HMGB1-induced Inflammatory Responses Research Articles

M2-polarized macrophages prevent preterm birth and improve neonatal survival and immunity

Abstract Preterm birth (PTB), commonly preceded by preterm labor, is the leading cause of neonatal morbidity and mortality worldwide. Most cases of preterm labor are associated with sterile intra-amniotic inflammation (SIAI), an inflammatory condition without detectable microorganisms. To date, no successful strategies to treat SIAI have been developed. Herein, we present mechanistic proof that treatment with M2-polarized macrophages (M2 MΦ) can effectively prevent PTB [(HMGB1, n = 28 vs. HMGB1+M2 MΦ, n = 29) (p<0.05)] and neonatal mortality [(HMGB1, n = 20 litters vs. HMGB1+M2 MΦ, n = 14 litters) (p<0.001)] induced by the ultrasound-guided intra-amniotic injection of the alarmin HMGB1 in mice. M2 MΦ halt the premature pathway of labor by infiltrating maternal and fetal compartments, where they inhibit NLRP3 inflammasome activation triggered by HMGB1. Furthermore, M2 MΦ dampen the HMGB1-induced inflammatory response in the amniotic cavity and fetal lung. Notably, neonates exposed to HMGB1 in utero display a reduced capacity to clear bacterial infection and gut microbiome dysbiosis, which are restored by M2 MΦ treatment [(HMGB1, n = 10 vs. HMGB1+ M2 MΦ, n = 10) (p<0.001 and p<0.01, respectively)]. Our findings provide cogent evidence that M2 MΦ can serve as a cellular strategy to mitigate PTB and decrease neonatal mortality.

Da-Cheng-Qi decoction improves severe acute pancreatitis-associated acute lung injury by interfering with intestinal lymphatic pathway and reducing HMGB1-induced inflammatory response in rats

Context Da-Cheng-Qi Decoction (DCQD) has a significant effect on Severe Acute Pancreatitis-Associated Acute Lung Injury (SAP-ALI). Objective To explore the mechanism of DCQD in the treatment of SAP-ALI based on intestinal barrier function and intestinal lymphatic pathway. Materials and methods Forty-five Sprague-Dawley rats were divided into three groups: sham operation, model, and DCQD. The SAP model was induced by a retrograde infusion of 5.0% sodium taurocholate solution (1 mg/kg) at a constant rate of 12 mL/h using an infusion pump into the bile-pancreatic duct. Sham operation and model group were given 0.9% normal saline, while DCQD group was given DCQD (5.99 g/kg/d) by gavage 1 h before operation and 1, 11 and 23 h after operation. The levels of HMGB1, RAGE, TNF-α, IL-6, ICAM-1, d-LA, DAO in blood and MPO in lung were detected using ELISA. The expression of HMGB1, RAGE, NF-κB p65 in mesenteric lymph nodes and lung were determined. Results Compared with SAP group, DCQD significantly reduced the histopathological scoring of pancreatic tissue (SAP, 2.80 ± 0.42; DCQD, 2.58 ± 0.52), intestine (SAP, 3.30 ± 0.68; DCQD, 2.50 ± 0.80) and lung (SAP, 3.30 ± 0.68; DCQD, 2.42 ± 0.52). DCQD reduced serum HMGB1 level (SAP, 134.09 ± 19.79; DCQD, 88.05 ± 9.19), RAGE level (SAP, 5.05 ± 1.44; DCQD, 2.13 ± 0.54). WB and RT-PCR showed HMGB1-RAGE pathway was inhibited by DCQD (p < 0.01). Discussion and conclusions DCQD improves SAP-ALI in rats by interfering with intestinal lymphatic pathway and reducing HMGB1-induced inflammatory response.

Interplay between RAGE and TLR4 Regulates HMGB1-Induced Inflammation by Promoting Cell Surface Expression of RAGE and TLR4.

