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Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation
American Journal of Emergency Medicine xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
American Journal of Emergency Medicine journal homepage: www.elsevier.com/locate/ajem
Original Contribution
Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation Huibao Long, MD, Bincan Xu, MM, Yanling Luo, MM, Keqin Luo, MM ⁎ Department of Emergency, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China
a r t i c l e
i n f o
Article history: Received 15 December 2015 Received in revised form 29 December 2015 Accepted 29 December 2015 Available online xxxx
a b s t r a c t Background: NLRP3 inflammasome activation is recently reported to be linked to the pathogenesis of sepsis. Artemisinin is shown to play beneficial effects in sepsis. However, the impacts of artemisinin on burn sepsis have not been investigated. This study is designed to investigate the role of artemisinin in burn sepsis and the involvement of NLRP3 inflammasome activation. Methods: Male BALB/c mice were randomly divided into sham burn, burn, burn sepsis, and artemisinin treated groups. Inflammatory cytokines were measured by enzyme-linked immunosorbent assay. Adhesion molecules and neutrophil infiltration in lung and heart were detected by real-time polymerase chain reaction. Mortality rates were monitored. Artemisinin was added to Raw 264.7 cells that were stimulated with burn sepsis serum in the presence/absence of an inhibitor of NLRP3 inflammasome, 3, 4-methylenedioxy-β-nitrostyrene. Interleukin (IL) 1β and IL-18 messenger RNA expression as well as NLRP3 and caspase 1 protein were measured. Results: Production of inflammatory cytokines in serum, levels of adhesion molecules and neutrophil infiltration in lung and heart, and mortality rate of burn septic mice were significantly higher than those of control. These effects were attenuated by artemisinin. Artemisinin down-regulated protein levels of NLRP3 and caspase 1 and inhibited the increases of IL-1β and IL-18 messenger RNA expression from Raw 264.7 cells that were stimulated with burn sepsis serum. These effects of artemisinin were not further strengthened in the presence of 4-methylenedioxy-β-nitrostyrene. Conclusion: Artemisinin protects mice from burn sepsis by attenuating the inflammatory response and alleviating inflammatory infiltration in vital organs, likely through inhibiting the activation of NLRP3 inflammasome. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Sepsis is a major cause of death in patients who have sustained a severe burn injury, despite advances in burn treatment and critical care [1]. Sepsis after a burn is a result of multiple factors, such as excessive inflammation, immunological disorder, dysfunction in coagulation, and others. Current strategies in the treatment of sepsis include antiinflammatory and antimicrobial therapies, shock prevention, use of corticosteroids, insulin therapy, glycemic control, and others [2]. A better understanding of the underlying mechanisms or development of new therapies is still needed to help better control this devastating condition. Recent studies have suggested a role of the inflammasome, particularly the nucleotide-binding domain and leucine-rich repeat containing family, pyrin containing 3 (NLRP3), inflammasome in the pathogenesis
⁎ Corresponding author at: Department of Emergency, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan-Jiangxi Road, Guangzhou 510120, China. Tel./fax: +86 02081332648. E-mail addresses: [email protected] (H. Long), [email protected] (B. Xu), [email protected] (Y. Luo), [email protected] (K. Luo).
of sepsis [3]. Inflammasomes are multimeric protein complexes that assemble in the cytosol after sensing dangerous stimuli, which can serve as a scaffold to recruit the inactive zymogen procaspase 1 [4]. NLRP3 inflammasome, one of the best characterized inflammasomes, senses various danger signals from both exogenous and endogenous origin. Upon activation, NLRP3 inflammasome recruits and activates procaspase 1 to its mature form, which, in turn, cleaves the precursors of interleukin (IL) 1β and IL-18 to their activated forms [5]. NLRP3 inflammasome is linked to many inflammatory conditions or diseases, including sepsis. Inhibition of NLRP3 inflammasome activation is associated with beneficial effects in sepsis and septic shock [3,6]. Artemisinin is extracted from the plant Artemisia apiacea, a Chinese traditional herb. Except for its well-characterized use as an antimalarial drug, artemisinin has been reported to have many other effects [7]. Artemisinin possesses strong inhibitory effects against viruses, such as herpes viruses and hepatitis B and C viruses [8,9]. In addition, artemisinin is reported to display antifungal activities, anti-inflammation, and anticancer effects [10–12]. Although artemisinin has been shown to be protective in some models of sepsis [13], its role in burn sepsis, which may differ in many mays from the management of sepsis in the general critical care population, has not been investigated.
http://dx.doi.org/10.1016/j.ajem.2015.12.075 0735-6757/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
2
H. Long et al. / American Journal of Emergency Medicine xxx (2016) xxx–xxx
In this study, we aim to test the hypothesis that artemisinin protects mice from burn sepsis by attenuating the inflammatory response and alleviating inflammatory infiltration in vital organs, which is probably associated with inhibiting the activation of the NLRP3 inflammasome.
purified MPO in the kit. Data are expressed as units of activity per gram of tissue (units per gram).
