-L-Lysine in complete media containing 10% FBS and maintained for 48 h. Cells were then stimulated with LPS (100 ng mL 1) or HMGB1 (1 lg mL 1) for 1 h with or without treatment with AG (5 or 10 lM) for 1 h. For cytoskeletal staining, cells were fixed in 4% formaldehyde in PBS (v/v) for 15 min at room temperature, and for immunostaining, cells were permeabilized in 0.05% Triton X-100 in PBS for 15 min and blocked in blocking buffer (5% BSA in PBS) overnight at 4 °C. Cells were incubated with primary rabbit monoclonal NF-jB p65 antibody, anti-rabbit alexa 488, and Factin-labelled fluorescein phalloidin (F 432; Molecular Probes, Invitrogen) overnight at 4 °C. Nuclei were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), and cells were visualized by confocal microscopy at a 63 9 magnification (TCS-Sp5, Leica Microsystem, Wetzlar, Germany).
Caecal ligation and puncture (CLP) Cell–Cell adhesion assay THP-1 cell adherence to HUVECs was evaluated by fluorescent labelling of THP-1 cells as previously described (Akeson & Woods 1993). Briefly, THP-1 cells (1.5 9 106 per mL, 200 lL per well) were labelled with Vybrant DiD dye and then added to washed and stimulated HUVECs. Human umbilical vein endothelial cell monolayers were treated with HMGB1 (1 lg mL 1) for 16 h, followed by treatment with AG for 6 h. THP-1 cells were allowed to adhere, and non-adherent THP-1 cells were removed by washing. Adherent THP-1 cell percentages were calculated using the formula: % adherence = (adherent signal/ total signal) 9 100. Results are expressed as the mean of at least three independent experiments.
ELISA of NF-jB, ERK1/2, tumour necrosis factor (TNF)a and interleukin-1b (IL-1b), IL-6 and keratinocytederived protein (KC, homolog to human IL-8) Total and phosphorylated p65 NF-jB (#7174, #7173, Cell Signaling Technology), total and phosphorylated Akt (#7170, #7252 Cell Signaling Technology), and total and phosphorylated ERK1/2 (R&D Systems, Minneapolis, MN, USA) activities in nuclear lysates were determined using ELISA kits. The concentrations of TNF-a in cell culture supernatants and IL1b, IL-6 and KC in mouse plasma were determined using ELISA kits (R&D Systems). Values were measured using an ELISA plate reader (Tecan Austria GmbH).
Immunofluorescence staining Human umbilical vein endothelial cells were grown to confluence on glass cover slips coated with 0.05%
For induction of sepsis, male mice were anesthetized with zoletil (tiletamine and zolazepam, 1 : 1 mixture, 30 mg kg 1) and rompun (xylazine, 10 mg kg 1). The CLP-induced sepsis model was prepared as described previously (Wang et al. 2004). In brief, a 2 cm midline incision was placed to allow exposure of the caecum and adjoining intestine. The caecum was then tightly ligated using a 3.0-silk suture at 5.0 mm from the caecal tip and punctured once using a 22-gauge needle. The caecum was then gently squeezed to extrude a small amount of faeces from perforation sites and returned to the peritoneal cavity. The laparotomy site was then sutured with 4.0 silk. In sham control animals, the caecum was exposed but not ligated or punctured and then returned to the abdominal cavity. This protocol was approved in advance by the Animal Care Committee at Kyungpook National University.
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 less than 0.05 (P < 0.05) were considered significant.
Results and discussion In this study, the effects of andrographolide (AG, Fig. 1) on the release of HMGB1 and the HMGB1mediated vascular barrier disruptive response were determined in vitro and in vivo. Emodin-6-O-b-D-glucoside (EG) was used as a positive control (Lee et al. 2013).
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Figure 1 Structure of Andropholide (AG).
Effect of AG on LPS and CLP-mediated release of HMGB1 Previous studies have demonstrated that LPS stimulates release of HMGB1 in murine macrophages and human endothelial cells (Mullins et al. 2004, El Gazzar 2007, Bae & Rezaie 2011). In agreement with the previous results, LPS (100 ng mL 1) stimulated the release of HMGB1 by HUVECs (Fig. 2a). To investigate the effects of AG on LPS-mediated release of HMGB1, endothelial cells were stimulated with 100 ng mL 1 LPS for 16 h before treatment of cells with increasing concentrations of AG for 6 h. Emodin-6-O-D-b-glucoside (10 lM, EG) was used as a positive control (Lee et al. 2013). The results shown in Figure 2a indicate that AG inhibits release of HMGB1 by LPS in endothelial cells, with the optimal effect occurring at a concentration above 10 lM AG. However, treatment with AG alone did not affect HMGB1 release (Figure 2a). To confirm this effect in vivo, we next subjected mice to severe sepsis in a standardized model of caecal ligation and puncture (CLP) as this model closely resembles human sepsis (Buras et al. 2005). As shown in Figure 2b, AG induced marked inhibition of CLP-induced release of HMGB1 in mice. Because the average weight of a mouse is 20 g and the average blood volume is 2 mL, the injected AG (3.5 or 7 lg per mouse) produced a concentration maximum of 5 or 10 lM in peripheral blood. Next, we investigated the effects of AG on expression of the HMGB1 receptors, TLR2, TLR4 and RAGE in HUVECs. As shown in Figure 2c, HMGB1 induced 2.4-fold expression of TLR-2, TLR4 and RAGE in HUVECs, and treatment with AG resulted in marked inhibition of receptor expression. In addition, synthesized HMGB1 by LPS enhanced the expressions of HMGB1 receptors, and anti-HMGB1 180
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antibody inhibited their expressions (Fig. 2d). To exclude the possibility that inhibition of HMGB1 release was due to cytotoxicity caused by AG, cellular viability assays were performed in HUVECs treated with AG for 24 h. At the concentrations used (up to 20 lM), AG did not affect cell viability (Fig. 2e). High plasma concentrations of HMGB1 in patients with inflammatory diseases are known to be related to poor prognosis and high mortality. In addition, the pharmacological inhibition of HMGB1 is known to result in improved survival in animal models of acute inflammation in response to endotoxin challenge (Sama et al. 2004). Therefore, the prevention of LPS or CLP-induced release of HMGB1 by AG suggests that AG might be used for the treatment of vascular inflammatory diseases.
