High Mobility Group Box Chromosomal Protein 1 Research Articles

HMGB1 can activate cartilage progenitor cells in response to cartilage injury through the CXCL12/CXCR4 pathway.

Recent studies have suggested that cartilage progenitor cells (CPCs) could be activated and differentiated into chondrocytes to produce matrix and to restore the integrity of damaged cartilage after injury. However, the mechanism involved in CPC activation upon damage is still unclear. This study aims to investigate the role of high mobility group box chromosomal protein 1 (HMGB1) in both activation and migration of CPCs during cartilage injury. Explants harvested from mature bovine stifle joints were used for impact injury. The proliferation and migration of CPCs were examined via confocal imaging. Gene and protein expression of Hmbg1, Cxcl12, and Cxcr4 was also examined by quantitative polymerase chain reaction (qPCR), ELISA, and western blot. Each experiment was repeated 3 times. ANOVA and Student's t-test were performed for statistical analysis. HMGB1 released from dead and damaged chondrocytes after an impact injury could activate CPCs in the superficial zone of cartilage and promote their migration and proliferation to injury sites. However, the block of HMGB1 activation with its specific binding inhibitor glycyrrhizin inhibits the proliferation and migration of CPCs. Further investigations demonstrate that HMGB1 promotes CPCs migration through the pathway of C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4. Quantitative analysis of HMGB1 in cell culture medium also indicates that CPCs may have a self-activation property after the HMGB1 released from dead cells has been exhausted. HMGB1 is a pivotal factor that could enhance the migration and proliferation of CPCs through the CXCL12/CXCR4 pathway after cartilage injury, which could provide useful information for cartilage repair and osteoarthritis treatment.

Recombinant Human Thrombomodulin Reduces Mortality and Acute Lung Injury Caused by Septic Peritonitis in Rats.

This study aimed to investigate the therapeutic effects of recombinant human thrombomodulin (rhTM) on acute lung injury (ALI) caused by sepsis in rats. Rats that underwent cecal ligation and puncture (CLP) were treated with or without rhTM, and then mortality was analyzed. In another set of experiments, ALI was assessed. Furthermore, microthrombosis in the lungs was investigated by immunohistochemistry. Moreover, plasma inflammatory and anti-inflammatory cytokines, such as TNF-α, high-mobility group box chromosomal protein 1 (HMGB-1), and IL-10, were evaluated by ELISA. Production of TNF-α and HMGB-1 by isolated tissue macrophages (Mφs) was assessed in vitro. Mortality after CLP was significantly improved by rhTM treatment. In addition, rhTM treatment improved the wet/dry weight ratio of the lungs, the pulmonary microvascular permeability, and the lung injury scores in animals that underwent CLP. Microthrombosis was detected in the lungs after CLP. These pathophysiological changes were blunted by rhTM treatment. Increased plasma TNF-α and HMGB-1 levels were blunted by rhTM treatment; however, the anti-inflammatory cytokine IL-10 was significantly greater in the rhTM(+) group than in the rhTM(-) group. Increased TNF-α and HMGB-1 production by the tissue Mφs stimulated with LPS were significantly blunted by rhTM treatment in vitro, but the production of IL-10 by the tissue Mφs was not changed in the cells incubated with rhTM. Overall, rhTM improved the mortality caused by septic peritonitis. The possible mechanisms are most likely anti-inflammatory and anticoagulant effects, which lead to the prevention of ALI.

Association of HMGBP1 expression with clinical periimplant parameters among smokers and never-smokers with and without peri-implantitis.

With our study we aimed at investigating the levels of high mobility group box chromosomal protein-1 (HMGB-1), tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β in periimplant crevicular fluid (PICF) of smokers and never-smokers, with and without periimplantitis, and correlate these levels with the clinical and radiographic periimplant parameters. Sixty participants (n=15/group) were recruited and divided into 4 groups: cigarette smokers with periimplantitis (CSPI); cigarette smokers without periimplantitis (CSNPI); never-smokers with periimplantitis (NSPI); and never-smokers without periimplantitis (NSNPI). Clinical and radiographic periimplant parameters, including plaque scores (PS), bleeding on probing (BOP), probing depth (PD) and crestal bone level (CBL), were assessed. Crevicular levels of HMGB-1, TNF-α, and IL-1β were quantified using human enzyme linked immunosorbent assay. p-values were generated using Kruskal-Wallis' test for comparison between the study groups, while correlations between HMGB-1, TNF-α, IL-1β levels and clinical variables were analyzed using Spearman rank correlation coefficient analysis. Bleeding on probing was least in NSNPI and CSNPI followed by CSPI and NSPI (p<0.05). The highest PD and CBL was recorded for CSPI and NSPI groups, while the least PD and CBL were recorded among non-periimplantitis groups. HMGB-1 and IL-1β were found to be significantly highest in CSPI groups followed by NSPI and CSNPI groups with no statistically significant difference between CSPI and NSPI groups (p<0.05). CSPI groups reported the highest TNF-α levels in the PICF in comparison to other groups (p<0.05). A significant negative correlation was observed between plaque scores (p=0.0187) and CBL (p=0.0049) in NSNPI and CSPI groups with HMGB-1, respectively. A significant positive correlation was seen for HMGB-1 in groups CSPI (p=0.0023) and NSPI (p=0.0018) for BOP. In CSPI group, a significant positive correlation was observed between TNF-α and PD (p=0.0443). On correlating IL-1β, a significant positive correlation was observed for CBL in CSPI (p=0.0006) and NSPI (p=0.0275) groups, respectively. HMGB-1 could play a significant role in periimplant inflammatory response and inflammation. Higher crevicular fluid HMGB-1 levels are indicative of a possible surrogate biomarker for peri-implantitis.

