The role of HMGB1 in COVID-19-induced cytokine storm and its potential therapeutic targets: A review.

Hyperinflammation characterized by elevated proinflammatory cytokines known as 'cytokine storms' is the major cause of high severity and mortality seen in COVID-19 patients. The pathology behind the cytokine storms is currently unknown. Increased HMGB1 levels in serum/plasma of COVID-19 patients were reported by many studies, which positively correlated with the level of proinflammatory cytokines. Dead cells following SARS-CoV-2 infection might release a large amount of HMGB1 and RNA of SARS-CoV-2 into extracellular space. HMGB1 is a well-known inflammatory mediator. Additionally, extracellular HMGB1 might interact with SARS-CoV-2 RNA because of its high capability to bind with a wide variety of molecules including nucleic acids and could trigger massive proinflammatory immune responses. This review aimed to critically explore the many possible pathways by which HMGB1-SARS-CoV-2 RNA complexes mediate proinflammatory responses in COVID-19. The contribution of these pathways to impair host immune responses against SARS-CoV-2 infection leading to a cytokine storm was also evaluated. Moreover, since blocking the HMGB1-SARS-CoV-2 RNA interaction might have therapeutic value, some of the HMGB1 antagonists have been reviewed. The HMGB1- SARS-CoV-2 RNA complexes might trigger endocytosis via RAGE which is linked to lysosomal rupture, PRRs activation, and pyroptotic death. High levels of the proinflammatory cytokines produced might suppress many immune cells leading to uncontrolled viral infection and cell damage with more HMGB1 released. Altogether these mechanisms might initiate a proinflammatory cycle leading to a cytokine storm. HMGB1 antagonists could be considered to give benefit in alleviating cytokine storms and serve as a potential candidate for COVID-19 therapy.

Glycyrrhizin ameliorating sterile inflammation induced by low-dose radiation exposure.

Glycyrrhizin (GL) is a direct inhibitor of HMGB1 which acts as an alarmin when excreted into the extracellular space. High-dose radiation in radiotherapy induces collateral damage to the normal tissue, which can be mitigated by GL inhibiting HMGB1. The purpose of this study was to assess changes in HMGB1 and pro-inflammatory cytokines and to evaluate the protective effect of GL after low-dose radiation exposure. BALB/c mice were irradiated with 0.1 Gy (n = 10) and 1 Gy (n = 10) with GL being administered to half of the mice (n = 5, respectively) before irradiation. Blood and spleen samples were harvested and assessed for oxidative stress, HMGB1, pro-inflammatory cytokines, and cell viability. HMGB1 and pro-inflammatory cytokines increased and cell viability decreased after irradiation in a dose-dependent manner. Oxidative stress also increased after irradiation, but did not differ between 0.1 Gy and 1 Gy. With the pretreatment of GL, oxidative stress, HMGB1, and all of the pro-inflammatory cytokines decreased while cell viability was preserved. Our findings indicate that even low-dose radiation can induce sterile inflammation by increasing serum HMGB1 and pro-inflammatory cytokines and that GL can ameliorate the sterile inflammatory process by inhibiting HMGB1 to preserve cell viability.

Influence of Herbal Medicines on HMGB1 Release, SARS-CoV-2 Viral Attachment, Acute Respiratory Failure, and Sepsis. A Literature Review.

By attaching to the angiotensin converting enzyme 2 (ACE2) protein on lung and intestinal cells, Sudden Acute Respiratory Syndrome (SARS-CoV-2) can cause respiratory and homeostatic difficulties leading to sepsis. The progression from acute respiratory failure to sepsis has been correlated with the release of high-mobility group box 1 protein (HMGB1). Lack of effective conventional treatment of this septic state has spiked an interest in alternative medicine. This review of herbal extracts has identified multiple candidates which can target the release of HMGB1 and potentially reduce mortality by preventing progression from respiratory distress to sepsis. Some of the identified mixtures have also been shown to interfere with viral attachment. Due to the wide variability in chemical superstructure of the components of assorted herbal extracts, common motifs have been identified. Looking at the most active compounds in each extract it becomes evident that as a group, phenolic compounds have a broad enzyme inhibiting function. They have been shown to act against the priming of SARS-CoV-2 attachment proteins by host and viral enzymes, and the release of HMGB1 by host immune cells. An argument for the value in a nonspecific inhibitory action has been drawn. Hopefully these findings can drive future drug development and clinical procedures.

