Inhibited High Mobility Group Box 1 Release Research Articles

Glycyrrhizin protects against focal cerebral ischemia via inhibition of T cell activity and HMGB1-mediated mechanisms.

BackgroundGlycyrrhizin (Gly) protects against brain injury induced by stroke. We studied whether Gly achieves its protection by inhibiting T cell activity and high-mobility group box 1 (HMGB1) release in the ischemic brain.MethodsStroke was induced by transient middle cerebral artery occlusion in rats and mice. Gly was injected intraperitoneally before or after stroke. We measured infarction, neuroinflammatory cells, gene expressions of interferon-γ (IFNγ), IL-4, and IL-10 in CD4 T cells, HMGB1 release, and T cell proliferation in cultured splenocytes.ResultsGly treatment reduced infarctions and neuroinflammation characterized by the infiltration of CD68-positive macrophages and myeloperoxidase-positive neutrophils, which corresponds to a reduction in the number of T cells and their subsets, CD4 and CD8 T cells, in the ischemic brain, as measured by flow cytometry. Unlike in wild-type animals, Gly did not offer protection in nude rats and severe combined immunodeficient (SCID) mice who had no T cells, while Gly reduced infarction in both nude rats and SCID mice whose T cells were reconstituted, suggesting that T cells should be the target of Gly. In addition, Gly administration inhibited T cell proliferation stimulated by ConA in in vitro assays and inhibited HMGB1 release from the ischemic brain. Furthermore, Gly attenuated gene expression of IFNγ, but not IL-4 and IL-10 in CD4 T cells. Lastly, HMGB1 promoted T cell proliferation stimulated by ConA, which was inhibited by the addition of Gly.ConclusionsGly blocks infarction by inhibiting IFNγ-mediated T cell activity, which is at least partly modulated by HMGB1 activity.

Inhibiting HMGB1 with Glycyrrhizic Acid Protects Brain Injury after DAI via Its Anti-Inflammatory Effect.

High-mobility group box 1 (HMGB1), a nuclear protein that has endogenous cytokine-like activity, is involved in several neurological diseases by mediating inflammatory response. In this study, a lateral head rotation device was used to establish a rat diffuse axonal injury (DAI) model. The dynamic expression of HMGB1, apoptosis-associated proteins, and proinflammatory cytokines were detected by Western blot, and neuronal apoptosis was observed by TUNEL staining. The extracellular release of HMGB1 and the accumulation of β-APP were observed by immunofluorescence and immunohistochemistry, respectively. The brain injury was indicated by modified neurological severity score (mNSS), brain water content (BWC), and the extravasation of Evans blue. We showed that HMGB1 level obviously decreased within 48 h after DAI, accompanied by neuronal apoptosis, the activation of caspases 3 and 9, and the phosphorylation of BCL-2. Inhibiting HMGB1 with glycyrrhizic acid (GL) can suppress the activation of apoptosis-associated proteins and inhibit the expression of proinflammatory cytokines, which ameliorated motor and cognitive deficits, reduced neuronal apoptosis, and protected the integrity of blood brain barrier (BBB) and axonal injury after experimental DAI in rats. Thus, HMGB1 may be involved in the inflammatory response after DAI, and inhibition of HMGB1 release with GL can notably alleviate the brain injury after DAI.

Alarmin High Mobility Group Box 1 (HMGB1) Activates Alternative NFkB Signaling in Bone Marrow Macrophages in the Leukemia Microenvironment