Effect of AG on LPS or HMGB1-mediated barrier disruption A permeability assay was performed for determination of the effects of AG on the barrier integrity of HUVECs. Treatment with AG (10 lM) alone did not result in the alteration of barrier integrity (Fig. 3a). On the other hand, LPS is known to induce cleavage and disruption of endothelial membrane barriers (Berman et al. 1993, Goldblum et al. 1993). Human umbilical vein endothelial cells were treated with various concentrations of AG for 6 h after addition of LPS (100 ng mL 1). As shown in Figure 3a, AG induced dose-dependent downregulation of LPS-mediated membrane disruption. HMGB1 is also known to induce cleavage and disruption of barrier integrity (Yang et al. 2005, Wolfson et al. 2011). Therefore, HUVECs were treated with various concentrations of AG for 6 h after addition of HMGB1 (1 lg mL 1). As shown in Figure 3b, treatment with AG resulted in decreased HMGB1-mediated membrane disruption in a dose-dependent manner. To confirm this effect in vivo, HMGB1- or CLP-induced vascular permeability in mice was evaluated. As shown in Figure 3c, AG induced marked inhibition of the peritoneal leakage of dye induced by HMGB1 or CLP. HMGB1 is known to induce pro-inflammatory responses by promoting phosphorylation of p38 MAPK (Qin et al. 2009, Sun et al. 2009). To determine whether AG inhibits HMGB1-induced activation of p38 MAPK in HUVECs, cells were activated with HMGB1 and then incubated with AG, followed by determination of phosphorylated p38 MAPK levels. As shown in Figure 3d, HMGB1 induced upregulated expression of phosphorylated p38, which was significantly inhibited by treatment with AG. These findings demonstrate inhibition of HMGB1-mediated endothelial disruption and maintenance of human endothelial
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Figure 2 Effects of AG on HMGB1 release and receptor expression. (a) Human umbilical vein endothelial cells (HUVECs) were treated with the indicated concentrations of AG or emodin-6-O-D-b-glucoside (10 lM, EG) for 6 h after stimulation with LPS 100 ng mL 1 for 16 h; HMGB1 release was then measured by ELISA. (b) Male C57BL/6 mice underwent CLP and were administered AG or EG (9.0 lg per mouse, i.v.) intravenously at 12 h after CLP (n = 5). Mice were killed 24 h after CLP. Serum HMGB1 levels were measured by ELISA. (c) Confluent HUVECs were incubated with HMGB1 (1 lg mL 1) for 16 h; cells were then treated with or without AG or 10 lM EG for 6 h. Expression of receptors, such as TLR-2 (white bar), TLR-4 (grey bar) and RAGE (black bar), on HUVECs was measured by cell-based ELISA. (d) The same as (c) except that cells were incubated with PBS (white bar), LPS (100 ng mL 1, grey bar) for 16 h with or without anti-HMGB1 neutralizing antibody (black bar). (e) The effects of AG on cellular viability were measured using MTT assays. Results are expressed as the mean SEM of three independent experiments. *P < 0.05 vs. LPS alone (a, d), CLP (b) or HMGB1 alone (c).
cell barrier integrity by AG in mice treated with HMGB1. Cytoskeletal proteins are important for the maintenance of cell integrity and shape (Schnittler et al. 2001). In addition, redistribution of the actin cytoskeleton, detachment of cells and loss of cell–cell contact due to cytokine stimulation are all associated with increased endothelial monolayer permeability (Friedl et al. 2002, Petrache et al. 2003). Therefore, we next examined the effects of AG on HUVEC actin cytoskeletal arrangement by immunofluorescence staining of HUVEC monolayers with F-actin labelled fluorescein phalloidin. Control HUVECs exhibited a random distribution of F-actin throughout cells with some localization of actin filament bundles at cell boundaries (Fig. 3e). Barrier disruption by HMGB1 (1 lg mL 1) was manifested by the formation of
paracellular gaps (shown by arrows) in HUVECs. A similar cytoskeletal arrangement was induced by LPS (100 ng mL 1) (data not shown). In addition, treatment with AG (10 lM) resulted in inhibited formation of HMGB1-induced paracellular gaps with formation of dense F-actin rings (Fig. 3e). These results suggested that treatment with AG led to maintenance of HMGB1-mediated morphological changes and gap formation of endothelial cells associated with F-actin redistribution, thereby increasing vascular barrier integrity.
AG inhibited expression of CAMs and proinflammatory responses Previous studies have demonstrated that HMGB1 mediates inflammatory responses by increasing the cell
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Figure 3 Effects of AG on HMGB1-mediated permeability in vitro and in vivo. (a, b) Human umbilical vein endothelial cells (HUVECs) were stimulated with LPS (a, 100 ng mL 1 for 4 h) or HMGB1 (b, 1 lg mL 1, 16 h) and then treated with different concentrations of AG or 10 lM EG for 6 h. Then, permeability was monitored by measuring the flux of Evans blue-bound albumin across HUVECs. (c) The effects of AG or EG (9.0 lg per mouse, i.v.) on HMGB1- (2 lg per mouse, i.v., white bar) or CLP- (at 24 h after CLP, black bar) induced vascular permeability in mice were examined by measuring the amount of Evans blue in peritoneal washings (expressed lg per mouse, n = 5). (d) HUVECs were activated with HMGB1 and were then treated with AG or 10 lM EG for 6 h. The effects of AG on HMGB1-mediated expression of phospho-p38 were measured by ELISA. (e) Staining for F-actin. HUVEC monolayers grown on glass coverslips were stimulated with HMGB1 for 1 h, followed by treatment with AG for 6 h, and immunofluorescence staining for F-actin. Arrows indicate intercellular gaps. Results are expressed as the mean SEM of three independent experiments. *P < 0.05 vs. LPS (a), HMGB1 (b, c, d) or CLP (d).
surface expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin on the surfaces of endothelial cells, thereby promoting adhesion and migration of leucocytes across endothelium to sites of inflammation (Andersson et al. 2000). To determine the effects of AG on expression of CAMs in HMGB1stimulated endothelial cells, we monitored expression of VCAM-1, ICAM-1 and E-selectin on HMGB1-stimulated AG pre-treated HUVECs. As shown in Figure 4a, AG suppressed the expression of VCAM-1, ICAM-1 and E-selectin. Adhesion of leucocytes to endothelial cells and transendothelial migration (TEM) of leucocytes are important steps in the proinflammatory response (Hansson & Libby 2006). Therefore, we attempted to determine whether expression of CAMs corresponded to enhanced leucocyte binding, and whether AG could block adhesion of monocytes to 182
HMGB1-stimulated HUVECs. We found that AG effectively inhibited binding of leucocytes to HMGB1stimulated HUVECs (Fig. 4b). Further studies revealed an association of leucocyte to HUVEC binding with the subsequent TEM of leucocytes and that AG effectively inhibited this step (Fig. 4c). To confirm this effect in vivo, HMGB1- or CLP-induced leucocytes and neutrophils migration was examined in mice. HMGB1 and CLP-induced significant stimulation of leucocytes and neutrophils migration into the peritoneal cavities of mice, and AG at doses of 5–10 lM significantly inhibited this migration (Fig. 4d and e). These results indicate that AG not only inhibits the endotoxin-mediated release of HMGB1 by endothelial cells, but that it also downregulates the pro-inflammatory signalling effect of released HMGB1 and thereby inhibits the amplification of inflammatory pathways
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Figure 4 Effect of AG on HMGB1mediated pro-inflammatory responses. Human umbilical vein endothelial cells (HUVECs) were stimulated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG or 10 lM EG for 6 h. HMGB1-mediated (a) expression of VCAM-1 (white box), ICAM-1 (grey box), and E-selectin (black box) in HUVECs, (b) adherence of monocytes to HUVEC monolayers, and (c) migration of monocytes through HUVEC monolayers were analysed. (d, e) Male C57BL/6 mice were stimulated with HMGB1 (2 lg per mouse, i.v., white bar) or CLP-operated mice (at 24 h after CLP, black bar) were treated with AG or EG (9 lg per mouse, i.v.). HMGB1- or CLPmediated migration of leucocytes (d) and neutrophils (e) into the peritoneal cavity of mice was analysed. Results are expressed as the mean SEM of three independent experiments. *P < 0.05 vs. HMGB1 (a–d) or CLP (d, e).