HMGB1 A box protects neurons by potently inhibiting both microglia and T cell-mediated inflammation in a mouse Parkinson's disease model.

In the subacute Parkinson's disease (PD) mice model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), injection of HMGB1 competitive inhibitor protein HMGB1 A box and the ethyl pyruvate (EP) that inhibit the release of HMGB1 from cells restored the number of dopaminergic neurons and TH+ fibers in the SN and striatum. Our data show that A box up-regulated CD200-CD200R signal of microglia inhibited the activation of microglia mediated by HMGB1, and the production of TNF-α, IL-1ß and IL-6 in vivo and in vitro mixed culture system. Microglia overexpressing CD200R produced less inflammatory chemokines and reduced the loss of TH+ neurons. In addition, HMGB1 A box decreased the level of CCL5 and significantly inhibited the infiltration of almost all T cells including Th17 and the proportion of Th17 in CD4+ T cells. In vitro MPP+ induced model and HMGB1-stimulated mesencephalic cell system activated microglia induced the differentiation of naïve T cells to Th17, and A box significantly inhibited this process. To sum up, our results show that HMGB1 A box targeting HMGB1, which effectively reduces the activation of microglia in MPTP PD model by restoring CD200-CD200R signal inhibit microglia mediated neuroinflammation and the differentiation of T cells to Th17.

Neutrophil Extracellular Traps (NETs) and Damage-Associated Molecular Patterns (DAMPs): Two Potential Targets for COVID-19 Treatment.

COVID-19 is a pandemic disease caused by the new coronavirus SARS-CoV-2 that mostly affects the respiratory system. The consequent inflammation is not able to clear viruses. The persistent excessive inflammatory response can build up a clinical picture that is very difficult to manage and potentially fatal. Modulating the immune response plays a key role in fighting the disease. One of the main defence systems is the activation of neutrophils that release neutrophil extracellular traps (NETs) under the stimulus of autophagy. Various molecules can induce NETosis and autophagy; some potent activators are damage-associated molecular patterns (DAMPs) and, in particular, the high-mobility group box 1 (HMGB1). This molecule is released by damaged lung cells and can induce a robust innate immunity response. The increase in HMGB1 and NETosis could lead to sustained inflammation due to SARS-CoV-2 infection. Therefore, blocking these molecules might be useful in COVID-19 treatment and should be further studied in the context of targeted therapy.

Bioactive fractions from Securidaca inappendiculata alleviated collagen-induced arthritis in rats by regulating metabolism-related signaling.

Securidaca inappendiculata is a xanthone rich medicinal plant that has been used in the treatment of inflammation and autoimmune diseases like rheumatoid arthritis (RA) for centuries; however, the material base and mechanism of action responsible for its anti-arthritis effect still remains elusive. The objective of this study is to evaluate the therapeutic effects of xanthone-enriched extract of the plant against collagen-induced arthritis (CIA) in rats and explore the underlying mechanisms. The xanthone-deprived fraction (XDF) and xanthone-rich fraction (XRF) were obtained by using a resin adsorption coupled with acid-base treatment method, and their chemical composition difference was characterized by UPLC-MS/MS analysis. Effects of the two on CIA were analyzed using radiographic, histological, and immunohistochemical analyses. The results indicated that XRF alleviated joint structures destructions with the higher efficacy than XDF, and decreased levels of TNF-α, IL-6, and anti-cyclic citrullinated peptide antibody in CIA rats significantly. Furthermore, XRF inhibited nicotinamide phosphoribosyl transferase (NAMPT) mediated fat biosynthesis and utilization indicated by clinical evidences and metabonomics analysis, which thereby disrupted energy-metabolism feedback. In addition, Toll-like Receptor 4 and High Mobility Group Protein 1 expressions were downregulated in XRF-treated CIA rats. Collective evidences suggest NAMPT could be an ideal target for RA treatments and reveal a novel antirheumatic mechanism of S. inappendiculata by regulating NAMPT controlled fat metabolism.

Aloin Reduces HMGB1-Mediated Septic Responses and Improves Survival in Septic Mice by Activation of the SIRT1 and PI3K/Nrf2/HO-1 Signaling Axis.