Receptor for advanced glycation end-products (RAGE) and TLR4 play an important role in the inflammatory response against High-mobility group box 1 protein (HMGB1), a late proinflammatory cytokine and a damage-associated molecular pattern. As cell surface receptors, both RAGE and TLR4 are constantly trafficking between the cytoplasm and plasma membrane. However, whether TLR4 is related to the intracellular transport of RAGE in HMGB1-induced inflammation remains unknown. In this study, we demonstrated that HMGB1 not only increased RAGE expression in both the cytoplasm and plasma membrane but also upregulated the expression of TLR4 in the plasma membrane. Knocking out of RAGE led to decreased MAPK activation, TLR4 cellular membrane expression, and corresponding inflammatory cytokine generation. Meanwhile, inhibiting MAPK activation also decreased TLR4 surface expression. These results indicated that HMGB1 may bind to cell surface RAGE receptors on the cell surface, leading to MAPK activation, thus promoting TLR4 translocation on the cell surface, but does not regulate its transcription and translation. In contrast, TLR4 can increase the transcription and translation of RAGE, which translocates to the cell surface and is able to bind to more HMGB1. The cell surface receptors TLR4 and RAGE bind to HMGB1, leading to the transcription and secretion of inflammatory cytokines. Finally, we also observed these results in the mice pseudofracture model, which is closely related to HMGB1-induced inflammatory response. All these results demonstrated that the interplay between RAGE and TLR4 are critical for HMGB1-induced inflammatory response.

Escin suppresses HMGB1-induced overexpression of aquaporin-1 and increased permeability in endothelial cells.

Escin, a natural triterpene saponin mixture obtained from the horse chestnut tree (Aesculus hippocastanum), has been used for the treatment of chronic venous insufficiency (CVI), hemorrhoids, and edema. However, it is unclear how escin protects against endothelial barrier dysfunction induced by pro‐inflammatory high mobility group protein 1 (HMGB1). Here, we report that escin can suppress (a) HMGB1‐induced overexpression of the aquaporin‐1 (AQP1) water channel in endothelial cells and (b) HMGB1‐induced increases in endothelial cell permeability. This is the first report that escin inhibits AQP1 and alleviates barrier dysfunction in HMGB1‐induced inflammatory response.

Regulation of Acetylation in High Mobility Group Protein B1 Cytosol Translocation.

High mobility group protein B1 (HMGB1) is a nonhistone that mainly binds to nucleus DNA. As an important late inflammatory transmitter, extracellular HMGB1 is involved in the inflammatory immune response, tumor growth, infiltration, and metastasis. HMGB1 is actively released by activated inflammatory cells or passively released by necrotic cells. Then the released extracellular HMGB1 further induces monocytes/macrophages, neutrophils, and dendritic cells to secrete inflammatory cytokines. Therefore, HMGB1 can not only act as a proinflammatory factor to directly involve in tissue damage, but also acts as an inflammatory medium to aggravate the inflammatory cascade reaction. Studies have shown that the post-translational modification (PTM) participated in the process of HMGB1 cytosol translocation and extracellular release. The acetylation modification is the most common PTM for localization sequence of HMGB1, and the affinity of HMGB1 to DNA depends on the degree of acetylation for HMGB1. The acetylation can weaken the binding of HMGB1 to DNA, which means less HMGB1 cytosol translocation and extracellular release. This article reviews the acetylation regulation mechanisms of cytosol translocation and extracellular release of HMGB1 and provides a therapeutic strategy for controlling HMGB1-induced inflammatory responses in the future.

Umbelliferone alleviates hepatic injury in diabetic db/db mice via inhibiting inflammatory response and activating Nrf2-mediated antioxidant.

