High Mobility Group Box 1 mRNA Research Articles

MiR-142-3p Overexpression Increases Chemo-Sensitivity of NSCLC by Inhibiting HMGB1-Mediated Autophagy

Background: Non-small-cell lung cancer (NSCLC) is a deadly cancer with high mortality rate. Drug resistance represents a main obstacle in NSCLC treatment. High mobility group box-1 (HMGB1) protein promotes drug resistance in NSCLC cells by activating protective autophagy. Methods: In the current study, we investigated the regulatory role of microRNA-142-3p (miR-142-3p) in HMGB1-mediated autophagy of NSCLC cells and its impact on drug resistance of NSCLC in vitro and in vivo. HMGB1 was identified as a putative target gene of miR-142-3p by in silico analysis. Our luciferase reporter assay results confirmed that miR-142-3p directly targets the 3’-UTR of HMGB1 in NSCLC cells. Results: MiR-142-3p overexpression suppressed while miR-142-3p knockdown increased HMGB1 mRNA and protein expression. Starvation induced HMGB1 expression and activated autophagy in NSCLC cells. The starvation-induced autophagy was inhibited by miR-142-3p overexpression or HMGB1 knockdown. Moreover, miR-142-3p overexpression or HMGB1 knockdown increased PI3K, Akt, and mTOR phosphorylation. Inhibition of PI3K or mTOR restored starvation-induced autophagy inhibited by miR-142-3p overexpression or HMGB1 knockdown. Conclusions: These results demonstrated that miR-142-3p regulates starvation-induced autophagy of NSCLC cells by directly downregulating HMGB1 and subsequently activating the PI3K/Akt/mTOR pathway. Further, miR-142-3p overexpression inhibited anticancer drug-induced autophagy and increased chemo-sensitivity of NSCLC in vitro and in vivo. These findings shed light on the therapeutic potential of miR-142-3p in combating acquired NSCLC chemo-resistance.

The Effects of Remifentanil on Expression of High Mobility Group Box 1 in Septic Rats.

High mobility group box 1 (HMGB1) is a pivotal mediator of sepsis progression. Remifentanil, an opioid agonist, has demonstrated anti-inflammatory effects in septic mice. However, it is not yet known whether remifentanil affects the expression of HMGB1. We investigated the effects of remifentanil on HMGB1 expression and the underlying mechanism in septic rats. Forty-eight male Sprague-Dawley rats were randomly divided into 3 groups; a sham group, a cecal ligation and puncture (CLP) group, and a CLP with remifentanil treatment (Remi) group. The rat model of CLP was used to examine plasma concentrations of proinflammatory cytokines, tissue HMGB1 mRNA and the activity of nuclear factor (NF)-κB in the liver, lungs, kidneys, and ileum. Pathologic changes and immunohistochemical staining of NF-κB in the liver, lungs, and kidneys tissue were observed. We found that remifentanil treatment suppressed the level of serum interleukin (IL)-6 and tumor necrosis factor (TNF)-α 6 hours after CLP, and serum HMGB1 24 hours after CLP. HMGB1 mRNA levels and the activity of NF-κB in multiple organs decreased by remifentanil treatment 24 hours after CLP. Remifentanil treatment also attenuated nuclear expression of NF-κB in immunohistochemical staining and mitigated pathologic changes in multiple organs. Altogether, these results suggested that remifentanil inhibited expression of HMGB1 in vital organs and release of HMGB1 into plasma. The mechanism was related to the inhibitory effect of remifentanil on the release of proinflammatory cytokines and activation of NF-κB.

MicroRNA-126 suppresses inflammation in endothelial cells under hyperglycemic condition by targeting HMGB1