High mobility group box 1 (HMGB1) is recognized as a late mediator of sepsis, and the inhibition of HMGB1 release and recovery of vascular barrier integrity have emerged as attractive therapeutic strategies for the management of sepsis. We tested the hypothesis that aloin induces sirtuin 1 (SIRT1) and heme oxygenase (HO)-1, which inhibit HMGB1 release in lipopolysaccharide (LPS)-stimulated cells, thereby inhibiting HMGB1-induced hyperpermeability and increasing the survival of septic mice. Aloin was administered after LPS or HMGB1 challenge, and the antiseptic activity of aloin was determined from measurements of permeability, activation of pro-inflammatory proteins and production of markers for tissue injury in HMGB1-activated human umbilical vein endothelial cells (HUVECs) and a cecal ligation and puncture (CLP)-induced sepsis mouse model. Aloin significantly reduced HMGB1 release in LPS-activated HUVECs via SIRT1-mediated HMGB1 deacetylation and the PI3K/Nrf2/heme oxygenase (HO)-1 signaling axis. Aloin also suppressed the production of tumor necrosis factor (TNF)- α and interleukin (IL)-6, as well as the activation of nuclear factor (NF)- κ B and extracellular signal-regulated kinase 1/2 (ERK 1/2) by HMGB1. Moreover, aloin restored HMGB1-mediated vascular disruption and inhibited vascular hyperpermeability in mice. In addition, treatment with aloin reduced the CLP-induced release of HMGB1, sepsis-related mortality and tissue injury in vivo. Our results suggest that aloin reduces HMGB1 release and sepsis-related mortality by activating SIRT1 and PI3K/Nrf2/HO-1 signals, indicating that aloin has potential for the treatment of sepsis.

JH-4 reduces HMGB1-mediated septic responses and improves survival rate in septic mice.

Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity are emerging as attractive therapeutic strategies for the management of severe vascular inflammatory diseases. Recently, we found that JH-4, a synthesized decursin derivative, exhibited a strong anti-Hutchinson-Gilford progeria syndrome by efficiently blocking progerin-lamin A/C binding. In this study, we examined the effects of JH-4 on HMGB1-mediated septic responses and the survival rate in a mouse sepsis model. The anti-inflammatory activities of JH-4 were monitored based on its effects on lipopolysaccharide- or cecal ligation and puncture (CLP)-mediated release of HMGB1. The antiseptic activities of JH-4 were determined by measuring permeability, leukocyte adhesion, migration, and the activation of proinflammatory proteins in HMGB1-activated human umbilical vein endothelial cells and mice. JH-4 inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. JH-4 also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with JH-4 reduced CLP-induced release of HMGB1, sepsis-related mortality, and pulmonary injury in vivo. Our results indicate that JH-4 is a possible therapeutic agent to treat various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.

Berberine attenuates ischemia-reperfusion injury through inhibiting HMGB1 release and NF-κB nuclear translocation.

Inflammatory damage plays an important role in cerebral ischemic pathogenesis and represents a new target for treatment of stroke. Berberine is a natural medicine with multiple beneficial biological activities. In this study, we explored the mechanisms underlying the neuroprotective action of berberine in mice subjected transient middle cerebral artery occlusion (tMCAO). Male mice were administered berberine (25, 50 mg/kg/d, intragastric; i.g.), glycyrrhizin (50 mg/kg/d, intraperitoneal), or berberine (50 mg/kg/d, i.g.) plus glycyrrhizin (50 mg/kg/d, intraperitoneal) for 14 consecutive days before tMCAO. The neurological deficit scores were evaluated at 24 h after tMCAO, and then the mice were killed to obtain the brain samples. We showed that pretreatment with berberine dose-dependently decreased the infarct size, neurological deficits, hispathological changes, brain edema, and inflammatory mediators in serum and ischemic cortical tissue. We revealed that pretreatment with berberine significantly enhanced uptake of 18F-fluorodeoxyglucose of ischemic hemisphere comparing with the vehicle group at 24 h after stroke. Furthermore, pretreatment with berberine dose-dependently suppressed the nuclear-to cytosolic translocation of high-mobility group box1 (HMGB1) protein, the cytosolic-to nuclear translocation of nuclear factor kappa B (NF-κB) and decreased the expression of TLR4 in ischemic cortical tissue. Moreover, co-administration of glycyrrhizin and berberine exerted more potent suppression on the HMGB1/TLR4/NF-κB pathway than berberine or glycyrrhizin administered alone. These results demonstrate that berberine protects the brain from ischemia-reperfusion injury and the mechanism may rely on its anti-inflammatory effects mediated by suppressing the activation of HMGB1/TLR4/NF-κB signaling.