2. Methods and materials
Real-time polymerase chain reaction (PCR) was performed as previously described [17]. Total RNA was extracted using the RNeasy 96 Kit (Qiagen, Hamburg, Germany) according to the manufacturer's instructions. The complementary DNA was synthesized and was amplified using the SYBR green PCR master mix (Applied Biosystems, Foster City, CA). Fluorescence was monitored and analyzed in an ABI 7500 system (Applied Biosystems). β-Actin was used as an internal control. All samples were analyzed in duplicate. The cycle threshold values were obtained, and the fold changes of gene expression were calculated by the 2-ΔΔCt method [18]. The sequences of the primers are as follows: intercellular adhesion molecule (ICAM) 1 sense 5′-GGCTGGCATTGTTCTCTA-3′ and antisense 5′TCCTCAGTCACCTCTACC-3′, vascular cell adhesion molecule (VCAM) 1 sense 5′-GCGAGTCACCATTGTTCT-3′ and antisense 5′-GCCACTGA ATTGAATCTCTG-3′, IL-1β sense 5′-CTTCAGGCAGGCAGTATC-3′ and antisense 5′-CAGCAGGTTATCATCATCATC-3′, IL-18 sense 5′-ACTCTTGCGT CAACTTCA-3′ and antisense 5′-GTCCTCTTACTTCACTGTCT-3′, β-actin sense 5′-GTCAGAAGGACTCCTATGTG-3′ and antisense 5′-ACGCAGCTCA TTGTAGAAG-3′.
2.1. Animals BALB/c mice, the most widely used inbred strains used in animal experimentation, were chosen in this study for their easy accessibility and usefulness in research of immunology. Male BALB/c mice weighing 20 ± 1 g were purchased from Laboratory Animal Center of Chinese Academy of Medical Sciences, Beijing, China. All animals were housed conventionally in an air-conditioned room, under a constant temperature (25°C) and humidity (50%-60%), with a 12-hour/12-hour light/ dark cycle (6:00 PM to 6:00 AM). Animals were provided with food and water ad libitum and were housed for at least 7 days before initiating the experiments. The research protocol was approved by the Committee of Scientific Research of Sun Yat-Sen Memorial Hospital, Guangzhou, China. 2.2. Burn sepsis model and treatment Mice were randomly divided into 4 groups: sham burn, burn, burn sepsis, and artemisinin treated groups (10 mice in each group). Mice were anesthetized by intraperitoneal injection with 50 mg/kg pentobarbital sodium (Sigma-Aldrich, St Louis, MO). The hairs of the dorsal area were shaved and depilated. Mice were placed in a template exposing 25% of the total body surface area to 100°C water for 10 seconds, to create a full-thickness burn injury [14]. Animals were then resuscitated by intraperitoneal injection with 40 mL/kg 0.9% saline and placed in individual cages with free access to food and water. Mice in the sham burn group were treated in the same manner except that they were immersed in lukewarm water. To create a burn sepsis model, burned animals were subjected to intraperitoneal inoculation of Pseudomonas aeruginosa (2 × 105 CFU/mL, no. 27853; American Type Culture Collection, Manassas, VA) at 1 day postburn as previously described [15]. Mice in the artemisinin treated group were injected intraperitoneally with 40 mg/kg artemisinin (in dimethylsulfoxide; Sigma-Aldrich, St Louis, MO) immediately after the burn sepsis model was created and once daily for 3 days. Animals were euthanized at 5 days postburn for measurement of the proinflammatory cytokine levels and inflammatory infiltration in the lung and heart. Another 36 animals were used to monitor the mortality rate in burn, burn sepsis, and artemisinin treated groups (12 mice each) until 9 days postburn. 2.3. Enzyme-linked immunosorbent assay Interleukin 1β, IL-6, tumor necrosis factors (TNF) α, and macrophage chemoattractant protein (MCP) 1 levels in serum were measured with enzyme-linked immunosorbent assay kits purchased from R&D Systems (Minneapolis, MN). All procedures were performed according to the manufacturer's instructions. 2.4. Myeloperoxidase assay Myeloperoxidase (MPO) activities in the lung and heart were measured by a commercial kit purchased from Nanjing Jiancheng Bioengineering Institute, Jiangsu, China. The procedures are as previously described [16]. In brief, tissue samples were homogenized and centrifuged. Twenty microliters of the supernatant was incubated with 200 μL of substrate buffer. The optical density changes over a 2-minute period were read at 460 nm by a microplate reader. Myeloperoxidase activities were obtained using a standard curve of
2.5. Quantitative real-time polymerase chain reaction