MicroRNA-126(miR-126) targets involved in inflammation need to be identified. In this study, we aim to investigate whether high-mobility group box 1(HMGB1), an inflammation-related gene, is the target of miR-126 in diabetic vascular endothelium. The diabetic apoE−/− mice model, a classical diabetic atherosclerosis model, was established. The aorta of diabetic apoE−/− mice showed decrease of miR-126 and elevation of HMGB1 and inflammation. Next, we employed several in vitro experiments to address the role of miRNA-126 on the regulation of HMGB1 in endothelial cells under hyperglycemic and inflammatory conditions. Manipulation of miRNA levels in human umbilical vein endothelial cells (HUVECs) was achieved by transfecting cells with miR-126 mimic and antagomir. Overexpression of miR-126 could decrease the expression of downstream components of HMGB1 including TNF-α, ROS, and NADPH oxidase activity in HUVECs under hyperglycemic condition. Nevertheless, such phenomenon was completely reversed by miR-126 antagomir. The expression of HMGB1 protein rather than HMGB1 mRNA was down-regulated after transfection with miR-126 mimic, which indicated the modulation of HMGB1 mediated by miR-126 was at the posttranslational level. Luciferase reporter assay confirmed the 3′-UTR of HMGB1 gene was a direct target of miR-126. Western blot analysis also indicated that overexpression of miR-126 contributed to the elevation of p-eNOS, eNOS and p-AKT expressions, respectively. In summary, our findings suggest that miR-126 may suppress inflammation and ROS production in endothelial cells treated by high glucose through modulating the expression of HMGB1. Our study provides a novel pathogenic link between dysregulated miRNA expression and inflammation in diabetic vascular endothelium.

Glycyrrhizin Reduces HMGB1 and Bacterial Load in Pseudomonas aeruginosa Keratitis.

PurposeHigh mobility group box 1 (HMGB1) contributes to poor disease outcome in Pseudomonas aeruginosa keratitis. This study tests the prophylactic effect of treatment with HMGB1 inhibitors, glycyrrhizin (GLY) and its derivative, carbenoxolone (CBX), for Pseudomonas keratitis.MethodsWe treated C57BL/6 (B6) mice subconjunctivally with GLY or CBX, infected with a noncytotoxic clinical isolate (KEI 1025) or a cytotoxic strain (ATCC 19660) of P. aeruginosa, and injected intraperitoneally with either agent. Clinical score, photography with a slit lamp, real-time RT-PCR, ELISA, myeloperoxidase (MPO) assay, bacterial plate count, histopathology, and absorbance assays were used to assess treatment efficacy and bacteriostatic activity.ResultsAfter KEI 1025 infection, GLY treatment reduced HMGB1 (mRNA and protein levels) and improved disease outcome with significant reduction in mRNA levels of IL-1β, TLR4, CXCL2, and IL-12; protein expression (IL-1β, CXCL2); neutrophil infiltrate; and bacterial load. Treatment with GLY enhanced antimicrobial proteins, including CRAMP and mBD2, but not mBD3. Glycyrrhizin also reduced clinical scores and improved disease outcome in corneas infected with strain 19660. However, neither HMGB1 mRNA or protein levels were reduced, but rather, CXCL2 expression (mRNA and protein), neutrophil infiltrate, and bacterial load were reduced statistically. Treatment with GLY initiated 6 hours after infection reduced plate count; GLY also was bacteriostatic for KEI 1025 and ATCC 19660.ConclusionsGlycyrrhizin reduces HMGB1 and is protective against P. aeruginosa–induced keratitis with a clinical isolate that is noncytotoxic. It was similar, but less effective when used after infection with a cytotoxic strain, which did not reduce HMGB1.

Treatment effects of oxaliplatin combined with gemcitabine on colorectal cancer and its influence on HMGB1 expression.

In the present study, we analyzed the supra-additive effect of oxaliplatin in combination with gemcitabine on terminal colorectal cancer and its influence on high-mobility group box 1 (HMGB1) expression. A total of 86 patients with terminal colorectal cancer were enrolled in this study. Patients received oxaliplatin in combination with gemcitabine. Immunohistochemistry was used to determined the subcellular localization of HMGB1 in cancer tissues as well as in para-carcinoma tissues, and RT-PCR and western blot analysis were used to assess the mRNA and protein expression level, respectively. The total effective rate was analyzed based on WHO tumor chemotherapy reference where complete remission (CR) implies the complete disappearance of the tumor; partial remission (PR) implies that the tumor size was reduced at least by 50%; stable disease (SD) implies that the tumor size remained unchanged and progressive disease (PD) implies an increase in the size of the tumor by ≥25%. We identified 20 cases of CR, 37 cases of PR, 12 cases of SD and 17 cases of PD. The total effective rate (CR+PR) was 66.3%. HMGB1 expression rate in the cancer tissues in the effective group was significantly lower than that in the ineffective group. Positive expression of HMGB1 protein was mainly localized in the karyon. Survival time in the patients with positive HMGB1 expression was significantly shorter than that in the patients with negative HMGB1 expression. In the effective group, HMGB1 mRNA and protein expression levels were obviously lower than those in the ineffective group. We conclude that the reduced expression rate of HMGB1 in terminal colorectal cancer cases was probably related to the effects of oxaliplatin combined with gemcitabine.

