- Email: [email protected]
Ketamine attenuates high mobility group box-1–induced inflammatory responses in endothelial cells
Accepted Manuscript Ketamine attenuates HMGB1-induced inflammatory responses in endothelial cells Zhaohui Liu, M.D., Zhengping Wang, Ph.D., Guangwei Han, M.D., Lina Huang, Ph.D., Jihong Jiang, Ph.D., Shitong Li, M.D. PII:
S0022-4804(15)00871-9
DOI:
10.1016/j.jss.2015.08.032
Reference:
YJSRE 13501
To appear in:
Journal of Surgical Research
Received Date: 2 April 2015 Revised Date:
2 August 2015
Accepted Date: 19 August 2015
Please cite this article as: Liu Z, Wang Z, Han G, Huang L, Jiang J, Li S, Ketamine attenuates HMGB1induced inflammatory responses in endothelial cells, Journal of Surgical Research (2015), doi: 10.1016/ j.jss.2015.08.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ketamine attenuates HMGB1-induced inflammatory responses in endothelial cells Zhaohui Liu,M.D.,a,1 Zhengping Wang, Ph.D., b,1 Guangwei Han, M.D.,c
a
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Lina Huang, Ph.D.,c Jihong Jiang, Ph.D.,c and Shitong Li, M.D.c Department of Anesthesiology, Cangzhou Central Hospital, Hebei
Medical University, Hebei, China
Department of Anesthesiology, Shanghai Tenth People’s Hospital,
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b
c
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Tongji University School of Medicine, Shanghai, China
Department of Anesthesiology, Shanghai First People’s Hospital,
Shanghai Jiaotong University School of Medicine, Shanghai, China 1
These authors contributed equally to this study.
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Running title: Ketamine attenuates HMGB1-mediated inflammatory responses.
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Category: Sepsis
Author contributions Zhaohui Liu: conception, design, analysis and interpretation of data; writing the manuscript. Zhengping Wang: conception, design, analysis and interpretation of data; writing the manuscript. Guangwei Han: analysis and interpretation of data; data collection.
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Lina Huang: analysis and interpretation of data; data collection. Jihong Jiang: analysis and interpretation of data; data collection. Shitong Li: conception, design, analysis and interpretation of data; data
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collection; critical revision of the manuscript; statistical expertise; obtaining funding; supervision.
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Conflicts of Interest
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The authors report no proprietary or commercial interest in any product mentioned, or concept discussed, in this article.
Corresponding Author:
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Shitong Li,
No. 100 Haining Road, Shanghai, China Phone: +86 021 66307531
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FAX: +86 021 66301082
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Email: [email protected]
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Abstract Aim and objective High mobility group box-1 (HMGB1) acts as an inflammatory mediator and has been implicated in pathophysiological
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damage of vascular inflammatory diseases. Ketamine, an anesthetic agent with sedative and analgesic properties, has been shown to have potent anti-inflammatory effects in a variety of models of systemic inflammation.
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However, the effects of ketamine on HMGB1-mediated pro-inflammatory
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responses have not been fully investigated. In the present study, we investigated the effects of ketamine on HMGB1-activated endothelial cells and explored the underlying mechanisms.
Methods Human endothelial cells were incubated with or without
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HMGB1 (1 µg/mL) in the presence or absence of ketamine, an NF-κB inhibitor (PDTC), anti-TLR2/4 antibody, or small interfering RNA (si RNA). The anti-inflammatory activities of ketamine were determined by
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measuring solute flux, leukocyte adhesion and migration and activation of
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pro-inflammatory proteins in HMGB1-activated endothelial cells. The effect of ketamine on toll like receptor (TLR)-2/4 and NF-κB activation was evaluated using Enzyme-Linked Immunosorbent Assays, and Immunofluorescence Confocal Microscopy Assay. Results We found that ketamine inhibited the HMGB1-mediated barrier disruption, neutrophil adhesion and migration, and expression of cell adhesion molecules in a dose-dependent manner. Furthermore, ketamine
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down-regulated the toll like receptor (TLR)-2 and -4, expression in HMGB1-activated endothelial cells. Treatment with ketamine also significantly inhibited the activation of TLR2/4 and the nuclear
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translocation of NF-κB p50/p65. Furthermore, our study shows that the HMGB1-induced release of inflammatory mediators was suppressed by PDTC, anti-TLR2/4 antibody, and si RNA.
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Conclusion Our study has demonstrated that ketamine exerts anti
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-inflammatory effects in HMGB1-mediated pro-inflammatory responses in a dose-dependent manner. The mechanism responsible for these effects involves the TLR2/4 and NF-κB signaling pathway.
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Keywords Ketamine; HMGB1; endothelial cells; Inflammation; sepsis
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Introduction Systemic inflammation is one of the hallmarks of septic shock, and the microvascular endothelium provides an important site of regulation and
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amplification of this inflammatory responses[1]. Endothelial cell activation leads to alterations in hemostasis, increases in vascular permeability, cell swelling and loss of barrier function, leukocyte
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adherence with cell clumping, and microthrombi formation[2; 3].
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Microvascular injury is one of the characteristics of sepsis-associated tissue damage that may be manifested by single (eg, acute respiratory distress syndrome) or multiple organ failure syndromes[4]. Many inflammatory mediators of sepsis contribute to the development of an
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activated endothelium (eg, tumor necrosis factor-α [TNF-α] and interleukin 1β [IL-1β])[5]. Recently, high mobility group protein 1 (HMGB1) has been identified as a novel inflammatory cytokine and a
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late mediator of endotoxin lethality in mice[6].
High mobility group protein 1 (HMGB1), a secreted nuclear protein, is a potent mediator and associates with a variety of severe vascular inflammatory diseases such as sepsis and septic shock [6]. HMGB1, when nuclear, is involved in nucleosome stabilization and gene transcription[7]. Once released into extracellular milieu, HMGB1 can stimulate the secretion of pro-inflammatory cytokines from endothelial
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cells, monocytes, and macrophages, leading to a wide range of inflammatory responses [8; 9]. HMGB1 mediates cell signaling by binding to several transmembrane receptors, such as, receptor for
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advanced glycation end products (RAGE), toll like receptor (TLR)-2, and -4, and activates intracellular signal of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB[10-12]. High plasma levels of
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HMGB1 correlate with poor prognosis and increased mortality in patients
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with inflammatory diseases, and targeting HMGB1 with specific antagonists is protective in established preclinical inflammatory disease models including lethal endotoxemia or sepsis [13; 14]. Therefore, inhibiting HMGB1active release and/or blocking HMGB1
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pro-inflammatory activities could be a promising therapeutic target for vascular inflammatory diseases.
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Ketamine, an anesthetic agent with sedative and analgesic properties, has
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been shown to have potent anti-inflammatory effects in a variety of models of systemic inflammation, including endotoxemia, sepsis, ischemia, and burns[15-18]. These effects have been found in multiple organ systems and involve modulation of the molecular mediators of the inflammatory response, including transcription factors such as nuclear NF-κB and MAPKs[19-21]. Additionally, when given either before or after various pro-inflammatory insults, ketamine has been shown to
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diminish systemic production of cytokines, such as tumor necrosis factor-α (TNF-α), interleukins-1β (IL-1β), and interleukins-6(IL-6), and interferon-γ (IFN-γ), and improve survival[17; 20]. Furthermore, recent
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evidence suggests that ketamine improves end-organ dysfunction and has anti-inflammatory effects that appear to be present even at sub-anesthetic (sedative) doses[22; 23]. However, the anti-inflammatory activities of
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ketamine on HMGB1-mediated pro-inflammatory responses have not
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been well studied.
The present study addressed the question whether ketamine could attenuate HMGB1-induced pro-inflammatory response through inhibiting TLR2/4 expression and NF-κB activation, could protect vascular integrity
endothelial cells.
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and inhibit cell adhesion molecule expression in HMGB1-activated
Reagents
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Materials and methods
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Ketamine, Bacterial lipopolysaccharide (LPS, used at 100 ng/mL), Evans blue, crystal violet and fMLP (N-formyl-methionyl-leucyl-phenylalanine) were obtained from Sigma (St. Louis, MO, USA). Human recombinant HMGB1 was purchased from Invitrogen (Carlsbad, CA).
