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Magnesium sulfate inhibits the secretion of high mobility group box 1 from lipopolysaccharide-activated RAW264.7 macrophages in vitro
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Magnesium sulfate inhibits the secretion of high mobility group box 1 from lipopolysaccharide-activated RAW264.7 macrophages in vitro Zhaohui Liu, MD,a Junjie Zhang, PhD,a Xiaojing Huang, MD,b Lina Huang, PhD,a Shitong Li, MD,a and Zhengping Wang, MDb,* a b
Department of Anesthesiology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Department of Pain, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
article info
abstract
Article history:
Background: High mobility group box 1 (HMGB1) is an important inflammatory factor that is
Received 3 December 2011
closely related to mortality in patients with sepsis. High magnesium therapy has been
Received in revised form
proved to reduce sepsis-related mortality and sepsis-induced pathologic complications.
5 February 2012
These effects result from reduced expression and release of many inflammatory cytokines,
Accepted 7 February 2012
although it is not clear whether high magnesium affects the expression and release of
Available online 28 March 2012
HMGB1. In the present study, we explored the effect of magnesium sulfate on the expression and release of HMGB1 in lipopolysaccharide (LPS)-activated macrophages.
Keywords:
Methods: RAW264.7 cells were incubated with LPS in the presence or absence of various
Magnesium sulfate
concentrations of magnesium sulfate. An enzyme-linked immunosorbent assay was used
High mobility group box 1
to detect the levels of HMGB1 in the culture supernatant. Real-time polymerase chain
HMGB1 protein
reaction was used to assess the expression of HMGB1 mRNA. The nuclear/cytoplasm
Lipopolysaccharides
extraction kit was used to extract the nuclear and cytoplasmic proteins. Western blotting
NF-kB
was used to observe the changes in the translocation of HMGB1 from the nucleus to the cytoplasm. The nuclear factor (NF)-kB p50/p65 Transcription Factor Assay Kit was used to analyze NF-kB activity in the nuclear extract. Results: We found that magnesium sulfate inhibited translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular release in LPS-activated macrophages and also suppressed the expression of HMGB1 mRNA. Furthermore, magnesium sulfate inhibited the translocation of NF-kB from the cytoplasm to the nucleus in LPS-activated macrophages in a dose-dependent manner. Conclusions: Our study has demonstrated that magnesium sulfate inhibits the translocation of HMGB1 from the nucleus to the cytoplasm and the expression of HMGB1 mRNA in a dose-dependent manner. The mechanism responsible for these effects involves the NF-kB signaling pathway. Crown Copyright ª 2013 Published by Elsevier Inc. All rights reserved.
* Corresponding author. Department of Pain, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, No. 100 Haining Road, Hongkou District, Shanghai, China. Tel.: þ86 21 6324 0090, ext. 3022; fax: þ86 21 6324 0825. E-mail address: [email protected] (Z. Wang). 0022-4804/$ e see front matter Crown Copyright ª 2013 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.02.012
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1.
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Introduction
The pathogenesis of sepsis results from a systemic inflammatory response caused by infection, which is characterized by the excessive release of early pro-inflammatory cytokines (e.g., tumor necrosis factor-a, interleukin -1, interleukin-6) and late pro-inflammatory cytokines (e.g., high mobility group box 1 [HMGB1]). The excessive generation of these cytokines can lead to very serious pathologic consequences, including systemic capillary leak syndrome, tissue damage, and lethal multiple organ failure [1,2]. Therefore, the suppression of the excessive release of proinflammatory cytokines provides a new therapy option for the treatment of sepsis. As a nonhistone chromosomal protein, HMGB1 was originally described as an important regulator of genetic information; however, Wang et al. [3] subsequently found that HMGB1 is a lethal inflammatory cytokine involved with sepsis. HMGB1 can be actively released into the extracellular environment from immune cells, including macrophages, monocytes, natural killer cells, dendritic cells, endothelial cells, and platelets [3e6]. HMGB1 functions as a stimulus by binding receptors (receptor for advanced glycation end products, toll-like receptors 2 and 4) to stimulate the release of early proinflammatory cytokines (tumor necrosis factor-a, IL-1b, and IL-8) and elicit various inflammatory responses [7]. The administration of anti-HMGB1 antibodies or inhibitors significantly improved survival in septic rats and reduced multiple organ damage [8,9]; moreover, such antibodies had a good clinical effect in patients with septic shock [10]. Magnesium sulfate, a commonly used antiepileptic drug [11], has strong anti-inflammatory properties [12,13]. Magnesium sulfate reduces sepsis-related acute lung injury [14] and has a protective effect on ischemiaereperfusion injury [15,16]. Activation of a variety of immune cells (macrophages, lymphocytes, and neutrophils) was significantly inhibited by magnesium sulfate [13,17]. Recent data have confirmed that magnesium sulfate inhibits a variety of inflammatory molecules, including chemokines, cytokines, nitric oxide, and prostaglandin E2 [13]. These anti-inflammatory effects of magnesium sulfate are produced by inhibiting nuclear factor (NF)-kB activation and Ca2þ channel opening. However, the role of magnesium sulfate on HMGB1 expression and activity has not been discussed. Previous data have indicated that RAW264.7 cells, an immortalized murine macrophage-like cell line, are often used as alternatives for macrophages, because they readily express inflammatory molecules on stimulation by endotoxin [18,19]. It has been previously reported that lipopolysaccharide (LPS)-activated macrophages actively secrete HMGB1. Because overexpression and release of HMGB1 occurs in various inflammatory diseases, and because magnesium sulfate has an inhibitory effect on the NF-kB signaling pathway, we hypothesized that magnesium sulfate might inhibit overexpression of HMGB1 during the inflammatory process. Therefore, in the present study, we investigated whether magnesium sulfate inhibits the expression and release of HMGB1 from LPS-activated macrophages and explored the mechanisms of these antiinflammatory effects.