Paeoniflorin protects against liver ischemia/reperfusion injury in mice via inhibiting HMGB1-TLR4 signaling pathway.

Hepatic ischemia/reperfusion (I/R) injury is a major cause of high morbidity and mortality after liver resection, transplantation, and hemorrhagic shock. Paeoniflorin (PF), the main substance of glucosides in Radix Paeoniae Alba, has been widely used to treat various hepatic inflammatory diseases including I/R injury. However, the underlying mechanisms of PF on hepatic I/R injury remain further investigated. In this study, the liver I/R model was performed by clamping the portal vein and hepatic artery with an atraumatic clamp for 90 min followed by 6 hr reperfusion. PF (100 mg/kg) was given three times a day by gavage before I/R. The blood and hepatic samples were collected to evaluate liver injury and molecular indexes. The results showed that PF pretreatment significantly inhibited I/R-induced serum ALT and AST activities (40.3% and 53.8% those of I/R group, respectively), hepatic pathological damages and hepatic apoptosis (P < 0.01), and infiltration of neutrophils into liver. In addition, PF suppressed the production of pro-inflammatory cytokines (P < 0.01), decreased the expression of high mobility group box-1 (HMGB1), and down-regulated toll-like receptors 4 (TLR4) and phosphorylated ERK1/2, JNK1/2, p38, and NF-κB signal molecules expression in the I/R-operated mice. These findings indicated that PF played a protective role in liver I/R injury, and this protection was associated with inhibition of I/R-activated HMGB1-TLR4 signaling pathway to attenuate hepatic inflammation responses.

Disruption of the gut-liver axis in the pathogenesis of acute-on-chronic liver failure.

Acute-on-chronic liver failure (ACLF) is characterized by organ failure mediated by acute decompensation of cirrhosis. Recent studies have highlighted the importance of the gut-liver axis (GLS) and its association with ACLF pathogenesis. In this review, we discuss the mechanisms related to the alteration of the GLA and their involvement in ACLF pathogenesis and suggest some possible therapeutic options that could modulate the GLA dysfunction. This knowledge may provide information useful for the design of therapeutic strategies for gut dysbiosis and its complications in ACLF.

Sulforaphane Reduces HMGB1-Mediated Septic Responses and Improves Survival Rate in Septic Mice.

Sulforaphane (SFN), a natural isothiocyanate present in cruciferous vegetables such as broccoli and cabbage, is effective in preventing carcinogenesis, diabetes, and inflammatory responses. Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity is emerging as an attractive therapeutic strategy in the management of severe sepsis or septic shock. In this study, we examined the effects of SFN on HMGB1-mediated septic responses and survival rate in a mouse sepsis model. The anti-inflammatory activities of SFN were monitored based on its effects on lipopolysaccharide (LPS)- or cecal ligation and puncture (CLP)-mediated release of HMGB1. The antiseptic activities of SFN were determined by measuring permeability, leukocyte adhesion and migration, and the activation of pro-inflammatory proteins in HMGB1-activated human umbilical vein endothelial cells (HUVECs) and mice. SFN inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. SFN also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with SFN reduced CLP-induced release of HMGB1 and sepsis-related mortality and pulmonary injury in vivo. Our results indicate that SFN is a possible therapeutic agent that can be used to treat various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.

Subcritical water-hydrolyzed fish collagen ameliorates survival of endotoxemic mice by inhibiting HMGB1 release in a HO-1-dependent manner.