High-mobility group box chromosomal protein-1 deletion alleviates osteoporosis in OVX rat model via suppressing the osteoclastogenesis and inflammation

BackgroundOsteoporosis is a skeletal metabolic disease that constitutes a great threaten to human health. However, there is currently no gold standard for its treatment. High-mobility group box chromosomal protein-1 (HMGB-1) has been reported to play an important role in various orthopedic diseases. Till now, its role in osteoporosis remains elusive.MethodsRats underwent ovariectomy (OVX) were used to construct a postmenopausal model of osteoporosis. Then, rats were divided into sham groups without OVX surgery, OVX model group, HMGB-1 knockdown (HMGB-1 KD) OVX model groups. The expression of HMGB1 was evaluated by qRT-PCR and western blotting. Subsequently, the changes of trabeculae were evaluated by micro-computed tomography (CT) assay. Skeletal necrosis and metabolism were further analyzed by hematoxylin–eosin (HE) staining, Alcian blue staining and Masson’s trichrome staining. The contents of serum alkaline phosphatase (ALP) and osteocalcin were detected by ELISA assay. Expression of osteoclast-associated receptor (OSCAR) and tartrate-resistant acid phosphatase (TRAP) were determined to investigate the effects of HMGB-1 loss on osteoclastogenesis.ResultsSingle HMGB-1 deletion exerted no significant effect on rat trabeculae, serum ALP and osteocalcin. Noticeably, HMGB1 knockdown dramatically ameliorated OVX-induced changes in above indexes. Trabeculae structures of OVX rats were sparse with disorder arrangement, which were greatly recovered after HMGB-1 deletion. Enhanced osteoclastogenesis was observed in OVX rats by increasing number of TRAP + cells and expression of TRAP and OSCAR, and loss of HMGB1 ameliorated osteoclastogenesis in OVA rats. Moreover, HMGB-1 deletion antagonized OVX-evoked downregulation of osteoblast activity markers osterix (OSX), collagen type I alpha 1(COL1A1) and distal-less homeobox 2 (DLX2) protein. Furthermore, loss of HMGB-1 attenuated fluctuation of inflammatory factors in OVX rats. Additionally, HMGB-1 deficiency inhibited OVX-evoked activation of the Toll-like receptor (TLR) 4/NF-κB signaling pathway. Moreover, reactivating the TLR4 signaling further aggravated OVX-induced osteoporosis, which was reversed by HMGB1 knockdown.ConclusionHMGB-1 deletion alleviated OVX-triggered osteoporosis by suppressing osteoclastogenesis and inflammatory disorder via the inhibition of the TLR4 signaling. Therefore, HMGB-1 may be a promising therapeutic target for osteoporosis.

Down-regulated HDAC1 and up-regulated microRNA-124-5p recover myocardial damage of septic mice

ABSTRACT Studies have revealed the relationship between histone deacetylases (HDACs)/microRNAs (miRNAs) and sepsis, but little has ever investigated the mechanism of HDAC1/miR-124-5p in sepsis. Herein, we studied the impacts of HDAC1/miR-124-5p on myocardial damage of septic mice via regulating high-mobility group box chromosomal protein 1 (HMGB1). Septic mice were induced by cecal ligation and puncture. HDAC1, miR-124-5p and HMGB1 expression in myocardial tissues of septic mice were detected. Septic mice were injected with HDAC1 low expression-, miR-124-5p high expression- or HMGB1 low expression-related structures to observe cardiac function, inflammatory response, oxidative stress response, myocardial pathological changes and apoptosis in myocardial tissues of septic mice. The relationship of HDAC1/miR-124-5p/HMGB1 was verified. HDAC1 and HMGB1 expression were upregulated while miR-124-5p expression was decreased in myocardial tissues of septic mice. Restored miR-124-5p/depleted HDAC1 or HMGB1 recovered the cardiac function, improved cardiac function, inflammatory response, oxidative stress response, myocardial pathological changes and inhibit ed cardiomyocyte apoptosis in septic mice. HDAC1 bound to miR-124-5p which directly targeted HMGB1. This study suggests that down-regulated HDAC1 or up-regulated miR-124-5p recovers myocardial damage of septic mice via decreasing HMGB1.