2.6. Cell culture We chose a commonly used mice macrophage cell line, Raw 264.7 cells (ATCC) for in vitro study, to mimic the inflammatory response in burn sepsis. Cells were pretreated with artemisinin (5 μmol/L) or artemisinin plus 3, 4-methylenedioxy-β-nitrostyrene (MNS; 5 μmol/L; Abcam, Cambridge, UK) for 1 hour, and then were stimulated with 10% serum extracted from burn sepsis mice for 4 hours. The messenger RNA (mRNA) expression of IL-1β and IL-18 were detected by quantitative real-time PCR. Protein levels of NLRP3 and caspase 1 in cell lysates were determined by Western blot. 2.7. Western blot analysis Protein in cell lysates was separated by electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked by nonfat milk for 2 hours and then incubated with antiNLRP3 and anti–caspase 1 (both from Santa Cruz Biotechnology, Inc, Dallas, TX) diluted in Tris-buffered saline with Tween overnight at 4°C. Membranes were then incubated with secondary antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Immunoreactive bands were developed using an enhanced chemiluminescence reagent (Pierce Biotechnology, Rockford, IL) and exposed onto Kodak film (Eastman Kodak, Rochester, NY). Band densities were normalized using β-actin and quantified using a scanning densitometric analysis Image J software (National Institutes of Health, Bethesda, MD). 2.8. Statistical analysis Data were presented as means ± SDs. GraphPad prism software V.6.01 for Windows (GraphPad Software, La Jolla, CA) was used for analyses. Statistical analysis was performed by using 1-way analysis of variance followed by Tukey test. P b .05 was considered statistically significant. 3. Results 3.1. Artemisinin attenuated inflammatory cytokines production after burn sepsis Levels of IL-1β, IL-6, TNF-α, and MCP-1 in serum of burn septic mice were profoundly higher than those of burned and sham burned mice.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
H. Long et al. / American Journal of Emergency Medicine xxx (2016) xxx–xxx
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Fig. 1. Interleukin 1β, IL-6, TNF-α, and MCP-1 levels in serum of sham burned, burned, burn septic, and artemisinin treated mice were detected by enzyme-linked immunosorbent assay. Data were presented as means ± SD, n = 10 mice/group, ***P b .001, ****P b .0001.
The increases of these inflammatory cytokines were significantly inhibited by the treatment of artemisinin (Fig. 1). 3.2. Artemisinin inhibited adhesive molecule expressions in the lung and heart
sepsis serum. These increases were significantly abolished by artemisinin treatment. Moreover, the effects of artemisinin were not further strengthened in the presence of a specific NLRP3 inflammasome inhibitor, MNS (Fig. 5). 4. Discussion
The relative mRNA expressions of ICAM-1 and VCAM-1 in lung and heart were markedly elevated after burn sepsis, as compared to the burn and sham burn groups. These increases were significantly inhibited by artemisinin treatment (Fig. 2). 3.3. Artemisinin ameliorated neutrophil infiltration in the lung and heart and promoted survival in burn septic mice We used MPO assay to determine the level of neutrophil infiltration in lung and heart, 2 vital organs that are mostly influenced in burn sepsis. Myeloperoxidase activities in lung and heart of burn septic mice were remarkably higher than those of burned and sham burned mice. These increased neutrophil infiltrations were significantly ameliorated by the treatment of artemisinin (Fig. 3). In addition, the survival rate of burn septic mice treated with artemisinin was significantly higher than those of nontreated mice until 9 days postburn (Fig. 4). 3.4. The effects of artemisinin were likely mediated by inhibiting the NLRP3 inflammasome activation The protein levels of NLRP3 and caspase 1 as well as IL-1β and IL-18 mRNA expression were highly elevated after stimulating with burn
In this study, we investigated the role of artemisinin in burn sepsis using an experimental burn sepsis model. Although previous studies based on animal or clinical research have demonstrated a protective role of artemisinin in sepsis [11,13,19], few studies have focused on the effect of artemisinin in burn sepsis. Because of the unique physiologic and metabolic changes after burns, the characteristics of sepsis in burns are different in many ways from other kinds of sepsis, which makes the results of this study have certain significance. The effects of artemisinin beyond antimalarial have long been investigated, especially the anti-inflammatory effects. In the present study, artemisinin also showed strong anti-inflammatory effects, as evidenced by decreased inflammatory cytokines production, adhesive molecule expression, and neutrophil infiltration after artemisinin treatment. Previous studies have shown the anti-inflammatory effects of artemisinins are through inhibiting signaling pathways including Toll-like receptors, Syk tyrosine kinase, Phosphatidyl Inositol 3-kinase/Akt, Mitogen Activated Protein Kinase, nuclear factor kappa B, and others [20–22]. We found in this study that artemisinin inhibited NLRP3 inflammasome activation, as evidenced by decreased protein levels of NLRP3 and caspase 1 as well as IL-1β and IL-18 mRNA expression. This finding is consistent with a previous study, which demonstrated that artemisinin
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
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Fig. 2. The relative mRNA expression of ICAM-1 and VCAM-1 in lung and heart of sham burned, burned, burn septic, and artemisinin treated mice were detected by quantitative real-time PCR. Data were calculated by using the 2-ΔΔCt method, where Ct is cycle threshold. n = 10 mice/group, **P b .01, ***P b .001, ****P b .0001.