To investigate potential mechanisms underlying the bioactivity of hydrolyzed fish collagen, we examined the anti-inflammatory actions of subcritical water-hydrolyzed fish collagen (SWFC) in lipopolysaccharide (LPS)-triggered inflammation and endotoxemia. SWFC markedly inhibited LPS-stimulated release of high mobility group box 1 (HMGB1) in murine RAW264.7 macrophages, along with decreased cytosolic translocation of HMGB1. Both the protein and mRNA levels of heme oxygenase-1 (HO-1) were significantly upregulated in SWFC-treated RAW 264.7 cells in an Nrf2-dependent manner. In line with these effects of SWFC, both HO-1 siRNA and ZnPPIX (zinc protoporphyrin IX) actually attenuated the effects of SWFC on HMGB1 release stimulated by LPS, indicating a possible mechanism by which SWFC modulates HMGB1 release through HO-1 signaling. Notably, administration of SWFC improved the survival rates of LPS-injected endotoxemic mice, in which the serum level of HMGB1 was significantly reduced. Taken together, these results indicate that the anti-inflammatory activities of SWFC are achieved by inhibiting HMGB1 release induced by LPS in a HO-1-sensitive manner.

Inhibition of HMGB1 protects the retina from ischemia-reperfusion, as well as reduces insulin resistance proteins.

The role of inflammation in diabetic retinal amage is well accepted. While a number of cytokines and inflammatory mediators are responsible for these changes, upstream regulators are less well studied. Additionally, the role for these upstream mediators in retinal health is unclear. In this study, we hypothesized that inhibition of high mobility group box 1 (HMGB1) could restore normal insulin signaling in retinal endothelial cells (REC) grown in high glucose, as well as protect the retina against ischemia/reperfusion (I/R)-induced retinal damage. REC were grown in normal (5mM) or high glucose (25mM) and treated with Box A or glycyrrhizin, two different HMGB1 inhibitors. Western blotting was done for HMGB1, toll-like receptor 4 (TLR4), insulin receptor, insulin receptor substrate-1 (IRS-1), and Akt. ELISA analyses were done for tumor necrosis factor alpha (TNFα) and cleaved caspase 3. In addition, C57/B6 mice were treated with glycyrrhizin, both before and after ocular I/R. Two days following I/R, retinal sections were processed for neuronal changes, while vascular damage was measured at 10 days post-I/R. Results demonstrate that both Box A and glycyrrhizin reduced HMGB1, TLR4, and TNFα levels in REC grown in high glucose. This led to reduced cleavage of caspase 3 and IRS-1Ser307 phosphorylation, and increased insulin receptor and Akt phosphorylation. Glycyrrhizin treatment significantly reduced loss of retinal thickness and degenerate capillary numbers in mice exposed to I/R. Taken together, these results suggest that inhibition of HMGB1 can reduce retinal insulin resistance, as well as protect the retina against I/R-induced damage.

Glycyrrhizin, a Direct HMGB1 Antagonist, Ameliorates Inflammatory Infiltration in a Model of Autoimmune Thyroiditis via Inhibition of TLR2-HMGB1 Signaling.

BACKGROUND: High mobility group box-1 (HMGB1), a non-histone protein, plays an important role in autoimmune diseases. However, the significance of HMGB1 in the pathogenesis of autoimmune thyroiditis has not been reported. The purpose of this study was to explore whether HMGB1 participates in the pathogenesis of autoimmune thyroiditis, and whether glycyrrhizin (GL), a direct inhibitor of HMGB1, attenuates the severity of thyroid inflammatory infiltration in a murine model of autoimmune thyroiditis. METHODS: A total of 80 male NOD.H-2h4 mice were randomly divided into a control or iodine supplement (NaI) group at four weeks of age, and the control group was fed with regular water, whereas the NaI group was supplied with 0.005% sodium iodine water. Another 24 male NOD.H-2h4 mice were also randomized into three groups (eight mice per group) as follows: control, NaI, and GL treatment after iodine supplementation (NaI + GL). The NOD.H-2h4 mice were fed with 0.005% sodium iodide water for eight weeks to enhance autoimmune thyroiditis. After iodine treatment, the mice received intraperitoneal injections of GL for four weeks. The severity of lymphocytic infiltration in the thyroid gland was measured by histopathological studies. The serum levels of HMGB1, tumor necrosis factor alpha, interleukin (IL)-6, IL-1ß, and thyroglobulin antibody titers were measured using an enzyme-linked immunosorbent assay. HMGB1 expression was measured by immunohistochemical staining and real-time polymerase chain reaction. TLR2, HMGB1, MyD88, and nuclear transcription factor κB were measured by Western blot. RESULTS: The mRNA expression of HMGB1 was significantly higher at 8 and 16 weeks in the NaI group than it was in the control group. Serum levels of thyroglobulin antibodies, HMGB1, tumor necrosis factor alpha, IL-6, and IL-1ß were significantly increased in the NaI group, but they were dramatically attenuated with GL injection. The prevalence of thyroiditis and the infiltration of lymphocytes were significantly decreased in the NaI + GL group. GL administration also significantly reduced the protein expression of TLR2, MyD88, HMGB1 and nuclear transcription factor κB in the thyroid gland and attenuated the severity of thyroiditis. CONCLUSION: HMGB1 may play a crucial role in autoimmune thyroiditis by causing inflammatory infiltration, thus increasing the severity of autoimmune thyroiditis. GL effectively attenuated thyroiditis in the iodine-induced NOD.H-2h4 mice via a molecular mechanism related to the inhibition of TLR2-HMGB1 signaling.