Netrin-1 Ameliorates Postoperative Delirium-Like Behavior in Aged Mice by Suppressing Neuroinflammation and Restoring Impaired Blood-Brain Barrier Permeability.

Postoperative delirium (POD) is a common and serious postoperative complication in elderly patients, and its underlying mechanism is elusive and without effective therapy at present. In recent years, the neuroinflammatory hypothesis has been developed in the pathogenesis of POD, in which the damaged blood-brain barrier (BBB) plays an important role. Netrin-1 (NTN-1), an axonal guidance molecule, has been reported to have strong inflammatory regulatory and neuroprotective effects. We applied NTN-1 (45 μg/kg) to aged mice using a POD model with a simple laparotomy to assess their systemic inflammation and neuroinflammation by detecting interleukin-6 (IL-6), interleukin-10 (IL-10), and high mobility group box chromosomal protein-1 (HMGB-1) levels. We also assessed the reactive states of microglia and the permeability of the BBB by detecting cell junction proteins and the leakage of dextran. We found that a single dose of NTN-1 prophylaxis decreased the expression of IL-6 and HMGB-1 and upregulated the expression of IL-10 in the peripheral blood, hippocampus, and prefrontal cortex. Nerin-1 reduced the activation of microglial cells in the hippocampus and prefrontal cortex and improved POD-like behavior. NTN-1 also attenuated the anesthesia/surgery-induced increase in BBB permeability by upregulating the expression of tight junction-associated proteins such as ZO-1, claudin-5, and occludin. These findings confirm the anti-inflammatory and BBB protective effects of NTN-1 in an inflammatory environment in vivo and provide better insights into the pathophysiology and potential treatment of POD.

Mechanism of miR-181a-5p in Regulatory T/T-Helper 17 Immune Imbalance and Asthma Development in Mice with Allergic Rhinitis

Introduction: Allergic rhinitis (AR) is an immune disorder and also a risk factor of asthma. microRNAs (miRNAs) are implicated in autoimmune diseases, including RA. This study investigated effect of miR-181a-5p on regulatory T (Treg)/ T-helper (Th) 17 immune imbalance in AR. Methods: A murine model of AR was established and treated with lentivirus modified miR-181a-5p. The allergic symptoms of mice were examined. The contents of Th17-related cytokines (interferon [IFN]-γ and interleukin [IL]-6), Treg-related cytokine (IL-10), and Treg-specific nuclear transcription factor (Foxp3) in nasal mucosa and lung tissues were determined. The proportion of Treg and Th17 cells was analyzed by flow cytometry. The level of ovalbumin-specific immunoglobulin E in the serum, and the contents of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and IFN-γ, IL-6, and IL-10 in nasal lavage fluid were measured. The targeting relationship between miR-181a-5p and high mobility group box chromosomal protein 1 (HMGB1) was verified. HMGB1 and receptor for advanced glycation end products (RAGE) expression in RA were determined, and the interaction between HMGB1 and RAGE was detected. Results: miR-181a-5p expression was reduced in AR mice. miR-181a-5p overexpression attenuated allergic behaviors, alleviated Treg/Th17 imbalance, and delayed asthma development. HMGB1 and RAGE were elevated in AR mice. miR-181a-5p targeted HMGB1, and HMGB1 bound to RAGE, while miR-181a-5p overexpression reduced the binding between them. Activating HMGB1/RAGE reversed the protective effect of miR-181a-5p overexpression on AR and induced the development of asthma. Conclusion: miR-181a-5p overexpression reduced the binding of HMGB1 and RAGE by inhibiting HMGB1, thus alleviating Treg/Th17 immune imbalance and blocking AR from developing into asthma.

High Mobility Group Box Chromosomal Protein-1 Induces Myofibroblast Differentiation and Extracellular Matrix Production via RAGE, p38, JNK and AP-1 Signaling Pathways in Nasal Fibroblasts.