inhibited NLRP3 inflammasome activation in a mice neuroinflammation model [23]. The NLRP3 inflammasome has been linked to many inflammatory conditions or diseases, such as intestinal inflammation, traumatic brain injury, thromboembolic stroke, and cancer [24–26]. However, its role in sepsis has only recently been explored. Inhibition of NLRP3 inflammasome activation has been associated with improved outcome
in sepsis, septic shock, and other sepsis-related syndromes [3,6,27]. The NLRP3 inflammasome is a multiprotein complex that acts as a platform to trigger the activation of caspase 1 and the maturation of IL-1β and IL-18 [28]. Inhibition of NLRP3 inflammasome activation leads to reduced caspase 1 and IL-1β maturation, which may consequently attenuate downstream inflammatory signaling pathways. In our in vitro study, the increases of NLRP3 inflammasome activation after burn
Fig. 3. Myeloperoxidase activities in lung and heart of sham burned, burned, burn septic, and artemisinin treated mice were detected by a commercial kit according to the manufacturer's instructions. Data were presented as means ± SD, n = 10 mice/group, ****P b .0001.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
H. Long et al. / American Journal of Emergency Medicine xxx (2016) xxx–xxx
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serum challenge were significantly abolished by artemisinin treatment. Moreover, this effect of artemisinin was not further strengthened in the presence of a specific NLRP3 inflammasome inhibitor, MNS. These results suggested that the effect of artemisinin may largely dependent upon inhibiting the NLRP3 inflammasome activation.
5. Limitations
Fig. 4. Survival rates of mice in burned, burn septic, and artemisinin treated groups were analyzed using the Kaplan-Meier method and compared using the log-rank test. The survival rate of burn septic mice treated with artemisinin was significantly higher than those of nontreated mice until 9 days postburn (58.33% vs 16.67%; P = .0027). n = 12 mice/ group, **P b .01.
First, our study is limited by the small sample size. Second, the results of this study cannot be necessarily generalized to large animals or humans. Third, this study is limited to the lack of a group that was treated with antibiotics, and ongoing fluid resuscitation as is customary for sepsis. In conclusion, the results in this study provided evidence that artemisinin protected mice from burn sepsis by attenuating the inflammatory response and alleviating inflammatory infiltration in vital organs, which is likely mediated by inhibiting the activation of NLRP3 inflammasome.
Fig. 5. A and B, IL-1β and IL-18 relative mRNA expression in cell lysates were detected by quantitative real-time PCR. C, The protein levels of NLRP3 and cleaved caspase 1 were determined by Western blot. β-Actin was used as internal control. Data were presented as means ± SD. Results are representative of at least 3 independent experiments. **P b .01, ***P b .001, ****P b .0001; ns, nonsignificant.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
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Conflict of interest The authors declare no conflict of interests.
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[14] Shen CA, Fagan S, Fischman AJ, Carter EE, Chai JK, Lu XM, et al. Effects of glucagonlike peptide 1 on glycemia control and its metabolic consequence after severe thermal injury—studies in an animal model. Surgery 2011;149:635–44. [15] Liu QY, Yao YM, Yu Y, Dong N, Sheng ZY. Astragalus polysaccharides attenuate postburn sepsis via inhibiting negative immunoregulation of CD4+ CD25(high) T cells. PLoS One 2011;6:e19811. http://dx.doi.org/10.1371/journal.pone.0019811. [16] Xiao M, Li L, Li C, Zhang P, Hu Q, Ma L, et al. Role of autophagy and apoptosis in wound tissue of deep second-degree burn in rats. Acad Emerg Med 2014;21: 383–91. [17] Luo K, Long H, Xu B, Luo Y. Metallothionein ameliorates burn sepsis partly via activation of Akt signaling pathway in mice: a randomized animal study. World J Emerg Surg 2015;10:53. [18] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008;3:1101–8. [19] Moore CC, Jacob ST, Pinkerton R, Banura P, Meya DB, Reynolds SJ, et al. Treatment of severe sepsis with artemether-lumefantrine is associated with decreased mortality in Ugandan patients without malaria. Am J Trop Med Hyg 2009;80: 723–8. [20] Hou LF, He SJ, Li X, Yang Y, He PL, Zhou Y, et al. Oral administration of artemisinin analog SM934 ameliorates lupus syndromes in MRL/lpr mice by inhibiting Th1 and Th17 cell responses. Arthritis Rheum 2011;63:2445–55. [21] Hou LF, He SJ, Li X, Wan CP, Yang Y, Zhang XH, et al. SM934 treated lupus-prone NZB x NZW F1 mice by enhancing macrophage interleukin-10 production and suppressing pathogenic T cell development. PLoS One 2012;7:e32424. http://dx.doi.org/10. 