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such as the upregulation of CAMs, leucocytes adhesion and migration by HMGB1.
AG inhibited HMGB1-stimulated activation of NF-jB/ ERK/Akt and production of TNF-a and IL-1b HMGB1 and LPS both induced a significant increase in nuclear translocation of NF-jB and phosphorylation of Akt and p38 MAPK in human endothelial cells (Kawahara et al. 2008, Dagia et al. 2010, Andersson & Tracey 2011, Wang et al. 2013), and activation of NF-jB and ERK1/2 is required for pro-inflammatory responses (Marui et al. 1993, Lockyer et al. 1998, Rose et al. 2010). Previous studies have reported activation of NF-jB and ERK 1/2 by HMGB1 in vascular inflammatory responses (Park et al. 2006, Palumbo et al. 2007, Yang & Tracey 2010). Therefore, to investigate the potential effects of AG on activation of inflammatory signalling molecules and production of TNF-a and IL-1b in HMGB1-activated HUVECs, cells were activated with HMGB1 for 16 h, followed by incubation with AG for 6 h. Data showed that phosphorylation of NF-jB (Fig. 5a, g), ERK1/2 (Fig. 5b), and Akt (Fig. 5c), and production of TNF-a (Fig. 5d)
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and IL-1b (Fig. 5e) were increased by HMGB1, and these increases were significantly reduced by AG (Fig. 5). These results indicate that AG may regulate the most important signals involved in induction of proinflammatory responses in human endothelial cells.
Protective effect of AG in CLP-induced production of IL6/KC and septic mortality Sepsis, a systemic response to serious infection, has a poor prognosis when it is associated with organ dysfunction, hypoperfusion or hypotension (Cohen 2002, Bae 2012). Production of interleukin (IL) 6 and 8 is required for pro-inflammatory responses and important markers of mortality (Teiten et al. 2010). Based on the above-mentioned findings, we hypothesized that treatment with AG would result in reduced production of IL-6/KC and mortality in our CLP-induced sepsis mouse model. As shown in Figure 6a and b, production of IL-6 and KC in CLP-induced mice was reduced by treatment with AG. This result was consistent with the previous finding that the IL-6 levels were increased prior to death (Xiao et al. 2006). To determine whether AG protects mice from CLP-induced
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sepsis lethality, AG was administered to mice after CLP. Twenty-four hours after the operation, animals manifested signs of sepsis, including shivering, bristled hair and weakness. Administration of AG at dual dose (3.5 or 7.0 lg per mouse, 12 h after CLP) did not prevent CLP-induced death (data not shown); therefore, AG was administered two times (once 12 h after CLP and once 50 h after CLP), resulting in an increase in the survival rate from 30 to 60% according to Kaplan–Meier survival analysis (P < 0.0001, Fig. 6c). However, no beneficial effect was observed for a lower dose of AG (data not shown). This marked survival benefit achieved by administration of AG suggests that suppression of HMGB1 release and HMGB1-mediated inflammatory responses provides a therapeutic strategy for the management of sepsis and septic shock. Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. A wide array of pro-inflammatory cytokines including TNF-a, IL-1, IFN-c and macrophage migration inhibitory factor (MIF) individually or in combination contributes to the pathogenesis of lethal systemic inflammation (Bhatia et al. 2009). For instance, neutralizing antibodies against TNF (Tracey et al. 1987) reduce lethality in an 184
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Figure 5 Effects of AG on HMGB1stimulated activation of NF-jB/ERK/ AKT and production of TNF-a/IL-1b in human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were stimulated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG or 10 lM EG for 6 h. (a–c) HMGB1 (1 lg mL 1)-mediated activation of phospho- total NF-jB p65 (a), phospho- total ERK1/2 (b) or phospho- or total Akt (c) in HUVECs was analysed. White bar, phosphor form; black bar, total form. HMGB1 (1 lg mL 1)-mediated production of TNF-a (d) and IL-1b (e) in HUVECs was analysed. (f) Immunofluorescence microscopy analysis of the nuclear translocation of p65 in HUVECs. Human umbilical vein endothelial cells were stimulated (or not) for 1 h with 1 lg mL 1 HMGB1 and treated or not with 5 or 10 lM AG for 6 h. The subcellular localization of p65 was examined by IF staining. Results are expressed as the mean SEM of three independent experiments. *P < 0.05 vs. HMGB1.
animal model of endotoxaemic/bacteraemic shock. However, the early kinetics of systemic TNF accumulation makes it difficult to target in clinical setting (Tracey et al. 1987), prompting the investigation of other late proinflammatory mediators (such as HMGB1) as potential therapeutic target for inflammatory diseases. The prevailing theories of sepsis as a dys-regulated systemic inflammatory response are supported by extensive studies employing various animal models of sepsis, including endotoxaemia and peritonitis induced by caecal ligation and puncture (CLP) (Wichterman et al. 1980). In murine models of endotoxaemia and sepsis, HMGB1 is first detectable in the circulation eight hours after the onset of diseases, subsequently increasing to plateau levels from 16 to 32 h (Wang et al. 2001). This late appearance of circulating HMGB1 precedes and parallels with the onset of animal lethality from endotoxaemia or sepsis and distinguishes itself from TNF and other early proinflammatory cytokines (Wang et al. 2001). Therefore, in this study, AG was treated at 16 h after inflammatory challenge. In summary, our results demonstrate that AG inhibits both LPS and CLP-mediated release of HMGB1, expression of HMGB1 receptor (TLR2, TLR4 and RAGE) and HMGB1-mediated barrier disruption
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(b)
(c)
Figure 6 Effects of AG on production of IL-6/KC and lethality after CLP. (a, b) Male C57BL/6 mice underwent CLP and were administered AG or EG (9.0 lg per mouse, i.v.) intravenously at 12 h and 50 h after CLP (n = 5). Mice were killed 96 h after CLP. Serum IL-6 (a) or KC (b) levels were measured by ELISA. (c) Male C57BL/6 mice (n = 10) were administered i.v. AG (3.5 lg per mouse, □) or AG (7.0 lg per mouse, ■) at 12 and 50 h after CLP. Animal survival was monitored every 6 h after CLP for 126 h. Control CLP mice (●) and sham-operated mice (○) were administered sterile saline (n = 10). A Kaplan–Meier survival analysis was used for determination of overall survival rates vs. CLP-treated mice. *P < 0.05 vs. CLP.
through increases in barrier integrity and inhibition of CAM expression. In addition, AG reduces adhesion and migration of monocytes towards HUVECs. These barrier protective effects of AG were confirmed in a mouse model, in which treatment with AG resulted in reduction in HMGB1-induced mortality. Our findings indicate that AG may be regarded as a candidate for use in the treatment of severe vascular inflammatory diseases, such as septic shock.