Chronic rhinosinusitis is involved in myofibroblast differentiation and extracellular matrix (ECM) accumulation. High mobility group box chromosomal protein 1 (HMGB-1) is known to stimulate lung fibroblast to produce ECM in lung fibrosis. The aim of this study was to investigate whether HMGB-1 induces myofibroblast differentiation and ECM production in nasal fibroblasts and to identify the signal pathway. Human nasal fibroblasts were cultured. After stimulation with HMGB-1, expressions of α-smooth muscle actin (α-SMA) and fibronectin were determined by real-time PCR and western blot. Total collagen was measured by Sircol assay. To investigate signal pathway, various signal inhibitors and RAGE siRNA were used. HMGB-1 increased α-SMA and fibronectin in mRNA and protein levels. It also increased collagen production. RAGE siRNA inhibited HMGB-1-induced α-SMA and fibronectin, and production of collagen. Furthermore, the inhibitors of RAGE downstream molecules such as p38, JNK and AP-1 also blocked the HMGB-1-induced effects. HMGB-1 induces myofibroblast differentiation and ECM production in nasal fibroblast, which is mediated by RAGE, p38, JNK and AP-1 signal pathway. These results suggest that HMGB-1 may play an important role in tissue remodeling during chronic rhinosinusitis progression.

Omarigliptin Mitigates Lipopolysaccharide-Induced Neuroinflammation and Dysfunction of the Integrity of the Blood–Brain Barrier

The blood-brain barrier (BBB) is an important barrier that separates brain tissue from peripheral blood. The permeability of the BBB can be destroyed by external harmful factors, such as lipopolysaccharide (LPS), which contributes to neuroinflammation and central nervous system diseases. The present study aims to investigate the protective effects of Omarigliptin against LPS-induced neuroinflammation and the underlying mechanism using a series of both in vivo and in vitro experiments. A neuroinflammation model was established by intraperitoneal injection of LPS into mice. We found that administration of Omarigliptin reduced LPS-induced inflammatory responses by inhibiting the expressions of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Importantly, we found that Omarigliptin protected the integrity of the BBB against LPS by increasing the expression of the tight junction proteins claudin-1 and claudin-5. Our results also demonstrate that Omarigliptin reduced LPS-induced increase in expressions of matrix matalloproteinases-2 (MMP-2) and matrix matalloproteinases-9 (MMP-9) at both the mRNA and protein levels. Notably, Omarigliptin showed a powerful beneficial effect against LPS-induced cell damage in bEnd.3 brain endothelial cells by reducing the release of high mobility group box chromosomal protein 1 (HMGB-1). Consistently, Omarigliptin ameliorated LPS-induced exacerbation of endothelial permeability by increasing the expressions of claudin-1 and claudin-5 and reducing the expression of MMP-2 and MMP-9. Mechanistically, Omarigliptin inhibited the activation of the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88/nuclear factor κB (TLR4/Myd88/NF-κB) signaling pathway. On the basis of these findings, we concluded that Omarigliptin might mitigate LPS-induced neuroinflammation and dysfunction of the integrity of the blood-brain barrier.

HMGB-1/RAGE signaling inhibition by dioscin attenuates hippocampal neuron damage induced by oxygen-glucose deprivation/reperfusion.

Cerebral ischemia is one of the most common clinical diseases characterized by high morbidity and mortality. Neurocyte apoptosis and a cascade of inflammatory signals following cerebral ischemia-reperfusion injury (IRI) may contribute to secondary brain damage, resulting in severe neurological damage. It has been reported that dioscin, a natural steroid saponin, exerts anti-inflammatory properties against different diseases. The present study aimed to investigate the role of dioscin in oxygen-glucose deprivation/reperfusion (OGD/R) induction in hippocampal cells in vitro and in vivo. For the in vitro study, hippocampal cells were collected from rat embryos of gestational age of E18. The oxygen-glucose deprivation model in primary hippocampal neurons was used to mimic cerebral IRI in vitro. To select the optimum dioscin concentration and acting time, cell viability was evaluated by a Cell Counting Kit-8 (CCK-8) assay. Neurons subjected to OGD/R were treated with dioscin and the inflammatory cytokines, high mobility group box chromosomal protein 1 (HMGB-1)/receptor for advanced glycation end products (RAGE) signaling molecules and apoptosis-associated genes were determined. The intracellular reactive oxygen species (ROS) generation was detected. Furthermore, the effects of dioscin on the antioxidant defense mechanisms were evaluated by measuring the activity of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and the glutathione (GSH)/glutathione disulphide (GSSG) ratio. In addition, OGD/R-induced cells were transfected with pcDNA3.1-HMGB-1 and treated with dioscin, and the neuronal cell apoptosis rate was determined using a terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) assay. The mRNA and protein expression levels of the inflammatory factors were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. For the in vivo investigation, the oxidation and anti-oxidation system in rat hippocampal tissue was evaluated by detecting the expression of the aforementioned oxidative stress-associated proteins, 3-NT as well as 8-oxo-deoxyguanosine (8-OHdG). In the hippocampal region, the apoptotic rate was determined using a TUNEL assay. The results demonstrated that dioscin at a dose of 400 ng/ml significantly reversed the increase in the expression levels of the inflammatory factors and attenuated those of apoptotic cytokines induced by OGD/R. Additionally, dioscin notably reversed the OGD/R-mediated activation of the HMGB-1/RAGE signaling pathway in vitro and in vivo. Cell treatment with dioscin significantly attenuated ROS production and increased the activity of antioxidant enzymes. Additionally, increasing the expression of HMGB-1 inhibited the protective effects of dioscin on cell apoptosis in the OGD/R-induced neurons. Furthermore, HMGB-1 overexpression reversed the antiapoptotic and anti-inflammatory effects of dioscin on neurons. The results of the present study indicated that dioscin exerted anti-inflammatory, antiapoptotic and antioxidant effects via the HMGB-1/RAGE signaling pathway. These results suggest a novel perspective of the protective effects of dioscin as a prospective remedial factor for IRI.