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) that signals through pattern recognition receptors on monocyte/macrophages to recruit them to sites of tissue injury. In models of sepsis and autoimmunity, HMGB1 can have both immune-activating and regulatory effects depending on the receptor system engaged and the downstream pathway activated. Although chemotherapy-stressed tumor cells are known to release HMGB1, little is known about the effects of tumor-derived HMGB1 on macrophages in the tumor microenvironment.We have previously shown that primary human acute lymphoblastic leukemia cells release high levels of HMGB1 after treatment with anthracycline chemotherapy, and that leukemia-associated HMGB1 induces expression of the IL-1 converting enzyme (caspase-1), consistent with activation of the inflammasome. Paradoxically, inflammatory cytokine production (including IL-1beta) is reduced in this setting. In the present study, we test the hypothesis leukemia-associated HMGB1 activates alternative NFkB signaling through the repressor protein RelB which has been recently shown to silence inflammatory cytokine production via epigenetic effects in sepsis models.To establish proof-of-principle, we first treated primary human bone marrow macrophages (BMM) and THP1 monocytes with 200 ng/ml recombinant HMGB1 (r-HMGB1) in the presence or absence of bacterial lipopolysaccharide (LPS). As predicted, LPS alone increased baseline expression of total NFkB, total p65/RelA and phosphorylated p65/RelA mRNA (20.6 + 7.9-fold by qPCR ddCT, p<0.0001) and protein (measured by Western blot, in BMM and THP1. In contrast, r-HMGB1 treatment produced a significant increase in the expression of RelB mRNA (2.5 + 0.7 fold increase p<0.05) and protein without a significant change in the expression of tNFkB or p65/RelA. Treatment with both r-HMGB1 and LPS maintained this pattern of increased RelB expression without increased expression of p65/RelA, suggesting HMGB1 activity as the dominant effect. Increased RelB expression was unaffected by the addition of an antibody to TLR4, the primary receptor for LPS and a candidate receptor for HMGB1.Next, we determined whether induction of RelB expression also occurs in response to leukemia-derived HMGB1. Immunomagnetically selected ALL cells from the bone marrow of 3 pediatric patients and NALM 6 ALL cells were treated with 20 nM doxorubicin, washed, and placed in culture for 24 hours prior to the addition of BMM in direct coculture. In our model, ALL cells typically release 100-450 ng/ml HMGB1 under these conditions. Again, significantly increased expression of RelB mRNA (6.7 + 2.9 fold, p<0.001) and protein was observed in BMM cultured with doxorubicin-treated ALL cells compared to ALL cells treated with media control, with a corresponding decrease in phospho-IkBa protein expression and unchanged total NFkB, p65/RelA and phosphorylated p65/RelA. Pre-treatment of ALL cells with quercetin, a commercially available flavonoid antioxidant shown to inhibit HMGB1 release, inhibited protein expression of RelB in BMM in a dose-dependent fashion.These data indicate that tumor-derived HMGB1 activates alternative NFkB signaling and is capable of reprogramming the inflammatory response to LPS. Studies are ongoing to the determine downstream effects on macrophage antitumor function to optimize therapeutic reversal of HMGB1 effects and improve the potential for bone marrow macrophages to eradicate residual leukemia that remains after chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Deacetylation-mediated interaction of SIRT1-HMGB1 improves survival in a mouse model of endotoxemia.

Inflammatory signal-mediated release of high-mobility group box 1 (HMGB1) is a damage-associated molecular pattern or alarmin. The inflammatory functions of HMGB1 have been extensively investigated; however, less is known about the mechanisms controlling HMGB1 release. We show that SIRT1, the human homolog of the Saccharomyces cerevisiae protein silent information regulator 2, which is involved in cellular senescence and possibly the response to inflammation, forms a stable complex with HMGB1 in murine macrophage RAW264.7 cells. SIRT1 directly interacted with HMGB1 via its N-terminal lysine residues (28–30), and thereby inhibited HMGB1 release to improve survival in an experimental model of sepsis. By contrast, inflammatory stimuli such as lipopolysaccharide (LPS) and tumor necrosis factor-α promoted HMGB1 release by provoking its dissociation from SIRT1 dependent on acetylation, thereby increasing the association between HMGB1 and chromosome region maintenance 1, leading to HMGB1 translocation. In vivo infection with wild-type SIRT1 and HMGB1K282930R, a hypo-acetylation mutant, improved survival (85.7%) during endotoxemia more than infection with wild-type SIRT1 and HMGB1-expressing adenovirus, indicating that the acetylation-dependent interaction between HMGB1 and SIRT1 is critical for LPS-induced lethality. Taken together, we propose that SIRT1 forms an anti-inflammatory complex with HMGB1, allowing cells to bypass the response to inflammation.