2.
Methods
2.1.
Cell culture and stimulation
Murine macrophage-like RAW264.7 cells (Shanghai Institute of Cell Biology, The Chinese Academy of Sciences, Shanghai, China) were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 100 U/mL penicillin, and 100 mg/mL streptomycin in an atmosphere of humidified 5% carbon dioxide at 37 C. At 80e90% confluence, the cells were washed 3 times and then transferred to 6-well polystyrene culture plates at 1 106 cells/ well in 2 mL medium/well. After overnight incubation, the medium was removed and replaced with Roswell Park Memorial Institute 1640 medium containing 0.25% fetal calf serum (for experiments designed to measure HMGB1 in conditioned media). The RAW264.7 cells were incubated with LPS (500 ng/mL) in the absence or presence of graded concentrations of magnesium sulfate (1, 5, and 10 mmol/L). Cell-free supernatants were collected after 24 h of stimulation for determination of the HMGB1 levels. Total RNA was also extracted for determination of HMGB1 mRNA levels. The HMGB1 levels in the cytoplasm and nucleus were assayed by Western blot analysis to detect the intracellular translocation of HMGB1. The nuclear proteins of RAW264.7 cells were used to measure the NF-kB activity with the NF-kB p50/p65 transcription factor assay kit. For cell viability analysis, RAW264.7 cells were incubated with graded concentrations of magnesium sulfate. Cells cultured in media alone acted as a negative control in all tests.
2.2.
Enzyme-linked immunosorbent assay
The levels of HMGB1 in the culture medium were determined using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) and were performed according to the manufacturer’s instructions.
2.3.
Cell counting kit-8 assay
The viability of RAW264.7 cells was determined using the Cell Counting Kit-8 Assay Kit (Beyotime, Jiangsu, China), as previously reported. RAW264.7 cells were plated at a density of 104 cells/well in 96-well plates in 100 mL Roswell Park Memorial Institute 1640 medium. Next, 20 mL cell counting kit-8 assay was added to RAW264.7 cells in each microwell and incubated for 2 h at 37 C at the end of the 24-h incubation period. The absorbance of the colored solution was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA) at a test wavelength of 450 nm and a reference wavelength of 630 nm.
2.4.
Extraction of cytoplasmic and nuclear proteins
At 24 h after treatment, the RAW264.7 cells were harvested and washed 3 times with cold phosphate-buffered saline (PBS). The cytoplasmic and nuclear protein fractions were extracted using NE-PER extraction reagents according to the
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manufacturer’s protocol (Pierce Biotechnology, Rockford, IL). Cytoplasmic and nuclear protein extracts were used for Western blot analysis.
2.5.
Western blot analysis
The levels of HMGB1 in the cytoplasm and nucleus were assayed by Western blot analysis. The proteins were quantified using the Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China). The samples were denatured at 100 C for 5 min before adding dithiothreitol. An equal amount of protein was loaded in each well for electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto polyvinyldine fluoride microporous membranes (Millipore, Bedford, MA). Membranes containing the transferred proteins were blocked with PBS containing 0.1% Tween 20 (PBS-T) and 5% skim milk for 1 h at room temperature. After 3 washes with PBS-T, the membranes were then incubated with rabbit anti-HMGB1 polyclonal antibody (1:300; Abcam, San Diego, CA), anti-b-actin antibody (1:1,000; Santa Cruz Technology, Santa Cruz, CA), and anti-histone H3.1 antibody (1:1,000; SAB, Pearland, TX) for 1 h at room temperature. After 3 washes with PBS-T, the membranes were incubated with horseradish peroxidase-linked secondary antibodies (1:1,000; Golden Bridge, Beijing, China) for 1 h at room temperature. After 3 final washes in PBS-T and 2 washes in PBS, the membranes were impregnated with the ECL reagents (Amersham, Buckinghamshire, UK) and then exposed digitally with Image Reader LAS-4000 (FujiFilm, Tokyo, Japan) and quantified for statistical analysis using Multi Gauge, version 3.0, software (FujiFilm Life Science, Tokyo, Japan).
2.7.
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NF-kB binding assay
The DNA binding activity of NF-kB (p50/p65) was determined using the enzyme-linked immunosorbent assay-based nonradioactive NF-kB p50/p65 Transcription Factor Assay Kit (Chemicon, Temecula, CA), according to the manufacturer’s instructions. The absorbance at 450 nm was analyzed by an automated plate reader (Bio-Rad Laboratories).
2.8.
Statistical analysis
All data are presented as the mean SD of the results obtained from 3 replicates. Differences between each group were assessed by 1-way analysis of variance followed by NewmanKeuls tests. P < 0.05 was considered statistically significant. All data were analyzed using SPSS, version 13.0, statistical software (SPSS, Chicago, IL).
3.
Results
3.1. Effect of magnesium sulfate on HMGB1 levels secreted by cultured RAW264.7 cells after treatment with LPSs
Real-time polymerase chain reaction analysis
The supernatant was collected 24 h after LPS stimulation to determine the release of HMGB1 by enzyme-linked immunosorbent assay. We found that the HMGB1 levels in the culture supernatant increased after the administration of LPSs (500 ng/mL); however, the administration of magnesium sulfate significantly inhibited the release of HMGB1 in a dosedependent manner (Fig. 1).