Conflict of interest The authors declare no conflict of interests. This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government [MSIP] (Grant No. 2012-000940).
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Andrographolide inhibits HMGB1-induced inflammatory responses in human umbilical vein endothelial cells and in murine polymicrobial sepsis W. Lee,1,2* S. Ku,3* H. Yoo,1 K. Song1 and J. Bae1 1 College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea 2 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu, Korea 3 Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan, Korea
Received 7 October 2013, revision requested 6 December 2013, revision received 21 December 2013, accepted 24 February 2014 Correspondence: J. Bae, College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702701, Korea. E-mail: [email protected]
*First two authors contributed equally to this work.
Abstract Aim: Nuclear DNA-binding protein high-mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions, such as septic shock, upregulating pro-inflammatory cytokines. Andrographolide (AG) is isolated from the plant of Andrographis paniculata and used as a folk medicine for treatment of viral infection, diarrhoea, dysentery and fever. However, the effect of AG on HMGB1-induced inflammatory response has not been studied. Methods: Firstly, we accessed this question by monitoring the effects of post-treatment AG on lipopolysaccharide (LPS) and caecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Results: Post-treatment AG was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. AG also inhibited HMGB1-mediated hyperpermeability and leucocyte migration in septic mice. In addition, AG inhibited production of tumour necrosis factor-a (TNF-a) and activation of AKT, nuclear factor-jB (NF-jB) and extracellular-regulated kinases (ERK) 1/2 by HMGB1 in HUVECs. AG also induced downregulation of CLP-induced release of HMGB1, production of interleukin (IL) 1b/6/8 and mortality. Conclusion: Collectively, these results suggest that AG may be regarded as a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signalling pathway. Keywords Andrographolide, HMGB1, sepsis, vascular inflammation.
High-mobility group box chromosomal protein 1 (HMGB1) is a highly conserved ubiquitously expressed nuclear protein involved in nucleosome stabilization and gene transcription (Lotze & Tracey 2005). HMGB1 can be actively released by immune cells (activated monocytes, macrophages, neutrophils and platelets) into the extracellular environment (Degryse et al. 2001, Bianchi & Manfredi 2004, Ito et al. 2007). Extracellular HMGB1 secretes inflammatory stimulation function as a pro-inflammatory cytokine 176
from damaged and necrotic cells and triggers proinflammatory responses from macrophages and endothelial cells (Andersson & Tracey 2011). HMGB1 binds to several transmembrane receptors, such as receptor for advanced glycation end products (RAGE) and toll-like receptors (TLR)-2 and -4, and activates NF-jB and extracellular-regulated kinase (ERK) 1 and 2 (Hori et al. 1995, Park et al. 2004). Activation of endothelial cells by HMGB-1 leads to expression of cell adhesion molecules (CAMs), such as intercellular
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adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and E-selectin, which induces upregulated inflammation through recruitment of leucocytes (Bae & Rezaie 2011). Accumulation of HMGB1 occurs during sepsis, leading to multiple organ collapse and a lethal outcome (Yang et al. 2004). Therefore, it is a therapeutic target for clinical management of lethal systemic inflammatory diseases. Herbal medicines derived from plant extracts are increasingly utilized for the treatment of a wide variety of diseases. Recently, post-treatment of herbal extract could reduce the inflammatory responses (Park et al. 2012, Persson & Persson 2012). Andrographis paniculata, a popular herb, has been widely used in China as a folk medicine and is cultivated in various Asian countries. Andrographolide (AG) has numerous reported pharmacological properties and has been used extensively in the clinical setting for the treatment of infections, inflammation, colds, fever and diarrhoea. The anti-inflammatory activities of A. paniculata have been thoroughly studied (Chiou et al. 2000, Chao et al. 2009). In addition, andrographolide (AG), extracted and purified from A. paniculata, is currently prescribed for treatment of inflammationrelated diseases, such as laryngitis, upper respiratory tract infection and rheumatoid arthritis in China (Puri et al. 1993, Amroyan et al. 1999, Shen et al. 2000, 2002, Batkhuu et al. 2002). Studies on the biological effects of AG have demonstrated that AG decreases TNF-a-induced expression of ICAM-1 and monocyte adhesion in human endothelial cells (Habtemariam 1998, Yu et al. 2010). Treatment with AG resulted in reduced TNF-a-induced expression of ICAM-1 in HUVECs via the PI3K/Akt pathway and downstream target NF-jB activation (Chen et al. 2011). However, the effects of AG in HMGB1-mediated pro-inflammatory responses in HUVECs have not yet been studied. In this study, we described for the first time the effects of AG on HMGB1 release, HMGB1-mediated proinflammatory responses and the molecular mechanisms responsible for the barrier protective effects of AG in vitro and in vivo.
Materials and methods Reagents Andrographolide, 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, USA). Human recombinant HMGB1 was purchased from Abnova (Taipei City, Taiwan). Foetal bovine serum (FBS) and Vybrant DiD were purchased from Invitrogen (Carlsbad, CA, USA). Anti-HMGB1
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neutralizing antibody was purchased from BioLegend (Tokyo, Japan).
Cell culture Primary human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Bio Science (Charles City, IA, USA) and maintained as described previously (Bae & Rezaie 2008, Lee et al. 2012b). Briefly, cells were cultured to confluency at 37 °C and 5% CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science). THP-1 cells, a monocyte cell line, were maintained as previously described (Kim & Bae 2010).
Animals and husbandry Male C57BL/6 mice (6–7 week old, weighting 18–20 g) purchased from Orient Bio (Sungnam, KyungKiDo, Korea) were used in this study 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 hour light/dark cycle. During acclimatization, animals were supplied a normal rodent pellet diet and water ad libitum. All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University.
Cell viability assay Microculture Tetrazolium Test (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 AG. After a 48 h incubation period, cells were washed, and 100 lL of MTT (1 mg mL 1) 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 OD at 540 nm using a microplate reader (Tecan Austria GmbH, Gr€ odig/Salzburg, Austria).