1371/journal.pone.0032424. [22] Li X, Li TT, Zhang XH, Hou LF, Yang XQ, Zhu FH, et al. Artemisinin analogue SM934 ameliorates murine experimental autoimmune encephalomyelitis through enhancing the expansion and functions of regulatory T cell. PLoS One 2013;8: e74108. http://dx.doi.org/10.1371/journal.pone.0074108. [23] Shi JQ, Zhang CC, Sun XL, Cheng XX, Wang JB, Zhang YD, et al. Antimalarial drug artemisinin extenuates amyloidogenesis and neuroinflammation in APPswe/ PS1dE9 transgenic mice via inhibition of nuclear factor-kappaB and NLRP3 inflammasome activation. CNS Neurosci Ther 2013;19:262–8. [24] Chen GY, Nunez G. Inflammasomes in intestinal inflammation and cancer. Gastroenterology 2011;141:1986–99. [25] Abulafia DP, de Rivero Vaccari JP, Lozano JD, Lotocki G, Keane RW, Dietrich WD. Inhibition of the inflammasome complex reduces the inflammatory response after thromboembolic stroke in mice. J Cereb Blood Flow Metab 2009;29:534–44. [26] de Rivero Vaccari JP, Lotocki G, Alonso OF, Bramlett HM, Dietrich WD, Keane RW. Therapeutic neutralization of the NLRP1 inflammasome reduces the innate immune response and improves histopathology after traumatic brain injury. J Cereb Blood Flow Metab 2009;29:1251–61. [27] He L, Peng X, Zhu J, Chen X, Liu H, Tang C, et al. Mangiferin attenuate sepsis-induced acute kidney injury via antioxidant and anti-inflammatory effects. Am J Nephrol 2014;40:441–50. [28] Schroder K, Tschopp J. The inflammasomes. Cell 2010;140:821–32.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
Contents lists available at ScienceDirect
American Journal of Emergency Medicine journal homepage: www.elsevier.com/locate/ajem
Original Contribution
Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation Huibao Long, MD, Bincan Xu, MM, Yanling Luo, MM, Keqin Luo, MM ⁎ Department of Emergency, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China
a r t i c l e
i n f o
Article history: Received 15 December 2015 Received in revised form 29 December 2015 Accepted 29 December 2015 Available online xxxx
a b s t r a c t Background: NLRP3 inflammasome activation is recently reported to be linked to the pathogenesis of sepsis. Artemisinin is shown to play beneficial effects in sepsis. However, the impacts of artemisinin on burn sepsis have not been investigated. This study is designed to investigate the role of artemisinin in burn sepsis and the involvement of NLRP3 inflammasome activation. Methods: Male BALB/c mice were randomly divided into sham burn, burn, burn sepsis, and artemisinin treated groups. Inflammatory cytokines were measured by enzyme-linked immunosorbent assay. Adhesion molecules and neutrophil infiltration in lung and heart were detected by real-time polymerase chain reaction. Mortality rates were monitored. Artemisinin was added to Raw 264.7 cells that were stimulated with burn sepsis serum in the presence/absence of an inhibitor of NLRP3 inflammasome, 3, 4-methylenedioxy-β-nitrostyrene. Interleukin (IL) 1β and IL-18 messenger RNA expression as well as NLRP3 and caspase 1 protein were measured. Results: Production of inflammatory cytokines in serum, levels of adhesion molecules and neutrophil infiltration in lung and heart, and mortality rate of burn septic mice were significantly higher than those of control. These effects were attenuated by artemisinin. Artemisinin down-regulated protein levels of NLRP3 and caspase 1 and inhibited the increases of IL-1β and IL-18 messenger RNA expression from Raw 264.7 cells that were stimulated with burn sepsis serum. These effects of artemisinin were not further strengthened in the presence of 4-methylenedioxy-β-nitrostyrene. Conclusion: Artemisinin protects mice from burn sepsis by attenuating the inflammatory response and alleviating inflammatory infiltration in vital organs, likely through inhibiting the activation of NLRP3 inflammasome. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Sepsis is a major cause of death in patients who have sustained a severe burn injury, despite advances in burn treatment and critical care [1]. Sepsis after a burn is a result of multiple factors, such as excessive inflammation, immunological disorder, dysfunction in coagulation, and others. Current strategies in the treatment of sepsis include antiinflammatory and antimicrobial therapies, shock prevention, use of corticosteroids, insulin therapy, glycemic control, and others [2]. A better understanding of the underlying mechanisms or development of new therapies is still needed to help better control this devastating condition. Recent studies have suggested a role of the inflammasome, particularly the nucleotide-binding domain and leucine-rich repeat containing family, pyrin containing 3 (NLRP3), inflammasome in the pathogenesis
⁎ Corresponding author at: Department of Emergency, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan-Jiangxi Road, Guangzhou 510120, China. Tel./fax: +86 02081332648. E-mail addresses: [email protected] (H. Long), [email protected] (B. Xu), [email protected] (Y. Luo), [email protected] (K. Luo).