Total RNA was extracted from treated RAW264.7 cells by adding TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. First-strand cDNA was synthesized using 2 mg total cellular RNA as the template, 1 mL of 10 mM deoxyribonucleotide triphosphate mix, 5 mL of Moloney-murine leukemia virus 5 reaction buffer (with Mg2þ), 0.625 mL Recombinant RNasin, and 1 mL Moloneymurine leukemia virus (Promega, Madison, WI), in a final volume of 25 mL. Quantitative real-time polymerase chain reaction (PCR) was performed on a LightCycler 2.0 Real-Time PCR System (Roche Applied Science, Indianapolis, IN). The reaction contained 1 mL of the cDNA template, 2 mL of the forward primer, and 2 mL of the reverse primer (HMGB1 forward, CAC CGT GGG ACT ATT AGG AT; HMGB1 reverse, GCT CAC ACT TTT GGG GAT AC; b-actin forward, CCT CTA TGC CAA CAC AGT; b-actin reverse, AGC CAC CAA TCC ACA CAG), and 10 mL PCR Premix in a total volume of 20 mL. The PCR reaction involved initial denaturation (95 C, 5 min), followed by 40 cycles of denaturation (95 C, 5 s) and extension (60 C, 30 s), and then denaturation (95 C, 15 s), extension (72 C, 10 s), and a final denaturation (95 C, 15 s). The real-time PCR assays were conducted in triplicate for each sample to ensure experimental accuracy. The mean fold-change in the expression of HMGB1 mRNA in the experimental group compared with the control group was calculated using the 2DDCt method.
Fig. 1 e Effect of magnesium sulfate at different concentrations on HMGB1 release from RAW264.7 cells after 24 h stimulation with LPS. RAW264.7 cells were stimulated without or with LPSs (500 ng/mL) in the absence or presence of magnesium sulfate (1, 5, and 10 mmol/L) for 24 h. Culture supernatants were collected and assayed for HMGB1 with an enzyme-linked immunosorbent assay kit. Mean values ± SD (n [ 3). *P < 0.05 compared with the LPS-treated group; yP < 0.01 compared with the LPS-treated group. ND [ not detectable.
2.6.
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3.2. Effect of magnesium sulfate on viability of RAW264.7 cells RAW264.7 cell viability after treatment with graded magnesium sulfate was measured using the Cell Counting Kit-8 assay. The results showed that increasing concentrations of magnesium sulfate did not have a significant effect on RAW264.7 cell viability (Fig. 2).
3.3. Effect of magnesium sulfate on HMGB1 mRNA expression To gain additional insight into the mechanism responsible for the inhibition of magnesium sulfate on HMGB1 release, we next tested the effect of magnesium sulfate on HMGB1 transcription. We found that magnesium sulfate inhibited the LPS-induced increase in HMGB1 mRNA expression at the transcriptional level. The data indicated that the changes in HMGB1 levels in the cell culture supernatant are consistent with the changes in intracellular levels of HMGB1 mRNA expression (Fig. 3).
Fig. 3 e Effect of magnesium sulfate at different concentrations on HMGB1 mRNA expression in RAW264.7 cells after 24 h stimulation with LPSs. RAW264.7 cells were stimulated with or without LPS (500 ng/mL) in the absence or presence of magnesium sulfate (1, 5, and 10 mmol/L) for 24 h. Total RNA of RAW264.7 cells was extracted and assayed for HMGB1 mRNA using real-time PCR. Mean values ± SD (n [ 3). *P < 0.05 compared with LPS-treated group, yP < 0.01 compared with the LPS-treated group.
3.4. Effect of magnesium sulfate on translocation of HMGB1 from nucleus to cytoplasm
3.5. Effect of magnesium sulfate on LPS-induced increase in NF-kB activity
HMGB1 protein levels in the nucleus were significantly reduced after LPS stimulation. However, the addition of magnesium sulfate significantly reversed this effect. Likewise, the increased HMGB1 protein levels in the cytoplasm after LPS stimulation were reduced by treatment with magnesium sulfate. Thus, the addition of magnesium sulfate caused reduced translocation of HMGB1 from the nucleus to the cytoplasm (Fig. 4).
These results demonstrated that magnesium sulfate inhibited the expression of HMGB1 mRNA in LPS-activated RAW264.7 cells. Thus, we next explored whether the upstream NF-kB signal transduction pathway was also involved. Our results showed that the NF-kB activity was increased in RAW264.7 cells stimulated with LPSs (500 ng/mL). Magnesium sulfate significantly reduced the NF-kB activity in LPS-stimulated RAW264.7 cells in a dose-dependent manner (Fig. 5).
4.
Fig. 2 e Effect of magnesium sulfate at different concentrations on RAW264.7 cell viability after incubation for 24 h. RAW264.7 cells were incubated with graded concentrations of magnesium sulfate (1, 5, and 10 mmol/L) or cultured in Roswell Park Memorial Institute 1640 medium alone (with 0.4 mmol/L magnesium sulfate) for 24 h. Cell viability was measured using the Cell Counting Kit-8 assay. Mean values ± SD (n [ 3). P > 0.05 compared with the control group (0.4 mmol/L magnesium sulfate).