Competitive ELISA (enzyme-linked immunosorbent assay) for HMGB1 Concentration of HMGB1 was determined using the competitive ELISA method as previously described (Lee et al. 2012a). Briefly, 96-well flat plastic microtitre plates (Corning, NY, USA) were coated with HMGB1 protein in 20 mM carbonate–bicarbonate buffer (pH 9.6) containing 0.02% sodium azide, overnight at 4 °C. Plates were then rinsed three times in PBS-0.05% Tween 20 (PBS–T) and kept at 4 °C. Lyophilized culture media were pre-incubated with anti-HMGB1
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antibodies (Abnova, diluted 1 : 1000 in PBS-T) in 96well plastic round microtitre plates for 90 min at 37 °C, and these pre-incubated samples were then transferred to pre-coated plates and incubated for 30 min at room temperature. Plates were then rinsed three times in PBS-T, incubated for 90 min at room temperature with peroxidase-conjugated anti-rabbit IgG antibodies (diluted 1 : 2000 in PBS-T, Amersham Pharmacia Biotech, Uppsala, Sweden), re-rinsed three times with PBS-T and incubated for 60 min at room temperature in the dark with 200 lL substrate solution (100 lg mL 1 o-phenylenediamine and 0.003% H2O2). After stopping the reaction with 50 lL of 8N H2SO4, absorbance was read at 490 nm.
Expression of CAMs and receptors Expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin on HUVECs was determined by wholecell ELISA as previously described (Che et al. 2002, Kim et al. 2012). Briefly, confluent monolayers of HUVECs were treated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG for 6 h. The medium was removed, and cells were washed with PBS and fixed with 50 lL of 1% paraformaldehyde for 15 minutes at room temperature. After washing, 100 lL of mouse antihuman monoclonal antibodies (VCAM-1, ICAM-1, E-selectin, Temecula, CA, USA, 1 : 50 each) was added. After 1 h (37 °C, 5% CO2), the cells were washed three times, followed by addition of 100 lL of 1 : 2000 peroxidase-conjugated anti-mouse IgG antibodies (Sigma) for 1 h. The cells were washed again three times and developed using o-phenylenediamine substrate (Sigma). Colorimetric analysis was performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells. The same experimental procedures were used for monitoring the cell surface expression of TLR2, TLR4 and RAGE receptors using specific antibodies (A-9, H-80 and A-9, respectively) obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
ELISA for phosphorylated p38 mitogen-activated protein kinase (MAPK) Expression of phosphorylated p38 MAPK was quantified according to the manufacturer’s instructions using a commercially available ELISA kit (Cell Signaling Technology, Danvers, MA, USA).
Permeability assay in vitro Endothelial cell permeability in response to increasing concentrations of AG was quantified by spectrophoto178
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metric measurement of the flux of Evans blue-bound albumin across functional cell monolayers using a modified two-compartment chamber model, as previously described (Bae & Rezaie 2011). Human umbilical vein endothelial cells were plated (5 9 104/well) in 3-lm pore size, 12-mm diameter transwells for 3 days. Confluent monolayers of HUVECs were treated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG for 6 h. Transwell inserts were then washed with PBS (pH 7.4), followed by addition of 0.5 mL of Evans blue (0.67 mg mL 1) diluted in growth medium containing 4% BSA. Fresh growth medium was then added to the lower chamber, and the medium in the upper chamber was replaced with Evans blue/BSA. Ten minutes later, optical density was measured at 650 nm in the lower chamber.
Migration assay in vitro Migration assays were performed in 6.5 mm diameter transwell plates containing 8 lm pore size filters. Human umbilical vein endothelial cells (6 9 104) were cultured for 3 days to obtain confluent endothelial monolayers. Before addition of THP-1 cells to the upper compartment, cell monolayers were treated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG for 6 h. Transwell plates were then incubated at 37 °C, 5% CO2 for 2 h. Cells in the upper chamber were then aspirated, and non-migrating cells on top of the filter were removed using a cotton swab. THP-1 cells 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 twice per well, and for duplicate wells, nine randomly selected high power microscopic fields (HPF, 200 9 ) were counted and results are defined as migration indices.
In vivo permeability and leucocyte migration assays For in vivo study, male mice were anesthetized with zoletil (tiletamine and zolazepam, 1 : 1 mixture, 30 mg kg 1) and rompun (xylazine, 10 mg kg 1). CLPoperated mice or mice were pre-treated with HMGB1 (2 lg per mouse) intravenously, and after 16 h, AG (3.5 or 7.0 lg per mouse) was injected intravenously. After 6 h, 1% Evans blue dye solution in normal saline was injected intravenously into each mouse. Thirty minutes later, the mice were sacrificed and the peritoneal exudates were collected after being washed with 5 mL of normal saline and centrifuged at 200 g for 10 min. The absorbance of the supernatant was read at 650 nm. The vascular permeability was expressed in terms of dye (lg per mouse), which leaked into the peritoneal cavity according to a standard curve of Evans blue dye, as previously described (Lee et al. 2009, Bae et al. 2012).
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For assessment of leucocyte and neutrophils migration, CLP-operated mice or mice were treated with HMGB1 (2 lg per mouse, i.v.) in normal saline for 16 h followed by treatment with AG (3.5 or 7.0 lg per mouse, i.v.) for 6 h. The mice were then sacrificed, and the peritoneal cavities were washed with 5 mL of normal saline. Twenty microlitre of peritoneal fluid was mixed with 0.38 mL of Turk’s solution (0.01% crystal violet in 3% acetic acid), and the number of leucocytes was counted under a light microscope. For neutrophil migration to the peritoneal cavity, total counts were performed with a cell counter (Coulter AC T series analyzer; Coulter Corporation, Miami, Florida, USA), and differential cell counts were carried out on cytocentrifuge slides (Cytospin 3; Shandon Southern Products, Astmoore, UK) stained by the May–Gr€ umwald–Giemsa (Rosenfeld) method (Valerio et al. 2007). The results were expressed as neutrophils 9 106 per peritoneal cavity.
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Poly-L-Lysine in complete media containing 10% FBS and maintained for 48 h. Cells were then stimulated with LPS (100 ng mL 1) or HMGB1 (1 lg mL 1) for 1 h with or without treatment with AG (5 or 10 lM) for 1 h. For cytoskeletal staining, cells were fixed in 4% formaldehyde in PBS (v/v) for 15 min at room temperature, and for immunostaining, cells were permeabilized in 0.05% Triton X-100 in PBS for 15 min and blocked in blocking buffer (5% BSA in PBS) overnight at 4 °C. Cells were incubated with primary rabbit monoclonal NF-jB p65 antibody, anti-rabbit alexa 488, and Factin-labelled fluorescein phalloidin (F 432; Molecular Probes, Invitrogen) overnight at 4 °C. Nuclei were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), and cells were visualized by confocal microscopy at a 63 9 magnification (TCS-Sp5, Leica Microsystem, Wetzlar, Germany).