Cell culture
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Primary human umbilical vein endothelial cells (HUVECs) were obtained from the Typical Species Preservation Center of Wuhan University (Wuhan, Hubei, China) and maintained as described previously[24].
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Briefly, the cells were cultured to confluence at 37℃ and 5% CO2 in endothelial basal medium (EBM; Clonetics, Walkersville, MD)
supplemented with epidermal growth factor (10 ng/mL), hydrocortisone
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(1µg/mL), penicillin (100 U/mL), streptomycin (100 µg/mL),
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amphotericin (250 ng/mL), and 10% FCS. HUVECs of passage numbers 3 or 4 were used in the experiments. Freshly isolated neutrophils were kindly provided by Dr. Hui Liu (Shanghai Sixth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China). The
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human microvascular endothelial cell line HMEC-1, which were obtained from the Typical Species Preservation Center of Wuhan University (Wuhan, Hubei, China), is an SV-40-transformed microvascular
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endothelial cell line that retains the morphologic phenotype and
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functional characteristics of human microvascular endothelial cells[25; 26]. HMEC-1 cells were grown and maintained in endothelial basal medium supplemented with epidermal growth factor (10 ng/mL), hydrocortisone (1µg/mL), penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin (250 ng/mL), and 10% FCS. These cells were grown at 37℃ in a humidified 5% CO2 incubator and were subcultured
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at 50-80% confluence using 0.05% trypsin, 0.02% EDTA (Gibco, NY, USA).
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Measuring solute flux Solute flux was quantitated by spectrophotometric measurement of the flux of Evans blue-bound albumin across functional endothelial cells
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monolayers using a modified 2-compartment chamber model as
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previously described[27]. Briefly, Endothelial cells were plated (5× 104/well) in 3µm pore size and 12 mm diameter transwells for 3 days. The confluent monolayers were incubated with ketamine for 6 h followed by HMGB1 (1 µg/mL) for 16 h. Inserts were washed with PBS, pH 7.4,
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before adding 0.5 mL Evans blue (0.67 mg/mL) diluted in growth medium containing 4% BSA. Fresh growth medium was added to the lower chamber, and the medium in the upper chamber was replaced with
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Evans blue/BSA. After 10 min, the optical density at 650 nm was
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measured in the lower chamber. Experiments were performed in triplicate and repeated multiple times.
Cell viability assay Cell viability was determined using the Cell Counting Kit-8 assay (CCK-8; Beyotime, Jiangsu, China)[28-32]. Endothelial cells were grown in 96-well plates at a density of 5 × 103 cells/well. After 24 h, cells were
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washed with fresh medium and then treated with ketamine. After 24 h incubation, cells were rewashed and 100 µL of CCK-8reagent was added and incubated for 4 h. The absorbance of the solution was measured using
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a microplate reader (Bio-Rad Laboratories, Hercules, CA) at a test wavelength of 450 nm and a reference wavelength of 630 nm.
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Cell-cell adhesion assay
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Adherence of human neutrophils to endothelial cells was evaluated by fluorescent labeling of neutrophils as described previously [33]. Briefly, neutrophils were labeled with Vybrant DiD. Following two washes, cells were resuspended in adhesion medium (RPMI containing 2% FBS and 20
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mM HEPES) and added to confluent monolayers of endothelial cells in 96-well plates that were treated for 6 h with ketamine followed by HMGB1 (1 µg/mL for 16 h). Non-adherent neutrophils were washed off
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and the fluorescence of the adherent cells was measured. The percentage
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of adherent neutrophils was calculated by the formula: % adherence = (adherent signal / total signal) × 100[33].
Migration assay
Migration assays were performed in transwell plates. Endothelial cells were cultured for 3 days to obtain confluent endothelial monolayers. The cell monolayers were treated for 6 h with ketamine followed by HMGB1
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(1 µg/mL for 16 h) or fMLP (10-8 M for 2 h) and freshly isolated neutrophils were added to the upper compartment. After transwell plates were incubated for 2 h, neutrophils on the lower side of the filter were
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fixed with 8% glutaraldehyde and stained with 0.25% crystal violet in 20% methanol (w/v). Each experiment was repeated in duplicate wells
and, within each well, nine randomly selected microscopic high power
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fields (HPF, ×200) were counted and expressed as the migration index.
Expression of cell surface receptors
The expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin on endothelial
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cells was determined by whole-cell ELISA as described previously[34]. Briefly, confluent monolayers of endothelial cells were treated with ketamine for 6 h followed by HMGB1 (1 µg/mL) for 16 h. The medium
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was removed, and the cells were washed with PBS and fixed by adding
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50 µL of 1% paraformaldehyde for 15 min at room temperature. After washing, 100µL of mouse anti-human monoclonal antibodies (VCAM-1, ICAM-1, E-selectin; Sigma, CA, USA) were added. After 1 h (37℃, 5% CO2), the cells were washed three times and then 100µL of 1:2000 peroxidase conjugated anti-mouse IgG antibodies (Sigma) were added for 1 h. The cells were washed again three times and developed using o-phenylenediamine substrate (Sigma). Colorimetric analysis was
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performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells. The same experimental procedures were used to monitor the cell surface expression of TLR2, TLR4 and RAGE
Biotechnology Inc. (Santa Cruz, CA, USA).
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RNA interference
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receptors using specific antibodies obtained from Santa Cruz
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The expression of inflammatory mediators by endothelial cells in response to HMGB1 (1 µg/mL for 16 h) was evaluated following the knockdown of TLR2, TLR4 and NF-κB expression by pools of target-specific 20- to 25-nucleotide siRNAs obtained from Santa Cruz
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Biotechnology Inc. according to the manufacturer’s instruction and as described previously [27]. A non-targeting 20- to 25-nucleotide siRNA
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obtained from the same company was used as a negative control.
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Quantitative real-time polymerase chain reaction (qPCR) analysis Total RNA was extracted from endothelial cells using Trizol (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s instructions, and reverse-transcribed with MMLV reverse transcriptase (Promega, Madison, WI). Quantitative real-time polymerase chain reaction (qPCR) was performed using a LightCycler 2.0 Real-Time PCR System (Roche Applied Science, Indianapolis, IN). cDNA was amplified using specific
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primers for inflammatory mediators gene expression and the results were normalized to β-actin gene expression. The relative mean fold-change of inflammatory mediators gene expression in the experimental group was
Immunofluorescent staining for NF-κB p65
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calculated using the 2△△ Ct method and compared to the control group[35].
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To visually identify the translocation of nuclear NF-κB p65 translocation,
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HUVECs were plated on gelatin-coated glass-bottom microwell plates (MatTek, MA, USA) and cultured until confluence. After appropriate treatment, the cells were fixed and permeabilized for 15 min at room temperature in PBS with 3.7% formaldehyde and 0.5% Triton X-100.
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Then cells were washed in PBS twice, blocked in 5% BSA for 1 h, and incubated with NF-κB antibody (1:50) at 4 ºC overnight. After a thorough wash in PBS, the cells were stained with an FITC-conjugated secondary
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antibody (1:200) and conjugated rhodamine-phalloidin (1:100) for 1 h.
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Cells were further incubated with diamidino-2-phenylindole (DAPI, 1:1000) for 15 min and then washed with PBS again. Rhodamin-phalloidin and DAPI were used to stain F-actin and nuclear DNA, respectively, to reveal the location of the cytoplasm and nucleus in the HUVECs. The staining results were imaged using a Zeiss LSM780 laser confocal scanning microscope (Zeiss, Germany).
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Statistical analysis Results are expressed as the mean ± the standard deviation (S.D.) of at least three independent experiments. The statistical significance of
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differences between test groups was used for statistical comparison (SPSS, version 14.0, SPSS Science, Chicago, IL, USA). Statistical relevance was determined by analysis of variance (ANOVA) with
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P<0.05.