Discussion
Because HMGB1 is a critical inflammatory cytokine involved in inflammatory disorders, and NF-kB is widely involved in regulating the production of a variety of other inflammatory cytokines, we hypothesized that drugs blocking the NF-kB signaling pathway might play a protective role in the inflammatory process through a variety of mechanisms. Our study confirmed that magnesium sulfate inhibited the release of HMGB1 in a dose-dependent manner. The mechanism of inhibition of HMGB1 release involves inhibiting the translocation of HMGB1 from the nucleus to the cytoplasm, as well as inhibiting the LPS-induced upregulation of HMGB1 mRNA expression at the transcriptional level. These effects are achieved by inhibiting the NF-kB signaling pathway. Previous studies exploring the treatment of sepsis have been largely confined to the suppression of the early inflammatory cytokines during the super-acute inflammatory response. The use of antibodies specific to the early inflammatory cytokines (tumor necrosis factor-a and IL-1b) has had some success in avoiding the development of septic shock in an animal model [20e22]. However, this therapy for patients
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Fig. 4 e Effect of magnesium sulfate at different concentrations on the translocation of HMGB1 from the nucleus to the cytoplasm in RAW264.7 cells after 24 h stimulation with LPS. The nuclear and cytoplasmic proteins of RAW264.7 cells were extracted using the Nuclear/Cytoplasm Extraction Kit and assayed by Western blot analysis. Mean values ± SD (n [ 3). *P < 0.05 compared with the LPS-treated group, yP < 0.01 compared with the LPS-treated group.
with sepsis did not produce satisfactory clinical results [23,24], which might have been because of an inappropriate therapeutic window. The serum concentrations of HMGB1 increased late (16e24 h) in patients with sepsis and septic rats [3,25]. Moreover, it has been found that elevated levels are closely related to mortality in septic patients [3]. HMGB1 protein inhibitors and/or antagonists can significantly reduce the endotoxin-induced lethal endotoxemia and concurrent acute tissue damage, even when their application is after the peak concentration of the early inflammatory cytokines [8,9,26]. Therefore, inhibition of HMGB1 is considered to be a good target for reducing mortality and complications in patients with sepsis. Salem et al. [27] have reported that high magnesium therapy has a protective effect on septic rats, and Lee et al. [14] have reported that magnesium sulfate mitigates endotoxininduced acute lung injury in septic rats. Although they reported that magnesium sulfate significantly reduced acute lung injury in endotoxemia by slowing down the secretion of several early inflammatory cytokines, they did not investigate whether magnesium sulfate can inhibit HMGB1. Our results have confirmed that magnesium sulfate can reduce sepsisrelated complications by inhibiting the synthesis and release of HMGB1. Because HMGB1 is a key mediator involved in the development of hemorrhagic shock [28], stroke [29], pancreatitis [30], neuropathic pain [31], ischemiaereperfusion injury [32], transplantation [33], and, even, cancer [34], magnesium sulfate might play an important role in treating a number of clinical inflammatory diseases by inhibiting the synthesis and release of HMGB1 under pathologic conditions. Thus, our present study is important for understanding the antiinflammatory effect of magnesium sulfate on sepsis and many other inflammatory diseases. However, the exact mechanism of the anti-inflammatory effect of magnesium sulfate has not been fully clarified. Recent studies have shown that the inhibitory effects of magnesium sulfate on early inflammatory cytokines involve
the NF-kB signaling pathway [13]. Previous studies have confirmed that the NF-kB signaling pathway regulates the expression and release of HMGB1 [35]. Thus, our study supports the hypothesis that magnesium sulfate inhibits the NF-kB signaling pathway in a dose-dependent manner, which regulates the translocation of HMGB1 from the nucleus to the cytoplasm. Previous studies have also confirmed that L-type calcium channels [13] and N-methyl-D-aspartate (NMDA) receptors [14] are involved in regulating the release of inflammatory cytokines by the NF-kB signaling pathway, Therefore, magnesium might act through L-type calcium channels, NMDA receptors, and other upstream pathways that regulate HMGB1.
Fig. 5 e Magnesium sulfate reduces NF-kB activity in a dose-dependent manner. RAW264.7 cells were stimulated with or without LPS (500 ng/mL) in the absence or presence of magnesium sulfate (1, 5, and 10 mmol/L) for 24 h. The nuclear proteins were extracted, and NF-kB activity was determined using the NF-kB p50/p65 Transcription Factor Assay Kit (Chemicon). Mean values ± SD (n [ 3). *P < 0.05 compared with the LPS-treated group, y P < 0.01 compared with the LPS-treated group.
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Magnesium sulfate has been shown to have a protective effect on various cell types involved in the inflammation process [12,13,17]. Our study only focused on RAW264.7 cells, 1 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 required. We also need to study whether the protective effects of magnesium sulfate on the pathologic complications related to sepsis involves the inhibition of HMGB1. Furthermore, the effect of magnesium sulfate on upstream signaling pathways that regulate the expression of HMGB1, such as L-type calcium channels and NMDA receptors, needs to be investigated.
5.
Conclusions
Our study has confirmed that magnesium sulfate, as an antiinflammatory drug, inhibits both the translocation of HMGB1 from the nucleus to the cytoplasm and the LPS-induced expression of HMGB1 mRNA. These effects were achieved partially by inhibiting the NF-kB signaling pathway. Our findings will help us better understand the anti-inflammatory mechanisms of magnesium sulfate and also help to explore new clinical treatments of inflammatory disease.
