High Mobility Group Box 1 Gene Expression Research Articles

Evaluating the protective role of trimetazidine versus nano-trimetazidine in amelioration of bilateral renal ischemia/reperfusion induced neuro-degeneration: Implications of ERK1/2, JNK and Galectin-3 /NF-κB/TNF-α/HMGB-1 signaling

BackgroundRenal ischemia/reperfusion (I/R) is a primary culprit of acute kidney injury. Neurodegeneration can result from I/R, but the mechanisms are still challenging. We studied the implications of bilateral renal I/R on brain and potential involvement of the oxidative stress (OS) driven extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase (ERK1/2, JNK) and Galectin-3 (Gal-3)/nuclear factor Kappa B (NF-қB)/tumor necrosis factor-alpha (TNF-α), high mobility group box-1 (HMGB-1), and caspase-3 paths upregulation. We tested the impact of Nano-trimetazidine (Nano-TMZ) on these pathways being a target of its neuroprotective effects. MethodsStudy groups; Sham, I/R, TMZ+I/R, and Nano-TMZ+I/R. Kidney functions, cognition, hippocampal OS markers, Gal-3, NF-қB, p65 and HMGB-1 gene expression, TNF-α level, t-JNK/p-JNK and t-ERK/p-ERK proteins, caspase-3, glial fibrillary acidic protein (GFAP) and ionized calcium binding protein-1 (Iba-1) were assessed. ResultsNano-TMZ averted renal I/R-induced hippocampal impairment by virtue of its anti: oxidative, inflammatory, and apoptotic properties. ConclusionNano-TMZ is more than anti-ischemic.

Altered responsiveness to glutamatergic modulation by MK-801 and to repeated stress of immune challenge in female dopamine transporter knockout rats

Chronic stress is a key factor in psychiatric and neurological disorders often worsening disease symptoms. In this study, a unique animal model, the dopamine transporter knockout (DAT-KO) rat exhibiting behavioral signs resembling those occurring in mania, schizophrenia, attention deficit hyperactivity disorder, and obsessive-compulsive disorder was used. We have tested the hypothesis that the hyperdopaminergic state in DAT-KO rats (i) modulates behavioral response to the NMDA antagonist MK-801 (dizocilpine) and (ii) leads to abnormal endocrine and immune activation under subchronic stress induced by an immune challenge. Glutamatergic modulation with MK-801 induced a different behavioral pattern. While the WT rats responded to MK-801 injection with a robust rise in their locomotor activity, the hyperactive DAT-KO rats exhibited reduced locomotion. Signs of chronic stress including increased basal corticosterone and aldosterone but blunted anxiety were demonstrated in rats lacking the DAT. Repeated injections of increasing doses of lipopolysaccharide (LPS, 5 days) did not modify plasma prolactin concentrations which were however significantly lower in DAT-KO than in WT rats. Concentrations of plasma high mobility group box 1 (HMGB1) protein were significantly higher in LPS-treated DAT-KO than in WT rats. The gene expression of interleukin-6 in the anterior pituitary increased under the stress induced by the immune challenge in the WT but not the DAT-KO rats. The most evident differences between the genotypes were revealed in the spleen. The splenic gene expression of interleukin-1β, interleukin-6, and HMGB1 was lower and that of ferritin was higher in DAT-KO compared to WT rats. Obtained results emphasize the functional interaction of the endocrine and immune systems with monoamine and glutamatergic neurotransmission in the mechanisms leading to behavioral alterations and psychiatric disorders associated with dopamine dysfunction.

Ameliorative Effect of D-Carvone against Hepatic Ischemia-Reperfusion-Induced Injury in Rats.