Results
Effect of ketamine on LPS-mediated HMGB1 release in endothelial cells
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Previous studies have demonstrated that LPS stimulates HMGB1 release in murine macrophages and human endothelial cells[21; 34]. Following its release to intravascular spaces, HMGB1 is known to interact with
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specific cell surface receptors to amplify inflammatory responses by
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inducing the expression of pro-inflammatory cytokines[9]. In agreement with previous results, LPS (100 ng/mL) stimulated HMGB1 release by endothelial cells (Fig. 1C). To investigate the effect of ketamine on LPS-mediated HMGB1 release, endothelial cells were pretreated with increasing concentrations of ketamine for 6 h before stimulation with 100 ng/mL LPS for 16 h. The results presented in Fig. 1C indicated that ketamine inhibits HMGB1 release by endothelial cells, with optimal
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effect occurring at a concentration above 100 µM ketamine. To exclude the possibility that the inhibition of HMGB1 release was due to cytotoxicity caused by ketamine, cellular viability assays were performed
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in endothelial cells treated with ketamine for 24 h. At the concentrations used (up to 1000 µM), ketamine did not affect cell viability (Fig. 1B).
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Effect of ketamine on HMGB1-mediated barrier disruption in
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endothelial cells
To determine the effects of ketamine on barrier integrity in endothelial cells, solute flux was measured. Ketamine (up to 1000 µM) alone did not alter barrier integrity (Fig. 2). HMGB1 is known to cleave and disrupt
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barrier integrity[36]. Human endothelial cells were treated with various concentrations of ketamine for 6 h before adding HMGB1 (1 µg/mL). As shown in Fig. 2, ketamine decreased HMGB1-mediated membrane
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disruption in a dose-dependent manner.
Effects of ketamine on HMGB1-mediated cell adhesion molecule (CAM) expression, human neutrophil adhesion and migration in endothelial cells
Previous reports showed that HMGB1mediates inflammatory responses by increasing the cell surface expression of the cell adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of endothelial cells,
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thereby promoting adhesion and migration of leukocytes across the endothelium and to sites of inflamed tissues [5, 7, 28, 29]. Therefore, we tested whether ketamine could modulate HMGB1-mediated CAM
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expression. As presented in Fig. 3, HMGB1 up-regulated the cell surface expression of all three adhesion molecules (CAMs) and ketamine
inhibited these effects of HMGB1 in a concentration-dependent manner.
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The elevated expression of CAMs correlated well with enhanced binding
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of human neutrophils to HMGB1-activated endothelial cells and their subsequent migration. Ketamine down-regulated the adherence of human neutrophils to HMGB1-activated endothelial cells and their subsequent migration in a concentration-dependent manner (Fig. 4A, B), as well as to
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the chemotactic agent fMLP (Fig. 4C).
Ketamine down-regulates the expression of HMGB1 receptors in
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endothelial cells
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We next investigated the effect of HMGB1 on the stimulation of its own receptors and the effect of ketamine on the modulation of the expression of these receptors on endothelial cells. As shown in Fig. 5A-C, HMGB1 induced the expression of all three receptors, TLR2, TLR4 and RAGE, in endothelial cells, and ketamine significantly inhibited the stimulatory effect of HMGB1 on TLR2 and TLR4 but not RAGE.
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The TLR2/4 and NF-κB p65 inhibitor suppresses HMGB1-induced release of inflammatory mediators in endothelial cells To evaluate whether ketamine inhibited the inflammatory responses
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through TLR2/4 and NF-κB p65, endothelial cells were pretreated with or without anti-TLR2 antibody (5 µg/mL), anti-TLR4 antibody (5 µg/mL), PDTC (80 µM) for 1 h before adding ketamine (1000 µM) for 6 h, and
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then cells were stimulated with HMGB1 (1 µg/mL) for 16 h. The TLR2/4
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and NF-κB p65 inhibitor and ketamine all partially suppressed LPS-induced overexpression of ICAM-1, VCAM-1, and E-selectin (*P < 0.05, Fig. 6A-C). Furthermore, pretreating human endothelial cells with a combination of anti-TLR2/4 antibody/ PDTC and ketamine
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synergistically reversed the HMGB1-induced expression of ICAM-1, VCAM-1, and E-selectin (*P < 0.05, Fig. 6A-C). The results suggest that TLR2/4 and NF-κB p65 may be involved in ketamine’s reduction of
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HMGB1-induced inflammation in human endothelial cells.
TLR2/4 and NF-κB p65 siRNA suppresses HMGB1-induced expression of inflammatory mediators in endothelial cells We further confirmed the results by knocking down TLR2/4 and NF-κB p65 in endothelial cells using specific siRNA of TLR2/4 and NF-κB p65. We found that silencing TLR2/4 and NF-κB p65 with siRNA inhibited the expression levels of ICAM-1, VCAM-1, and E-selectin mRNA in
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HMGB1-stimulated endothelial cells (*P < 0.05, Fig. 7A-C) and the ketamine treatment strongly increased the inhibition effect of siRNA (#P
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< 0.05, Fig. 7A-C).
TLR2/4 and NF-κB p65 siRNA suppresses HMGB1-induced barrier disruption and human neutrophil adhesion and migration in
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endothelial cells
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To evaluate whether ketamine inhibited the barrier disruption and human neutrophil adhesion and migration through TLR2/4 and NF-κB p65, endothelial cells were knocked down for TLR2/4 and NF-κB p65 using specific siRNA before adding ketamine (1000 µM) for 6 h, and then cells
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were stimulated with HMGB1 (1 µg/mL) for 16 h. We found that silencing TLR2/4 and NF-κB p65 with siRNA inhibited the membrane disruption and human neutrophil adhesion and migration in
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HMGB1-stimulated endothelial cells (*P < 0.05, Fig. 8A-C) and the
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ketamine treatment strongly increased the inhibition effect of siRNA (#P < 0.05, Fig. 8A-C).
Ketamine attenuates HMGB1-induced nuclear translocation of NF-κB p65 in endothelial cells To evaluate whether ketamine inhibited the inflammatory responses through NF-κB in endothelial cells, nuclear translocation of NF-κB p65
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was observed using immunofluorescent staining. The visualizing images of NF-κB nuclear translocation using a confocal microscopic showed that, under control conditions, NF-κB (green staining) is located in the
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cytoplasmic region, and the nuclear area is stained with almost pure blue (DAPI), indicating little NF-κB in nuclei. NF-κB p65 nuclear
translocation in endothelial cells was demonstrated by the indigo color in
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the nuclear area after application of HMGB1 (Fig. 9). The
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HMGB1-induced NF-κB p65 translocation from the cytoplasm to the nucleus was decreased significantly by ketamine.
Discussion
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Because HMGB1 is a critical pro-inflammatory cytokine involved in inflammatory disorders, and TLR2/4 and NF-κB p65 are widely involved in regulating the production of a variety of inflammatory cytokines, we
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hypothesized that drugs blocking the TLR2/4 and NF-κB p65 signaling
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pathway might play a protective role in the HMGB1-mediated inflammatory processes through a variety of mechanisms. The present results confirmed that ketamine significantly inhibited the release of HMGB1 by lipopolysaccharide (LPS) and also suppressed the HMGB1-mediated barrier disruption, neutrophil adhesion and migration, and expression of cell adhesion molecules in HMGB1-activated endothelial cells in vitro. Cell viability was not affected, indicating that
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treatment with ketamine was anti-inflammatory rather than cytotoxic. The data also suggest that the inhibitory effects of ketamine occur via inhibition of TLR2/4 and NF-κB p65 signaling pathways. Therefore, the
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inhibition of HMGB1-activated inflammatory responses shown in this study suggests that ketamine could be beneficial in the treatment of
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vascular inflammatory diseases.
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However, the exact mechanism of the anti-inflammatory effects of ketamine on HMGB1-mediated inflammatory responses has not been fully clarified. Recent studies have shown that the inhibitory effects of ketamine on CLP-mediated inflammatory responses involve the TLR4
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signaling pathway [19; 20]. Previous studies have confirmed that the TLR4 signaling pathway regulates HMGB1-mediated inflammatory responses [11]. Thus, our study supports the hypothesis that ketamine
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inhibits the TLR4 signaling pathway in a dose-dependent manner, which
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regulates the HMGB1-mediated pro-inflammatory responses. Previous studies have also confirmed that p38 MAPK[37] are involved in regulating the release of inflammatory cytokines by the TLR4 signaling pathway. Therefore, ketamine might act through p38 MAPK, and other upstream pathways that regulate HMGB1-mediated inflammatory responses.