Acknowledgments This work was supported by a grant from Shanghai Health Bureau Foundation (grant 024015).
references
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Available online at www.sciencedirect.com
journal homepage: www.JournalofSurgicalResearch.com
Magnesium sulfate inhibits the secretion of high mobility group box 1 from lipopolysaccharide-activated RAW264.7 macrophages in vitro Zhaohui Liu, MD,a Junjie Zhang, PhD,a Xiaojing Huang, MD,b Lina Huang, PhD,a Shitong Li, MD,a and Zhengping Wang, MDb,* a b
Department of Anesthesiology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Department of Pain, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
article info
abstract
Article history:
Background: High mobility group box 1 (HMGB1) is an important inflammatory factor that is
Received 3 December 2011
closely related to mortality in patients with sepsis. High magnesium therapy has been
Received in revised form
proved to reduce sepsis-related mortality and sepsis-induced pathologic complications.
5 February 2012
These effects result from reduced expression and release of many inflammatory cytokines,
Accepted 7 February 2012
although it is not clear whether high magnesium affects the expression and release of
Available online 28 March 2012
HMGB1. In the present study, we explored the effect of magnesium sulfate on the expression and release of HMGB1 in lipopolysaccharide (LPS)-activated macrophages.
Keywords:
Methods: RAW264.7 cells were incubated with LPS in the presence or absence of various
Magnesium sulfate
concentrations of magnesium sulfate. An enzyme-linked immunosorbent assay was used
High mobility group box 1
to detect the levels of HMGB1 in the culture supernatant. Real-time polymerase chain
HMGB1 protein
reaction was used to assess the expression of HMGB1 mRNA. The nuclear/cytoplasm
Lipopolysaccharides
extraction kit was used to extract the nuclear and cytoplasmic proteins. Western blotting
NF-kB
was used to observe the changes in the translocation of HMGB1 from the nucleus to the cytoplasm. The nuclear factor (NF)-kB p50/p65 Transcription Factor Assay Kit was used to analyze NF-kB activity in the nuclear extract. Results: We found that magnesium sulfate inhibited translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular release in LPS-activated macrophages and also suppressed the expression of HMGB1 mRNA. Furthermore, magnesium sulfate inhibited the translocation of NF-kB from the cytoplasm to the nucleus in LPS-activated macrophages in a dose-dependent manner. Conclusions: Our study has demonstrated that magnesium sulfate inhibits the translocation of HMGB1 from the nucleus to the cytoplasm and the expression of HMGB1 mRNA in a dose-dependent manner. The mechanism responsible for these effects involves the NF-kB signaling pathway. Crown Copyright ª 2013 Published by Elsevier Inc. All rights reserved.
* Corresponding author. Department of Pain, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, No. 100 Haining Road, Hongkou District, Shanghai, China. Tel.: þ86 21 6324 0090, ext. 3022; fax: þ86 21 6324 0825. E-mail address: [email protected] (Z. Wang). 0022-4804/$ e see front matter Crown Copyright ª 2013 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.02.012
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1.
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Introduction
The pathogenesis of sepsis results from a systemic inflammatory response caused by infection, which is characterized by the excessive release of early pro-inflammatory cytokines (e.g., tumor necrosis factor-a, interleukin -1, interleukin-6) and late pro-inflammatory cytokines (e.g., high mobility group box 1 [HMGB1]). The excessive generation of these cytokines can lead to very serious pathologic consequences, including systemic capillary leak syndrome, tissue damage, and lethal multiple organ failure [1,2]. Therefore, the suppression of the excessive release of proinflammatory cytokines provides a new therapy option for the treatment of sepsis. As a nonhistone chromosomal protein, HMGB1 was originally described as an important regulator of genetic information; however, Wang et al. [3] subsequently found that HMGB1 is a lethal inflammatory cytokine involved with sepsis. HMGB1 can be actively released into the extracellular environment from immune cells, including macrophages, monocytes, natural killer cells, dendritic cells, endothelial cells, and platelets [3e6]. HMGB1 functions as a stimulus by binding receptors (receptor for advanced glycation end products, toll-like receptors 2 and 4) to stimulate the release of early proinflammatory cytokines (tumor necrosis factor-a, IL-1b, and IL-8) and elicit various inflammatory responses [7]. The administration of anti-HMGB1 antibodies or inhibitors significantly improved survival in septic rats and reduced multiple organ damage [8,9]; moreover, such antibodies had a good clinical effect in patients with septic shock [10]. Magnesium sulfate, a commonly used antiepileptic drug [11], has strong anti-inflammatory properties [12,13]. Magnesium sulfate reduces sepsis-related acute lung injury [14] and has a protective effect on ischemiaereperfusion injury [15,16]. Activation of a variety of immune cells (macrophages, lymphocytes, and neutrophils) was significantly inhibited by magnesium sulfate [13,17]. Recent data have confirmed that magnesium sulfate inhibits a variety of inflammatory molecules, including chemokines, cytokines, nitric oxide, and prostaglandin E2 [13]. These anti-inflammatory effects of magnesium sulfate are produced by inhibiting nuclear factor (NF)-kB activation and Ca2þ channel opening. However, the role of magnesium sulfate on HMGB1 expression and activity has not been discussed. Previous data have indicated that RAW264.7 cells, an immortalized murine macrophage-like cell line, are often used as alternatives for macrophages, because they readily express inflammatory molecules on stimulation by endotoxin [18,19]. It has been previously reported that lipopolysaccharide (LPS)-activated macrophages actively secrete HMGB1. Because overexpression and release of HMGB1 occurs in various inflammatory diseases, and because magnesium sulfate has an inhibitory effect on the NF-kB signaling pathway, we hypothesized that magnesium sulfate might inhibit overexpression of HMGB1 during the inflammatory process. Therefore, in the present study, we investigated whether magnesium sulfate inhibits the expression and release of HMGB1 from LPS-activated macrophages and explored the mechanisms of these antiinflammatory effects.