Background: D-carvone is a monoterpene that exists in the essential oils of several plant species. Hepatic ischemia-reperfusion (Hep I/R) takes place clinically during different scenarios of liver pathologies. The aim of the current investigation is to disclose the hepato-protective actions of carvone against Hep I/R-induced damage and to reveal the underlying mechanism. Material and methods: Rats were assigned into five groups: sham and carvone plus sham groups, in which rats were administered either saline or carvone orally for three weeks prior to the induction of Hep I/R. In the Hep I/R group, rats were administered saline orally prior to the Hep I/R induction operation. The carvone 25 plus Hep I/R and Carvone 50 plus Hep I/R groups were administered carvone (25 and 50 mg/kg, respectively) for three weeks, followed by the induction of Hep I/R. Results: Liver ischemic animals demonstrated impaired liver function, several histopathological variations, and reduced levels of antioxidant enzyme activities. Furthermore, the Hep I/R groups showed the elevated gene expression of high-mobility group box 1 (HMGB1), toll-like receptors 4 (TLR4), nuclear factor kappa B (NFκB), and LR family pyrin domain containing 3 (NLP3), with subsequent escalated adhesion molecule 1 (ICAM-1), neutrophil infiltration, and several inflammatory mediators, including interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), as well as apoptotic markers. Pretreatment with D-carvone alleviated ischemia/reperfusion-induced impaired liver function, diminished the histopathological deviations, and augmented the antioxidant enzymes. In addition, D-carvone mitigated the gene expression of HMGB1, TLR4, NFκB, and NLP3, with a subsequent reduction in ICAM-1, neutrophils infiltration, inflammatory mediators, and apoptotic markers. Conclusion: Rats pretreated with D-carvone exhibited hepato-protective actions against Hep I/R-induced damage via the downregulation of HMGB1, TLR4, NFκB, NLP3, associated inflammatory mediators, and apoptotic markers.

Effect of a 6-week Aerobic Exercise Program on High-mobility Group Box 1 Gene Expression in Aortic Tissue of Diabetic Rats

Aims: Type 2 diabetes is a rising problem and a significant risk factor for small and large vessel disease. The present study aims to investigate the effect of a 6-week aerobic exercise program on High-mobility Group Box 1 (HMGB1) gene expression in aortic tissue of diabetic rats. Methods & Materials: In this experimental study, 40 male rats aged 8 weeks were randomly selected. Once familiarized with the exercise protocol, they were divided into four groups of healthy-control (n=10), diabetic-control (n=10), healthy-exercise (n=10), and diabetic-exercise (n=10). Type 2 diabetes was first induced, and then the rats run on a treadmill for 6 weeks, 5 sessions per week. After 12-14 h of fasting and 72 h after the last session, aortic tissue sampling was performed for HMGB1 analysis by quantitative real-time polymerase chain reaction (q-RT PCR) technique. Data analysis was performed using 1-way ANOVA and Tukey post hoc test at the significant level of P<0.05. Findings: Induction of type 2 diabetes led to a significant increase in HMGB1 gene expression in rats (P=0.001), significantly reduced following aerobic exercise (P=0.003). Aerobic exercise also reduced the expression of the HMGB1 gene in healthy rats (P=0.000). Conclusion: Aerobic exercise may have a protective effect by reducing the HMGB1 gene expression in the aortic tissue of diabetic rats. It can be used as an effective non-pharmacological method to improve diabetes-induced inflammation and prevent vascular disorders.

Elevation of Proinflammatory Cytokine HMGB1 in the Synovial Fluid of Patients With Legg-Calvé-Perthes Disease and Correlation With IL-6.

ABSTRACTLegg‐Calvé‐Perthes disease (LCPD) is a childhood ischemic osteonecrosis (ON) of the femoral head associated with the elevation of proinflammatory cytokine interleukin‐6 (IL‐6) in the synovial fluid. Currently, there is no effective medical therapy for patients with LCPD. In animal models of ischemic ON, articular chondrocytes produce IL‐6 in response to ischemic ON induction and IL‐6 receptor blockade improves bone healing. High‐mobility group box 1 (HMGB1) is a damage‐associated molecular pattern released from dying cells. In addition, extracellular HMGB1 protein is a well‐known proinflammatory cytokine elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. The purpose of this study was to investigate IL‐6–related proinflammatory cytokines, including HMGB1, in the synovial fluid of patients with LCPD. Our working hypothesis was that HMGB1, produced by articular chondrocytes following ischemic ON, plays an important role in IL‐6 upregulation. Here, HMGB1 protein levels were significantly higher in the synovial fluid of patients with LCPD by threefold compared with controls (p < 0.05), and were highly correlated with IL‐6 levels (Pearson correlation coefficient 0.94, p < 0.001, R 2 = 0.87). In the mouse model of ischemic ON, both HMGB1 gene expression and protein levels were elevated in the articular cartilage. In vitro studies revealed a significant elevation of HMGB1 and IL‐6 proteins in the supernatants of human chondrocytes exposed to hypoxic and oxidative stresses. Overexpressed HMGB1 protein in the supernatants of chondrocytes synergistically increased IL‐6 protein. Silencing HMGB1 RNA in human chondrocytes significantly repressed inteleukin‐1β (IL‐1β) gene expression, but not IL‐6. Further, both IL‐1β and tumor necrosis factor‐α (TNF‐α) protein levels in the synovial fluid of patients with LCPD were significantly correlated with IL‐6 protein levels. Taken together, these results suggest that proinflammatory cytokines, HMGB1, tumor necrosis factor‐α (TNF‐α), and IL‐1β, are significantly involved with IL‐6 in the pathogenesis of LCPD. This study is clinically relevant because the availability of multiple therapeutic targets may improve the development of therapeutic strategy for LCPD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Decreased Expression of the High Mobility Group Box 1 (HMGB1) Gene in Peripheral Blood in Patients with Mild or Moderate Clostridioides difficile Infection.