The Alarmin HMGB1 Mediates Age-Induced Neuroinflammatory Priming.

Amplified neuroinflammatory responses following an immune challenge occur with normal aging and can elicit or exacerbate neuropathology. The mechanisms mediating this sensitized or "primed" immune response in the aged brain are not fully understood. The alarmin high mobility group box 1 (HMGB1) can be released under chronic pathological conditions and initiate inflammatory cascades. This led us to investigate whether HMGB1 regulates age-related priming of the neuroinflammatory response. Here, we show that HMGB1 protein and mRNA were elevated in the hippocampus of unmanipulated aged rats (24-month-old F344XBN rats). Furthermore, aged rats had increased HMGB1 in the CSF, suggesting increased HMGB1 release. We demonstrate that blocking HMGB1 signaling with an intracisterna magna (ICM) injection of the competitive antagonist to HMGB1, Box-A, downregulates basal expression of several inflammatory pathway genes in the hippocampus of aged rats. This indicates that blocking the actions of HMGB1 might reduce age-associated inflammatory priming. To test this hypothesis, we evaluated whether HMGB1 antagonism blocks the protracted neuroinflammatory and sickness response to peripheral Escherichia coli (E. coli) infection in aged rats. ICM pretreatment of aged rats with Box-A 24 h before E. coli infection prevented the extended hippocampal cytokine response and associated cognitive and affective behavioral changes. ICM pretreatment with Box-A also inhibited aging-induced potentiation of the microglial proinflammatory response to lipopolysaccharide ex vivo Together, these results suggest that HMGB1 mediates neuroinflammatory priming in the aged brain. Blocking the actions of HMGB1 appears to "desensitize" aged microglia to an immune challenge, thereby preventing exaggerated behavioral and neuroinflammatory responses following infection. The world's population is aging, highlighting a need to develop treatments that promote quality of life in aged individuals. Normal aging is associated with precipitous drops in cognition, typically following events that induce peripheral inflammation (e.g., infection, surgery, heart attack). Peripheral immune stimuli cause exaggerated immune responses in the aged brain, which likely underlie these behavioral deficits. Here, we investigated whether the alarmin high mobility group box 1 (HMGB1) mediates age-associated "priming" of the neuroinflammatory response. HMGB1 is elevated in aged rodent brain and CSF. Blocking HMGB1 signaling downregulated expression of inflammatory pathway genes in aged rat brain. Further, HMGB1 antagonism prevented prolonged infection-induced neuroinflammatory and sickness responses in aged rats. Overall, blocking HMGB1 "desensitized" microglia in the aged brain, thereby preventing pathological infection-elicited neuroinflammatory responses.

Downregulation of HMGB1 by miR-34a is sufficient to suppress proliferation, migration and invasion of human cervical and colorectal cancer cells.

High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein known to be highly expressed in human cervical (CaCx) and colorectal (CRC) cancers, and sustained high levels of HMGB1 contribute to tumourigenesis and metastasis. HMGB1-targeted cancer therapy is of recent interest, and there are not many studies on miRNA-mediated HMGB1 regulation in these cancers. Since miRNA-based therapeutics for cancer is gaining importance in recent years, it was of interest to predict miRNAs targeting HMGB1. Based on the identification of a potential miR-34a response element in HMGB1-3' untranslated region (3'UTR) and an inverse correlation between HMGB1 and miR-34a expression levels in CaCx and CRC tissues, from a subset of the local population as well as a large sampling from TCGA database, experiments were performed to validate HMGB1 as a direct target of miR-34a in CaCx and CRC cells. Ectopic expression of miR-34a decreased the wild-type HMGB1-3'UTR luciferase activity but not that of its mutant in 3'UTR luciferase assays. While forced expression of miR-34a in CaCx and CRC cells inhibited HMGB1 mRNA and protein levels, proliferation, migration and invasion, inhibition of endogenous miR-34a enhanced these tumourigenic properties. siRNA-mediated HMGB1 suppression imitated miR-34a expression in reducing proliferation and metastasis-related events. Combined with the disparity in expression of miR-34a and HMGB1 in clinical specimens, the current findings would help in not only understanding the complexity of miRNA-target regulatory mechanisms but also in designing novel therapeutic interventions in CaCx and CRC.