2.
Methods
2.1.
Cell culture and stimulation
Murine macrophage-like RAW264.7 cells (Shanghai Institute of Cell Biology, The Chinese Academy of Sciences, Shanghai, China) were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 100 U/mL penicillin, and 100 mg/mL streptomycin in an atmosphere of humidified 5% carbon dioxide at 37 C. At 80e90% confluence, the cells were washed 3 times and then transferred to 6-well polystyrene culture plates at 1 106 cells/ well in 2 mL medium/well. After overnight incubation, the medium was removed and replaced with Roswell Park Memorial Institute 1640 medium containing 0.25% fetal calf serum (for experiments designed to measure HMGB1 in conditioned media). The RAW264.7 cells were incubated with LPS (500 ng/mL) in the absence or presence of graded concentrations of magnesium sulfate (1, 5, and 10 mmol/L). Cell-free supernatants were collected after 24 h of stimulation for determination of the HMGB1 levels. Total RNA was also extracted for determination of HMGB1 mRNA levels. The HMGB1 levels in the cytoplasm and nucleus were assayed by Western blot analysis to detect the intracellular translocation of HMGB1. The nuclear proteins of RAW264.7 cells were used to measure the NF-kB activity with the NF-kB p50/p65 transcription factor assay kit. For cell viability analysis, RAW264.7 cells were incubated with graded concentrations of magnesium sulfate. Cells cultured in media alone acted as a negative control in all tests.
2.2.
Enzyme-linked immunosorbent assay
The levels of HMGB1 in the culture medium were determined using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) and were performed according to the manufacturer’s instructions.
2.3.
Cell counting kit-8 assay
The viability of RAW264.7 cells was determined using the Cell Counting Kit-8 Assay Kit (Beyotime, Jiangsu, China), as previously reported. RAW264.7 cells were plated at a density of 104 cells/well in 96-well plates in 100 mL Roswell Park Memorial Institute 1640 medium. Next, 20 mL cell counting kit-8 assay was added to RAW264.7 cells in each microwell and incubated for 2 h at 37 C at the end of the 24-h incubation period. The absorbance of the colored solution was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA) at a test wavelength of 450 nm and a reference wavelength of 630 nm.
2.4.
Extraction of cytoplasmic and nuclear proteins
At 24 h after treatment, the RAW264.7 cells were harvested and washed 3 times with cold phosphate-buffered saline (PBS). The cytoplasmic and nuclear protein fractions were extracted using NE-PER extraction reagents according to the
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manufacturer’s protocol (Pierce Biotechnology, Rockford, IL). Cytoplasmic and nuclear protein extracts were used for Western blot analysis.
2.5.
Western blot analysis
The levels of HMGB1 in the cytoplasm and nucleus were assayed by Western blot analysis. The proteins were quantified using the Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China). The samples were denatured at 100 C for 5 min before adding dithiothreitol. An equal amount of protein was loaded in each well for electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto polyvinyldine fluoride microporous membranes (Millipore, Bedford, MA). Membranes containing the transferred proteins were blocked with PBS containing 0.1% Tween 20 (PBS-T) and 5% skim milk for 1 h at room temperature. After 3 washes with PBS-T, the membranes were then incubated with rabbit anti-HMGB1 polyclonal antibody (1:300; Abcam, San Diego, CA), anti-b-actin antibody (1:1,000; Santa Cruz Technology, Santa Cruz, CA), and anti-histone H3.1 antibody (1:1,000; SAB, Pearland, TX) for 1 h at room temperature. After 3 washes with PBS-T, the membranes were incubated with horseradish peroxidase-linked secondary antibodies (1:1,000; Golden Bridge, Beijing, China) for 1 h at room temperature. After 3 final washes in PBS-T and 2 washes in PBS, the membranes were impregnated with the ECL reagents (Amersham, Buckinghamshire, UK) and then exposed digitally with Image Reader LAS-4000 (FujiFilm, Tokyo, Japan) and quantified for statistical analysis using Multi Gauge, version 3.0, software (FujiFilm Life Science, Tokyo, Japan).
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NF-kB binding assay
The DNA binding activity of NF-kB (p50/p65) was determined using the enzyme-linked immunosorbent assay-based nonradioactive NF-kB p50/p65 Transcription Factor Assay Kit (Chemicon, Temecula, CA), according to the manufacturer’s instructions. The absorbance at 450 nm was analyzed by an automated plate reader (Bio-Rad Laboratories).
2.8.
Statistical analysis
All data are presented as the mean SD of the results obtained from 3 replicates. Differences between each group were assessed by 1-way analysis of variance followed by NewmanKeuls tests. P < 0.05 was considered statistically significant. All data were analyzed using SPSS, version 13.0, statistical software (SPSS, Chicago, IL).
3.
Results
3.1. Effect of magnesium sulfate on HMGB1 levels secreted by cultured RAW264.7 cells after treatment with LPSs
Real-time polymerase chain reaction analysis
The supernatant was collected 24 h after LPS stimulation to determine the release of HMGB1 by enzyme-linked immunosorbent assay. We found that the HMGB1 levels in the culture supernatant increased after the administration of LPSs (500 ng/mL); however, the administration of magnesium sulfate significantly inhibited the release of HMGB1 in a dosedependent manner (Fig. 1).
