Chemiluminescence of non‐differentiated THP‐1 promonocytes: developing an assay for screening anti‐inflammatory milk proteins and peptides

Human leukemic THP‐1 promonocytes are widely used as a model for peripheral blood monocytes. However, superoxide production during respiratory burst (RB) of non‐differentiated THP‐1 (nd‐THP‐1) cells is very low. Here we present a rapid and low‐cost method for measuring the chemiluminescence (CL) of opsonized zymosan (OZ) induced RB which allows detection of Escherichia coli lipopolysaccharide (LPS) induced priming of nd‐THP‐1 cells on the basis of CL reaction kinetics. Maximum CL intensity obtained was 2.20 ± 0.25 and 1.30 ± 0.11 relative light units, while CL peak time was achieved at 18.1 ± 2.6 and 28.7 ± 1.3 min in primed and non‐primed cells, respectively. The priming of nd‐THP‐1 cells with LPS evoked typical TNF‐α and IL‐6 production. We tested the effects of bovine lactoferrin and protein fractions from Lactobacillus helveticus BGRA43 fermented milk for potential anti‐inflammatory effects on LPS primed nd‐THP‐1 cells. Four fractions were found to inhibit the OZ‐induced CL in a dose‐dependent manner (IC₅₀ 3–30 µg/mL), while lactoferrin inhibited CL to a lesser extent (IC₅₀ 270 µg/mL). These results suggest that measuring CL response of nd‐THP‐1 cells can serve as a method for screening anti‐inflammatory compounds which could be used in reducing the risk of phagocyte‐mediated inflammatory diseases. Copyright © 2010 John Wiley & Sons, Ltd.     

Aminooxyacetic acid attenuates post‐infarct cardiac dysfunction by balancing macrophage polarization through modulating macrophage metabolism in mice

Excessive activation of pro‐inflammatory M1 macrophages following acute myocardial infarction (MI) aggravates adverse cardiac remodelling and heart dysfunction. There are two break points in the tricarboxylic acid cycle of M1 macrophages, and aspartate‐arginosuccinate shunt compensates them. Aminooxyacetic acid (AOAA) is an inhibitor of aspartate aminotransferase in the aspartate‐arginosuccinate shunt. Previous studies showed that manipulating macrophage metabolism may control macrophage polarization and inflammatory response. In this study, we aimed to clarify the effects of AOAA on macrophage metabolism and polarization and heart function after MI. In vitro, AOAA inhibited lactic acid and glycolysis and enhanced ATP levels in classically activated M1 macrophages. Besides, AOAA restrained pro‐inflammatory M1 macrophages and promoted anti‐inflammatory M2 phenotype. In vivo, MI mice were treated with AOAA or saline for three consecutive days. Remarkably, AOAA administration effectively inhibited the proportion of M1 macrophages and boosted M2‐like phenotype, which subsequently attenuated infarct size as well as improved post‐MI cardiac function. Additionally, AOAA attenuated NLRP3‐Caspase1/IL‐1β activation and decreased the release of IL‐6 and TNF‐α pro‐inflammatory cytokines and reciprocally increased IL‐10 anti‐inflammatory cytokine level in both ischaemic myocardium and M1 macrophages. In conclusion, short‐term AOAA treatment significantly improves cardiac function in mice with MI by balancing macrophage polarization through modulating macrophage metabolism and inhibiting NLRP3‐Caspase1/IL‐1β pathway.     

Glycosaminoglycans modulate inflammation and apoptosis in LPS‐treated chondrocytes

Previous studies reported that hyaluronic acid (HA), chondroitin sulphate (CS) and heparan sulphate (HS) were able to reduce the inflammatory process in a variety of cell types after lypopolysaccharide (LPS) stimulation. The aim of this study was to investigate the anti‐inflammatory effect of glycosaminoglycans (GAGs) in mouse articular chondrocytes stimulated with LPS. Chondrocyte treatment with LPS (50 µg/ml) generated high levels of TNF‐α, IL‐1β, IL‐6, IFN‐γ, MMP‐1, MMP‐13, iNOS gene expression and their related proteins, increased NO concentrations (evaluated in terms of nitrites formation), NF‐κB activation and IkBα degradation as well as apoptosis evaluated by the increase in caspase‐3 expression and the amount of its related protein. The treatment of chondrocytes using two different doses (0.5 and 1.0 mg/ml) of HA, chondroitin‐4‐sulphate (C4S), chondroitin‐6‐sulphate (C6S), HS, keratan sulphate (KS) and dermatan sulphate (DS) produced a number of effects. HA exerted a very small anti‐inflammatory and anti‐apoptotic effect while it significantly reduced NO levels, although the effect on iNOS expression and activity was extremely slight. C4S and C6S reduced inflammation mediators and the apoptotic process. C6S failed to decrease NO production, although iNOS expression and activity were significantly reduced. HS, like C4S, was able to reduce all the effects stimulated by LPS treatment. KS and DS produced no reduction in any of the parameters considered. These results give further support to the hypothesis that GAGs actively participate in the regulation of inflammatory and apoptotic processes. J. Cell. Biochem. 106: 83–92, 2009. © 2008 Wiley‐Liss, Inc.     