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.

MicroRNA-451 protects against cardiomyocyte anoxia/reoxygenation injury by inhibiting high mobility group box 1 expression

High mobility groupbox1 (HMGB1) protein serves an important role in myocardial ischemia/reperfusion (I/R) injury. MicroRNAs (miRNAs) are a group of small non‑coding RNAs that regulate numerous signaling pathways involved in myocardial I/R injury. The present study aimed to investigate whether miR‑451 protects against cardiomyocyte anoxia/reoxygenation (A/R) injury by attenuating HMGB1 expression. Neonatal rat ventricular cardiomyocytes were prepared and then subjected to A/R injury. The effect of upregulation or downregulation of miR‑451 on cell viability, apoptosis, superoxide dismutase (SOD) activity, and the expression of cleaved‑caspase‑3 and HMGB1 were measured accordingly. A luciferase assay was performed to further confirm whether miR‑451 can directly recognize the 3'‑untranslatedregion of HMGB1 in HEK293cells. The expression of miR‑451 was significantly decreased in the cardiomyocytes during A/R, and upregulation of miR‑451 led to increased miR‑451 expression (P<0.05). Upregulation of miR‑451 significantly attenuated the loss of cardiomyocyte viability (P<0.05) and increased the intracellular levels of SOD during A/R (P<0.05). Furthermore, upregulation of miR‑451 significantly decreased the apoptosis of cardiomyocytes during A/R (P<0.05). The HMGB1 mRNA and protein expression levels were significantly downregulated in the Ad‑miR‑451 group compared with those in the A/R group (P<0.05). In addition, upregulation of miR‑451 reduced its translocation from the nucleus to the cytoplasm. The luciferase assay confirmed that HMGB1 mRNA is a direct target of miR‑451 in cardiomyocytes. In conclusion, the present study suggested that upregulation of miR‑451 could protect against A/R‑induced cardiomyocyte injury by inhibiting HMGB1 expression.

HMGB1 induces apoptosis and EMT in association with increased autophagy following H/R injury in cardiomyocytes.

Hypoxia/reoxygenation (H/R) is a critical factor in the pathogenesis of tissue injury following myocardial infarction (MI) which can lead to tissue damage and pathological remodeling. Therefore, it is necessary to try and prevent myocardial H/R injury in order to optimize the treatment of MI. This study aimed to explore the functions and molecular mechanisms of action of high mobility group box 1 (HMGB1) and its role in H/R injury to H9c2 cells. The mRNA expression of levels genes were detected by RT-qPCR. The protein levels were examined by western blot analysis. The Beclin 1 expression level was further determined by immunocytochemistry (ICC). In addition, an HMGB1 overexpression vector and a shRNA lentiviral vector were constructed in order to induce the overexpression and silencing of HMGB1, respectively. The apoptotic rate of the H9c2 cells was determined by flow cytometry. The expression of miR-210 was markedly increased following the exposure of the cells to H/R, thus indicating that the cell model of H/R injury was successfully established. In addition, an in vivo model of MI was also created using rats. The mRNA and protein level of HMGB1 was found to be upregulated in the myocardial tissue of the rats with MI and in the H9c2 cells subjected to H/R injury. HMGB1 promoted apoptosis by increasing the expression of cleaved caspase-3 and the apoptotic rate of the cells, while decreasing the expression of Bcl-2 during H/R in the H9c2 cells. HMGB1 promoted epithelial-to-mesenchymal transition (EMT) by reducing the protein level of the epithelial marker, E-cadherin, while increasing the expression of the mesenchymal markers, vimentin and fibroblast-specific protein (FSP), during H/R in the H9c2 cells. HMGB1 induced the apoptosis of the H9c2 cells and EMT following H/R in association with the induction of autophagy. HMGB1 induced autophagy by upregulating the expression of discoidin domain receptor 1 (DDR1) and downregulating the phosphorylation levels of mammalian target of rapamycin (mTOR). In conclusion, the findings of our study suggest that HMGB1 promotes apoptosis and EMT in association with the induction of autophagy through the upregulation of the expression of DDR1 and the downregulation of the phosphorylation of mTOR following H/R injury in H9c2 cells.