Linagliptin protects human chondrogenic ATDC5 cells against advanced glycation end products (AGEs)-induced apoptosis via a mitochondria-dependent pathway

Chondrocytes in joints are responsible for the formation and remodeling of articular cartilage. The accumulation of advanced glycation end products (AGEs) in cartilage is detrimental to the survival of chondrocytes. Linagliptin is one of the most commonly used anti-diabetes agents, and recent work indicates that it exerts an anti-inflammatory effect in different cell types. In this study, we showed that Linagliptin had a protective role in AGEs-induced chondrocyte injury. The presence of Linagliptin ameliorated AGEs-induced reactive oxygen species (ROS) induction and reduced cellular protein carboxyl content. Linagliptin mitigated AGEs-induced mitochondrial membrane potential (ΔΨm) reduction and NAPDH oxidase subunit NOX-4 induction, indicating that Linagliptin is a potent anti-ROS agent in chondrocytes. Additionally, Linagliptin inhibited AGEs-induced production of high mobility group box chromosomal protein 1 (HMGB-1), and the expression of matrix metalloproteases (MMPs)-2 and −9. Flow cytometry experimentation showed that Linagliptin inhibited AGEs-induced apoptotic subpopulation. Moreover, Linagliptin inhibited the AGEs-induced increased ratio of Bax to Bcl-2, translocation of cytochrome C from mitochondria to the cytoplasm, and release of cleaved caspase-3. Collectively, our data indicate that the anti-diabetes drug Linagliptin has a new role in rescuing chondrocyte from insult by AGEs, and may, therefore, have the potential to treat joint disorders.

Association of chemokine expression in anterior cruciate ligament deficient knee with patient characteristics: Implications for post-traumatic osteoarthritis

BackgroundStromal cell-derived factor-1a (SDF-1α) and high mobility group box chromosomal protein 1 (HMGB1) are chemokines that can drive post-traumatic osteoarthritis (PTOA) induced by anterior cruciate ligament (ACL) injury. However, the influence of patient characteristics on expression of those chemokines remains unclear. Our aim was to determine the relationship between chemokine expression in synovial fluid (SF) of the ACL-deficient (ACL-D) knee and patient characteristics including time from injury, sex, and age. MethodsSF samples were collected immediately prior to the first-time ACL reconstruction (ACLR) from 82 patients. Expression of SDF-1α and HMGB1 was measured with human-specific solid phase sandwich enzyme-linked immunosorbent assays. The expression levels between groups divided by time from injury, or age, or sex was compared using Student's t-test. The association of SDF-1α or HMGB1 levels with those variables was determined using regression analysis and Pearson product–moment correlation coefficient. ResultsRegression and correlation analysis indicated significant correlation between SDF-1α expression and time from injury in the cohort (r = −0.266, P = 0.016, n = 82) and in females (r = −0.386, P = 0.024, n = 34). Significant correlation was also observed between SDF-1α expression and age in the cohort (r = −0.224, P = 0.043, n = 82) and in males (r = −0.289, P = 0.046, n = 48). No significant correlation between HMGB1 expression and patient characteristics was detected. ConclusionsSDF-1α rather than HMGB1 might serve as a protein marker for monitoring the development of PTOA in the ACL-D knee, especially in female patients.

Impact of perioperative high mobility group box chromosomal protein 1 expression on long-term outcomes in patients with esophageal squamous cell carcinoma.