Vagal modulation of high mobility group box-1 protein mediates electroacupuncture-induced cardioprotection in ischemia-reperfusion injury.

Excessive release of high mobility group box-1 (HMGB1) protein from ischemic cardiomyocytes activates inflammatory cascades and enhances myocardial injury after reperfusion. Here we report evidence that electroacupuncture of mice at Neiguan acupoints can inhibit the up-regulation of cardiac HMGB1 following myocardial ischemia and attenuate the associated inflammatory responses and myocardial injury during reperfusion. These benefits of electroacupuncture were partially reversed by administering recombinant HMGB1 to the mice, and further potentiated by administering anti-HMGB1 antibody. Electroacupuncture-induced inhibition of HMGB1 release was markedly reduced by unilateral vagotomy or administration of nicotinic receptor antagonist, but not by chemical sympathectomy. The cholinesterase inhibitor neostigmine mimicked the effects of electroacupuncture on HMGB1 release and myocardial ischemia reperfusion injury. Culture experiments with isolated neonatal cardiomyocytes showed that acetylcholine, but not noradrenaline, inhibited hypoxia-induced release of HMGB1 via a α7nAchR-dependent pathway. These results suggest that electroacupuncture acts via the vagal nerve and its nicotinic receptor-mediated signaling to inhibit HMGB1 release from ischemic cardiomyocytes. This helps attenuate pro-inflammatory responses and myocardial injury during reperfusion.

Stearoyl lysophosphatidylcholine prevents lipopolysaccharide-induced extracellular release of high mobility group box-1 through AMP-activated protein kinase activation

Previous studies have suggested that stearoyl lysophosphatidlycholine (LPC) protects against lethal experimental sepsis by inhibiting lipopolysaccharide (LPS)-induced extracellular release of high-mobility group box 1 (HMGB1). However, limited information exists on the mechanism by which stearoyl-LPC suppresses the extracellular release of HMGB1 in monocyte/macrophages stimulated with LPS. In this study, we found that stearoyl-LPC increased the phosphorylation of AMP-activated protein kinase (AMPK) in macrophages. Exposure of LPS-stimulated macrophages to stearoyl-LPC decreased the extracellular release of HMGB1 in peritoneal macrophages, which were inhibited by the AMPK inhibitor, compound C. In addition, stearoyl-LPC-mediated suppression of HMGB1 release was abolished by siRNA-mediated knock-down of AMPKα1. Stearoyl-LPC increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of activated AMPK, in mice lungs and decreased HMGB1 levels in bronchoalveolar lavage fluids in mice administered LPS. These results reveal a novel mechanism by which stearoyl-LPC regulates LPS-mediated cellular translocation of HMGB1.

The Free Radical Scavenger NecroX-7 Attenuates Acute Graft-versus-Host Disease via Reciprocal Regulation of Th1/Regulatory T Cells and Inhibition of HMGB1 Release.

Graft-versus-host disease (GVHD) is a major complication associated with allogeneic hematopoietic stem cell transplantation. Despite the prominent role of the adaptive immune system, the importance of controlling the innate immune system in the pathogenesis of GVHD has recently been rediscovered. High-mobility group box 1 (HMGB1) is a crucial damage-associated molecular pattern signal that functions as a potent innate immune mediator in GVHD. In the present study, we investigated treatment of experimental GVHD through HMGB1 blockade using the compound cyclopentylamino carboxymethylthiazolylindole (NecroX)-7. Treated animals significantly attenuated GVHD-related mortality and inhibited severe tissue damage. These protective effects correlated with the decrease in HMGB1 expression and lower levels of reactive oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of TNF and IL-6, as well as the expression of TLR-4 and receptor for advanced glycation end products. We also observed increased regulatory T cell numbers, which may be associated with regulation of differentiation signals independent of HMGB1. Taken together, these data indicate that NecroX-7 protects mice against lethal GVHD by reciprocal regulation of regulatory T/Th1 cells, attenuating systemic HMGB1 accumulation and inhibiting HMGB1-mediated inflammatory response. Our results indicate the possibility of a new use for a clinical drug that is effective for the treatment of GVHD.

Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals

High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. We tested hypothesis that ascorbic acid (AscA) induces heme oxygenase (HO)-1 which inhibits HMGB1 release in lipopolysaccharide (LPS)-stimulated cells and increases survival of septic mice. AscA increased HO-1 protein expression in a concentration- and time-dependent manner via Nrf2 activation in RAW 264.7 cells. HO-1 induction by AscA was significantly reduced by Nrf2 siRNA-transfected cells. Mutation of cysteine to serine of keap-1 proteins (C151S, C273S, and C288S) lost the ability of HO-1 induction by AscA, due to failure of translocation of Nrf-2 to nucleus. The PI3 kinase inhibitor, LY294002, inhibited HO-1 induction by AscA. Oxyhemoglobin (HbO2), LY294002, and ZnPPIX (HO-1 enzyme inhibitor) reversed effect of AscA on HMGB1 release. Most importantly, administration of AscA (200mg/kg, i.p.) significantly increased survival in LPS-induced endotoxemic mice. In cecal ligation and puncture (CLP)-induced septic mice, AscA reduced hepatic injury and serum HMGB1 and plasminogen activator inhibitor (PAI)-1 in a ZnPPIX-sensitive manner. In addition, AscA failed to increase survival in Nrf2 knockout mice by LPS. Thus, we concluded that high dose of AscA may be useful in the treatment of sepsis, at least, by activation of Nrf2/HO-1 signals.

Hesperidin ameliorates lipopolysaccharide-induced acute lung injury in mice by inhibiting HMGB1 release.

Hesperidin (HDN), a flavanone glycoside, possesses anti-inflammatory properties and has been suggested to be able to modulate the lipopolysaccharide (LPS)-induced acute lung injury (ALI). High-mobility group box 1 (HMGB1) serves as an inflammatory cytokine when released extracellularly and is involved in the pathogenesis of diverse inflammatory disorders. The current study aimed to investigate the involvement of HMGB1 in HDN-induced immunoregulation of ALI. ALI in male BALB/c mice was induced by intranasal administration of LPS (0.5mg/kg). HDN (500mg/kg) was administered intragastrically 10days prior to LPS exposure. HDN significantly protected animals from LPS-induced ALI as evidenced by decreased elevation of the lung wet to dry weight ratio, total cells, neutrophils, macrophages, and myeloperoxidase (MPO) activity, associated with reduced lung histological damage. In the meantime, HDN pretreatment markedly inhibited the production of pro-inflammatory cytokines and chemokine, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Furthermore, HDN pretreatment dramatically inhibited the infiltration of macrophages and suppressed the expression and release of HMGB1 in vivo and in vitro. In addition, intranasal application of exogenous HMGB1 could result in lung injury which was also alleviated by HDN administration. These results suggest that HDN pretreatment protects mice from LPS-induced ALI via inhibiting the production of TNF-α and IL-6. Moreover, we found that HDN could inhibit the expression and release of HMGB1 via suppressing the infiltration of macrophages and production of MCP-1.

Role of high-mobility group box-1 in myocardial ischemia/reperfusion injury and the effect of ethyl pyruvate.