Effect of alprostadil on acute lung injury in septic rats

Objective To evaluate the effect of alprostadil on acute lung injury in septic rats. Methods Thirty adult male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 3 groups (n=10 each) using a random number table: sham operation group (group S), acute lung injury group (group ALI), and alprostadil group (group Q). The animals were anesthetized with intraperitoneal 1% pentobarbital sodium 5 ml/100 g. Sepsis was induced by cecal ligation and puncture.In group Q, alprostadil (10 μg/2 ml) 2 ml/kg was injected via the tail vein at 30 min before cecal ligation and puncture.The equal volume of normal saline was given in S and ALI groups.At 24 h after operation, blood samples were taken for determination of serum tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) concentrations by enzyme-linked immunosorbent assay.The animals were then sacrificed.The left lungs were immediately removed for microscopic examination, and the right lungs were immediately removed for determination of wet/dry lung weight ratio (W/D ratio), and expression of TNF-α mRNA and high mobility group box-1 (HMGB1) using real-time reverse transcriptase-polymerase chain reaction. Results Compared with group S, the concentrations of serum TNF-α and IL-6 were significantly increased, and the expression of TNF-α mRNA and HMGB1 mRNA was up-regulated, and W/D ratio was increased in ALI and Q groups (P<0.05). Compared with group ALI, the concentrations of serum TNF-α and IL-6 were significantly decreased, and the expression of TNF-α mRNA and HMGB1 mRNA was down-regulated, and W/D ratio was decreased in group Q (P<0.05). The pathological changes of left lungs were significantly attenuated in group Q as compared with group ALI. Conclusion Alprostadil can reduce acute lung injury in septic rats, and the mechanism may be related to down-regulation of HMGB1 expression and inhibition of inflammatory responses. Key words: Alprostadil; Sepsis; Respiratory distress syndrome, adult; High mobility group proteins; Systemic inflammatory respones syndrome

Electro-acupuncture on Lower He-sea Points: How Does it Affect Serum HMGB1 and Tissue α7 nAChR in Ulcerative Colitis Rats

Abstract Objective: To observe the expression levels of serum high mobility group box 1 (HMGB1) , nicotinic acetylcholine receptor α7 (α7 nAChR) and nuclear factor kappa-B mRNA(NF-κB mRNA) in colon tissue after electroacupuncture (EA) on Shangjuxu, Zusanli, Xiajuxu,Yanglingquan and Chengjin points, by comparison of the different effects we could thus explore the relative specificity of Shangjuxu on treating its corresponding viscera’ s disease. Methods: Seventy SD rats were randomly assigned to seven groups labeled by blank control (A), Model (B), Shang Ju Xu (C), Zu San Li (D), Xia Ju Xu (E), Yang Ling Quan (F), Cheng Jin (G), ten (half males and half females) in each group. Except for the blank control group, animals in other groups were modeled to ulcerative colitis (UC) by lavation of 2-4-6 trinitro-benzene-sulfonic acid (TNBS)/ethanol solution. After successful modeling animals in the five treatment groups were respectively treated with electroaccupuncture at corresponding acupoint for 10 consecutive days. Then histological changes in the colon were observed by light microscopy. ELISA method was used to check models’serum HMGB1 content, and α7 nAChR in the colon were detected by western blot , RT-PCR was used to detecte NF-κB mRNA. Results: ① Compared with the model group, the tissue injury changes in 5 treatment groups improved to a certain extent, and the colon tissue NF-κB mRNA expression of them decreasd signifcantly（P&lt;0.01),as well, the α7 nAChR expression of Shang Ju Xu group, Zu San Li group and Xia Ju Xu group increased signifcantly (P&lt;0.01 or P&lt;0.05), and their serum HMGB1 content made obvious decrease(P&lt;0.01 or P&lt;0.05), the α7 nAChR expression in Yang Ling Quan group was also increased significantly（P&lt;0.01). ② Compared with Shang Ju Xu group, the tissue injury changes of groups including Xia Ju Xu,Yang Ling Quan and Cheng Jin are much severe under light microscope, and the α7 nAChR expression of them decreased with significance（P&lt;0.01),both serum HMGB1 content and colon tissue NF-κB mRNA expression increased significantly as well（P&lt;0.01),the serum HMGB1 content of Zu San Li group also increased significantly（P&lt;0.01). Conclusion: ① Electroacupuncture on all five different acupoints can improve the UC through regulating HMGB1, α7 nAChR and NF-κB mRNA, and effectively inhibiting the immune response, reducing inflammatory response of colon and improving pathological changes of colonic mucosa. ②The effect of treating UC inflammation in rats when electroacupuncture at Shang Ju Xu was better than Zu San Li, Xiajuxu, Yang Ling Quan and Cheng Jin, which demonstrated that Shangjuxu treating UC has some relative specificity.