Caecal ligation and puncture (CLP) Cell–Cell adhesion assay THP-1 cell adherence to HUVECs was evaluated by fluorescent labelling of THP-1 cells as previously described (Akeson & Woods 1993). Briefly, THP-1 cells (1.5 9 106 per mL, 200 lL per well) were labelled with Vybrant DiD dye and then added to washed and stimulated HUVECs. Human umbilical vein endothelial cell monolayers were treated with HMGB1 (1 lg mL 1) for 16 h, followed by treatment with AG for 6 h. THP-1 cells were allowed to adhere, and non-adherent THP-1 cells were removed by washing. Adherent THP-1 cell percentages were calculated using the formula: % adherence = (adherent signal/ total signal) 9 100. Results are expressed as the mean of at least three independent experiments.
ELISA of NF-jB, ERK1/2, tumour necrosis factor (TNF)a and interleukin-1b (IL-1b), IL-6 and keratinocytederived protein (KC, homolog to human IL-8) Total and phosphorylated p65 NF-jB (#7174, #7173, Cell Signaling Technology), total and phosphorylated Akt (#7170, #7252 Cell Signaling Technology), and total and phosphorylated ERK1/2 (R&D Systems, Minneapolis, MN, USA) activities in nuclear lysates were determined using ELISA kits. The concentrations of TNF-a in cell culture supernatants and IL1b, IL-6 and KC in mouse plasma were determined using ELISA kits (R&D Systems). Values were measured using an ELISA plate reader (Tecan Austria GmbH).
Immunofluorescence staining Human umbilical vein endothelial cells were grown to confluence on glass cover slips coated with 0.05%
For induction of sepsis, male mice were anesthetized with zoletil (tiletamine and zolazepam, 1 : 1 mixture, 30 mg kg 1) and rompun (xylazine, 10 mg kg 1). The CLP-induced sepsis model was prepared as described previously (Wang et al. 2004). In brief, a 2 cm midline incision was placed to allow exposure of the caecum and adjoining intestine. The caecum was then tightly ligated using a 3.0-silk suture at 5.0 mm from the caecal tip and punctured once using a 22-gauge needle. The caecum was then gently squeezed to extrude a small amount of faeces from perforation sites and returned to the peritoneal cavity. The laparotomy site was then sutured with 4.0 silk. In sham control animals, the caecum was exposed but not ligated or punctured and then returned to the abdominal cavity. This protocol was approved in advance by the Animal Care Committee at Kyungpook National University.
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 less than 0.05 (P < 0.05) were considered significant.
Results and discussion In this study, the effects of andrographolide (AG, Fig. 1) on the release of HMGB1 and the HMGB1mediated vascular barrier disruptive response were determined in vitro and in vivo. Emodin-6-O-b-D-glucoside (EG) was used as a positive control (Lee et al. 2013).
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Figure 1 Structure of Andropholide (AG).
Effect of AG on LPS and CLP-mediated release of HMGB1 Previous studies have demonstrated that LPS stimulates release of HMGB1 in murine macrophages and human endothelial cells (Mullins et al. 2004, El Gazzar 2007, Bae & Rezaie 2011). In agreement with the previous results, LPS (100 ng mL 1) stimulated the release of HMGB1 by HUVECs (Fig. 2a). To investigate the effects of AG on LPS-mediated release of HMGB1, endothelial cells were stimulated with 100 ng mL 1 LPS for 16 h before treatment of cells with increasing concentrations of AG for 6 h. Emodin-6-O-D-b-glucoside (10 lM, EG) was used as a positive control (Lee et al. 2013). The results shown in Figure 2a indicate that AG inhibits release of HMGB1 by LPS in endothelial cells, with the optimal effect occurring at a concentration above 10 lM AG. However, treatment with AG alone did not affect HMGB1 release (Figure 2a). To confirm this effect in vivo, we next subjected mice to severe sepsis in a standardized model of caecal ligation and puncture (CLP) as this model closely resembles human sepsis (Buras et al. 2005). As shown in Figure 2b, AG induced marked inhibition of CLP-induced release of HMGB1 in mice. Because the average weight of a mouse is 20 g and the average blood volume is 2 mL, the injected AG (3.5 or 7 lg per mouse) produced a concentration maximum of 5 or 10 lM in peripheral blood. Next, we investigated the effects of AG on expression of the HMGB1 receptors, TLR2, TLR4 and RAGE in HUVECs. As shown in Figure 2c, HMGB1 induced 2.4-fold expression of TLR-2, TLR4 and RAGE in HUVECs, and treatment with AG resulted in marked inhibition of receptor expression. In addition, synthesized HMGB1 by LPS enhanced the expressions of HMGB1 receptors, and anti-HMGB1 180
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antibody inhibited their expressions (Fig. 2d). To exclude the possibility that inhibition of HMGB1 release was due to cytotoxicity caused by AG, cellular viability assays were performed in HUVECs treated with AG for 24 h. At the concentrations used (up to 20 lM), AG did not affect cell viability (Fig. 2e). High plasma concentrations of HMGB1 in patients with inflammatory diseases are known to be related to poor prognosis and high mortality. In addition, the pharmacological inhibition of HMGB1 is known to result in improved survival in animal models of acute inflammation in response to endotoxin challenge (Sama et al. 2004). Therefore, the prevention of LPS or CLP-induced release of HMGB1 by AG suggests that AG might be used for the treatment of vascular inflammatory diseases.