of sepsis [3]. Inflammasomes are multimeric protein complexes that assemble in the cytosol after sensing dangerous stimuli, which can serve as a scaffold to recruit the inactive zymogen procaspase 1 [4]. NLRP3 inflammasome, one of the best characterized inflammasomes, senses various danger signals from both exogenous and endogenous origin. Upon activation, NLRP3 inflammasome recruits and activates procaspase 1 to its mature form, which, in turn, cleaves the precursors of interleukin (IL) 1β and IL-18 to their activated forms [5]. NLRP3 inflammasome is linked to many inflammatory conditions or diseases, including sepsis. Inhibition of NLRP3 inflammasome activation is associated with beneficial effects in sepsis and septic shock [3,6]. Artemisinin is extracted from the plant Artemisia apiacea, a Chinese traditional herb. Except for its well-characterized use as an antimalarial drug, artemisinin has been reported to have many other effects [7]. Artemisinin possesses strong inhibitory effects against viruses, such as herpes viruses and hepatitis B and C viruses [8,9]. In addition, artemisinin is reported to display antifungal activities, anti-inflammation, and anticancer effects [10–12]. Although artemisinin has been shown to be protective in some models of sepsis [13], its role in burn sepsis, which may differ in many mays from the management of sepsis in the general critical care population, has not been investigated.
http://dx.doi.org/10.1016/j.ajem.2015.12.075 0735-6757/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
2
H. Long et al. / American Journal of Emergency Medicine xxx (2016) xxx–xxx
In this study, we aim to test the hypothesis that artemisinin protects mice from burn sepsis by attenuating the inflammatory response and alleviating inflammatory infiltration in vital organs, which is probably associated with inhibiting the activation of the NLRP3 inflammasome.
purified MPO in the kit. Data are expressed as units of activity per gram of tissue (units per gram).
2. Methods and materials
Real-time polymerase chain reaction (PCR) was performed as previously described [17]. Total RNA was extracted using the RNeasy 96 Kit (Qiagen, Hamburg, Germany) according to the manufacturer's instructions. The complementary DNA was synthesized and was amplified using the SYBR green PCR master mix (Applied Biosystems, Foster City, CA). Fluorescence was monitored and analyzed in an ABI 7500 system (Applied Biosystems). β-Actin was used as an internal control. All samples were analyzed in duplicate. The cycle threshold values were obtained, and the fold changes of gene expression were calculated by the 2-ΔΔCt method [18]. The sequences of the primers are as follows: intercellular adhesion molecule (ICAM) 1 sense 5′-GGCTGGCATTGTTCTCTA-3′ and antisense 5′TCCTCAGTCACCTCTACC-3′, vascular cell adhesion molecule (VCAM) 1 sense 5′-GCGAGTCACCATTGTTCT-3′ and antisense 5′-GCCACTGA ATTGAATCTCTG-3′, IL-1β sense 5′-CTTCAGGCAGGCAGTATC-3′ and antisense 5′-CAGCAGGTTATCATCATCATC-3′, IL-18 sense 5′-ACTCTTGCGT CAACTTCA-3′ and antisense 5′-GTCCTCTTACTTCACTGTCT-3′, β-actin sense 5′-GTCAGAAGGACTCCTATGTG-3′ and antisense 5′-ACGCAGCTCA TTGTAGAAG-3′.