High-mobility group box-1 (HMGB1) acts as a proinflammatory cytokine that triggers and amplifies the inflammation cascade following ischemia/reperfusion (I/R). Ethyl pyruvate (EP) has been reported to inhibit HMGB1 release in several I/R models. This study was designed to investigate the potential role of HMGB1 in a rat myocardial I/R model and to determine the effect of EP. Male Sprague Dawley rats were subjected to 30 min myocardial ischemia and 48 h reperfusion. In protocol 1, the rats were assigned to one of four groups (n=16 per group): Phosphate-buffered saline (PBS) or recombinant human HMGB1 (rhHMGB1) at three different doses (1, 10 or 100 μg/kg). In protocol 2, the rats were also assigned to one of four groups (n=16 per group): Sham, control, EP and EP + rhHMGB1. EP (40 mg/kg) or rhHMGB1 (100 μg/kg) was injected intravenously prior to reperfusion. Hemodynamic measurements were performed, and myocardial infarct size (IS) was calculated. Western blotting was conducted to evaluate HMGB1, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expression levels. In the protocol 1 rats, the IS was markedly increased in the rhHMGB1 (100 μg/kg) group compared with that in the PBS group, and this increase was accompanied by elevated levels of TNF-α and IL-6. In the protocol 2 rats, I/R resulted a 4.8-fold increase in HMGB1 expression with an increased IS and impaired cardiac function compared with the sham group. EP significantly inhibited the elevated HMGB1 level, suppressed the activated TNF-α and IL-6 and reduced cardiac dysfunction. This cardioprotection was abolished by rhHMGB1. In conclusion, accumulation of HMGB1 is deleterious to the heart following myocardial I/R. EP can exert a strong protective effect against myocardial I/R injury, and these benefits are associated with a reduction in HMGB1.

HMGB1 exacerbates renal tubulointerstitial fibrosis through facilitating M1 macrophage phenotype at the early stage of obstructive injury.

Previous studies have indicated that macrophage phenotype diversity is involved in the progression of renal fibrosis. However, the factors facilitating M1 or M2 phenotypes and the function of these polarized macrophages in kidney injury and fibrosis remain largely unknown. In the present study, we found that macrophages accumulated in the kidney interstitium exhibited mainly as the M1 phenotype at the early stage of unilateral ureter obstruction (UUO). High-mobility group box 1 (HMGB1) protein expressed and released from tubular epithelial cells and interstitial macrophages was essential for the M1 macrophage transition. HMGB1 significantly induced the expression of the M1 marker inducible nitric oxide synthase while decreasing the M2 marker IL-10 in macrophages. Moreover, a glycyrrhizic acid derivative, a blocker of HMGB1 release, reduced UUO-mediated kidney injury and ameliorated UUO-induced renal fibrosis. Interestingly and importantly, UUO caused a low pH value in the urine accumulated in the obstructed ureter, and the acidified urine induced HMGB1 release from tubular epithelial cells and macrophages in vitro. Our data demonstrate that HMGB1 is an essential contributor in facilitating M1 polarization at the early stage of UUO. Inhibition of HMGB1 release may alter macrophage phenotype and contribute to the protection of kidney tissue from injury and fibrosis.

Ligand-activated peroxisome proliferator-activated receptor-δ and -γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1.

Peroxisome proliferator-activated receptors (PPARs) inhibit lipopolysaccharide (LPS)-primed release of high mobility group box 1 (HMGB1), a late proinflammatory mediator, but the underlying molecular mechanism is not completely understood. In this study, we demonstrated that the inhibition of HMGB1 release by PPAR-δ and -γ is associated with the deacetylase activity of SIRT1. Ligand-activated PPAR-δ and -γ inhibited LPS-primed release of HMGB1, concomitant with elevation in SIRT1 expression and promoter activity. These effects were significantly reduced in the presence of small interfering (si)RNAs against PPAR, indicating that PPAR-δ and -γ are involved in both HMGB1 release and SIRT1 expression. In addition, modulation of SIRT1 expression and activity by siRNA or chemicals correspondingly influenced the effects of PPARs on HMGB1 release, suggesting a mechanism in which SIRT1 modulates HMGB1 release. Furthermore, we showed for the first time that HMGB1 acetylated in response to LPS or p300/CBP-associated factor (PCAF) is an effective substrate for SIRT1, and that deacetylation of HMGB1 is responsible for blockade of HMGB1 release in macrophages. Finally, acetylation of HMGB1 was elevated in mouse embryonic fibroblasts from SIRT1-knockout mice, whereas this increase was completely reversed by ectopic expression of SIRT1. These results indicate that PPAR-mediated upregulation of SIRT1 modulates the status of HMGB1 acetylation, which, in turn, has a critical role in the cellular response to inflammation through deacetylation-mediated regulation of HMGB1 release.