4'-O-β-D-glucosyl-5-O-methylvisamminol, an active ingredient of Saposhnikovia divaricata, attenuates high-mobility group box 1 and subarachnoid hemorrhage-induced vasospasm in a rat model.

BackgroundHigh-mobility group box 1 (HMGB1) was observed to be an important extracellular mediator involved in vascular inflammation associated with subarachnoid hemorrhage (SAH). This study is of interest to examine the efficacy of 4′-O-β-d-glucosyl-5-O-methylvisamminol (4OGOMV), C22H28O10, on the alternation of cytokines and HMGB1 in an animal model.MethodsA rodent double hemorrhage SAH model was employed. Administration with 4OGOMV was initiated 1 h after animals were subjected to SAH. Basilar arteries (BAs) were harvested and cortexes examined for HMGB1 mRNA, protein expression (Western blot) and monocyte chemoattractant protein-1 (MCP-1) immunostaining. Cerebrospinal fluid samples were collected to examine IL-1β, IL-6, IL-8 and MCP-1 (rt-PCR).ResultsMorphological findings revealed endothelial cell deformity, intravascular elastic lamina torture, and smooth muscle necrosis in the vessels of SAH groups. Correspondently, IL-1β, IL-6 and MCP-1 in the SAH-only and SAH-plus vehicle groups was also elevated. 4OGOMV dose-dependently reduced HMGB1 protein expression when compared with the SAH groups.(p < 0.01) Likewise, 400 μg/kg 4OGOMV reduced IL-1β, MCP-1 and HMGB1 mRNA levels as well as MCP-1(+) monocytes when compared with the SAH groups..Conclusion4OGOMV exerts its neuro-protective effect partly through the dual effect of inhibiting IL-6 and MCP-1 activation and also reduced HMGB1 protein, mRNA and MCP-1(+) leukocytes translocation. This study lends credence to validating 4OGOMV as able to attenuate pro-inflammatory cytokine mRNA, late-onset inflammasome, and cellular basis in SAH-induced vasospasm.

Rhinacanthin-C, A Fat-Soluble Extract from Rhinacanthus nasutus, Modulates High-Mobility Group Box 1-Related Neuro-Inflammation and Subarachnoid Hemorrhage-Induced Brain Apoptosis in a Rat Model