Effect of AG on LPS or HMGB1-mediated barrier disruption A permeability assay was performed for determination of the effects of AG on the barrier integrity of HUVECs. Treatment with AG (10 lM) alone did not result in the alteration of barrier integrity (Fig. 3a). On the other hand, LPS is known to induce cleavage and disruption of endothelial membrane barriers (Berman et al. 1993, Goldblum et al. 1993). Human umbilical vein endothelial cells were treated with various concentrations of AG for 6 h after addition of LPS (100 ng mL 1). As shown in Figure 3a, AG induced dose-dependent downregulation of LPS-mediated membrane disruption. HMGB1 is also known to induce cleavage and disruption of barrier integrity (Yang et al. 2005, Wolfson et al. 2011). Therefore, HUVECs were treated with various concentrations of AG for 6 h after addition of HMGB1 (1 lg mL 1). As shown in Figure 3b, treatment with AG resulted in decreased HMGB1-mediated membrane disruption in a dose-dependent manner. To confirm this effect in vivo, HMGB1- or CLP-induced vascular permeability in mice was evaluated. As shown in Figure 3c, AG induced marked inhibition of the peritoneal leakage of dye induced by HMGB1 or CLP. HMGB1 is known to induce pro-inflammatory responses by promoting phosphorylation of p38 MAPK (Qin et al. 2009, Sun et al. 2009). To determine whether AG inhibits HMGB1-induced activation of p38 MAPK in HUVECs, cells were activated with HMGB1 and then incubated with AG, followed by determination of phosphorylated p38 MAPK levels. As shown in Figure 3d, HMGB1 induced upregulated expression of phosphorylated p38, which was significantly inhibited by treatment with AG. These findings demonstrate inhibition of HMGB1-mediated endothelial disruption and maintenance of human endothelial
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Figure 2 Effects of AG on HMGB1 release and receptor expression. (a) Human umbilical vein endothelial cells (HUVECs) were treated with the indicated concentrations of AG or emodin-6-O-D-b-glucoside (10 lM, EG) for 6 h after stimulation with LPS 100 ng mL 1 for 16 h; HMGB1 release was then measured by ELISA. (b) Male C57BL/6 mice underwent CLP and were administered AG or EG (9.0 lg per mouse, i.v.) intravenously at 12 h after CLP (n = 5). Mice were killed 24 h after CLP. Serum HMGB1 levels were measured by ELISA. (c) Confluent HUVECs were incubated with HMGB1 (1 lg mL 1) for 16 h; cells were then treated with or without AG or 10 lM EG for 6 h. Expression of receptors, such as TLR-2 (white bar), TLR-4 (grey bar) and RAGE (black bar), on HUVECs was measured by cell-based ELISA. (d) The same as (c) except that cells were incubated with PBS (white bar), LPS (100 ng mL 1, grey bar) for 16 h with or without anti-HMGB1 neutralizing antibody (black bar). (e) The effects of AG on cellular viability were measured using MTT assays. Results are expressed as the mean SEM of three independent experiments. *P < 0.05 vs. LPS alone (a, d), CLP (b) or HMGB1 alone (c).
cell barrier integrity by AG in mice treated with HMGB1. Cytoskeletal proteins are important for the maintenance of cell integrity and shape (Schnittler et al. 2001). In addition, redistribution of the actin cytoskeleton, detachment of cells and loss of cell–cell contact due to cytokine stimulation are all associated with increased endothelial monolayer permeability (Friedl et al. 2002, Petrache et al. 2003). Therefore, we next examined the effects of AG on HUVEC actin cytoskeletal arrangement by immunofluorescence staining of HUVEC monolayers with F-actin labelled fluorescein phalloidin. Control HUVECs exhibited a random distribution of F-actin throughout cells with some localization of actin filament bundles at cell boundaries (Fig. 3e). Barrier disruption by HMGB1 (1 lg mL 1) was manifested by the formation of
paracellular gaps (shown by arrows) in HUVECs. A similar cytoskeletal arrangement was induced by LPS (100 ng mL 1) (data not shown). In addition, treatment with AG (10 lM) resulted in inhibited formation of HMGB1-induced paracellular gaps with formation of dense F-actin rings (Fig. 3e). These results suggested that treatment with AG led to maintenance of HMGB1-mediated morphological changes and gap formation of endothelial cells associated with F-actin redistribution, thereby increasing vascular barrier integrity.
AG inhibited expression of CAMs and proinflammatory responses Previous studies have demonstrated that HMGB1 mediates inflammatory responses by increasing the cell
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Figure 3 Effects of AG on HMGB1-mediated permeability in vitro and in vivo. (a, b) Human umbilical vein endothelial cells (HUVECs) were stimulated with LPS (a, 100 ng mL 1 for 4 h) or HMGB1 (b, 1 lg mL 1, 16 h) and then treated with different concentrations of AG or 10 lM EG for 6 h. Then, permeability was monitored by measuring the flux of Evans blue-bound albumin across HUVECs. (c) The effects of AG or EG (9.0 lg per mouse, i.v.) on HMGB1- (2 lg per mouse, i.v., white bar) or CLP- (at 24 h after CLP, black bar) induced vascular permeability in mice were examined by measuring the amount of Evans blue in peritoneal washings (expressed lg per mouse, n = 5). (d) HUVECs were activated with HMGB1 and were then treated with AG or 10 lM EG for 6 h. The effects of AG on HMGB1-mediated expression of phospho-p38 were measured by ELISA. (e) Staining for F-actin. HUVEC monolayers grown on glass coverslips were stimulated with HMGB1 for 1 h, followed by treatment with AG for 6 h, and immunofluorescence staining for F-actin. Arrows indicate intercellular gaps. Results are expressed as the mean SEM of three independent experiments. *P < 0.05 vs. LPS (a), HMGB1 (b, c, d) or CLP (d).
surface expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin on the surfaces of endothelial cells, thereby promoting adhesion and migration of leucocytes across endothelium to sites of inflammation (Andersson et al. 2000). To determine the effects of AG on expression of CAMs in HMGB1stimulated endothelial cells, we monitored expression of VCAM-1, ICAM-1 and E-selectin on HMGB1-stimulated AG pre-treated HUVECs. As shown in Figure 4a, AG suppressed the expression of VCAM-1, ICAM-1 and E-selectin. Adhesion of leucocytes to endothelial cells and transendothelial migration (TEM) of leucocytes are important steps in the proinflammatory response (Hansson & Libby 2006). Therefore, we attempted to determine whether expression of CAMs corresponded to enhanced leucocyte binding, and whether AG could block adhesion of monocytes to 182
HMGB1-stimulated HUVECs. We found that AG effectively inhibited binding of leucocytes to HMGB1stimulated HUVECs (Fig. 4b). Further studies revealed an association of leucocyte to HUVEC binding with the subsequent TEM of leucocytes and that AG effectively inhibited this step (Fig. 4c). To confirm this effect in vivo, HMGB1- or CLP-induced leucocytes and neutrophils migration was examined in mice. HMGB1 and CLP-induced significant stimulation of leucocytes and neutrophils migration into the peritoneal cavities of mice, and AG at doses of 5–10 lM significantly inhibited this migration (Fig. 4d and e). These results indicate that AG not only inhibits the endotoxin-mediated release of HMGB1 by endothelial cells, but that it also downregulates the pro-inflammatory signalling effect of released HMGB1 and thereby inhibits the amplification of inflammatory pathways
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Figure 4 Effect of AG on HMGB1mediated pro-inflammatory responses. Human umbilical vein endothelial cells (HUVECs) were stimulated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG or 10 lM EG for 6 h. HMGB1-mediated (a) expression of VCAM-1 (white box), ICAM-1 (grey box), and E-selectin (black box) in HUVECs, (b) adherence of monocytes to HUVEC monolayers, and (c) migration of monocytes through HUVEC monolayers were analysed. (d, e) Male C57BL/6 mice were stimulated with HMGB1 (2 lg per mouse, i.v., white bar) or CLP-operated mice (at 24 h after CLP, black bar) were treated with AG or EG (9 lg per mouse, i.v.). HMGB1- or CLPmediated migration of leucocytes (d) and neutrophils (e) into the peritoneal cavity of mice was analysed. Results are expressed as the mean SEM of three independent experiments. *P < 0.05 vs. HMGB1 (a–d) or CLP (d, e).