Dioscorealide B suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF‐κB and ERK1/2 activation

Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti‐inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α) production in lipopolysaccharides (LPS)‐induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS‐induced NO production and cytokine expression through the activation of nuclear factor‐κB (NF‐κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess's reagent, DB reduced NO level with an IC₅₀ value of 2.85 ± 0.62 µM that was due to the significant suppression of LPS‐induced iNOS mRNA expression as well as IL‐1β, IL‐6, and IL‐10 mRNA at a concentration of 6 µM. At the signal transduction level, DB significantly inhibited NF‐κB binding activity, as determined using pNFκB‐Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5–6 µM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, IL‐6, and IL‐10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF‐κB and MAPK/ERK signaling pathway in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 109: 1057–1063, 2010. © 2010 Wiley‐Liss, Inc.     

Macrophages modulate the viability and growth of human mesenchymal stem cells

Following myocardial infarction, tissue repair is mediated by the recruitment of monocytes and their subsequent differentiation into macrophages. Recent findings have revealed the dynamic changes in the presence of polarized macrophages with pro‐inflammatory (M1) and anti‐inflammatory (M2) properties during the early (acute) and late (chronic) stages of cardiac ischemia. Mesenchymal stem cells (MSCs) delivered into the injured myocardium as reparative cells are subjected to the effects of polarized macrophages and the inflammatory milieu. The present study investigated how cytokines and polarized macrophages associated with pro‐inflammatory (M1) and anti‐inflammatory (M2) responses affect the survival of MSCs. Human MSCs were studied using an in vitro platform with individual and combined M1 and M2 cytokines: IL‐1β, IL‐6, TNF‐α, and IFN‐γ (for M1), and IL‐10, TGF‐β1, TGF‐β3, and VEGF (for M2). In addition, polarization molecules (M1: LPS and IFN‐γ; M2: IL‐4 and IL‐13) and common chemokines (SDF‐1 and MCP‐1) found during inflammation were also studied. Indirect and direct co‐cultures were conducted using M1 and M2 polarized human THP‐1 monocytes. M2 macrophages and their associated cytokines supported the growth of hMSCs, while M1 macrophages and their associated cytokines inhibited the growth of hMSCs in vitro under certain conditions. These data imply that an anti‐inflammatory (M2) environment is more accommodating to the therapeutic hMSCs than a pro‐inflammatory (M1) environment at specific concentrations. J. Cell. Biochem. 114: 220–229, 2012. © 2012 Wiley Periodicals, Inc.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of an acute moderate‐exercise session on metabolic and inflammatory profile of PPAR‐α knockout mice

Peroxisome proliferator‐activated receptors (PPARs) play a major role in metabolism and inflammatory control. Exercise can modulate PPAR expression in skeletal muscle, adipose tissue, and macrophages. Little is known about the effects of PPAR‐α in metabolic profile and cytokine secretion after acute exercise in macrophages. In this context, the aim of this study was to understand the influence of PPAR‐α on exercise‐mediated immune metabolic parameters in peritoneal macrophages. Mice C57BL/6 (WT) and PPAR‐α knockout (KO) were examined in non‐exercising control (n = 4) or 24 hours after acute moderate exercise (n = 8). Metabolic parameters (glucose, non‐esterified fatty acids, total cholesterol [TC], and triacylglycerol [TG]) were assessed in serum. Cytokine concentrations (IL‐1β, IL‐6, IL‐10, TNF‐α, and MCP‐1) were measured from peritoneal macrophages cultured or not with LPS (2.5 μg/mL) and Rosiglitazone (1 μM). Exercised KO mice exhibited low glucose concentration and higher TC and TG in serum. At baseline, no difference in cytokine production between the genotypes was observed. However, IL‐1β was significantly higher in KO mice after LPS stimulus. IL‐6 and IL‐1β had increased concentrations in KO compared with WT, even after exercise. MCP‐1 was not restored in exercised KO LPS group. Rosiglitazone was not able to reduce proinflammatory cytokine production in KO mice at baseline level or associated with exercise. Acute exercise did not alter mRNA expression in WT mice. Conclusion: PPAR‐α seems to be needed for metabolic glucose homeostasis and anti‐inflammatory effect of acute exercise. Its absence may induce over‐expression of pro‐inflammatory cytokines in LPS stimulus. Moreover, moderate exercise or PPAR‐γ agonist did not reverse this response.     