Cytokines are mediators of inflammation induced in the course of Clostridioides difficile infection (CDI). High Mobility Group Box 1 (HMGB1) is a cytokine playing an important role in the pathogenesis of numerous inflammatory and autoimmune diseases. The aim of the study was to assess the HMGB1 gene expression in the course of CDI. We have performed a prospective case-control study- including 55 adult patients, among them 27 with CDI, who were hospitalized from October 2018 to February 2020 and 28 healthy volunteers. We assessed: a complete blood count with differential leukocyte count, blood creatinine, albumin, and C-reactive protein (CRP) levels. Then, the expression of the HMGB1 gene was evaluated using quantitative Real-Time PCR. Patients with CDI were found to have a significant increase in white blood cells (WBC), neutrophil count, and CRP levels, they also exhibited decreased levels of albumin compared with controls. The HMGB1 gene expression was significantly lower among patients with CDI compared with the control group and significantly, inversely correlated with CRP level in blood. In conclusion, we have observed a decreased expression of the HMGB1 gene in peripheral blood of patients with mild or moderate CDI, which hypothetically could reflect their diminished capability to fight the pathogen.

Metformin inhibits high glucose-induced smooth muscle cell proliferation and migration.

We investigated the protective effects and mechanism of action of metformin on high glucose-induced smooth muscle cell proliferation and migration. Vascular smooth muscle cells (VSMCs) were subjected to a series of concentrations (0-10 mM) of metformin. CCK-8, wound healing, and transwell assays were performed. Correlations between metformin concentration and high-mobility group box 1 (HMGB1) and miR-142-3p levels were assessed. In addition, miR-142-3p mimic and siRNA were used to investigate VSMC migration in the presence or absence of metformin. In the high-glucose condition, metformin decreased cell growth and inhibited cell migration. HMGB1 gene expression correlated negatively with metformin concentration, whereas miR-142-3p expression correlated positively with metformin concentration. In addition, mimic-induced miR-142-3p elevation resulted in decreased HMGB1 and LC3II levels and elevated p62 levels in the high-glucose condition, whereas miR-142-3p knockdown had the reverse effects, and metformin abolished those effects. Metformin inhibits high glucose–induced VSMC hyperproliferation and increased migration by inducing miR-142-3p-mediated inhibition of HMGB1 expression via the HMGB1-autophagy related pathway.

Glycyrrhizin Attenuates the Process of Epithelial-to-Mesenchymal Transition by Modulating HMGB1 Initiated Novel Signaling Pathway in Prostate Cancer Cells.

High mobility group box 1 (HMGB1) is upregulated in nearly every tumor type. Importantly, clinical evidence also proposed that HMGB1 is particularly increased in metastatic prostate cancer patients. Besides, a growing number of studies highlighted that HMGB1 could be a successful therapeutic target for prostate cancer patients. Glycyrrhizin is a novel pharmacological inhibitor of HMGB1 that may repress prostate cancer metastasis. This research was aimed to investigate the effect of glycyrrhizin on inhibition of HMGB1-induced epithelial-to-mesenchymal transition (EMT), a key step of tumor metastasis, in prostate cancer cells. In this study, HMGB1 knock-downed DU145 prostate cancer cells were used. Silencing the HMGB1 gene expression triggered a change of cell morphology to a more epithelial-like shape, which was accompanied by a reduction of Cdc42/GSK-3β/Snail and induction of E-cadherin levels estimated by immunoblotting. Furthermore, HMGB1 facilitated cell migration and invasion via downstream signaling, whereas HMGB1 targeting by 10 mM ethyl pyruvate effectively inhibited EMT characteristics. Interestingly, cell migration capacity induced by HMGB1 in DU145 cells was abolished in a dose-dependent effect of 25-200 μM glycyrrhizin treatment. In conclusion, glycyrrhizin successfully inhibited HMGB1-induced EMT phenomenon, which suggested that glycyrrhizin may serves as a therapeutic agent for metastatic prostate cancer.