High-mobility group box 1 (HMGB1) was shown to be a major extracellular mediator involved in relayed neuro-inflammation in animals after subarachnoid hemorrhage (SAH). It is of interest to examine the effect of rhinacanthin-C (RCT-C, C25H30O5) on pro-inflammatory cytokines/HMGB1 in an SAH-related early brain injury model. A rodent double SAH model was used. RCT-C was administered orally at 100, 200, and 400 μmol/kg/day. Cerebral spinal fluid samples were obtained to assess interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor α using a real-time polymerase chain reaction. Basilar arteries were harvested and cerebral cortex was examined for HMGB1 mRNA and protein expression (western blot) and caspases (real-time polymerase chain reaction). An intrathecal injection of 1 ng of HMGB-1 recombinant protein was given in the 400 μmol/kg/day RCT-C plus SAH groups. The levels of IL-1β, IL-6, and tumor necrosis factor α mRNA were significantly increased in animals subject to SAH, compared with the healthy controls, but were absent in the RCT-C groups. Cleaved caspase-9a as well as HMGB-1 mRNA and protein were significantly reduced in the 400 μmol/kg/day RCT-C treatment groups. Similarly, administration of RCT-C reduced HMGB-1 mRNA and protein expression (P <0.01). RCT-C exerts a neuroprotective effect by reducing cleaved caspase-3- and caspase-9a-related apoptosis. Decreased HMGB-1 mRNA and protein expression in the RCT-C groups corresponds to its anti-inflammatory effect. HMGB-1 recombinant protein administration impaired the neuroprotective and immunosuppressive effect of RCT-C. This finding lends credence that RCT-C modulates the HMGB-1-related pathway and attenuates brain apoptosis in the pathogenesis of SAH.

Endothelin-1 Upregulates the Expression of High Mobility Group Box 1 in Human Bronchial Epithelial Cells

Both endothelin-1 (ET-1) and high mobility group box 1 (HMGB1) reportedly are closely involved in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). In this study, we explored the regulatory effects of ET-1 on the expression of HMGB1 in human bronchial epithelial cells (HBEpCs). Primary HBEpCs were treated with ET-1 with or without transcription inhibitor actinomycin D, ETA receptor blocker BQ123, ETB receptor blocker BQ788, focal adhesion kinase (FAK) inhibitor or shRNA, or different kinase inhibitors. ET-1 increased the HMGB1 mRNA level in a statistically significant dose- and time-dependent manner within 8 hours of treatment, which was reflected in the dose-dependent induction of the HMGB1 protein level and the FAK activity. BQ123 and FAK inhibitor or shRNA, but not BQ788, completely abolished the promoting effect of ET-1 on the expression of HMGB1. Luciferase reporter assays revealed that neither ET-1 nor ETA nor FAK inhibition had any significant effect on the HMGB1 gene promoter activity. In the presence of the transcription inhibitor actinomycin D, the HMGB1 mRNA level markedly decreased over time, and ET-1 dose-dependently rescued the HMGB1 mRNA level. This effect of ET-1 was completely abolished by BQ123 and FAK inhibitor or shRNA, but not by BQ788. In conclusion, this study provides the first evidence that ET-1 upregulates the expression of HMGB1 in HBEpCs by increasing the stability of HMGB1 mRNA via the ETA receptor by a FAK-dependent mechanism. It adds new insights into the molecular mechanisms underlying ALI/ARDS.

Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells

High-mobility group box 1 (HMGB1) is a nonhistone protein secreted by airway epithelial cells in hyperinflammatory diseases such as asthma. In order to down-regulate HMGB1 expression in airway epithelial cells, siRNA directed against HMGB1 was delivered through nanocomplexes based on a cationic copolymer of poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) by using H441 cells. Two copolymers were used in these experiments bearing respectively spermine side chains (PHEA-Spm) and both spermine and PEG2000 chains (PHEA-PEG-Spm).PHEA-Spm and PHEA-PEG-Spm derivatives complexed dsDNA oligonucleotides with a w/w ratio of 1 and higher as shown by a gel retardation assay. PHEA-Spm and PHEA-PEG-Spm siRNA polyplexes were sized 350–650nm and 100–400nm respectively and ranged from negativity/neutrality (at 0.5 ratio) to positivity (at 5 ratio) as ζ potential. Polyplexes formed either at a ratio of 0.5 (partially complexing) or at the ratio of 5 (fully complexing) were tested in subsequent experiments. Epifluorescence revealed that nanocomplexes favored siRNA entry into H441 cells in comparison with naked siRNA. As determined by flow cytometry and a trypan blue assay, PHEA-Spm and PHEA-PEG-Spm allowed siRNA uptake in 42–47% and 30% of cells respectively, however only with PHEA-Spm at w/w ratio of 5 these percentages were significantly higher than those obtained with naked siRNA (20%). Naked siRNA or complexed scrambled siRNA did not exert any effect on HMGB1mRNA levels, whereas PHEA-Spm/siRNA at the w/w ratio of 5 down-regulated HMGB1 mRNA up to 58% of control levels (untransfected cells). PEGylated PHEA-Spm/siRNA nanocomplexes were able to down-regulate HMGB1 mRNA levels up to 61% of control cells. MTT assay revealed excellent biocompatibility of copolymer/siRNA polyplexes with cells.In conclusion, we have found optimal conditions for down-regulation of HMGB1 by siRNA delivery mediated by polyaminoacidic polymers in airway epithelial cells in the absence of cytotoxicity. Functional and in-vivo studies are warranted.