Ketamine inhibits LPS-induced HGMB1 release in vitro and in vivo.

High mobility group box 1 (HMGB1) has been identified to be a critical mediator of severe sepsis. Ketamine has been shown to reduce sepsis-induced pathological complications. These effects are because of the reduced expression and release of several inflammatory mediators. However, whether ketamine affects the expression and release of HMGB1 is not known. We investigated the effect of ketamine on HMGB1 release in lipopolysaccharide (LPS)-induced macrophages in vitro and in cecal ligation and puncture (CLP)-induced septic rats in vivo, and determined its molecular mechanism of action. RAW264.7 cells or primary macrophages were incubated with or without LPS (500 ng/mL) in the presence or absence of ketamine, a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580), a nuclear factor-kappa B (NF-κB) inhibitor (pyrimidine dithiocarbamate), or small interfering RNA. The protein and expression levels of inflammatory mediators, such as HMGB1, tumor necrosis factor-α, and interleukin-1β were measured using enzyme-linked immunosorbent assays and real-time polymerase chain reaction. The effect of ketamine on NF-κB and p38 MAPK activation was evaluated using enzyme-linked immunosorbent assays, Western blot analysis, and electrophoretic mobility shift assay. Western blotting was used to observe changes in translocation of HMGB1 from the nucleus to cytoplasm. In addition, CLP-induced septic rats were treated with ketamine (0.5, 5, 10 mg/kg) or saline (10 mL/kg) 3h after sepsis, and the levels of HMGB1 and functional parameters of multiple organs were determined using several detection kits. Seven-day survival was also assessed. Ketamine inhibited HMGB1 release in LPS-activated RAW264.7 cells and CLP-induced septic rats. Translocation of HMGB1 from the nucleus to cytosol and expression of HMGB1 mRNA were inhibited significantly by ketamine. Ketamine inhibited the translocation of NF-κB from the cytoplasm to the nucleus and phosphorylation of p38 MAPK in LPS-activated RAW264.7 cells. Rats treated with ketamine improved survival in rats and significantly reduced CLP-induced dysfunction/injury of organs. Ketamine suppresses LPS-induced HMGB1 release in LPS-activated RAW264.7 cells and a CLP-induced model of sepsis in rats by partially inhibiting NF-κB/p38 MAPK pathways. Ketamine increased survival time induced by CLP and reduced organ dysfunction in septic peritonitis.

Levobupivacaine inhibits lipopolysaccharide-induced high mobility group box 1 release in vitro and in vivo

BackgroundThe aim of the study was to investigate whether levobupivacaine (LB) suppressed lipopolysaccharide (LPS)-induced high mobility group box 1 (HMGB1) release in vitro and in vivo, and to determin its molecular mechanisms of action. Materials and methodsRAW264.7 cells were treated with LPS and LB for 24 h. Levels of HMGB1, nuclear factor-kappa B (NF-κB) and phosphorylated p38 mitogen-activated protein kinase (MAPK) were measured by Enzyme-linked immunosorbent assay and Western blotting; the levels of HMGB1 messenger RNA were measured by real-time polymerase chain reaction. In addition, cecal ligation and puncture–induced septic C57BL/6 received LB infusion, and the levels of HMGB1 and functional parameters of multiple organs determined using several detection kits. ResultsLB inhibited HMGB1 release in vitro and in vivo. Furthermore, LB inhibited the translocation of NF-κB and phosphorylation of p38 MAPK in vitro. Mice treated with LB infusion improved survival in mice and significantly reduced cecal ligation and puncture–induced dysfunction of organs. ConclusionsLB suppresses LPS-induced HMGB1 release in vitro and in vivo by partially inhibiting NF-κB/p38 MAPK pathways. LB can rescue mice from sepsis and protect against organ dysfunction in septic mice.