Effect of Shenqi Yangxin decoction on high mobility group box 1 and inflammatory signal pathway in a rat model of dilated cardiomyopathy

OBJECTIVETo investigate the effects of Shenqi Yangxin decoction (SQYXD) on heart function in a rat model of dilated cardiomyopathy (DCM) and its potential mechanisms. METHODSSprague-Dawley rats were randomly divided into normal (10 rats) and DCM (150 rats) groups. DCM was induced by an intraperitoneal injection of adriamycin. Then, DCM baseline group was randomly selected sixteen DCM rats. The remaining DCM rats were randomly divided into DCM control, perindopril, metoprolol, and SQYXD groups. Cardiac function and histological analysis plus biochemical measurement of serum levels of brain natriuretic peptide (BNP), and inflammatory factors were measured. The mRNA and protein expression levels of high-mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and nuclear factor-κB (NF-κB) were determined. Myocardial metabolism imaging was performed on the normal, SQYXD and DCM control groups to evaluate the effectiveness of treatments. RESULTSRats in the DCM control group exhibited dilated left ventricular diameter, impaired cardiac function, disorganized sarcomere, impaired glucose metabolism, increased heart weight index, and increased levels of BNP, which were improved by treatment with SQYXD. In addition, hearts from rats in the DCM baseline group exhibited significantly higher levels of HMGB1, TLR-4, RAGE, NF-κB, tumor necrosis factor-α, interleukin-1, interleukin-6, interleukin-10, compared with the normal group. Interestingly, the mRNA level of HMGB1 in the DCM baseline group was positively correlated with that of TLR-4, RAGE, NF-κB, BNP, and LVEDD, but negatively correlated with LVEF. SQYXD inhibited the upregulation of HMGB1 expression and its downstream inflammatory factors. CONCLUSIONShenqi Yangxin decoction effectively reduced the dilated left ventricular diameter and improved heart function in dilated cardiomyopathy. The mechanisms underlying the action on DCM involve regulating the gene and protein expression of HMGB1 and its inflammatory signal pathways in the DCM rat model.

172. Vitamin D decreases gene and protein expression of NLRP3 inflammasome in placental explants cultured with hydrogen peroxide from women with preeclampsia

Introduction Pregnancies complicated by preeclampsia (PE) are associated with placental oxidative stress and related to the activation of NLRP3 inflammasome, an important complex for activation of caspase-1, interleukin-1 beta (IL-1β) and high-mobility group box-1 (HMGB1). The hyperactivation of NLRP3 inflammasome in placental tissues may be involved in the huge systemic inflammatory state in PE, which could be modulate by administration of substances with anti-inflammatory properties such as vitamin D (VD). Objectives This study aimed to evaluate vitamin D immunomodulatory effect on the NLRP3 inflammassome in placental explants from normotensive (NT) and preeclamptic women cultured with hydrogen peroxide. Methods Placental explants from 7 PE and 7 NT women in the third trimester of pregnancy were obtained from women undergoing elective cesarean section. Explants were cultured with or without H2O2 and H2O2 plus VD. NLRP3, HMGB1, caspase-1 and IL-1β gene expression were analysed by qPCR. Protein expression were determined by western blotting or ELISA. Results Endogenous gene expression of NLRP3 and HMGB1 were significantly higher in PE than in NT explants. NLRP3, caspase-1, IL-1β and HMGB1 gene expression were higher in cultures treated with H2O2 compared to control cultures, while explants treated with H2O2 plus VD led to lower gene expression in NT and PE groups. High protein expression of NLRP3, caspase-1, IL-1β and HMGB1 was detected in explants cultured with H2O2 compared to controls whereas cultures treated with H2O2 plus VD decreased these protein expression compared to H2O2 stimuli. Conclusion Vitamin D was able to decrease the gene and protein expression of NLRP3 inflammasome and HMGB1 production in placental explants cultured with H2O2 in PE and NT groups. These results suggest that vitamin D may play an immunomodulatory role in the systemic inflammatory response present in preeclampsia. Financial support FAPESP 2016/18155-9 and 2016/23452-2.