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Ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist, is widely used as an intravenous anesthetic agent[38]. Among nonvolatile anesthetics, ketamine has more stable hemodynamics than other
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anesthetic agents; it is used in various clinical areas by systemic treatment, including pediatric anesthesia, traumatic anesthesia, and obstetrics; and is quite widely serviceable in operating rooms and intensive care units[39].
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The side effects of ketamine systemic treatment include behavioral and
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psychological changes when used a large dose of ketamine[38].
Ketamine has been shown to have a protective effect on various cell types involved in the inflammation process [21; 40-44]. Our study only focused
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on endothelial cells, one of the more commonly used cells in the study of the inflammation process. Additional study of the role of HMGB1 in other types of immune cells under similar pathologic conditions is
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required. We also need to study whether the protective effects of
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ketamine on the pathologic complications related to sepsis involves the inhibition of HMGB1. Furthermore, the effect of ketamine on upstream signaling pathways that regulate HMGB1-mediated inflammatory responses, such as p38 MAPK, needs to be investigated.
Our study has confirmed that ketamine, as an anti-inflammatory drug, inhibits HMGB1-mediated pro-inflammatory responses by increasing
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barrier integrity and inhibiting CAM expression. These effects were achieved partially by inhibiting the TLR2/4 and NF-κB p65 signaling pathway. Our findings will help us better understand the
clinical treatments of vascular inflammatory disease.
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Conflict of interest
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anti-inflammatory mechanisms of ketamine and also help to explore new
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The authors declare that there are no conflicts of interest.
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of subanesthetic doses of ketamine on hemodynamic and immunologic variables in dogs with experimentally induced endotoxemia. Am J Vet Res 2008;69:228.
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[24] Ku SK, Bae JS. Concentration dependent anti-inflammatory effects thrombin on polyphosphate-mediated inflammatory responses in vitro and in vivo. Inflamm Res 2013;62:609.
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[25] Ades EW, Candal FJ, Swerlick RA, et al. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol 1992;99:683.
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vitro and in vivo. J Surg Res 2014;192:582. [30] Wu Y, Liu Y, Huang H, et al. Dexmedetomidine inhibits inflammatory reaction in lung tissues of septic rats by suppressing TLR4/NF-kappaB pathway. Mediators Inflamm 2013;2013:562154. [31] Liu Z, Zhang J, Huang X, Huang L, Li S, Wang Z. Magnesium sulfate inhibits the secretion of high mobility group box 1 from
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lipopolysaccharide-activated RAW264.7 macrophages in vitro. J Surg Res 2013;179:e189. [32] Liu Z, Chang Y, Zhang J, et al. Magnesium deficiency promotes
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[38] Liu FL, Chen TL, Chen RM. Mechanisms of ketamine-induced immunosuppression. Acta Anaesthesiol Taiwan 2012;50:172. [39] Haas DA, Harper DG. Ketamine: a review of its pharmacologic
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properties and use in ambulatory anesthesia. Anesth Prog
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lipopolysaccharide-induced astrocytes activation by suppressing
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TLR4/NF-kB pathway. Cell Physiol Biochem 2012;30:609. [41] Zeng J, Xia S, Zhong W, Li J, Lin L. In vitro and in vivo effects of ketamine on generation and function of dendritic cells. J Pharmacol Sci 2011;117:170.
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[43] Wang F, Meng Y, Zhang Y, et al. Ketamine reduces
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[44] Tan Y, Wang Q, She Y, Bi X, Zhao B. Ketamine reduces LPS-induced HMGB1 via activation of the Nrf2/HO-1 pathway and NF-kappaB suppression. J Trauma Acute Care Surg 2015;78:784.
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Figure Legend Fig. 1 Effect of ketamine on the release of HMGB1 in endothelial
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cells. A The chemical structure of ketamine. B Human endothelial cells were stimulated with LPS (100 ng/mL) for 16 h after treating the cell
monolayer with the indicated concentrations of ketamine for 6 h. The
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release of HMGB1 was measured by ELISA.C Effect of ketamine on
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cellular viability was measured by CCK-8 assay. All results are shown as means ± SD of three different experiments. *P<0.05 compared with the LPS-treated group, #P < 0.01 compared with the LPS -treated group.
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Fig. 2 Effect of ketamine on HMGB1-mediated barrier disruption in endothelial cells. Human endothelial cells were incubated with HMGB1 (1 µg/mL, for 16 h) to induce barrier disruption after treatment with
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various concentrations of ketamine for 6 h. Solute flux was monitored
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from the flux of Evans blue-bound albumin across human endothelial cells. All results are shown as the means ± SD of three different experiments. *P<0.05 compared with the HMGB1-treated group, #P < 0.01 compared with the HMGB1-treated group.
Fig. 3 Effect of ketamine on HMGB1-mediated CAM expression in endothelial cells. HMGB1-mediated (1µg/mL, for 16 h) expression of
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VCAM-1 (A), ICMA-1 (B) and E-selectin (C) in endothelial cells was analyzed after treating monolayers with indicated concentrations of ketamine for 6 h. All results are shown as the means ± SD of three
#
P < 0.01 compared with the HMGB1-treated group.
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different experiments. *P<0.05 compared with the HMGB1-treated group,
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Fig. 4 Effect of ketamine on HMGB1-mediated cell adhesion and
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TEM in endothelial cells. A HMGB1-mediated (1µg/mL, for 16 h) adherence of human neutrophils to endothelial cells monolayers was analyzed after treating endothelial cells with ketamine for 6 h. HMGB1-mediated (1µg/mL, for 16 h, B) or chemotactic agent mediated
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(fMLP, C) migration of human neutrophils through endothelial cells monolayers was analyzed after treating endothelial cells with ketamine. All results are shown as the means ± SD of three different experiments.
P < 0.01 compared with the HMGB1 (A, B) or fMLP (C)-treated group.
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#
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*P<0.05 compared with the HMGB1 (A, B) or fMLP (C)-treated group,
Fig. 5 Analysis of the HMGB1-mediated expression of pattern recognition receptors on endothelial cells. Human endothelial cells were incubated with HMGB1 (1µg/mL, for 16 h) with or without pretreating the cells with ketamine for 6 h. The expression of TLR2 (A), TLR4 (B) and RAGE (C) on endothelial cells was measured by a cell
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based ELISA. All results are shown as means ± SD of three different experiments. *P<0.05 compared with the HMGB1-treated group, #P <
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0.01 compared with the HMGB1-treated group.
Fig. 6 Effect of TLR2/4 and NF-κB inhibitors on HMGB1-induced overexpression of inflammatory mediators in endothelial cells.
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Human endothelial cells were pretreated with or without the anti-TLR2
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antibody (5 µg/mL), anti-TLR4 antibody (5 µg/mL), PDTC (80 µM) for 1 h before adding ketamine (Ket: 1000 µM) for 6 h, and subsequently treated with HMGB1 (1 µg/mL) for 16 h. The conditioned media were collected to measure the concentration of VCAM-1, ICAM-1, and
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E-selectin. All results are shown as means ± SD of three different experiments. *P<0.05 compared with the HMGB1-treated group, #P <
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group.
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0.05 compared with HMGB1, ketamine (Ket), and inhibitors -treated
Fig. 7 Effect of TLR2/4 and NF-κB siRNA on HMGB1-induced inflammatory mediators mRNA expression in endothelial cells. Human endothelial cells were transfected with siRNA for TLR2/4 and NF-κB p65 or control, and then incubated with ketamine (Ket: 1000 µM) for 6 h, and subsequently treated with HMGB1 (1 µg/mL) for 16 h. The expression of (A) VCAM-1 mRNA, (B) ICAM-1 mRNA and (C)
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E-selectin mRNA was assessed by qPCR following treatment with HMGB1. Data are expressed as mean ± SD from three independent experiments. *P<0.05 compared with the HMGB1-treated group, #P <
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0.05 compared with HMGB1, ketamine (Ket), and siRNA-treated group.