The current study was designed to investigate the protective effect and possible mechanisms of umbelliferone (Umb) on liver injury in diabetic C57BL/KsJ-db/db (dbdb) mice. Mice were divided into five groups: wild-type mice group (WY), dbdb mice group, dbdb mice + Metformin (100 mg/kg) group, dbdb mice + Umb (20, 40 mg/kg) group. Blood glucose regulation was assessed by an oral glucose tolerance test (OGTT). At 28 days after drug administration, blood samples were obtained for the analysis of lipids and enzymes related to hepatic function, including alanine aminotransferase (ALT), aspartate aminotransaminase (AST) and total cholesterol (TC) and triglyceride (TG). Expression levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and oxidative stress indicators (SOD and MDA) were measured with ELISA kit. The expressions of high-mobility group box 1 (HMGB1), Toll-like receptor (TLR) 4 (TLR4), Myd88, NF-κB, IκB, Nrf2, and HO-1 proteins were also evaluated by Western blotting analysis. The results showed that Umb significantly restored the blood glucose in OGTT, and inhibited the levels of insulin, TG, TC, as well as activities of ALT and AST. Moreover, Umb inhibited diabetic inflammation through down-regulating the expression of HMGB1, TLR4, NF-κB, and IκB. In addition, Umb alleviated oxidative damage in the liver by activating Nrf2-mediated signal pathway. These findings demonstrated that Umb exhibited protective effect against diabetic live injury, which may be through inhibiting HMGB1-induced inflammatory response and activating Nrf2-mediated antioxidant.

Human α1-antitrypsin attenuates injury-induced Inflammation through interacting with high mobility group box-1 (HMGB1)

Abstract HMGB1 is involved in pathologies such as cellular injury and autoimmunity. Indeed, upon binding to immune-related receptors, HMGB1 promotes an inflammatory response, such as that might take part in pancreatic islet destruction during both autoimmune diabetes and islet transplant rejection. Specifically, elevated circulating HMGB1 levels were reported in patients with type 1 diabetes, and increased release of HMGB1 was found to correlate with injury degree and islet allograft survival. The anti-inflammatory and immune-modulatory acute-phase protein human α1-antitrypsin (hAAT) promotes islet survival in both transplantation and autoimmune diabetes models. While AAT binds to damage-released proteins, such as gp96 and HSP70, its tissue protective mechanism remains poorly understood. We now extend this observation that AAT protective activity is related to its interaction with HMGB1. Clinical-grade AAT (Glassia, Kamada Ltd., Israel), diminished recombinant HMGB1-induced inflammatory responses in mouse pancreatic islets (while restoring disrupted insulin inflammation-mediated release). Similar behavior was depicted in HMGB1-stimulated peritoneal macrophages introduced to hAAT. In addition, hAAT modulated HMGB1-inducible surface expression by altering its distribution. Lastly, we show by immunoprecipitation that HMGB1 associated proteins was positive for the presence of AAT, this is also supported by a direct ELISA. Our findings suggest that hAAT is a naturally-occurring regulator of inflammatory responses mediated by extracellular HMGB1, such that preclude outcomes of islet transplantation. hAAT may therefore be considered for therapy of cell damage-mediated pathologies in a clinically-safe manner.

A novel synthetic derivative of squamosamide FLZ inhibits the high mobility group box 1 protein-mediated neuroinflammatory responses in murine BV2 microglial cells

High mobility group box 1 (HMGB1) is a critical pro-inflammatory cytokine that contributes to the pathogenesis of various human diseases. FLZ, a squamosamide derivative, has been demonstrated to have neuroprotective effects in Parkinson's disease models and shows strong anti-inflammatory activity, while the precise mechanism remains unclear. Here, we investigated the anti-inflammatory mechanism of FLZ on HMGB1-mediated inflammatory responses. The effects of FLZ on HMGB1 release from microglial cells induced by lipopolysaccharide were first explored by Western blot assay and ELISA. Then, co-immunoprecipition was used to study FLZ's effect on the interaction between HMGB1 and its receptor TLR4. Finally, we employed HMGB1 to simulate pro-inflammatory responses and then studied the inhibitory effects of FLZ on its bioactivity. FLZ has a significant inhibitory effect on HMGB1 release while it exerts no inhibitory effect on the binding between HMGB1 and TLR4. After the recognition of HMGB1 by TLR4, NF-κB signaling pathway is activated. FLZ could efficaciously alleviate HMGB1-induced inflammatory responses via the suppression of TLR4/MyD88/NF-κB signaling pathway. FLZ could inhibit HMGB1 release as well as HMGB1-induced inflammatory responses, HMGB1 might be one of the FLZ anti-inflammatory targets, and interfering at this inflammatory mediator may have benefit effects on neurodegenerative disorders, such as Parkinson's disease.