Total RNA was extracted from treated RAW264.7 cells by adding TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. First-strand cDNA was synthesized using 2 mg total cellular RNA as the template, 1 mL of 10 mM deoxyribonucleotide triphosphate mix, 5 mL of Moloney-murine leukemia virus 5 reaction buffer (with Mg2þ), 0.625 mL Recombinant RNasin, and 1 mL Moloneymurine leukemia virus (Promega, Madison, WI), in a final volume of 25 mL. Quantitative real-time polymerase chain reaction (PCR) was performed on a LightCycler 2.0 Real-Time PCR System (Roche Applied Science, Indianapolis, IN). The reaction contained 1 mL of the cDNA template, 2 mL of the forward primer, and 2 mL of the reverse primer (HMGB1 forward, CAC CGT GGG ACT ATT AGG AT; HMGB1 reverse, GCT CAC ACT TTT GGG GAT AC; b-actin forward, CCT CTA TGC CAA CAC AGT; b-actin reverse, AGC CAC CAA TCC ACA CAG), and 10 mL PCR Premix in a total volume of 20 mL. The PCR reaction involved initial denaturation (95 C, 5 min), followed by 40 cycles of denaturation (95 C, 5 s) and extension (60 C, 30 s), and then denaturation (95 C, 15 s), extension (72 C, 10 s), and a final denaturation (95 C, 15 s). The real-time PCR assays were conducted in triplicate for each sample to ensure experimental accuracy. The mean fold-change in the expression of HMGB1 mRNA in the experimental group compared with the control group was calculated using the 2DDCt method.
Fig. 1 e Effect of magnesium sulfate at different concentrations on HMGB1 release from RAW264.7 cells after 24 h stimulation with LPS. RAW264.7 cells were stimulated without or with LPSs (500 ng/mL) in the absence or presence of magnesium sulfate (1, 5, and 10 mmol/L) for 24 h. Culture supernatants were collected and assayed for HMGB1 with an enzyme-linked immunosorbent assay kit. Mean values ± SD (n [ 3). *P < 0.05 compared with the LPS-treated group; yP < 0.01 compared with the LPS-treated group. ND [ not detectable.
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3.2. Effect of magnesium sulfate on viability of RAW264.7 cells RAW264.7 cell viability after treatment with graded magnesium sulfate was measured using the Cell Counting Kit-8 assay. The results showed that increasing concentrations of magnesium sulfate did not have a significant effect on RAW264.7 cell viability (Fig. 2).
3.3. Effect of magnesium sulfate on HMGB1 mRNA expression To gain additional insight into the mechanism responsible for the inhibition of magnesium sulfate on HMGB1 release, we next tested the effect of magnesium sulfate on HMGB1 transcription. We found that magnesium sulfate inhibited the LPS-induced increase in HMGB1 mRNA expression at the transcriptional level. The data indicated that the changes in HMGB1 levels in the cell culture supernatant are consistent with the changes in intracellular levels of HMGB1 mRNA expression (Fig. 3).
Fig. 3 e Effect of magnesium sulfate at different concentrations on HMGB1 mRNA expression in RAW264.7 cells after 24 h stimulation with LPSs. RAW264.7 cells were stimulated with or without LPS (500 ng/mL) in the absence or presence of magnesium sulfate (1, 5, and 10 mmol/L) for 24 h. Total RNA of RAW264.7 cells was extracted and assayed for HMGB1 mRNA using real-time PCR. Mean values ± SD (n [ 3). *P < 0.05 compared with LPS-treated group, yP < 0.01 compared with the LPS-treated group.
3.4. Effect of magnesium sulfate on translocation of HMGB1 from nucleus to cytoplasm
3.5. Effect of magnesium sulfate on LPS-induced increase in NF-kB activity
HMGB1 protein levels in the nucleus were significantly reduced after LPS stimulation. However, the addition of magnesium sulfate significantly reversed this effect. Likewise, the increased HMGB1 protein levels in the cytoplasm after LPS stimulation were reduced by treatment with magnesium sulfate. Thus, the addition of magnesium sulfate caused reduced translocation of HMGB1 from the nucleus to the cytoplasm (Fig. 4).
These results demonstrated that magnesium sulfate inhibited the expression of HMGB1 mRNA in LPS-activated RAW264.7 cells. Thus, we next explored whether the upstream NF-kB signal transduction pathway was also involved. Our results showed that the NF-kB activity was increased in RAW264.7 cells stimulated with LPSs (500 ng/mL). Magnesium sulfate significantly reduced the NF-kB activity in LPS-stimulated RAW264.7 cells in a dose-dependent manner (Fig. 5).
4.
Fig. 2 e Effect of magnesium sulfate at different concentrations on RAW264.7 cell viability after incubation for 24 h. RAW264.7 cells were incubated with graded concentrations of magnesium sulfate (1, 5, and 10 mmol/L) or cultured in Roswell Park Memorial Institute 1640 medium alone (with 0.4 mmol/L magnesium sulfate) for 24 h. Cell viability was measured using the Cell Counting Kit-8 assay. Mean values ± SD (n [ 3). P > 0.05 compared with the control group (0.4 mmol/L magnesium sulfate).