Fig. 8 Effect of TLR2/4 and NF-κB siRNA on HMGB1-induced
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barrier disruption, adherence and migration in endothelial cells. (A)
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Human endothelial cells were transfected with siRNA for TLR2/4 and NF-κB p65 or control, and then incubated with HMGB1 (1 µg/mL, for 16 h) to induce barrier disruption after treatment with ketamine (Ket: 1000 µM) for 6 h. Solute flux was monitored from the flux of Evans
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blue-bound albumin across human endothelial cells. Human endothelial cells were transfected with siRNA for TLR2/4 and NF-κB p65 or control, HMGB1-mediated (1µg/mL, for 16 h) adherence (B) or migration(C) of
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human neutrophils to endothelial cells monolayers was analyzed after
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treating endothelial cells with ketamine (Ket: 1000 µM) for 6 h. Data are expressed as mean ± SD from three independent experiments. *P<0.05 compared with the HMGB1-treated group, #P < 0.05 compared with HMGB1, ketamine (Ket), and siRNA-treated group.
Fig.9 Effect of ketamine on HMGB1-induced nuclear translocation of NF-κB p65 in endothelial cells. HUVECs were pretreated with ketamine
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(1000 µM) in the presence or absence of HMGB1 (1 µg/mL) for 1 h. The nuclear NF-κB translocation is visualized by immunofluorescence staining of NF-κB (green). F-actin and nuclei are labeled with phalloidin
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(red) and DAPI (blue), respectively. The enlarged, representative single cell images are shown in A and corresponding multi-cellular images are displayed in B. The enlarged cell is marked using a white arrow. All
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results are shown as means ± SD of three different experiments. *P<0.05
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HMGB1-treated group.
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compared with the HMGB1-treated group, #P < 0.01 compared with the
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S0022-4804(15)00871-9
DOI:
10.1016/j.jss.2015.08.032
Reference:
YJSRE 13501
To appear in:
Journal of Surgical Research
Received Date: 2 April 2015 Revised Date:
2 August 2015
Accepted Date: 19 August 2015
Please cite this article as: Liu Z, Wang Z, Han G, Huang L, Jiang J, Li S, Ketamine attenuates HMGB1induced inflammatory responses in endothelial cells, Journal of Surgical Research (2015), doi: 10.1016/ j.jss.2015.08.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ketamine attenuates HMGB1-induced inflammatory responses in endothelial cells Zhaohui Liu,M.D.,a,1 Zhengping Wang, Ph.D., b,1 Guangwei Han, M.D.,c
a
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Lina Huang, Ph.D.,c Jihong Jiang, Ph.D.,c and Shitong Li, M.D.c Department of Anesthesiology, Cangzhou Central Hospital, Hebei
Medical University, Hebei, China
Department of Anesthesiology, Shanghai Tenth People’s Hospital,
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b
c
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Tongji University School of Medicine, Shanghai, China
Department of Anesthesiology, Shanghai First People’s Hospital,
Shanghai Jiaotong University School of Medicine, Shanghai, China 1
These authors contributed equally to this study.
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Running title: Ketamine attenuates HMGB1-mediated inflammatory responses.
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Category: Sepsis
Author contributions Zhaohui Liu: conception, design, analysis and interpretation of data; writing the manuscript. Zhengping Wang: conception, design, analysis and interpretation of data; writing the manuscript. Guangwei Han: analysis and interpretation of data; data collection.
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Lina Huang: analysis and interpretation of data; data collection. Jihong Jiang: analysis and interpretation of data; data collection. Shitong Li: conception, design, analysis and interpretation of data; data
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collection; critical revision of the manuscript; statistical expertise; obtaining funding; supervision.
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Conflicts of Interest
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The authors report no proprietary or commercial interest in any product mentioned, or concept discussed, in this article.
Corresponding Author:
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Shitong Li,
No. 100 Haining Road, Shanghai, China Phone: +86 021 66307531
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FAX: +86 021 66301082
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Email: [email protected]
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Abstract Aim and objective High mobility group box-1 (HMGB1) acts as an inflammatory mediator and has been implicated in pathophysiological
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damage of vascular inflammatory diseases. Ketamine, an anesthetic agent with sedative and analgesic properties, has been shown to have potent anti-inflammatory effects in a variety of models of systemic inflammation.
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However, the effects of ketamine on HMGB1-mediated pro-inflammatory
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responses have not been fully investigated. In the present study, we investigated the effects of ketamine on HMGB1-activated endothelial cells and explored the underlying mechanisms.
Methods Human endothelial cells were incubated with or without
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HMGB1 (1 µg/mL) in the presence or absence of ketamine, an NF-κB inhibitor (PDTC), anti-TLR2/4 antibody, or small interfering RNA (si RNA). The anti-inflammatory activities of ketamine were determined by
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measuring solute flux, leukocyte adhesion and migration and activation of
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pro-inflammatory proteins in HMGB1-activated endothelial cells. The effect of ketamine on toll like receptor (TLR)-2/4 and NF-κB activation was evaluated using Enzyme-Linked Immunosorbent Assays, and Immunofluorescence Confocal Microscopy Assay. Results We found that ketamine inhibited the HMGB1-mediated barrier disruption, neutrophil adhesion and migration, and expression of cell adhesion molecules in a dose-dependent manner. Furthermore, ketamine
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down-regulated the toll like receptor (TLR)-2 and -4, expression in HMGB1-activated endothelial cells. Treatment with ketamine also significantly inhibited the activation of TLR2/4 and the nuclear
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translocation of NF-κB p50/p65. Furthermore, our study shows that the HMGB1-induced release of inflammatory mediators was suppressed by PDTC, anti-TLR2/4 antibody, and si RNA.
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Conclusion Our study has demonstrated that ketamine exerts anti
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-inflammatory effects in HMGB1-mediated pro-inflammatory responses in a dose-dependent manner. The mechanism responsible for these effects involves the TLR2/4 and NF-κB signaling pathway.
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Keywords Ketamine; HMGB1; endothelial cells; Inflammation; sepsis
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Introduction Systemic inflammation is one of the hallmarks of septic shock, and the microvascular endothelium provides an important site of regulation and
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amplification of this inflammatory responses[1]. Endothelial cell activation leads to alterations in hemostasis, increases in vascular permeability, cell swelling and loss of barrier function, leukocyte
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adherence with cell clumping, and microthrombi formation[2; 3].
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Microvascular injury is one of the characteristics of sepsis-associated tissue damage that may be manifested by single (eg, acute respiratory distress syndrome) or multiple organ failure syndromes[4]. Many inflammatory mediators of sepsis contribute to the development of an
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activated endothelium (eg, tumor necrosis factor-α [TNF-α] and interleukin 1β [IL-1β])[5]. Recently, high mobility group protein 1 (HMGB1) has been identified as a novel inflammatory cytokine and a
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late mediator of endotoxin lethality in mice[6].
High mobility group protein 1 (HMGB1), a secreted nuclear protein, is a potent mediator and associates with a variety of severe vascular inflammatory diseases such as sepsis and septic shock [6]. HMGB1, when nuclear, is involved in nucleosome stabilization and gene transcription[7]. Once released into extracellular milieu, HMGB1 can stimulate the secretion of pro-inflammatory cytokines from endothelial
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cells, monocytes, and macrophages, leading to a wide range of inflammatory responses [8; 9]. HMGB1 mediates cell signaling by binding to several transmembrane receptors, such as, receptor for
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advanced glycation end products (RAGE), toll like receptor (TLR)-2, and -4, and activates intracellular signal of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB[10-12]. High plasma levels of
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HMGB1 correlate with poor prognosis and increased mortality in patients
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with inflammatory diseases, and targeting HMGB1 with specific antagonists is protective in established preclinical inflammatory disease models including lethal endotoxemia or sepsis [13; 14]. Therefore, inhibiting HMGB1active release and/or blocking HMGB1
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pro-inflammatory activities could be a promising therapeutic target for vascular inflammatory diseases.
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Ketamine, an anesthetic agent with sedative and analgesic properties, has
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been shown to have potent anti-inflammatory effects in a variety of models of systemic inflammation, including endotoxemia, sepsis, ischemia, and burns[15-18]. These effects have been found in multiple organ systems and involve modulation of the molecular mediators of the inflammatory response, including transcription factors such as nuclear NF-κB and MAPKs[19-21]. Additionally, when given either before or after various pro-inflammatory insults, ketamine has been shown to
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diminish systemic production of cytokines, such as tumor necrosis factor-α (TNF-α), interleukins-1β (IL-1β), and interleukins-6(IL-6), and interferon-γ (IFN-γ), and improve survival[17; 20]. Furthermore, recent
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evidence suggests that ketamine improves end-organ dysfunction and has anti-inflammatory effects that appear to be present even at sub-anesthetic (sedative) doses[22; 23]. However, the anti-inflammatory activities of
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ketamine on HMGB1-mediated pro-inflammatory responses have not
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been well studied.