Quinic acid derivatives from Salicornia herbacea alleviate HMGB1-mediated endothelial dysfunction

The high mobility group box 1 protein (HMGB1) has been targeted in the discovery of dietary supplements or medicinal resources to treat vascular inflammatory diseases. Salicornia herbacea has been utilized as a seasoned vegetable, salad, and traditional medicinal resource as well. Two new (1 and 2) and four known (3–6) caffeoylated quinic acids (CQAs) were isolated from the crude extract of S. herbacea exhibiting anti-HMGB1 activity. The isolated CQAs were further evaluated for their potential to ameliorate HMGB1-mediated vascular barrier disruption. Compounds 1, 3, 5, and 6 exerted vascular protective activity against HMGB1-induced inflammatory responses in both cellular and animal models and their mechanisms were also addressed. The active CQAs increased survival rates of cecal ligation and puncture-induced severe septic models to 20–40%. This study may serve the groundwork for commercializing CQAs as functional food components for prevention and treatment of pathogenic conditions related to endothelial hyperpermeability.

Role of moesin in HMGB1-stimulated severe inflammatory responses.

Sepsis is a life-threatening condition that arises when the body's response to infection causes systemic inflammation. High-mobility group box 1 (HMGB1), as a late mediator of sepsis, enhances hyperpermeability, and it is therefore a therapeutic target. Despite extensive research into the underlying mechanisms of sepsis, the target molecules controlling vascular leakage remain largely unknown. Moesin is a cytoskeletal protein involved in cytoskeletal changes and paracellular gap formation. The objectives of this study were to determine the roles of moesin in HMGB1-mediated vascular hyperpermeability and inflammatory responses and to investigate the mechanisms of action underlying these responses. Using siRNA knockdown of moesin expression in primary human umbilical vein endothelial cells (HUVECs), moesin was found to be required in HMGB1-induced F-actin rearrangement, hyperpermeability, and inflammatory responses. The mechanisms involved in moesin phosphorylation were analysed by blocking the binding of the HMGB1 receptor (RAGE) and inhibiting the Rho and MAPK pathways. HMGB1-treated HUVECs exhibited an increase in Thr558 phosphorylation of moesin. Circulating levels of moesin were measured in patients admitted to the intensive care unit with sepsis, severe sepsis, and septic shock; these patients showed significantly higher levels of moesin than healthy controls, which was strongly correlated with disease severity. High blood moesin levels were also observed in cecal ligation and puncture (CLP)-induced sepsis in mice. Administration of blocking moesin antibodies attenuated CLP-induced septic death. Collectively, our findings demonstrate that the HMGB1-RAGE-moesin axis can elicit severe inflammatory responses, suggesting it to be a potential target for the development of diagnostics and therapeutics for sepsis.

Ginsenosides Inhibit HMGB1-induced Inflammatory Responses in HUVECs and in Murine Polymicrobial Sepsis

Ginsenosides Inhibit HMGB1-induced Inflammatory Responses in HUVECs and in Murine Polymicrobial Sepsis

Exendin-4 inhibits HMGB1-induced inflammatory responses in HUVECs and in murine polymicrobial sepsis.