Discussion
Because HMGB1 is a critical inflammatory cytokine involved in inflammatory disorders, and NF-kB is widely involved in regulating the production of a variety of other inflammatory cytokines, we hypothesized that drugs blocking the NF-kB signaling pathway might play a protective role in the inflammatory process through a variety of mechanisms. Our study confirmed that magnesium sulfate inhibited the release of HMGB1 in a dose-dependent manner. The mechanism of inhibition of HMGB1 release involves inhibiting the translocation of HMGB1 from the nucleus to the cytoplasm, as well as inhibiting the LPS-induced upregulation of HMGB1 mRNA expression at the transcriptional level. These effects are achieved by inhibiting the NF-kB signaling pathway. Previous studies exploring the treatment of sepsis have been largely confined to the suppression of the early inflammatory cytokines during the super-acute inflammatory response. The use of antibodies specific to the early inflammatory cytokines (tumor necrosis factor-a and IL-1b) has had some success in avoiding the development of septic shock in an animal model [20e22]. However, this therapy for patients
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Fig. 4 e Effect of magnesium sulfate at different concentrations on the translocation of HMGB1 from the nucleus to the cytoplasm in RAW264.7 cells after 24 h stimulation with LPS. The nuclear and cytoplasmic proteins of RAW264.7 cells were extracted using the Nuclear/Cytoplasm Extraction Kit and assayed by Western blot analysis. Mean values ± SD (n [ 3). *P < 0.05 compared with the LPS-treated group, yP < 0.01 compared with the LPS-treated group.
with sepsis did not produce satisfactory clinical results [23,24], which might have been because of an inappropriate therapeutic window. The serum concentrations of HMGB1 increased late (16e24 h) in patients with sepsis and septic rats [3,25]. Moreover, it has been found that elevated levels are closely related to mortality in septic patients [3]. HMGB1 protein inhibitors and/or antagonists can significantly reduce the endotoxin-induced lethal endotoxemia and concurrent acute tissue damage, even when their application is after the peak concentration of the early inflammatory cytokines [8,9,26]. Therefore, inhibition of HMGB1 is considered to be a good target for reducing mortality and complications in patients with sepsis. Salem et al. [27] have reported that high magnesium therapy has a protective effect on septic rats, and Lee et al. [14] have reported that magnesium sulfate mitigates endotoxininduced acute lung injury in septic rats. Although they reported that magnesium sulfate significantly reduced acute lung injury in endotoxemia by slowing down the secretion of several early inflammatory cytokines, they did not investigate whether magnesium sulfate can inhibit HMGB1. Our results have confirmed that magnesium sulfate can reduce sepsisrelated complications by inhibiting the synthesis and release of HMGB1. Because HMGB1 is a key mediator involved in the development of hemorrhagic shock [28], stroke [29], pancreatitis [30], neuropathic pain [31], ischemiaereperfusion injury [32], transplantation [33], and, even, cancer [34], magnesium sulfate might play an important role in treating a number of clinical inflammatory diseases by inhibiting the synthesis and release of HMGB1 under pathologic conditions. Thus, our present study is important for understanding the antiinflammatory effect of magnesium sulfate on sepsis and many other inflammatory diseases. However, the exact mechanism of the anti-inflammatory effect of magnesium sulfate has not been fully clarified. Recent studies have shown that the inhibitory effects of magnesium sulfate on early inflammatory cytokines involve
the NF-kB signaling pathway [13]. Previous studies have confirmed that the NF-kB signaling pathway regulates the expression and release of HMGB1 [35]. Thus, our study supports the hypothesis that magnesium sulfate inhibits the NF-kB signaling pathway in a dose-dependent manner, which regulates the translocation of HMGB1 from the nucleus to the cytoplasm. Previous studies have also confirmed that L-type calcium channels [13] and N-methyl-D-aspartate (NMDA) receptors [14] are involved in regulating the release of inflammatory cytokines by the NF-kB signaling pathway, Therefore, magnesium might act through L-type calcium channels, NMDA receptors, and other upstream pathways that regulate HMGB1.
Fig. 5 e Magnesium sulfate reduces NF-kB activity in a dose-dependent manner. RAW264.7 cells were stimulated with or without LPS (500 ng/mL) in the absence or presence of magnesium sulfate (1, 5, and 10 mmol/L) for 24 h. The nuclear proteins were extracted, and NF-kB activity was determined using the NF-kB p50/p65 Transcription Factor Assay Kit (Chemicon). Mean values ± SD (n [ 3). *P < 0.05 compared with the LPS-treated group, y P < 0.01 compared with the LPS-treated group.
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Magnesium sulfate has been shown to have a protective effect on various cell types involved in the inflammation process [12,13,17]. Our study only focused on RAW264.7 cells, 1 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 required. We also need to study whether the protective effects of magnesium sulfate on the pathologic complications related to sepsis involves the inhibition of HMGB1. Furthermore, the effect of magnesium sulfate on upstream signaling pathways that regulate the expression of HMGB1, such as L-type calcium channels and NMDA receptors, needs to be investigated.
5.
Conclusions
Our study has confirmed that magnesium sulfate, as an antiinflammatory drug, inhibits both the translocation of HMGB1 from the nucleus to the cytoplasm and the LPS-induced expression of HMGB1 mRNA. These effects were achieved partially by inhibiting the NF-kB signaling pathway. Our findings will help us better understand the anti-inflammatory mechanisms of magnesium sulfate and also help to explore new clinical treatments of inflammatory disease.
Acknowledgments This work was supported by a grant from Shanghai Health Bureau Foundation (grant 024015).
references
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