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such as the upregulation of CAMs, leucocytes adhesion and migration by HMGB1.
AG inhibited HMGB1-stimulated activation of NF-jB/ ERK/Akt and production of TNF-a and IL-1b HMGB1 and LPS both induced a significant increase in nuclear translocation of NF-jB and phosphorylation of Akt and p38 MAPK in human endothelial cells (Kawahara et al. 2008, Dagia et al. 2010, Andersson & Tracey 2011, Wang et al. 2013), and activation of NF-jB and ERK1/2 is required for pro-inflammatory responses (Marui et al. 1993, Lockyer et al. 1998, Rose et al. 2010). Previous studies have reported activation of NF-jB and ERK 1/2 by HMGB1 in vascular inflammatory responses (Park et al. 2006, Palumbo et al. 2007, Yang & Tracey 2010). Therefore, to investigate the potential effects of AG on activation of inflammatory signalling molecules and production of TNF-a and IL-1b in HMGB1-activated HUVECs, cells were activated with HMGB1 for 16 h, followed by incubation with AG for 6 h. Data showed that phosphorylation of NF-jB (Fig. 5a, g), ERK1/2 (Fig. 5b), and Akt (Fig. 5c), and production of TNF-a (Fig. 5d)
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and IL-1b (Fig. 5e) were increased by HMGB1, and these increases were significantly reduced by AG (Fig. 5). These results indicate that AG may regulate the most important signals involved in induction of proinflammatory responses in human endothelial cells.
Protective effect of AG in CLP-induced production of IL6/KC and septic mortality Sepsis, a systemic response to serious infection, has a poor prognosis when it is associated with organ dysfunction, hypoperfusion or hypotension (Cohen 2002, Bae 2012). Production of interleukin (IL) 6 and 8 is required for pro-inflammatory responses and important markers of mortality (Teiten et al. 2010). Based on the above-mentioned findings, we hypothesized that treatment with AG would result in reduced production of IL-6/KC and mortality in our CLP-induced sepsis mouse model. As shown in Figure 6a and b, production of IL-6 and KC in CLP-induced mice was reduced by treatment with AG. This result was consistent with the previous finding that the IL-6 levels were increased prior to death (Xiao et al. 2006). To determine whether AG protects mice from CLP-induced
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sepsis lethality, AG was administered to mice after CLP. Twenty-four hours after the operation, animals manifested signs of sepsis, including shivering, bristled hair and weakness. Administration of AG at dual dose (3.5 or 7.0 lg per mouse, 12 h after CLP) did not prevent CLP-induced death (data not shown); therefore, AG was administered two times (once 12 h after CLP and once 50 h after CLP), resulting in an increase in the survival rate from 30 to 60% according to Kaplan–Meier survival analysis (P < 0.0001, Fig. 6c). However, no beneficial effect was observed for a lower dose of AG (data not shown). This marked survival benefit achieved by administration of AG suggests that suppression of HMGB1 release and HMGB1-mediated inflammatory responses provides a therapeutic strategy for the management of sepsis and septic shock. Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. A wide array of pro-inflammatory cytokines including TNF-a, IL-1, IFN-c and macrophage migration inhibitory factor (MIF) individually or in combination contributes to the pathogenesis of lethal systemic inflammation (Bhatia et al. 2009). For instance, neutralizing antibodies against TNF (Tracey et al. 1987) reduce lethality in an 184
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Figure 5 Effects of AG on HMGB1stimulated activation of NF-jB/ERK/ AKT and production of TNF-a/IL-1b in human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were stimulated with HMGB1 (1 lg mL 1) for 16 h followed by treatment with AG or 10 lM EG for 6 h. (a–c) HMGB1 (1 lg mL 1)-mediated activation of phospho- total NF-jB p65 (a), phospho- total ERK1/2 (b) or phospho- or total Akt (c) in HUVECs was analysed. White bar, phosphor form; black bar, total form. HMGB1 (1 lg mL 1)-mediated production of TNF-a (d) and IL-1b (e) in HUVECs was analysed. (f) Immunofluorescence microscopy analysis of the nuclear translocation of p65 in HUVECs. Human umbilical vein endothelial cells were stimulated (or not) for 1 h with 1 lg mL 1 HMGB1 and treated or not with 5 or 10 lM AG for 6 h. The subcellular localization of p65 was examined by IF staining. Results are expressed as the mean SEM of three independent experiments. *P < 0.05 vs. HMGB1.
animal model of endotoxaemic/bacteraemic shock. However, the early kinetics of systemic TNF accumulation makes it difficult to target in clinical setting (Tracey et al. 1987), prompting the investigation of other late proinflammatory mediators (such as HMGB1) as potential therapeutic target for inflammatory diseases. The prevailing theories of sepsis as a dys-regulated systemic inflammatory response are supported by extensive studies employing various animal models of sepsis, including endotoxaemia and peritonitis induced by caecal ligation and puncture (CLP) (Wichterman et al. 1980). In murine models of endotoxaemia and sepsis, HMGB1 is first detectable in the circulation eight hours after the onset of diseases, subsequently increasing to plateau levels from 16 to 32 h (Wang et al. 2001). This late appearance of circulating HMGB1 precedes and parallels with the onset of animal lethality from endotoxaemia or sepsis and distinguishes itself from TNF and other early proinflammatory cytokines (Wang et al. 2001). Therefore, in this study, AG was treated at 16 h after inflammatory challenge. In summary, our results demonstrate that AG inhibits both LPS and CLP-mediated release of HMGB1, expression of HMGB1 receptor (TLR2, TLR4 and RAGE) and HMGB1-mediated barrier disruption
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Figure 6 Effects of AG on production of IL-6/KC and lethality after CLP. (a, b) Male C57BL/6 mice underwent CLP and were administered AG or EG (9.0 lg per mouse, i.v.) intravenously at 12 h and 50 h after CLP (n = 5). Mice were killed 96 h after CLP. Serum IL-6 (a) or KC (b) levels were measured by ELISA. (c) Male C57BL/6 mice (n = 10) were administered i.v. AG (3.5 lg per mouse, □) or AG (7.0 lg per mouse, ■) at 12 and 50 h after CLP. Animal survival was monitored every 6 h after CLP for 126 h. Control CLP mice (●) and sham-operated mice (○) were administered sterile saline (n = 10). A Kaplan–Meier survival analysis was used for determination of overall survival rates vs. CLP-treated mice. *P < 0.05 vs. CLP.
through increases in barrier integrity and inhibition of CAM expression. In addition, AG reduces adhesion and migration of monocytes towards HUVECs. These barrier protective effects of AG were confirmed in a mouse model, in which treatment with AG resulted in reduction in HMGB1-induced mortality. Our findings indicate that AG may be regarded as a candidate for use in the treatment of severe vascular inflammatory diseases, such as septic shock.
Conflict of interest The authors declare no conflict of interests. This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government [MSIP] (Grant No. 2012-000940).
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