Anti-high mobility group box 1 antibody suppresses local inflammatory reaction and facilitates olfactory nerve recovery following injury

BackgroundRefractory olfactory dysfunction is a common finding in head trauma due to olfactory nerve injury. Anti-inflammatory treatment using steroids is known to contribute to functional recovery of the central and peripheral nervous systems in injury models, while there is a concern that steroids can induce side effects. The present study examines if the inhibition of proinflammatory cytokine, high mobility group box 1 (HMGB1), can facilitate olfactory functional recovery following injury.MethodsOlfactory nerve transection (NTx) was performed in OMP-tau-lacZ mice to establish injury models. We measured HMGB1 gene expression in the olfactory bulb using semi-quantitative polymerase chain reaction (PCR) assays and examined HMGB1 protein localization in the olfactory bulb using immunohistochemical staining. Anti-HMGB1 antibody was intraperitoneally injected immediately after the NTx and histological assessment of recovery within the olfactory bulb was performed at 5, 14, 42, and 100 days after the drug injection. X-gal staining labeled OMP in the degenerating and regenerating olfactory nerve fibers, and immunohistochemical staining detected the presence of reactive astrocytes and macrophages/microglia. Olfactory function was assessed using both an olfactory avoidance behavioral test and evoked potential recording.ResultsHMGB1 gene and protein were significantly expressed in the olfactory bulb 12 h after NTx. Anti-HMGB1 antibody-injected mice showed significantly smaller areas of injury-associated tissue, fewer astrocytes and macrophages/microglia and an increase in regenerating nerve fibers. Both an olfactory avoidance behavioral test and evoked potential recordings showed improved functional recovery in the anti-HMGB1 antibody-injected mice.ConclusionsThese findings suggest that inhibition of HMGB1 could provide a new therapeutic strategy for the treatment of olfactory dysfunction following head injuries.

Expression analysis of HMGB1 in histological samples of malignant pleural mesothelioma.

High mobility group box 1 (HMGB1) is a chromatin structural protein, expressed ubiquitously in the nuclei of mammalian cells. When transported extracellularly, it acts as a tumour suppressor and oncogenic protein. In malignant pleural mesothelioma (MPM), high serum levels of HMGB1 have been related to a poor prognosis. Conversely, the significance of HMGB1 expression in MPM tissues is still unclear. Biopsy samples from 170 patients with MPM were assessed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to evaluate HMGB1 protein and gene expression. The expression level of HMGB1 protein was scored using a semiquantitative system that sums the intensity (0-3) and the percentage (from 0 to 4) of positively stained cells in nuclei, cytoplasm and in both. The final score was considered as high (>3) or low (<3) expression. Gene expression levels were calculated using the ΔΔCt method. High expression levels of HMGB1 as total (P = 0.0011) and cytoplasmic score (P = 0.0462) were related to a worse disease-specific survival (DSS) in the entire cohort and in the clinicopathological subgroups. No significant correlation was found between HMGB1 gene expression and DSS. These findings indicate that HMGB1 may be a useful prognostic biomarker in MPM when detected by immunohistochemistry. Conversely, as it is also expressed in normal and reactive mesothelial cells, HMGB1 cannot be considered a diagnostic biomarker in histological samples of mesothelioma.

Induction of systemic inflammation by hyaluronan and hsp70 in women with pre-eclampsia

Pre-eclampsia (PE) is a human pregnancy syndrome with abnormal activation of the innate immune response. The study evaluated the involvement of molecular structures called damage-associated molecular patterns (DAMPs), such as hyaluronan (HA) and heat shock proteins (Hsp) on NLRP1 and NLRP3 inflammasomes activation in peripheral blood monocytes. Twenty pre-eclamptic women, 20 normotensive pregnant women (NT) and 20 non-pregnant women (NP) were studied. Enzyme immunoassay was employed for the determination of HA, Hsp70 and High mobility group Box 1 (HMGB1) in plasma, as well as for the detection of Interleukin-1β (IL-1β), IL-18 and tumor necrosis factor alpha (TNF-α) in the supernatant of monocytes cultured with or without HA and Hsp70. The inflammasomes induction was evaluated by the quantification of mRNA for NLRP1, NLRP3, caspase-1, IL-1β, IL-18, HMGB1 and TNF-α by qPCR in monocyte culture. The results showed significantly higher plasma levels of HA, Hsp70 and HMGB1 in pre-eclamptic women than in NT and NP women. Monocytes from women with PE showed endogenous activation of NLRP1 and NLRP3 inflammasomes, and expressed high amounts of IL-1β, IL-18, HMGB1 and TNF-α. The stimulation of monocytes with HA increased the gene expression of NLRP1, NLRP3, caspase-1, TNF-α, IL-1β, HMGB1 and IL-18 and the production of IL-1β in pre-eclamptic women. Monocytes cultured with Hsp70 produced elevated levels of IL-1β and TNF-α through a mechanism independent of inflammasomes activation. These results suggest the participation of these DAMPs in the systemic inflammatory response that is characteristic of PE.