Isoproterenol-mediated heme oxygenase-1 induction inhibits high mobility group box 1 protein release and protects against rat myocardial ischemia/reperfusion injury in vivo

Isoproterenol (ISO) has been reported to inhibit high mobility group box 1 (HMGB1) protein release via heme oxygenase-1 (HO-1) induction in lipopolysaccharide (LPS)-activated RAW 264.7 cells and increase the survival rate of cecal ligation and puncture (CLP)-induced septic mice. Therefore, it was examined whether ISO-mediated HO-1 induction inhibits HMGB1 release in cardiac myocytes and attenuates myocardial ischemia/reperfusion (I/R) injury in rats. Anesthetized male rats were pretreated with ISO [intraperitoneal (i.p.) injection of 10 mg/kg] prior to ischemia in the absence and/or presence of zinc protoporphyrin IX (ZnPPIX, i.p., 10 mg/kg), which is an inhibitor of HO-1, and then subjected to ischemia for 30 min followed by reperfusion for 24 h. The myocardial I/R injury and oxidative stress were assessed. In addition, the HO-1 protein and HMGB1 expression were measured by western blot analysis. ISO significantly attenuated the myocardial I/R injury, reduced oxidative stress, and induced HO-1 and reduced HMGB1 release. However, all these effects caused by ISO were significantly reversed in the presence of ZnPPIX. These results suggested that ISO has a pivotal role in the protective effects on myocardial I/R injury. This protection mechanism is possibly due to the inhibition of HMGB1 release via the induction of HO-1.

Baicalein inhibits HMGB1 release and MMP-2/-9 expression in lipopolysaccharide-induced cardiac hypertrophy.

Myocardial dysfunction, a common complication after sepsis, significantly contributes to the death of patients with septic shock. In the search for potentially effective drugs to decrease mortality from sepsis, we investigated the cardioprotective effects of baicalein, a flavonoid present in the root of Scutellaria baicalensis, on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and matrix metalloproteinase-2 and -9 (MMP-2/-9) expression. We found that baicalein significantly attenuated LPS-induced cardiac hypertrophy and counteracted reactive oxygen species (ROS) generation in neonatal rat cardiomyocytes. In addition, pretreatment with baicalein inhibited LPS-induced early (e.g., tumor necrosis factor-α (TNF-α) and interleukin-6) and late (e.g., high mobility group box 1 (HMGB1) pro-inflammatory cytokine release, inducible nitric oxide synthase (iNOS) expression and NO production. Finally, baicalein also significantly down-regulated the expression of MMP-2/-9 and attenuated HMGB1 translocation from the nucleus to the cytoplasm. These results suggest that baicalein can protect cardiomyocytes from LPS-induced cardiac injury via the inhibition of ROS and inflammatory cytokine production. These cardioprotective effects are possibly mediated through the inhibition of the HMGB1 and MMP-2/-9 signaling pathways.

Anti-inflammatory effects of rutin on HMGB1-induced inflammatory responses in vitro and in vivo

High mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions. Rutin (RT), an active flavonoid compound, is well known to possess potent antiplatelet, antiviral and antihypertensive properties. In this study, we investigated the anti-inflammatory effects of RT against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) induced by HMGB1 and the associated signaling pathways. The anti-inflammatory activities of RT were determined by measuring permeability, monocytes adhesion and migration, and activation of pro-inflammatory proteins in HMGB1-activated HUVECs and mice. We found that RT potently inhibited HMGB1 release, down-regulated HMGB1-dependent inflammatory responses in human endothelial cells, and inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with RT resulted in reduced cecal ligation and puncture-induced release of HMGB1 and sepsis-related mortality. Further studies revealed that RT suppressed the production of tumor necrosis factor-α and interleukin 6 and the activation of nuclear factor-κB and extracellular regulated kinases 1/2 by HMGB1. Collectively, these results indicate that RT could be a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Chloroquine inhibits HMGB1 inflammatory signaling and protects mice from lethal sepsis