Exendin-4 (EX4) has been reported to attenuate myocardial ischemia and reperfusion (I/R) injury and inflammatory and oxidative responses. Nuclear DNA-binding protein high-mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions. However, the effect of EX4 on HMGB1-induced inflammatory response has not been studied. First, we accessed this question by monitoring the effects of posttreatment EX4 on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of proinflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Posttreatment EX4 was found to suppress LPS-mediated release of HMGB1 and inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. EX4 also induced downregulation of CLP-induced release of HMGB1, production of IL-6, and mortality. Collectively, these results suggest that EX4 may be regarded as a candidate therapeutic agent for treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Orientin inhibits HMGB1-induced inflammatory responses in HUVECs and in murine polymicrobial sepsis.

High mobility group box-1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions. Orientin has been known to have anxiolytic and antioxidative activities. However, the effect of orientin on HMGB1-induced inflammatory response has not been studied. We assessed this question by monitoring the effects of post-treatment orientin and its derivatives on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Post-treatment orientin was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. Orientin inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. Orientin also induced down-regulation of CLP-induced release of HMGB1 and mortality. Collectively, these results suggest that orientin may be regarded as a candidate therapeutic agent for treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Andrographolide inhibits HMGB1‐induced inflammatory responses in human umbilical vein endothelial cells and in murine polymicrobial sepsis

Nuclear DNA-binding protein high-mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions, such as septic shock, upregulating pro-inflammatory cytokines. Andrographolide (AG) is isolated from the plant of Andrographis paniculata and used as a folk medicine for treatment of viral infection, diarrhoea, dysentery and fever. However, the effect of AG on HMGB1-induced inflammatory response has not been studied. Firstly, we accessed this question by monitoring the effects of post-treatment AG on lipopolysaccharide (LPS) and caecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Post-treatment AG was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. AG also inhibited HMGB1-mediated hyperpermeability and leucocyte migration in septic mice. In addition, AG inhibited production of tumour necrosis factor-α (TNF-α) and activation of AKT, nuclear factor-κB (NF-κB) and extracellular-regulated kinases (ERK) 1/2 by HMGB1 in HUVECs. AG also induced downregulation of CLP-induced release of HMGB1, production of interleukin (IL) 1β/6/8 and mortality. Collectively, these results suggest that AG may be regarded as a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signalling pathway.

Factor Xa inhibits HMGB1-induced septic responses in human umbilical vein endothelial cells and in mice.

Nuclear DNA-binding protein high mobility group box 1 (HMGB1) acts as a late mediator of severe vascular inflammatory conditions, such as sepsis. Activated factor X (FXa) is an important player in the coagulation cascade responsible for thrombin generation, and it influences cell signalling in various cell types by activating protease-activated receptors (PARs). However, the effect of FXa on HMGB1-induced inflammatory response has not been studied. First, we addressed this issue by monitoring the effects of post-treatment with FXa on lipopolysaccharide (LPS)- and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Post-treatment with FXa was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. FXa also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. In addition, FXa inhibited the production of tumour necrosis factor-α and interleukin (IL)-1β. FXa also facilitated the downregulation of CLP-induced release of HMGB1, production of IL-6, and mortality. Collectively, these results suggest that FXa may be regarded as a candidate therapeutic agent for treating vascular inflammatory diseases by inhibiting the HMGB1 signalling pathway.

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.

Anti-septic effects of glyceollins in HMGB1-induced inflammatory responses in vitro and in vivo

The ubiquitous nuclear protein High mobility group box 1 (HMGB1) is released by activated macrophages and human umbilical vein endothelial cells (HUVECs), and functions as a late mediator of experimental sepsis. Glyceollins (GCLs) are active compounds from Aspergillus sojae which have been reported for anti-cancer, anti-diabetes, and anti-inflammatory activities. We investigated here, the antiseptic effects and underlying mechanisms of GCLs against HMGB1-mediated septic responses in HUVECs and mice. According to the results, GCLs effectively inhibited lipopolysaccharide-induced release of HMGB1, and suppressed HMGB1-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. In addition, GCLs suppressed the production of tumor necrosis factor-α and interleukin 6 and activation of nuclear factor-κB and extracellular regulated kinases 1/2 by HMGB1. Collectively, these results indicate that GCLs could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Anti-septic Effects of Pellitorine in HMGB1-Induced Inflammatory Responses In Vitro and In Vivo