A Dalbergia odorifera extract improves the survival of endotoxemia model mice by inhibiting HMGB1 release.

BACKGROUND: Dalbergia odorifera T. Chen (Leguminosae) is an indigenous medicinal herb that is widely used as a popular remedy in northern and eastern Asia. However, the cellular mechanisms underlying the biological activity of D. odorifera are not fully elucidated. METHODS: Anti-inflammatory effect of D. odorifera extract (DOE) was determined through intraperitoneal injection in a mouse model of endotoxemia induced by lipopolysaccharide (LPS). RAW 264.7 cells, a murine macrophage, were also treated with LPS to generate a cellular model of inflammation, and investigated the anti-inflammatory activity and underlying mechanisms of DOE and its constituent isoliquiritigenin. RESULTS: DOE dose-dependently inhibited LPS-induced release of high mobility group box 1 (HMGB1), a late proinflammatory cytokine, and decreased cytosolic translocation of HMGB1 in RAW264.7 cells. This inhibitory effect of DOE on HMGB1 release was observed in cells treated with DOE before or after LPS treatment, suggesting that DOE is effective for both treatment and prevention. In addition, DOE significantly inhibited LPS-induced formation of nitric oxide (NO) and expression of inducible NO synthase (iNOS) in a dose-dependent manner. These effects of DOE were accompanied by suppression of HMGB1 release triggered by LPS, suggesting a possible mechanism by which DOE modulates HMGB1 release through NO signaling. Isoriquiritigenin, a constituent of DOE, also attenuated LPS-triggered NO formation and HMGB1 release in RAW264.7 cells, indicating that isoriquiritigenin is an indexing molecule for the anti-inflammatory properties of DOE. Furthermore, c-Jun N-terminal kinase, but not extracellular signal-regulated kinase and p38, mediated DOE-dependent inhibition of HMGB1 release and NO/iNOS induction in RAW 264.7 cells exposed to LPS. Notably, administration of DOE ameliorated survival rates in a mouse model of endotoxemia induced by LPS, where decreased level of circulating HMGB1 was observed. CONCLUSION: These results suggest that DOE confers resistance to LPS-triggered inflammation through NO-mediated inhibitory effects on HMGB1 release.

Inhibitory effects of 3α-hydroxy-lup-20(29)-en-23, 28-dioic acid on lipopolysaccharide-induced TNF-α, IL-1ß, and the high mobility group box 1 release in macrophages.

We investigated the anti-inflammatory effects of 3α-hydroxy-lup-20(29)-en-23, 28-dioic acid (HLEDA)-a lupane-type triterpene isolated from leaves of Acanthopanax gracilistylus W. W.Smith (AGS), as well as the underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW264.7 cells. Our results demonstrated that HLEDA concentration-dependently reduced the production of nitric oxide (NO), significantly suppressed LPS-induced expression of TNF-α and IL-1ß at the mRNA and protein levels in RAW264.7 cells. Further analysis revealed that HLEDA could reduce the secretion of High Mobility Group Box 1 (HMGB1). Additionally, the results showed that HLEDA efficiently decreased nuclear factor-kappaB (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα. These results suggest that HLEDA exerts anti-inflammatory properties in LPS-induced macrophages, possibly through inhibition of the NF-κB signaling pathway, which mediates the expression of pro-inflammatory cytokines. These results warrant further studies that would concern candidate therapy for diseases, such as fulminant hepatitis and rheumatology of triterpenoids in AGS.

13-Ethylberberine reduces HMGB1 release through AMPK activation in LPS-activated RAW264.7 cells and protects endotoxemic mice from organ damage.