PDX regulates inflammatory cell infiltration via resident macrophage in LPS‐induced lung injury

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti‐inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT‐PCR, and ELISA was used to measure MIP‐2, MCP‐1, TNF‐α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 10⁶) were cultured with 1 μg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS‐induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP‐1, MIP‐2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF‐α/MIP‐2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS‐stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.     

Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.     

Lancemaside A inhibits lipopolysaccharide‐induced inflammation by targeting LPS/TLR4 complex

In our previous study, lancemaside A isolated from Codonopsis lanceolata (family Campanulaceae) ameliorated colitis in mice. In this study, the anti‐inflammatory effects of lancemaside A was investigated in lipopolysaccharide (LPS)‐stimulated mice and their peritoneal macrophage cells. Lancemaside A suppressed the production of pro‐inflammatory cytokines, TNF‐α and IL‐1β, in vitro and in vivo. Lancemaside A also down‐regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), as well as the inflammatory mediators, nitric oxide (NO), and PGE₂. Lancemaside A also inhibited the expression of IL‐1 receptor‐associated kinase‐4 (IRAK‐4), the phosphorylation of IKK‐β and IκB‐α, the nuclear translocation of NF‐κB and the activation of mitogen‐activated protein kinases in LPS‐stimulated peritoneal macrophages. Furthermore, lancemaisde A inhibited the interaction between LPS and TLR4, as well as IRAK‐4 expression in peritoneal macrophages. Based on these findings, lancemaside A expressed anti‐inflammatory effects by regulating both the binding of LPS to TLR4 on macrophages. J. Cell. Biochem. 111: 865–871, 2010. © 2010 Wiley‐Liss, Inc.     

Natural antiviral compound silvestrol modulates human monocyte‐derived macrophages and dendritic cells

Outbreaks of infections with viruses like Sars‐CoV‐2, Ebola virus and Zika virus lead to major global health and economic problems because of limited treatment options. Therefore, new antiviral drug candidates are urgently needed. The promising new antiviral drug candidate silvestrol effectively inhibited replication of Corona‐, Ebola‐, Zika‐, Picorna‐, Hepatis E and Chikungunya viruses. Besides a direct impact on pathogens, modulation of the host immune system provides an additional facet to antiviral drug development because suitable immune modulation can boost innate defence mechanisms against the pathogens. In the present study, silvestrol down‐regulated several pro‐ and anti‐inflammatory cytokines (IL‐6, IL‐8, IL‐10, CCL2, CCL18) and increased TNF‐α during differentiation and activation of M1‐macrophages, suggesting that the effects of silvestrol might cancel each other out. However, silvestrol amplified the anti‐inflammatory potential of M2‐macrophages by increasing expression of anti‐inflammatory surface markers CD206, TREM2 and reducing release of pro‐inflammatory IL‐8 and CCL2. The differentiation of dendritic cells in the presence of silvestrol is characterized by down‐regulation of several surface markers and cytokines indicating that differentiation is impaired by silvestrol. In conclusion, silvestrol influences the inflammatory status of immune cells depending on the cell type and activation status.     

8‐Methoxypsoralen protects bovine mammary epithelial cells against lipopolysaccharide‐induced inflammatory injury via suppressing JAK/STAT and NF‐κB pathway

Bovine mastitis is the most common disease in dairy cattle. Bacterial infections are the main cause of mastitis. Lipopolysaccharide (LPS), a major structural component of the cell wall of Escherichia coli, is a good inducer used to replicate inflammation models. 8‐Methoxypsoralen (8‐MOP), a formerly considered photosensitizing agent, has been used in immunotherapy. This study investigated the protective effects of 8‐MOP on LPS‐induced inflammatory injury in bovine mammary epithelial cells (BMECs). LPS treatment (50 μg/mL for 12 hr) caused a decrease in cell viability, morphological damage, and cell apoptosis. Pretreatment with 8‐MOP at concentrations of 25 and 50 μg/ml significantly attenuated LPS‐induced inflammation in BMECs. qRT‐PCR analysis revealed that the messenger RNA expression of inflammatory cytokines and chemokine (interleukin‐1β [IL‐1β], IL‐6, tumor necrosis factor‐α, and IL‐8) was suppressed by 8‐MOP in LPS‐stimulated BMECs. Western blot analysis showed that 8‐MOP could also reduce the protein levels of cyclooxygenase‐2 and promote the translocation of high‐mobility group box 1 from the nucleus to cytoplasm. Furthermore, the anti‐inflammatory property of 8‐MOP was mediated by inhibiting nuclear factor kappa‐light‐chain‐enhancer of activated B cells activation and STAT1 phosphorylation. Taken together, 8‐MOP could protect cells from inflammatory injury induced by LPS, and may be a potential agent against bovine mastitis.     