Sepsis is caused by an overwhelming immune response to bacterial infection. The discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal sepsis has prompted investigation into the development of new therapeutics which specifically target this protein. Here, we show that chloroquine, an anti-malarial drug, prevents lethality in mice with established endotoxemia or sepsis. This effect is still observed even if administration of chloroquine is delayed. The protective effects of chloroquine were mediated through inhibition of HMGB1 release in macrophages, monocytes, and endothelial cells, thereby preventing its cytokine-like activities. As an inhibitor of autophagy, chloroquine specifically inhibited HMGB1-induced Iκ-B degradation and NF-κB activation. These findings define a novel mechanism for the anti-inflammatory effects of chloroquine and also suggest a new potential clinical use for this drug in the setting of sepsis.

Inhibitory effects of epi-sesamin on HMGB1-induced vascular barrier disruptive responses in vitro and in vivo

Nuclear DNA-binding protein high mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions, such as sepsis and septic shock. Epi-sesamin (ESM), an important component of Asarum sieboldii roots, is known to exhibit anti-allergic, anti-nociceptive, and anti-fungal effects. However, little is known of its effects on HMGB1-mediated inflammatory responses. Here, we investigated this issue by monitoring the effects of ESM on lipopolysaccharide (LPS) or cecal ligation and the puncture (CLP)-mediated release of HMGB1, and on modulation of HMGB1-mediated inflammatory responses. ESM potently inhibited HMGB1 release, down-regulated HMGB1-dependent inflammatory responses in human endothelial cells, and inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with ESM resulted in reduced CLP-induced release of HMGB1 and sepsis-related mortality. Of particular interest, ESM inhibition of HMGB1-mediated anti-inflammatory activity was more potent than that by sesamin (SM), likely due to differences between their three-dimensional structures. These results indicate that ESM could be a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Tanshinone IIA sodium sulfonate facilitates endocytic HMGB1 uptake

Our seminal discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal systemic inflammation has prompted a new field of investigation for the development of experimental therapeutics. We previously reported that a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS), selectively inhibited endotoxin-induced HMGB1 release and conferred protection against lethal endotoxemia and sepsis. To investigate the underlying mechanisms by which TSN-SS effectively inhibits HMGB1 release, we examined whether TSN-SS stimulates HMGB1 uptake by macrophages and whether genetic depletion of HMGB1 receptors [e.g., toll-like receptors (TLR)2, TLR4, or the receptor for advanced glycation end product (RAGE)] or pharmacological inhibition of endocytosis impairs TSN-SS-facilitated HMGB1 cellular uptake. TSN-SS stimulated internalization of exogenous HMGB1 protein into macrophage cytoplasmic vesicles that subsequently co-localized with microtubule-associated protein light chain 3 (LC3)-positive punctate structures (likely amphisomes). Meanwhile, it time-dependently elevated cellular levels of internalized HMGB1, leading to elevated LC3-II production and aggregation. Although genetic depletion of TLR2, TLR4, and/or RAGE did not impair TSN-SS-mediated HMGB1 uptake, specific inhibitors of the clathrin- and caveolin-dependent endocytosis significantly impaired TSN-SS-mediated HMGB1 uptake. Co-treatment with a lysosomal inhibitor, bafilomycin A1, led to enhanced accumulation of endogenous LC3-II and internalized exogenous HMGB1 in TSN-SS/rHMGB1-treated macrophages. Taken together, these findings suggest that TSN-SS may facilitate HMGB1 endocytic uptake, and subsequently delivered it to LC3-positive vacuoles (possibly amphisomes) for degradation via a lysosome-dependent pathway.