HMGB1/RAGE Signaling and Pro-Inflammatory Cytokine Responses in Non-HIV Adults with Active Pulmonary Tuberculosis.

BackgroundWe aimed to study the pathogenic roles of High-Mobility Group Box 1 (HMGB1) / Receptor-for-Advanced-Glycation-End-products (RAGE) signaling and pro-inflammatory cytokines in patients with active pulmonary tuberculosis (PTB).MethodsA prospective study was conducted among non-HIV adults newly-diagnosed with active PTB at two acute-care hospitals (n = 80); age-and-sex matched asymptomatic individuals (tested for latent TB) were used for comparison (n = 45). Plasma concentrations of 8 cytokines/chemokines, HMGB1, soluble-RAGE, and transmembrane-RAGE expressed on monocytes/dendritic cells, were measured. Gene expression (mRNA) of HMGB1, RAGE, and inflammasome-NALP3 was quantified. Patients’ PBMCs were stimulated with recombinant-HMGB1 and MTB-antigen (lipoarabinomannan) for cytokine induction ex vivo.ResultsIn active PTB, plasma IL-8/CXCL8 [median(IQR), 6.0(3.6–15.1) vs 3.6(3.6–3.6) pg/ml, P<0.001] and IL-6 were elevated, which significantly correlated with mycobacterial load, extent of lung consolidation (rs +0.509, P<0.001), severity-score (rs +0.317, P = 0.004), and fever and hospitalization durations (rs +0.407, P<0.001). IL-18 and sTNFR1 also increased. Plasma IL-8/CXCL8 (adjusted OR 1.12, 95%CI 1.02–1.23 per unit increase, P = 0.021) and HMGB1 (adjusted OR 1.42 per unit increase, 95%CI 1.08–1.87, P = 0.012) concentrations were independent predictors for respiratory failure, as well as for ICU admission/death. Gene expression of HMGB1, RAGE, and inflammasome-NALP3 were upregulated (1.2−2.8 fold). Transmembrane-RAGE was increased, whereas the decoy soluble-RAGE was significantly depleted. RAGE and HMGB1 gene expressions positively correlated with cytokine levels (IL-8/CXCL8, IL-6, sTNFR1) and clinico-/radiographical severity (e.g. extent of consolidation rs +0.240, P = 0.034). Ex vivo, recombinant-HMGB1 potentiated cytokine release (e.g. TNF-α) when combined with lipoarabinomannan.ConclusionIn patients with active PTB, HMGB1/RAGE signaling and pro-inflammatory cytokines may play important roles in pathogenesis and disease manifestations. Our clinico-immunological data can provide basis for the development of new strategies for disease monitoring, management and control.

High-mobility group box 1 enhances the inflammatory process in diabetic lung

Diabetes mellitus generates metabolic changes associated with inflammatory events that may eventually affect all body tissues. Both high-mobility group box 1 (HMGB1) and β-catenin are active players in inflammation. The study aimed to determine whether HMGB1 modulates the β-catenin activity in supporting inflammation, using an experimental type 1 diabetes mouse model. The protein and gene expression of HMGB1 were significantly increased (2-fold) in the diabetic lung compared to control and were positively correlated with the HMGB1 levels detected in serum. Co-immunoprecipitation of HMGB1 with RAGE co-exists with activation of PI3K/AKT1 and NF-kB signaling pathways. At the same time β-catenin was increased in nuclear fraction (3.5 fold) while it was down-regulated in diabetic plasma membrane (2-fold). There was no difference of β-catenin gene expression between the control and diabetic mice. β-Catenin phosphorylation at Ser552 was higher in diabetic nuclear fraction, suggesting that AKT1 activation promotes β-catenin nuclear translocation. In addition, c-Jun directly binds β-catenin indicating the transcriptional activity of β-catenin in diabetes, sustained by significantly COX2 increase by 6-fold in the cytosolic extract of diabetic lung compared to control. Taken together, the data support the new concept that HMGB1 maintains the inflammation through RAGE/AKT1/β-catenin pathway in the diabetic lung.