5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.     

Severely Obese Have Greater LPS‐stimulated TNF‐α Production Than Normal Weight African‐American Women

Obesity is associated with an increase in chronic, low‐grade inflammation which has been implicated in the development of type 2 diabetes mellitus and cardiovascular disease. The purpose of this study was to determine whether obesity was associated with an elevation of whole blood lipopolysaccharide (LPS)‐stimulated tumor necrosis factor‐α (TNF‐α) production. African‐American women were recruited from a larger study and assigned to one of five groups based on BMI: normal weight (NORM; BMI 20–25, n = 7), overweight (OVER; BMI 25–30, n = 12), class 1 obese (OB1; BMI 30–35, n = 19), class 2 obese (OB2; BMI 35–40, n = 10), or class 3 obese (OB3; BMI >40, n = 17). Body composition was determined via a whole body dual‐energy X‐ray absorptiometry (DXA) scan. Venous blood samples were collected following an overnight fast (>8 h), and stimulated with five doses of LPS (Salmonella enteriditis): 80, 40, 20, 10, and 5 µg/ml for 24 h in a 37 °C, 5% CO₂ incubator. Following stimulation, TNF‐α was measured using enzyme‐linked immunosorbent assay. OB3 produced 365% more TNF‐α than NORM at an LPS dose of 20 µg/ml (P < 0.05). When maximal TNF‐α production was assessed regardless of LPS dose, OB3 produced 230% more than NORM and OVER produced 190% more than NW (P = 0.001). Total and trunk fat mass and BMI were significantly correlated with maximal TNF‐α production and LPS = 20 µg/ml. Our findings are consistent with previous reports suggesting a relationship between increased adiposity and inflammatory marker production. This is one of the first studies to focus on African‐American women, who have higher rates of obesity.     

MiR‐16 regulates mouse peritoneal macrophage polarization and affects T‐cell activation

MiR‐16 is a tumour suppressor that is down‐regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR‐16 on macrophage polarization and subsequent T‐cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon‐γ and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL)‐4. The identity of polarized macrophages was determined by profiling cell‐surface markers by flow cytometry and cytokine production by ELISA. Macrophages were infected with lentivirus‐expressing miR‐16 to assess the effects of miR‐16. Effects on macrophage–T cell interactions were analysed by co‐culturing purified CD4⁺ T cells with miR‐16‐expressing peritoneal macrophages, and measuring activation marker CD69 by flow cytometry and cytokine secretion by ELISA. Bioinformatics analysis was applied to search for potential miR‐16 targets and understand its underlying mechanisms. MiR‐16‐induced M1 differentiation of mouse peritoneal macrophages from either the basal M0‐ or M2‐polarized state is indicated by the significant up‐regulation of M1 marker CD16/32, repression of M2 marker CD206 and Dectin‐1, and increased secretion of M1 cytokine IL‐12 and nitric oxide. Consistently, miR‐16‐expressing macrophages stimulate the activation of purified CD4⁺ T cells. Mechanistically, miR‐16 significantly down‐regulates the expression of PD‐L1, a critical immune suppressor that controls macrophage–T cell interaction and T‐cell activation. MiR‐16 plays an important role in shifting macrophage polarization from M2 to M1 status, and functionally activating CD4⁺ T cells. This effect is potentially mediated through the down‐regulation of immune suppressor PD‐L1.     

Dexmedetomidine protects against high mobility group box 1‐induced cellular injury by inhibiting pyroptosis

Dexmedetomidine (DEX) is a widely used clinical anesthetic with proven anti‐inflammatory effects. Both high mobility group box 1 (HMGB1) and pyroptosis play an important role in the inflammatory response to infection and trauma. Thus far, there have been no studies published addressing the effect of DEX on HMGB1 and pyroptosis. In order to fill this gap in the literature, bone marrow‐derived macrophages (BMDMs) were exposed to HMGB1 (4 µg/mL) with or without DEX (50 μM) pretreatment. The production of pro‐inflammatory cytokines [such as tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), and IL‐18], phosphorylation of extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) and P38, and the activation of caspase‐1 were measured by enzyme immunosorbent assay, western blot analysis, confocal microscope, and flow cytometry, respectively. We found that DEX protected against HMGB1‐induced cell death of BMDMs. In addition, DEX suppressed the generation of TNF‐α, IL‐1β, and IL‐18 as well as the phosphorylation of ERK1/2 and P38. Moreover, DEX inhibited caspase‐1 activation and decreased pyroptosis. Taken together, these findings demonstrate the protective effect of DEX in mediating HMGB1‐induced cellular injury, thus indicating that DEX may be a potential therapeutic candidate for the management of infection and trauma‐derived inflammation.