High mobility group box 1 (HMGB1) acts as a late mediator of vascular inflammatory conditions. Pellitorine (PT), an active amide compound from Asarum sieboldii, is known to possess antibacterial and anticancer properties. In this study, we investigated the anti-septic effects of PT against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) induced by HMGB1 and the associated signaling pathways. According to our findings, treatment with PT resulted in inhibited release of HMGB1, down-regulation of HMGB1-dependent inflammatory responses in HUVECs, and inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with PT resulted in reduced cecal ligation and puncture (CLP)-induced release of HMGB1 and sepsis-related mortality. PT 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 the potential of PT as a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Identification of Pharmacological Modulators of HMGB1-Induced Inflammatory Response by Cell-Based Screening

High mobility group box 1 (HMGB1), a highly conserved, ubiquitous protein, is released into the circulation during sterile inflammation (e.g. arthritis, trauma) and circulatory shock. It participates in the pathogenesis of delayed inflammatory responses and organ dysfunction. While several molecules have been identified that modulate the release of HMGB1, less attention has been paid to identify pharmacological inhibitors of the downstream inflammatory processes elicited by HMGB1 (C23-C45 disulfide C106 thiol form). In the current study, a cell-based medium-throughput screening of a 5000+ compound focused library of clinical drugs and drug-like compounds was performed in murine RAW264.7 macrophages, in order to identify modulators of HMGB1-induced tumor-necrosis factor alpha (TNFα) production. Clinically used drugs that suppressed HMGB1-induced TNFα production included glucocorticoids, beta agonists, and the anti-HIV compound indinavir. A re-screen of the NIH clinical compound library identified beta-agonists and various intracellular cAMP enhancers as compounds that potentiate the inhibitory effect of glucocorticoids on HMGB1-induced TNFα production. The molecular pathways involved in this synergistic anti-inflammatory effect are related, at least in part, to inhibition of TNFα mRNA synthesis via a synergistic suppression of ERK/IκB activation. Inhibition of TNFα production by prednisolone+salbutamol pretreatment was also confirmed in vivo in mice subjected to HMGB1 injection; this effect was more pronounced than the effect of either of the agents administered separately. The current study unveils several drug-like modulators of HMGB1-mediated inflammatory responses and offers pharmacological directions for the therapeutic suppression of inflammatory responses in HMGB1-dependent diseases.

Anti‐inflammatory activities of isorhamnetin‐3‐O‐galactoside against HMGB1‐induced inflammatory responses in both HUVECs and CLP‐induced septic mice

High mobility group box 1 (HMGB1) protein is a crucial nuclear cytokine that elicits severe vascular inflammatory diseases. Oenanthe javanica (water dropwort) extract has anti-arrhythmic, neuroprotective and anti-diabetic activity. However, isorhamnetin-3-O-galactoside (I3G), an active compound from O. javanica, is not researched well for its biological activity. Here, we investigated the anti-inflammatory activities of I3G by monitoring the effects of I3G on the lipopolysaccharide (LPS) or cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1 or CLP-mediated modulation of inflammatory responses. I3G potently inhibited the release of HMGB1 and down-regulated HMGB1-dependent inflammatory responses in human endothelial cells. I3G also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. Further studies revealed that I3G suppressed the production of tumor necrosis factor-α and activation of nuclear factor-κB by HMGB1. In addition, I3G reduced CLP-induced HMGB1 release and sepsis-related mortality. Given these results, I3G should be viewed as a candidate therapeutic agent for the treatment of severe vascular inflammatory diseases such as sepsis or septic shock via inhibition of the HMGB1 signaling pathway.