The present study addressed the question whether ketamine could attenuate HMGB1-induced pro-inflammatory response through inhibiting TLR2/4 expression and NF-κB activation, could protect vascular integrity
endothelial cells.
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and inhibit cell adhesion molecule expression in HMGB1-activated
Reagents
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Materials and methods
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Ketamine, Bacterial lipopolysaccharide (LPS, used at 100 ng/mL), Evans blue, crystal violet and fMLP (N-formyl-methionyl-leucyl-phenylalanine) were obtained from Sigma (St. Louis, MO, USA). Human recombinant HMGB1 was purchased from Invitrogen (Carlsbad, CA).
Cell culture
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Primary human umbilical vein endothelial cells (HUVECs) were obtained from the Typical Species Preservation Center of Wuhan University (Wuhan, Hubei, China) and maintained as described previously[24].
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Briefly, the cells were cultured to confluence at 37℃ and 5% CO2 in endothelial basal medium (EBM; Clonetics, Walkersville, MD)
supplemented with epidermal growth factor (10 ng/mL), hydrocortisone
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(1µg/mL), penicillin (100 U/mL), streptomycin (100 µg/mL),
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amphotericin (250 ng/mL), and 10% FCS. HUVECs of passage numbers 3 or 4 were used in the experiments. Freshly isolated neutrophils were kindly provided by Dr. Hui Liu (Shanghai Sixth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China). The
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human microvascular endothelial cell line HMEC-1, which were obtained from the Typical Species Preservation Center of Wuhan University (Wuhan, Hubei, China), is an SV-40-transformed microvascular
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endothelial cell line that retains the morphologic phenotype and
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functional characteristics of human microvascular endothelial cells[25; 26]. HMEC-1 cells were grown and maintained in endothelial basal medium supplemented with epidermal growth factor (10 ng/mL), hydrocortisone (1µg/mL), penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin (250 ng/mL), and 10% FCS. These cells were grown at 37℃ in a humidified 5% CO2 incubator and were subcultured
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at 50-80% confluence using 0.05% trypsin, 0.02% EDTA (Gibco, NY, USA).
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Measuring solute flux Solute flux was quantitated by spectrophotometric measurement of the flux of Evans blue-bound albumin across functional endothelial cells
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monolayers using a modified 2-compartment chamber model as
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previously described[27]. Briefly, Endothelial cells were plated (5× 104/well) in 3µm pore size and 12 mm diameter transwells for 3 days. The confluent monolayers were incubated with ketamine for 6 h followed by HMGB1 (1 µg/mL) for 16 h. Inserts were washed with PBS, pH 7.4,
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before adding 0.5 mL Evans blue (0.67 mg/mL) diluted in growth medium containing 4% BSA. Fresh growth medium was added to the lower chamber, and the medium in the upper chamber was replaced with
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Evans blue/BSA. After 10 min, the optical density at 650 nm was
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measured in the lower chamber. Experiments were performed in triplicate and repeated multiple times.
Cell viability assay Cell viability was determined using the Cell Counting Kit-8 assay (CCK-8; Beyotime, Jiangsu, China)[28-32]. Endothelial cells were grown in 96-well plates at a density of 5 × 103 cells/well. After 24 h, cells were
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washed with fresh medium and then treated with ketamine. After 24 h incubation, cells were rewashed and 100 µL of CCK-8reagent was added and incubated for 4 h. The absorbance of the solution was measured using
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a microplate reader (Bio-Rad Laboratories, Hercules, CA) at a test wavelength of 450 nm and a reference wavelength of 630 nm.
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Cell-cell adhesion assay
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Adherence of human neutrophils to endothelial cells was evaluated by fluorescent labeling of neutrophils as described previously [33]. Briefly, neutrophils were labeled with Vybrant DiD. Following two washes, cells were resuspended in adhesion medium (RPMI containing 2% FBS and 20
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mM HEPES) and added to confluent monolayers of endothelial cells in 96-well plates that were treated for 6 h with ketamine followed by HMGB1 (1 µg/mL for 16 h). Non-adherent neutrophils were washed off
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and the fluorescence of the adherent cells was measured. The percentage
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of adherent neutrophils was calculated by the formula: % adherence = (adherent signal / total signal) × 100[33].
Migration assay
Migration assays were performed in transwell plates. Endothelial cells were cultured for 3 days to obtain confluent endothelial monolayers. The cell monolayers were treated for 6 h with ketamine followed by HMGB1
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(1 µg/mL for 16 h) or fMLP (10-8 M for 2 h) and freshly isolated neutrophils were added to the upper compartment. After transwell plates were incubated for 2 h, neutrophils on the lower side of the filter were
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fixed with 8% glutaraldehyde and stained with 0.25% crystal violet in 20% methanol (w/v). Each experiment was repeated in duplicate wells
and, within each well, nine randomly selected microscopic high power
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fields (HPF, ×200) were counted and expressed as the migration index.
Expression of cell surface receptors
The expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin on endothelial
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cells was determined by whole-cell ELISA as described previously[34]. Briefly, confluent monolayers of endothelial cells were treated with ketamine for 6 h followed by HMGB1 (1 µg/mL) for 16 h. The medium
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was removed, and the cells were washed with PBS and fixed by adding
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50 µL of 1% paraformaldehyde for 15 min at room temperature. After washing, 100µL of mouse anti-human monoclonal antibodies (VCAM-1, ICAM-1, E-selectin; Sigma, CA, USA) were added. After 1 h (37℃, 5% CO2), the cells were washed three times and then 100µL of 1:2000 peroxidase conjugated anti-mouse IgG antibodies (Sigma) were added for 1 h. The cells were washed again three times and developed using o-phenylenediamine substrate (Sigma). Colorimetric analysis was
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performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells. The same experimental procedures were used to monitor the cell surface expression of TLR2, TLR4 and RAGE
Biotechnology Inc. (Santa Cruz, CA, USA).
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RNA interference
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receptors using specific antibodies obtained from Santa Cruz
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The expression of inflammatory mediators by endothelial cells in response to HMGB1 (1 µg/mL for 16 h) was evaluated following the knockdown of TLR2, TLR4 and NF-κB expression by pools of target-specific 20- to 25-nucleotide siRNAs obtained from Santa Cruz
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Biotechnology Inc. according to the manufacturer’s instruction and as described previously [27]. A non-targeting 20- to 25-nucleotide siRNA
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obtained from the same company was used as a negative control.
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Quantitative real-time polymerase chain reaction (qPCR) analysis Total RNA was extracted from endothelial cells using Trizol (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s instructions, and reverse-transcribed with MMLV reverse transcriptase (Promega, Madison, WI). Quantitative real-time polymerase chain reaction (qPCR) was performed using a LightCycler 2.0 Real-Time PCR System (Roche Applied Science, Indianapolis, IN). cDNA was amplified using specific
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primers for inflammatory mediators gene expression and the results were normalized to β-actin gene expression. The relative mean fold-change of inflammatory mediators gene expression in the experimental group was
Immunofluorescent staining for NF-κB p65
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calculated using the 2△△ Ct method and compared to the control group[35].
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To visually identify the translocation of nuclear NF-κB p65 translocation,
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HUVECs were plated on gelatin-coated glass-bottom microwell plates (MatTek, MA, USA) and cultured until confluence. After appropriate treatment, the cells were fixed and permeabilized for 15 min at room temperature in PBS with 3.7% formaldehyde and 0.5% Triton X-100.
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Then cells were washed in PBS twice, blocked in 5% BSA for 1 h, and incubated with NF-κB antibody (1:50) at 4 ºC overnight. After a thorough wash in PBS, the cells were stained with an FITC-conjugated secondary
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antibody (1:200) and conjugated rhodamine-phalloidin (1:100) for 1 h.
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Cells were further incubated with diamidino-2-phenylindole (DAPI, 1:1000) for 15 min and then washed with PBS again. Rhodamin-phalloidin and DAPI were used to stain F-actin and nuclear DNA, respectively, to reveal the location of the cytoplasm and nucleus in the HUVECs. The staining results were imaged using a Zeiss LSM780 laser confocal scanning microscope (Zeiss, Germany).