High mobility group box chromosomal protein-1 (HMGB-1) is a potential late mediator of sepsis and a possible risk factor for postoperative pulmonary complications after esophagectomy. This study aimed to determine the relationship between HMGB-1 and clinicopathological factors and long-term prognosis after esophagectomy for esophageal cancer. We measured perioperative serum HMGB-1 levels using ELISA and HMGB-1 protein by immunohistochemistry expression in resected specimens. Postoperative serum HMGB-1 levels were significantly higher than preoperative levels. Preoperative serum HMGB-1 levels were significantly higher in patients with more intraoperative bleeding, longer intensive care unit stays, and postoperative pneumonia. Postoperative serum HMGB-1 levels were significantly higher in older patients and those with longer operation time and more intraoperative bleeding. There were significant differences in long-term outcomes according to postoperative but not preoperative serum HMGB-1 levels. Multivariate analysis demonstrated that advanced pathological stage, postoperative pulmonary complications, and higher postoperative serum HMGB-1 levels were independently associated with relapse-free survival and overall survival. Preoperative serum HMGB-1 levels were significantly higher in patients with high HMGB-1 expression than those with low HMGB-1 expression by immunohistochemistry, whereas such statistical differences were not observed in postoperative serum HMGB-1. There were no differences in relapse-free survival and overall survival according to HMGB-1 expression by immunohistochemistry. Serum HMGB-1 levels were significantly increased after esophagectomy for esophageal cancer. Elevated postoperative serum HMGB-1, which was associated not only with poor long-term but also short-term outcomes such as postoperative complications, might serve as a potential marker for prognosis in esophageal cancer.

Inhibitions of HMGB1 and TLR4 alleviate DINP-induced asthma in mice.

We studied the effects of high mobility group box chromosomal protein 1 (HMGB1) and toll-like receptor (TLR4) in diisonoyl phthalate (DINP)-induced asthma. Mice with DINP-induced asthma were treated with a TLR4-signaling inhibitor or anti-HMGB1 antibody, and various markers of asthma were measured 24 h later. DINP increased airway hyperresponsiveness, numbers of cells in BALF, numbers of inflammatory cells (leukocytes, lymphocytes, monocytes, eosinophils, neutrophils, basophils) in blood, mucus production, pulmonary fibrosis, Th2 type cytokine levels in BALF, and lung cell apoptosis. On the other hand, administrations of TLR4-signaling inhibitors (TAK-242) or anti-HMGB1 antibodies to a mouse model of DINP-induced asthma reduced biological markers of asthma. These results show TLR4 and HMGB1 both contribute to DINP-induced asthma, and that the inhibitions of TLR4 or HMGB1 offer potential means of treating asthma induced by phthalates like DINP.

High Efficiency Removal of Cytokines and HMGB-1 by Continuous Hemofiltration With a Dual Layered Polyethersulfone Membrane: An Ex Vivo Study.

Cytokines and high mobility group box chromosomal protein-1 (HMGB-1) play key roles in inflammatory conditions. While hemofiltration has been shown to remove cytokines, removal of cytokines and HMGB-1 by hemofiltration using a polyethersulfone membrane has not been reported. This study aimed to test the hypothesis that the polyethersulfone membrane will achieve higher removal performance for substances including inflammatory cytokines compared to other hemofilters, while retaining low albumin removal capacity. Subjects were eight healthy volunteers. We collected 400 mL each of blood samples into containers with heparin and added 30 mg of lipopolysaccharide to spike cytokines and HMGB-1. After incubation at 39ºC for 12 h, each blood sample was circulated through a hemofiltration circuit with a polyethersulfone hemofilter (2.1 m2 or 1.1 m2 ) at a filtration flow rate of 2 L/h. Measurement samples were collected from arterial, venous, and ultrafiltrate sampling points. Concentrations of cytokines (IL-1ß, IL-4, IL-6, IL-8, IL- 10, and tumor necrosis factor [TNF-a]), HMGB-1, and albumin were determined at each time point (1, 4, 8, 12, and 24h). High sieving coefficients (SCs) above 0.8 were obtained for all cytokines except for TNF-a as well as HMGB-1, whereas the SC for albumin was less than 0.04 with both hemofilters. The hemofilter with a larger membrane area achieved significantly higher clearances for TNF-a and HMGB-1, and slower decreases in SCs over time for IL-1ß, IL-6, IL-8, TNF-a, and albumin. Continuous hemofiltration with a polyethersulfone membrane achieved high efficiency removal of cytokines and HMGB-1, without excessive removal of albumin.

Association of High Mobility Group Box Chromosomal Protein 1 and Receptor for Advanced Glycation End Products Serum Concentrations With Extraglandular Involvement and Disease Activity in Sjögren's Syndrome.