High mobility group box 1 (HMGB1), a highly conserved non-histone DNA-binding protein, plays an important role in the pathogenesis of sepsis. Previously, the authors reported 13-ethylberberine (13-EBR) has anti-inflammatory and antibacterial effects. However, the effect of 13-EBR on HMGB1 release was not investigated. In the present study, it was hypothesized 13-EBR might reduce HMGB1 release by activating AMPK under septic conditions. The results obtained showed 13-EBR significantly reduced HMGB1 release from LPS-activated RAW264.7 cells, and that this reduction was reversed by silencing p38, or AMPK, or by co-treating cells with p38 MAPKinase inhibitor. 13-EBR increased the phosphorylations of p38 and AMPK, and the phosphorylation of p38 by 13-EBR was inhibited by AMPK-siRNA, indicating AMPK acted upstream of p38. In the lung tissues of LPS-treated mice, 13-EBR administration significantly increased p-AMPK but reduced inducible nitric oxide synthase (iNOS) protein levels. Hematoxylin and eosin staining revealed 13-EBR significantly reduced LPS-induced lung and liver damage. In addition, 13-EBR inhibited NF-kB in LPS-activated RAW264.7 cells, and in LPS-treated mice, 13-EBR administration significantly increased survival. Furthermore, co-administration of 13-EBR plus LPS prevented LPS-induced aortic rings hypocontractile response to phenylephrine in vitro. Taken together, these results indicate 13-EBR might offer a means of treating sepsis through AMPK activation.

Blockade of high mobility group box 1 augments antitumor T-cell response induced by peptide vaccination as a co-adjuvant.

High mobility group box 1 (HMGB1) is a member of the family of damage-associated molecular patterns, which cause inflammation and trigger innate immunity through Toll-like receptors 2/4 and the receptor for advanced glycation end products. We examined the effect of glycyrrhizin, a selective inhibitor of HMGB1, on the induction of CTLs in mice. B6 mice, either OT-1 spleen cell-transferred or untransferred, were immunized with an s.c. injection of OVA257-264 peptide with topical imiquimod, and glycyrrhizin was mixed with the antigen peptide. Proliferation of OT-1 cells after immunization was enhanced by glycyrrhizin. The effect of glycyrrhizin was confirmed in other adjuvant systems, such as CpG oligonucleotide and monophosphoryl lipid A, but glycyrrhizin was not effective in Freund's incomplete adjuvant system. The augmenting effects of glycyrrhizin were also observed in other synthetic HMGB1 inhibitors, gabexate mesilate, nafamostat, and sivelestat. Thus, the effects are common to the HMGB1 inhibitors. Induction of CTLs detected by γ-interferon enzyme-linked immunospot assay was similarly augmented by glycyrrhizin. In a therapeutic vaccine model, glycyrrhizin inhibited the growth of s.c. transplanted EG.7 tumors. Expression of inflammatory cytokines in the skin inoculation site was downregulated by glycyrrhizin. These results suggest that HMGB1 inhibitors might be useful as a co-adjuvant for peptide vaccination with an innate immunity receptor-related adjuvant.

Effects and mechanisms of cavidine protecting mice against LPS-induced endotoxic shock.

LPS sensitized mice are usually considered as an experimental model of endotoxin shock. The present study aims to evaluate effects of cavidine on LPS-induced endotoxin shock. Mice were intraperitoneally administrated with cavidine (1, 3 and 10mg/kg) or DEX (5mg/kg) at 1 and 12h before injecting LPS (30mg/kg) intraperitoneally. Blood samples, liver, lung and kidney tissues were harvested after LPS injection. The study demonstrated that pretreatment with cavidine reduced the mortality of mice during 72h after endotoxin injection. In addition, cavidine administration significantly attenuated histological pathophysiology features of LPS-induced injury in lung, liver and kidney. Furthermore, cavidine administration inhibited endotoxin-induced production of pro-inflammatory cytokines including TNF-α, IL-6 and HMGB1. Moreover, cavidine pretreatment attenuated the phosphorylation of mitogen-activated protein kinase primed by LPS. In summary, cavidine protects mice against LPS-induced endotoxic shock via inhibiting early pro-inflammatory cytokine TNF-α, IL-6 and late-phase cytokine HMGB1, and the modulation of HMGB1 may be related with MAPK signal pathway.