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Statistical analysis Results are expressed as the mean ± the standard deviation (S.D.) of at least three independent experiments. The statistical significance of
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differences between test groups was used for statistical comparison (SPSS, version 14.0, SPSS Science, Chicago, IL, USA). Statistical relevance was determined by analysis of variance (ANOVA) with
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P<0.05.
Results
Effect of ketamine on LPS-mediated HMGB1 release in endothelial cells
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Previous studies have demonstrated that LPS stimulates HMGB1 release in murine macrophages and human endothelial cells[21; 34]. Following its release to intravascular spaces, HMGB1 is known to interact with
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specific cell surface receptors to amplify inflammatory responses by
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inducing the expression of pro-inflammatory cytokines[9]. In agreement with previous results, LPS (100 ng/mL) stimulated HMGB1 release by endothelial cells (Fig. 1C). To investigate the effect of ketamine on LPS-mediated HMGB1 release, endothelial cells were pretreated with increasing concentrations of ketamine for 6 h before stimulation with 100 ng/mL LPS for 16 h. The results presented in Fig. 1C indicated that ketamine inhibits HMGB1 release by endothelial cells, with optimal
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effect occurring at a concentration above 100 µM ketamine. To exclude the possibility that the inhibition of HMGB1 release was due to cytotoxicity caused by ketamine, cellular viability assays were performed
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in endothelial cells treated with ketamine for 24 h. At the concentrations used (up to 1000 µM), ketamine did not affect cell viability (Fig. 1B).
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Effect of ketamine on HMGB1-mediated barrier disruption in
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endothelial cells
To determine the effects of ketamine on barrier integrity in endothelial cells, solute flux was measured. Ketamine (up to 1000 µM) alone did not alter barrier integrity (Fig. 2). HMGB1 is known to cleave and disrupt
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barrier integrity[36]. Human endothelial cells were treated with various concentrations of ketamine for 6 h before adding HMGB1 (1 µg/mL). As shown in Fig. 2, ketamine decreased HMGB1-mediated membrane
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disruption in a dose-dependent manner.
Effects of ketamine on HMGB1-mediated cell adhesion molecule (CAM) expression, human neutrophil adhesion and migration in endothelial cells
Previous reports showed that HMGB1mediates inflammatory responses by increasing the cell surface expression of the cell adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of endothelial cells,
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thereby promoting adhesion and migration of leukocytes across the endothelium and to sites of inflamed tissues [5, 7, 28, 29]. Therefore, we tested whether ketamine could modulate HMGB1-mediated CAM
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expression. As presented in Fig. 3, HMGB1 up-regulated the cell surface expression of all three adhesion molecules (CAMs) and ketamine
inhibited these effects of HMGB1 in a concentration-dependent manner.
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The elevated expression of CAMs correlated well with enhanced binding
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of human neutrophils to HMGB1-activated endothelial cells and their subsequent migration. Ketamine down-regulated the adherence of human neutrophils to HMGB1-activated endothelial cells and their subsequent migration in a concentration-dependent manner (Fig. 4A, B), as well as to
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the chemotactic agent fMLP (Fig. 4C).
Ketamine down-regulates the expression of HMGB1 receptors in
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endothelial cells
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We next investigated the effect of HMGB1 on the stimulation of its own receptors and the effect of ketamine on the modulation of the expression of these receptors on endothelial cells. As shown in Fig. 5A-C, HMGB1 induced the expression of all three receptors, TLR2, TLR4 and RAGE, in endothelial cells, and ketamine significantly inhibited the stimulatory effect of HMGB1 on TLR2 and TLR4 but not RAGE.
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The TLR2/4 and NF-κB p65 inhibitor suppresses HMGB1-induced release of inflammatory mediators in endothelial cells To evaluate whether ketamine inhibited the inflammatory responses
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through TLR2/4 and NF-κB p65, endothelial cells were pretreated with or without anti-TLR2 antibody (5 µg/mL), anti-TLR4 antibody (5 µg/mL), PDTC (80 µM) for 1 h before adding ketamine (1000 µM) for 6 h, and
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then cells were stimulated with HMGB1 (1 µg/mL) for 16 h. The TLR2/4
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and NF-κB p65 inhibitor and ketamine all partially suppressed LPS-induced overexpression of ICAM-1, VCAM-1, and E-selectin (*P < 0.05, Fig. 6A-C). Furthermore, pretreating human endothelial cells with a combination of anti-TLR2/4 antibody/ PDTC and ketamine
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synergistically reversed the HMGB1-induced expression of ICAM-1, VCAM-1, and E-selectin (*P < 0.05, Fig. 6A-C). The results suggest that TLR2/4 and NF-κB p65 may be involved in ketamine’s reduction of
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HMGB1-induced inflammation in human endothelial cells.
TLR2/4 and NF-κB p65 siRNA suppresses HMGB1-induced expression of inflammatory mediators in endothelial cells We further confirmed the results by knocking down TLR2/4 and NF-κB p65 in endothelial cells using specific siRNA of TLR2/4 and NF-κB p65. We found that silencing TLR2/4 and NF-κB p65 with siRNA inhibited the expression levels of ICAM-1, VCAM-1, and E-selectin mRNA in
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HMGB1-stimulated endothelial cells (*P < 0.05, Fig. 7A-C) and the ketamine treatment strongly increased the inhibition effect of siRNA (#P
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< 0.05, Fig. 7A-C).
TLR2/4 and NF-κB p65 siRNA suppresses HMGB1-induced barrier disruption and human neutrophil adhesion and migration in
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endothelial cells
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To evaluate whether ketamine inhibited the barrier disruption and human neutrophil adhesion and migration through TLR2/4 and NF-κB p65, endothelial cells were knocked down for TLR2/4 and NF-κB p65 using specific siRNA before adding ketamine (1000 µM) for 6 h, and then cells
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were stimulated with HMGB1 (1 µg/mL) for 16 h. We found that silencing TLR2/4 and NF-κB p65 with siRNA inhibited the membrane disruption and human neutrophil adhesion and migration in
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HMGB1-stimulated endothelial cells (*P < 0.05, Fig. 8A-C) and the
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ketamine treatment strongly increased the inhibition effect of siRNA (#P < 0.05, Fig. 8A-C).
Ketamine attenuates HMGB1-induced nuclear translocation of NF-κB p65 in endothelial cells To evaluate whether ketamine inhibited the inflammatory responses through NF-κB in endothelial cells, nuclear translocation of NF-κB p65
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was observed using immunofluorescent staining. The visualizing images of NF-κB nuclear translocation using a confocal microscopic showed that, under control conditions, NF-κB (green staining) is located in the
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cytoplasmic region, and the nuclear area is stained with almost pure blue (DAPI), indicating little NF-κB in nuclei. NF-κB p65 nuclear
translocation in endothelial cells was demonstrated by the indigo color in
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the nuclear area after application of HMGB1 (Fig. 9). The
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HMGB1-induced NF-κB p65 translocation from the cytoplasm to the nucleus was decreased significantly by ketamine.
Discussion
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Because HMGB1 is a critical pro-inflammatory cytokine involved in inflammatory disorders, and TLR2/4 and NF-κB p65 are widely involved in regulating the production of a variety of inflammatory cytokines, we
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hypothesized that drugs blocking the TLR2/4 and NF-κB p65 signaling
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pathway might play a protective role in the HMGB1-mediated inflammatory processes through a variety of mechanisms. The present results confirmed that ketamine significantly inhibited the release of HMGB1 by lipopolysaccharide (LPS) and also suppressed the HMGB1-mediated barrier disruption, neutrophil adhesion and migration, and expression of cell adhesion molecules in HMGB1-activated endothelial cells in vitro. Cell viability was not affected, indicating that
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treatment with ketamine was anti-inflammatory rather than cytotoxic. The data also suggest that the inhibitory effects of ketamine occur via inhibition of TLR2/4 and NF-κB p65 signaling pathways. Therefore, the
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inhibition of HMGB1-activated inflammatory responses shown in this study suggests that ketamine could be beneficial in the treatment of
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vascular inflammatory diseases.