Down-regulation of HMGB1 expression by shRNA constructs inhibits the bioactivity of urothelial carcinoma cell lines via the NF-κB pathway.

The high mobility group box 1 (HMGB1), which is a highly conserved and evolutionarily non-histone nuclear protein, has been shown to associate with a variety of biological important processes, such as transcription, DNA repair, differentiation, and extracellular signalling. High HMGB1 expression has been reported in many cancers, such as prostate, kidney, ovarian, and gastric cancer. However, there have been few studies of the function of HMGB1 in the malignant biological behaviour of bladder urothelial carcinoma (BUC), and the potential mechanism of HMGB1 in the pathogenesis of BUC remains unclear. Thus, in this study, we constructed plasmid vectors that are capable of synthesizing specific shRNAs targeting HMGB1 and transfected them into BUC cells to persistently suppress the endogenous gene expression of HMGB1. The expression of HMGB1, the bioactivity of BUC cells, including proliferation, apoptosis, cell cycle distribution, migration and invasion, and the effects of HMGB1 knockdown on downstream signalling pathways were investigated. Our data suggest that HMGB1 promotes the malignant biological behaviour of BUC, and that this effect may be partially mediated by the NF-κB signalling pathway. HMGB1 may serve as a potential therapeutic target for BUC in the future.

Effect of ulinastatin on the expression and distribution of high mobility group box 1 in human colon carcinoma cells in vitro.

The aim of the present study was to investigate the in vitro effects of ulinastatin (UTI) on the proliferation, invasion, apoptosis, expression and distribution of high mobility group box 1 (HMGB1) and the expression of nuclear factor κB (NF‑κB) in human colon carcinoma LoVo cells. The cells were divided into control (untreated), UTI1 (400 U/ml UTI), UTI2 (800 U/ml UTI) and UTI3 (1,600 U/ml UTI) groups. The cell proliferation, invasion, apoptosis and the gene and protein expression of HMGB1 and NF‑κB were detected using a tetrazolium assay, Transwell cell invasion assays, a caspase‑3 activity assay, western blot analysis and reverse transcription quantitative polymerase chain reaction, respectively. The distribution of HMGB1 was detected using immunofluorescence. LoVo cell proilferation decreased the most in the UTI3 group followed, in order, by the UTI2, UTI1 and control groups. UTI inhibited invasion in LoVo cells and the inhibitory effect was enhanced as the UTI concentration increased. The activity of caspase‑3 increased the least in the control group followed, in order, by the UTI1, UTI2 and UTI3 groups. UTI inhibited the expression of HMGB1 and NF‑κB, and decreased the cytoplasmic distribution of HMGB1. Thus, UTI inhibited LoVo cell proliferation and induced LoVo cell apoptosis, the mechanism of which may be associated with a decreased in the expression of HMGB1 and NF‑κB, and the cytoplasmic distribution of HMGB1.

Human kallistatin administration reduces organ injury and improves survival in a mouse model of polymicrobial sepsis

Kallistatin, a plasma protein, has been shown to exert multi-factorial functions including inhibition of inflammation, oxidative stress and apoptosis in animal models and cultured cells. Kallistatin levels are reduced in patients with sepsis and in lipopolysaccharide (LPS)-induced septic mice. Moreover, transgenic mice expressing kallistatin are more resistant to LPS-induced mortality. Here, we investigated the effects of human kallistatin on organ injury and survival in a mouse model of polymicrobial sepsis. In this study, mice were injected intravenously with recombinant kallistatin (KS3, 3 mg/kg; or KS10, 10 mg/kg body weight) and then rendered septic by caecal ligation and puncture 30 min later. Kallistatin administration resulted in a > 10-fold reduction of peritoneal bacterial counts, and significantly decreased serum tumour necrosis factor-α, interleukin-6 and high mobility group box-1 (HMGB1) levels. Kallistatin also inhibited HMGB1 and toll-like receptor-4 gene expression in the lung and kidney. Administration of kallistatin attenuated renal damage and decreased blood urea nitrogen and serum creatinine levels, but increased endothelial nitric oxide synthase and nitric oxide levels in the kidney. In cultured endothelial cells, human kallistatin via its heparin-binding site inhibited HMGB1-induced nuclear factor-κB activation and inflammatory gene expression. Moreover, kallistatin significantly reduced apoptosis and caspase-3 activity in the spleen. Furthermore, kallistatin treatment markedly improved the survival of septic mice by 23% (KS3) and 41% (KS10). These results indicate that kallistatin is a unique protecting agent in sepsis-induced organ damage and mortality by inhibiting inflammation and apoptosis, as well as enhancing bacterial clearance in a mouse model of polymicrobial sepsis.