To assess serum levels of high mobility group box chromosomal protein 1 (HMGB-1) and the soluble receptor for advanced glycation end products (sRAGE) in patients with Sjögren's syndrome (SS) and explore correlations with disease activity. Thirty-nine patients with SS and 21 healthy controls were included in this cross-sectional study. Clinical and laboratory values were obtained from all patients. Disease activity was assessed using the European League Against Rheumatism SS Disease Activity Index (ESSDAI). Serum samples were collected and HMGB-1 and sRAGE levels were measured using enzyme-linked immunosorbent assay (ELISA), and HMGB-1 concentrations were semiquantified by Western blotting. In ELISA, HMGB-1 serum levels did not differ between healthy controls and patients with SS (P = 0.783). When measured by semiquantitative Western blotting, HMGB-1 levels were increased in patients with SS compared to healthy controls (P = 0.012). HMGB-1 serum levels detected by Western blotting were higher in patients with extraglandular manifestations (P = 0.003) and were correlated with ESSDAI disease activity (r = 0.544, P < 0.0001). Furthermore, sRAGE was elevated in the sera of patients with SS (P = 0.003) compared to healthy controls and was also correlated with the ESSDAI (r = 0.545, P = 0.002). Serum levels of total HMGB-1 and sRAGE were elevated in patients with SS compared to healthy controls and correlated with disease activity as measured by the ESSDAI. Patients with extraglandular involvement had high serum levels of HMGB-1.

Glycyrrhizin Treatment Facilitates Extinction of Conditioned Fear Responses After a Single Prolonged Stress Exposure in Rats

Background/Aims: Impaired fear memory extinction is widely considered a key mechanism of post-traumatic stress disorder (PTSD). Recent studies have suggested that neuroinflammation after a single prolonged stress (SPS) exposure may play a critical role in the impaired fear memory extinction. Studies have shown that high mobility group box chromosomal protein 1 (HMGB-1) is critically involved in neuroinflammation. However, the role of HMGB-1 underlying the development of impairment of fear memory extinction is still not known. Methods: Thus, we examined the levels of HMGB-1 in the basolateral amygdala (BLA) following SPS using Western blot and evaluated the levels of microglia and astrocytes activation in the BLA after SPS using immunohistochemical staining. We then examined the effects of pre-SPS intra-BLA administration of glycyrrhizin, an HMGB1 inhibitor, or LPS-RS, a competitive TLR4 antagonist, on subsequent post-SPS fear extinction. Results: We found that SPS treatment prolonged the extinction of contextual fear memory after the SPS. The impairment of SPS-induced extinction of contextual fear memory was associated with increased HMGB1 and Toll-like receptor 4 (TLR4) levels in the BLA. Additionally, the impairment of SPS-induced extinction of contextual fear memory was associated with increased activation of microglia and astrocyte in the BLA. Intra-BLA administrations of glycyrrhizin (HMGB-1 inhibitor) or LPS-RS (TLR4 antagonist) can prevent the development of SPS-induced fear extinction impairment. Conclusion: Taken together, these results suggested that SPS treatment may not only produce short term effects on the HMGB1/TLR4-mediated pro-inflammation, but alter the response of microglia and astrocytes to the exposure to fear associated contextual stimuli.

Abstract 3999: Cross talk between tumor cell derived HMGB1 and adaptive B cells in the tumor microenvironment of esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is the most aggressive type of cancer among gastrointestinal cancers. The lack of effective anti-tumor immunity strategy for ESCC patients imposes urgent demand for new treatment paradigm. Tumor microenvironment (TME) is characterized by diverse cell populations (cancerous, immune cells, and pleiotropic nature of cytokines and inhibitory receptors) whose alternations are closely related with tumor progression. High mobility group box chromosomal protein 1 (HMGB1) has been recognized to be expressed in various tumor cells, and importantly it has also been identified as one of the key factors regulating inflammatory reactions. By comparing differentially expressed genes between ESCC tumors (T) and their corresponding non-tumorous (NT) tissues high-throughput transcriptome sequencing system (RNA-Seq, Illumina HiSeq 2500), we found that the ligands of TIM-3 (an inhibitory receptor), HMGB1 and galectin-9, but not, cell-surface phosphatidylserine, were overexpressed in all ESCC samples when compared to their NT counterparts. We further characterized HMGB1 and galectin-9 gene expression changes in 37 matched primary ESCC T and NT control samples by qRT-PCR. We found that HMGB-1 and galectin-9 mRNA is constitutively expressed in all samples, while ESCC T tissue significantly expressed ~2fold higher of HMGB-1, but not galectin-9, mRNA as compared to paired NT tissue. Histopathological characterization of ESCC tissue by sequential single staining for B-cells (CD20) and plasma cells (CD138), demonstrating that B cell infiltrate was dominated by CD20+ B cells lacking of terminal differentiation into plasma cells. Additionally, CD20+ B cells infiltrates were negative for CD27 (marker for memory B cells) and IgD (marker for naïve B cells). Additionally, we isolated CD20+ B cells from healthy peripheral blood mononuclear cells (PBMC), and stimulated with/without HMGB-1/IgM. Conditional medium (CM) were collected for foci formation and we found that ESCC cell lines KYSE30 and KYSE180 have better capacity for proliferation when culture with activated B cells CM as compared to unstimulated B cells. Collectively, we believe that the suboptimal clinical outcomes relies on CD20+HMGB1 receptor+ B cells that are exposed to high HMGB1 concentrations in the ESCC microenvironment may eventually become pro-tumor B cells subset. Citation Format: Ngar Woon Kam, Xin-Yuan Guan, Dora Lai-Wan Kwong. Cross talk between tumor cell derived HMGB1 and adaptive B cells in the tumor microenvironment of esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3999. doi:10.1158/1538-7445.AM2017-3999