2.1. Animals BALB/c mice, the most widely used inbred strains used in animal experimentation, were chosen in this study for their easy accessibility and usefulness in research of immunology. Male BALB/c mice weighing 20 ± 1 g were purchased from Laboratory Animal Center of Chinese Academy of Medical Sciences, Beijing, China. All animals were housed conventionally in an air-conditioned room, under a constant temperature (25°C) and humidity (50%-60%), with a 12-hour/12-hour light/ dark cycle (6:00 PM to 6:00 AM). Animals were provided with food and water ad libitum and were housed for at least 7 days before initiating the experiments. The research protocol was approved by the Committee of Scientific Research of Sun Yat-Sen Memorial Hospital, Guangzhou, China. 2.2. Burn sepsis model and treatment Mice were randomly divided into 4 groups: sham burn, burn, burn sepsis, and artemisinin treated groups (10 mice in each group). Mice were anesthetized by intraperitoneal injection with 50 mg/kg pentobarbital sodium (Sigma-Aldrich, St Louis, MO). The hairs of the dorsal area were shaved and depilated. Mice were placed in a template exposing 25% of the total body surface area to 100°C water for 10 seconds, to create a full-thickness burn injury [14]. Animals were then resuscitated by intraperitoneal injection with 40 mL/kg 0.9% saline and placed in individual cages with free access to food and water. Mice in the sham burn group were treated in the same manner except that they were immersed in lukewarm water. To create a burn sepsis model, burned animals were subjected to intraperitoneal inoculation of Pseudomonas aeruginosa (2 × 105 CFU/mL, no. 27853; American Type Culture Collection, Manassas, VA) at 1 day postburn as previously described [15]. Mice in the artemisinin treated group were injected intraperitoneally with 40 mg/kg artemisinin (in dimethylsulfoxide; Sigma-Aldrich, St Louis, MO) immediately after the burn sepsis model was created and once daily for 3 days. Animals were euthanized at 5 days postburn for measurement of the proinflammatory cytokine levels and inflammatory infiltration in the lung and heart. Another 36 animals were used to monitor the mortality rate in burn, burn sepsis, and artemisinin treated groups (12 mice each) until 9 days postburn. 2.3. Enzyme-linked immunosorbent assay Interleukin 1β, IL-6, tumor necrosis factors (TNF) α, and macrophage chemoattractant protein (MCP) 1 levels in serum were measured with enzyme-linked immunosorbent assay kits purchased from R&D Systems (Minneapolis, MN). All procedures were performed according to the manufacturer's instructions. 2.4. Myeloperoxidase assay Myeloperoxidase (MPO) activities in the lung and heart were measured by a commercial kit purchased from Nanjing Jiancheng Bioengineering Institute, Jiangsu, China. The procedures are as previously described [16]. In brief, tissue samples were homogenized and centrifuged. Twenty microliters of the supernatant was incubated with 200 μL of substrate buffer. The optical density changes over a 2-minute period were read at 460 nm by a microplate reader. Myeloperoxidase activities were obtained using a standard curve of
2.5. Quantitative real-time polymerase chain reaction
2.6. Cell culture We chose a commonly used mice macrophage cell line, Raw 264.7 cells (ATCC) for in vitro study, to mimic the inflammatory response in burn sepsis. Cells were pretreated with artemisinin (5 μmol/L) or artemisinin plus 3, 4-methylenedioxy-β-nitrostyrene (MNS; 5 μmol/L; Abcam, Cambridge, UK) for 1 hour, and then were stimulated with 10% serum extracted from burn sepsis mice for 4 hours. The messenger RNA (mRNA) expression of IL-1β and IL-18 were detected by quantitative real-time PCR. Protein levels of NLRP3 and caspase 1 in cell lysates were determined by Western blot. 2.7. Western blot analysis Protein in cell lysates was separated by electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked by nonfat milk for 2 hours and then incubated with antiNLRP3 and anti–caspase 1 (both from Santa Cruz Biotechnology, Inc, Dallas, TX) diluted in Tris-buffered saline with Tween overnight at 4°C. Membranes were then incubated with secondary antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Immunoreactive bands were developed using an enhanced chemiluminescence reagent (Pierce Biotechnology, Rockford, IL) and exposed onto Kodak film (Eastman Kodak, Rochester, NY). Band densities were normalized using β-actin and quantified using a scanning densitometric analysis Image J software (National Institutes of Health, Bethesda, MD). 2.8. Statistical analysis Data were presented as means ± SDs. GraphPad prism software V.6.01 for Windows (GraphPad Software, La Jolla, CA) was used for analyses. Statistical analysis was performed by using 1-way analysis of variance followed by Tukey test. P b .05 was considered statistically significant. 3. Results 3.1. Artemisinin attenuated inflammatory cytokines production after burn sepsis Levels of IL-1β, IL-6, TNF-α, and MCP-1 in serum of burn septic mice were profoundly higher than those of burned and sham burned mice.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
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Fig. 1. Interleukin 1β, IL-6, TNF-α, and MCP-1 levels in serum of sham burned, burned, burn septic, and artemisinin treated mice were detected by enzyme-linked immunosorbent assay. Data were presented as means ± SD, n = 10 mice/group, ***P b .001, ****P b .0001.