Inflammation triggers high mobility group box 1 (HMGB1) secretion in adipose tissue, a potential link to obesity

BackgroundLow grade inflammation is one of the major metabolic disorders in case of obesity due to variable secretion of adipose derived cytokines called adipokines. Recently the nuclear protein HMGB1 was identified as an inflammatory alarmin in obesity associated diseases. However HMGB1 role in adipose tissue inflammation is not yet studied. ObjectivesThe aim of this study was to prove the expression of HMGB1 in human adipose tissue and to assess the levels of expression between normo-weight and obese individuals. Furthermore we determined which type of cells within adipose tissue is involved in HMGB1 production under inflammatory signal. MethodsWestern-blot was performed on protein lysates from human normo-weight and obese adipose tissue to study the differential HMGB1 expression. Human normo-weight adipose tissue, adipose-derived stromal cells (ASCs) and adipocytes were cultured and stimulated with LPS to induce inflammation. HMGB1, IL-6 and MCP-1 secretion and gene expression were quantified by ELISA and Q-PCR respectively, as well as cell death by LDH assay. HMGB1 translocation during inflammation was tracked down by immunofluorescence in ASCs. ResultsHMGB1 was expressed 2-fold more in adipose tissue from obese compared to normo-weight individuals. LPS led to an up-regulation in HMGB1 secretion and gene expression in ASCs, while no change was noticed in adipocytes. Moreover, this HMGB1 release was not attributable to any cell death. In LPS-stimulated ASCs, HMGB1 translocation from nucleus to cytoplasm was detectable at 12h and the nuclear HMGB1 was completely drained out after 24h of treatment. ConclusionThe expression level studies between adipose tissue from normo-weight and obese individuals together with in vitro results strongly suggest that adipose tissue secretes HMGB1 in response to inflammatory signals which characterized obesity.

High levels of circulating HMGB1 as a biomarker of acute liver failure in patients with viral hepatitis E

Viral hepatitis E clinically ranges between acute self-limiting hepatitis (AVH) and acute liver failure (ALF). The varied clinical course of the disease possibly thought to be immune-mediated. High-mobility group box 1 (HMGB1) is a non-histone chromosomal nuclear protein with recently discovered pro-inflammatory and immunomodulatory action. Its presence in abundance within hepatocytes is thought provoking in patients with hepatitis. The present study was designed to elucidate the role of circulating HMGB1 and its gene expression in patients with viral hepatitis E. Blood samples were obtained from AVH (n=38), ALF (n=34) and healthy controls (HC, n=30). The HMGB1 levels were estimated in serum by quantitative-micro-ELISA. Gene expression levels were studied in the patient's PBMCs by real-time PCR. Lymphocyte proliferation was estimated by colorimetric-MTT assay. Mean circulating HMGB1 levels in HC, AVH and ALF patients were found to be 12.04±2.23, 112.6±13.33 and 225.3±15.04ng/ml respectively. The levels were significantly higher in ALF than AVH and HC (P<0.0001). Moreover, 88.2% of ALF patients with >250ng/ml of circulating HMGB1 had a fatal outcome. The gene expression of HMGB1 in the PBMCs of ALF and AVH patients were comparable. A positive correlation was observed between HMGB1 level and INR. A significantly low lymphocyte proliferation was observed in ALF patients (P=0.008). Massive necrosis of hepatocytes in ALF patients might predispose to excessive accumulation of extracellular HMGB1 leading to suppression of T-cell proliferation. Therefore, it is proposed that excessive circulating HMGB1 might play an important role in immunosuppression and fulminant course of the disease following HEV infection.