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However, the exact mechanism of the anti-inflammatory effects of ketamine on HMGB1-mediated inflammatory responses has not been fully clarified. Recent studies have shown that the inhibitory effects of ketamine on CLP-mediated inflammatory responses involve the TLR4
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signaling pathway [19; 20]. Previous studies have confirmed that the TLR4 signaling pathway regulates HMGB1-mediated inflammatory responses [11]. Thus, our study supports the hypothesis that ketamine
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inhibits the TLR4 signaling pathway in a dose-dependent manner, which
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regulates the HMGB1-mediated pro-inflammatory responses. Previous studies have also confirmed that p38 MAPK[37] are involved in regulating the release of inflammatory cytokines by the TLR4 signaling pathway. Therefore, ketamine might act through p38 MAPK, and other upstream pathways that regulate HMGB1-mediated inflammatory responses.
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Ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist, is widely used as an intravenous anesthetic agent[38]. Among nonvolatile anesthetics, ketamine has more stable hemodynamics than other
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anesthetic agents; it is used in various clinical areas by systemic treatment, including pediatric anesthesia, traumatic anesthesia, and obstetrics; and is quite widely serviceable in operating rooms and intensive care units[39].
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The side effects of ketamine systemic treatment include behavioral and
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psychological changes when used a large dose of ketamine[38].
Ketamine has been shown to have a protective effect on various cell types involved in the inflammation process [21; 40-44]. Our study only focused
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on endothelial cells, one of the more commonly used cells in the study of the inflammation process. Additional study of the role of HMGB1 in other types of immune cells under similar pathologic conditions is
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required. We also need to study whether the protective effects of
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ketamine on the pathologic complications related to sepsis involves the inhibition of HMGB1. Furthermore, the effect of ketamine on upstream signaling pathways that regulate HMGB1-mediated inflammatory responses, such as p38 MAPK, needs to be investigated.
Our study has confirmed that ketamine, as an anti-inflammatory drug, inhibits HMGB1-mediated pro-inflammatory responses by increasing
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barrier integrity and inhibiting CAM expression. These effects were achieved partially by inhibiting the TLR2/4 and NF-κB p65 signaling pathway. Our findings will help us better understand the
clinical treatments of vascular inflammatory disease.
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Conflict of interest
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anti-inflammatory mechanisms of ketamine and also help to explore new
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The authors declare that there are no conflicts of interest.
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Figure Legend Fig. 1 Effect of ketamine on the release of HMGB1 in endothelial
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cells. A The chemical structure of ketamine. B Human endothelial cells were stimulated with LPS (100 ng/mL) for 16 h after treating the cell
monolayer with the indicated concentrations of ketamine for 6 h. The
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release of HMGB1 was measured by ELISA.C Effect of ketamine on
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cellular viability was measured by CCK-8 assay. All results are shown as means ± SD of three different experiments. *P<0.05 compared with the LPS-treated group, #P < 0.01 compared with the LPS -treated group.
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Fig. 2 Effect of ketamine on HMGB1-mediated barrier disruption in endothelial cells. Human endothelial cells were incubated with HMGB1 (1 µg/mL, for 16 h) to induce barrier disruption after treatment with
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various concentrations of ketamine for 6 h. Solute flux was monitored
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from the flux of Evans blue-bound albumin across human endothelial cells. All results are shown as the means ± SD of three different experiments. *P<0.05 compared with the HMGB1-treated group, #P < 0.01 compared with the HMGB1-treated group.
Fig. 3 Effect of ketamine on HMGB1-mediated CAM expression in endothelial cells. HMGB1-mediated (1µg/mL, for 16 h) expression of
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VCAM-1 (A), ICMA-1 (B) and E-selectin (C) in endothelial cells was analyzed after treating monolayers with indicated concentrations of ketamine for 6 h. All results are shown as the means ± SD of three
#
P < 0.01 compared with the HMGB1-treated group.
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different experiments. *P<0.05 compared with the HMGB1-treated group,
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Fig. 4 Effect of ketamine on HMGB1-mediated cell adhesion and
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TEM in endothelial cells. A HMGB1-mediated (1µg/mL, for 16 h) adherence of human neutrophils to endothelial cells monolayers was analyzed after treating endothelial cells with ketamine for 6 h. HMGB1-mediated (1µg/mL, for 16 h, B) or chemotactic agent mediated
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(fMLP, C) migration of human neutrophils through endothelial cells monolayers was analyzed after treating endothelial cells with ketamine. All results are shown as the means ± SD of three different experiments.
P < 0.01 compared with the HMGB1 (A, B) or fMLP (C)-treated group.
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#
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*P<0.05 compared with the HMGB1 (A, B) or fMLP (C)-treated group,
Fig. 5 Analysis of the HMGB1-mediated expression of pattern recognition receptors on endothelial cells. Human endothelial cells were incubated with HMGB1 (1µg/mL, for 16 h) with or without pretreating the cells with ketamine for 6 h. The expression of TLR2 (A), TLR4 (B) and RAGE (C) on endothelial cells was measured by a cell
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based ELISA. All results are shown as means ± SD of three different experiments. *P<0.05 compared with the HMGB1-treated group, #P <
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0.01 compared with the HMGB1-treated group.
Fig. 6 Effect of TLR2/4 and NF-κB inhibitors on HMGB1-induced overexpression of inflammatory mediators in endothelial cells.
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Human endothelial cells were pretreated with or without the anti-TLR2
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antibody (5 µg/mL), anti-TLR4 antibody (5 µg/mL), PDTC (80 µM) for 1 h before adding ketamine (Ket: 1000 µM) for 6 h, and subsequently treated with HMGB1 (1 µg/mL) for 16 h. The conditioned media were collected to measure the concentration of VCAM-1, ICAM-1, and
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E-selectin. All results are shown as means ± SD of three different experiments. *P<0.05 compared with the HMGB1-treated group, #P <
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group.
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0.05 compared with HMGB1, ketamine (Ket), and inhibitors -treated
Fig. 7 Effect of TLR2/4 and NF-κB siRNA on HMGB1-induced inflammatory mediators mRNA expression in endothelial cells. Human endothelial cells were transfected with siRNA for TLR2/4 and NF-κB p65 or control, and then incubated with ketamine (Ket: 1000 µM) for 6 h, and subsequently treated with HMGB1 (1 µg/mL) for 16 h. The expression of (A) VCAM-1 mRNA, (B) ICAM-1 mRNA and (C)
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E-selectin mRNA was assessed by qPCR following treatment with HMGB1. Data are expressed as mean ± SD from three independent experiments. *P<0.05 compared with the HMGB1-treated group, #P <
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0.05 compared with HMGB1, ketamine (Ket), and siRNA-treated group.
Fig. 8 Effect of TLR2/4 and NF-κB siRNA on HMGB1-induced
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barrier disruption, adherence and migration in endothelial cells. (A)
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Human endothelial cells were transfected with siRNA for TLR2/4 and NF-κB p65 or control, and then incubated with HMGB1 (1 µg/mL, for 16 h) to induce barrier disruption after treatment with ketamine (Ket: 1000 µM) for 6 h. Solute flux was monitored from the flux of Evans
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blue-bound albumin across human endothelial cells. Human endothelial cells were transfected with siRNA for TLR2/4 and NF-κB p65 or control, HMGB1-mediated (1µg/mL, for 16 h) adherence (B) or migration(C) of
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human neutrophils to endothelial cells monolayers was analyzed after
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treating endothelial cells with ketamine (Ket: 1000 µM) for 6 h. Data are expressed as mean ± SD from three independent experiments. *P<0.05 compared with the HMGB1-treated group, #P < 0.05 compared with HMGB1, ketamine (Ket), and siRNA-treated group.
Fig.9 Effect of ketamine on HMGB1-induced nuclear translocation of NF-κB p65 in endothelial cells. HUVECs were pretreated with ketamine
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(1000 µM) in the presence or absence of HMGB1 (1 µg/mL) for 1 h. The nuclear NF-κB translocation is visualized by immunofluorescence staining of NF-κB (green). F-actin and nuclei are labeled with phalloidin
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(red) and DAPI (blue), respectively. The enlarged, representative single cell images are shown in A and corresponding multi-cellular images are displayed in B. The enlarged cell is marked using a white arrow. All
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results are shown as means ± SD of three different experiments. *P<0.05
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compared with the HMGB1-treated group, #P < 0.01 compared with the
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