Uric Acid Induces Endothelial Dysfunction by Activating the HMGB1/RAGE Signaling Pathway.

Uric acid (UA) is a risk factor for endothelial dysfunction, a process in which inflammation may play an important role. UA increases high mobility group box chromosomal protein 1 (HMGB1) expression and extracellular release in endothelial cells. HMGB1 is an inflammatory cytokine that interacts with the receptor for advanced glycation end products (RAGE), inducing an oxidative stress and inflammatory response, which leads to endothelial dysfunction. In this study, human umbilical vein endothelial cells (HUVECs) were incubated with a high concentration of UA (20 mg/dL) after which endothelial function and the expression of HMGB1, RAGE, nuclear factor kappa B (NF-κB), inflammatory cytokines, and adhesion molecules were evaluated. UA inhibited endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production in HUVECs, increased intracellular HMGB1 expression and extracellular HMGB1 secretion, and upregulated RAGE expression. UA also activated NF-κB and increased the level of inflammatory cytokines. Blocking RAGE significantly suppressed the upregulation of RAGE and HMGB1 and prevented the increase in DNA binding activity of NF-κB and the levels of inflammatory cytokines. It also blocked the decrease in eNOS expression and NO production induced by UA. Our results suggest that high concentrations of UA cause endothelial dysfunction via the HMGB1/RAGE signaling pathway.

TLR2 Plays a Critical Role in HMGB1-Induced Glomeruli Cell Proliferation Through the FoxO1 Signaling Pathway in Lupus Nephritis.

The objective of this study was to examine the role and possible mechanisms of toll-like receptor 2 (TLR2) in high-mobility group box chromosomal protein 1 (HMGB1)-induced mouse mesangial cell (MMC) proliferation and glomeruli proliferation of MRL/Fas(lpr) mice. First, the expression of proliferating cell nuclear antigen (PCNA), TLR2 and Forkhead box protein O1 (FoxO1) messenger RNA (mRNA) and protein in the glomeruli of MRL/Fas(lpr) mice was quantified, and the correlation with cell proliferation of glomeruli was analyzed. Then, lipopolysaccharide (LPS), TLR2 neutralization antibody, and small hairpin TLR2 (shTLR2) were used to confirm the role of TLR2 in HMGB1-induced MMC proliferation. Furthermore, wild-type FoxO1 (WT-FoxO1) vector was used to investigate the effect of FoxO1 pathway on HMGB1-induced MMC proliferation. Finally, electroporation was used to knockdown TLR2 in the glomeruli of MRL/Fas(lpr) mice, and renal function, FoxO1, and PCNA expression were detected. The results showed that the TLR2 expression was upregulated and FoxO1 expression was decreased in the glomeruli of MRL/Fas(lpr) mice, and these effects were significantly correlated with cell proliferation of the glomeruli. In vitro, the TLR2 neutralization antibody and the WT-FoxO1 vector, both reduced the MMC proliferation levels induced by HMGB1. The TLR2 neutralization antibody also blocked the HMGB1-dependent activation of the FoxO1 pathway and cell proliferation. In addition, transfection with shTLR2 decreased the proliferation levels and PCNA expression induced by HMGB1. In vivo, treatment with shTLR2 significantly reduced the PCNA expression in the glomeruli of MRL/Fas(lpr) mice and improved renal function. In addition, treatment with shTLR2 or blocking of TLR2 also reduced the translocation of FoxO1. Thus, TLR2 plays a critical role in HMGB1-induced glomeruli cell proliferation through the FoxO1 signaling pathway in lupus nephritis.