The increases of these inflammatory cytokines were significantly inhibited by the treatment of artemisinin (Fig. 1). 3.2. Artemisinin inhibited adhesive molecule expressions in the lung and heart
sepsis serum. These increases were significantly abolished by artemisinin treatment. Moreover, the effects of artemisinin were not further strengthened in the presence of a specific NLRP3 inflammasome inhibitor, MNS (Fig. 5). 4. Discussion
The relative mRNA expressions of ICAM-1 and VCAM-1 in lung and heart were markedly elevated after burn sepsis, as compared to the burn and sham burn groups. These increases were significantly inhibited by artemisinin treatment (Fig. 2). 3.3. Artemisinin ameliorated neutrophil infiltration in the lung and heart and promoted survival in burn septic mice We used MPO assay to determine the level of neutrophil infiltration in lung and heart, 2 vital organs that are mostly influenced in burn sepsis. Myeloperoxidase activities in lung and heart of burn septic mice were remarkably higher than those of burned and sham burned mice. These increased neutrophil infiltrations were significantly ameliorated by the treatment of artemisinin (Fig. 3). In addition, the survival rate of burn septic mice treated with artemisinin was significantly higher than those of nontreated mice until 9 days postburn (Fig. 4). 3.4. The effects of artemisinin were likely mediated by inhibiting the NLRP3 inflammasome activation The protein levels of NLRP3 and caspase 1 as well as IL-1β and IL-18 mRNA expression were highly elevated after stimulating with burn
In this study, we investigated the role of artemisinin in burn sepsis using an experimental burn sepsis model. Although previous studies based on animal or clinical research have demonstrated a protective role of artemisinin in sepsis [11,13,19], few studies have focused on the effect of artemisinin in burn sepsis. Because of the unique physiologic and metabolic changes after burns, the characteristics of sepsis in burns are different in many ways from other kinds of sepsis, which makes the results of this study have certain significance. The effects of artemisinin beyond antimalarial have long been investigated, especially the anti-inflammatory effects. In the present study, artemisinin also showed strong anti-inflammatory effects, as evidenced by decreased inflammatory cytokines production, adhesive molecule expression, and neutrophil infiltration after artemisinin treatment. Previous studies have shown the anti-inflammatory effects of artemisinins are through inhibiting signaling pathways including Toll-like receptors, Syk tyrosine kinase, Phosphatidyl Inositol 3-kinase/Akt, Mitogen Activated Protein Kinase, nuclear factor kappa B, and others [20–22]. We found in this study that artemisinin inhibited NLRP3 inflammasome activation, as evidenced by decreased protein levels of NLRP3 and caspase 1 as well as IL-1β and IL-18 mRNA expression. This finding is consistent with a previous study, which demonstrated that artemisinin
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
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Fig. 2. The relative mRNA expression of ICAM-1 and VCAM-1 in lung and heart of sham burned, burned, burn septic, and artemisinin treated mice were detected by quantitative real-time PCR. Data were calculated by using the 2-ΔΔCt method, where Ct is cycle threshold. n = 10 mice/group, **P b .01, ***P b .001, ****P b .0001.
inhibited NLRP3 inflammasome activation in a mice neuroinflammation model [23]. The NLRP3 inflammasome has been linked to many inflammatory conditions or diseases, such as intestinal inflammation, traumatic brain injury, thromboembolic stroke, and cancer [24–26]. However, its role in sepsis has only recently been explored. Inhibition of NLRP3 inflammasome activation has been associated with improved outcome
in sepsis, septic shock, and other sepsis-related syndromes [3,6,27]. The NLRP3 inflammasome is a multiprotein complex that acts as a platform to trigger the activation of caspase 1 and the maturation of IL-1β and IL-18 [28]. Inhibition of NLRP3 inflammasome activation leads to reduced caspase 1 and IL-1β maturation, which may consequently attenuate downstream inflammatory signaling pathways. In our in vitro study, the increases of NLRP3 inflammasome activation after burn
Fig. 3. Myeloperoxidase activities in lung and heart of sham burned, burned, burn septic, and artemisinin treated mice were detected by a commercial kit according to the manufacturer's instructions. Data were presented as means ± SD, n = 10 mice/group, ****P b .0001.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
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serum challenge were significantly abolished by artemisinin treatment. Moreover, this effect of artemisinin was not further strengthened in the presence of a specific NLRP3 inflammasome inhibitor, MNS. These results suggested that the effect of artemisinin may largely dependent upon inhibiting the NLRP3 inflammasome activation.
5. Limitations
Fig. 4. Survival rates of mice in burned, burn septic, and artemisinin treated groups were analyzed using the Kaplan-Meier method and compared using the log-rank test. The survival rate of burn septic mice treated with artemisinin was significantly higher than those of nontreated mice until 9 days postburn (58.33% vs 16.67%; P = .0027). n = 12 mice/ group, **P b .01.
First, our study is limited by the small sample size. Second, the results of this study cannot be necessarily generalized to large animals or humans. Third, this study is limited to the lack of a group that was treated with antibiotics, and ongoing fluid resuscitation as is customary for sepsis. In conclusion, the results in this study provided evidence that artemisinin protected mice from burn sepsis by attenuating the inflammatory response and alleviating inflammatory infiltration in vital organs, which is likely mediated by inhibiting the activation of NLRP3 inflammasome.
Fig. 5. A and B, IL-1β and IL-18 relative mRNA expression in cell lysates were detected by quantitative real-time PCR. C, The protein levels of NLRP3 and cleaved caspase 1 were determined by Western blot. β-Actin was used as internal control. Data were presented as means ± SD. Results are representative of at least 3 independent experiments. **P b .01, ***P b .001, ****P b .0001; ns, nonsignificant.
Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075
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Conflict of interest The authors declare no conflict of interests.
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Please cite this article as: Long H, et al, Artemisinin protects mice against burn sepsis through inhibiting NLRP3 inflammasome activation, Am J Emerg Med (2016), http://dx.doi.org/10.1016/j.ajem.2015.12.075