Expression and significance of high mobility group box 1 in the renal tissues of urosepsis rabbits

Objective To investigate the expression and significance of high mobility group box 1 (HMGB1)in the renal tissues of rabbits with urosepsis. Methods Male rabbits were randomly divided into two group:sham group and urosepsis group.The rabbit urosepsis model was established by artificial high pressure in the infected pelvis.The blood white blood cells(WBC)count,serum levels of blood urea nitrogen(BUN), creatinine(Cr), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and the expression of HMGB1 mRNA in the renal tissues of the two groups were observed at 12th,24th and 36th h after operation. Results As compared with sham group, the WBC count[36 h(×109/L):17. 22±0. 32 vs.9. 54±0. 24], serum levels of BUN[36 h(mmol/L):11. 71±0. 35 vs.7. 14±0. 12]and Cr[36 h (μmol/L):136. 10±7. 39 vs.82. 74±2. 18]in urosepsis group were significantly increased at every time point(P< 0. 05). The serum levels of TNF-αand IL-6 were first increased and then decreased in urosepsis group as compared with sham group at 12th h[TNF-α(ng/L): 124. 6±4. 42 vs.18. 18± 1. 26; IL-6(ng/L):120. 5±6. 99 vs.51. 65±2. 26] and 24 h[TNF-α(ng/L): 96. 94±7. 95 vs. 19. 04±0. 89; IL-6(ng/L):83. 22±4. 23 vs.53. 95±2. 26].The expression of HMGB1 mRNA was gradually increased from 12th to 36th h in urosepsis group as compared with sham group at the same time point(P<0. 05). Conclusion In the progression of acute kidney injury induced by urosepsis, the expression of HMGB1 mRNA was gradually increased in renal tissues. Key words: Urosepsis; High mobility group box 1; Acute kidney injury; Inflammatory factor

Lung endothelial barrier protection by resveratrol involves inhibition of HMGB1 release and HMGB1-induced mitochondrial oxidative damage via an Nrf2-dependent mechanism

High-mobility group box 1 (HMGB1) contributes to lung vascular hyperpermeability during ventilator-induced lung injury. We aimed to determine whether the natural antioxidant resveratrol protected against HMGB1-induced endothelial hyperpermeability both in vitro and in vivo. We found that HMGB1 decreased vascular endothelial (VE)-cadherin expression and increased endothelial permeability, leading to mitochondrial oxidative damage in primary cultured mouse lung vascular endothelial cells (MLVECs). Both the mitochondrial superoxide dismutase 2 mimetic MnTBAP and resveratrol blocked HMGB1-induced mitochondrial oxidative damage, VE-cadherin downregulation, and endothelial hyperpermeability. In in vivo studies, anesthetized male ICR mice were ventilated for 4h using low tidal volume (6ml/kg) or high tidal volume (HVT; 30ml/kg) ventilation. The mice were injected intraperitoneally with resveratrol immediately before the onset of ventilation. We found that resveratrol attenuated HVT-associated lung vascular hyperpermeability and HMGB1 production. HVT caused a significant increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and Nrf2 target gene expression in lung tissues, which was further enhanced by resveratrol treatment. HMGB1 had no effect on Nrf2 activation, whereas resveratrol treatment activated the Nrf2 signaling pathway in HMGB1-treated MLVECs. Moreover, Nrf2 knockdown reversed the inhibitory effects of resveratrol on HMGB1-induced mitochondrial oxidative damage and endothelial hyperpermeability. The inhibitory effect of resveratrol on cyclic stretch-induced HMGB1 mRNA expression in primary cultured MLVECs was also abolished by Nrf2 knockdown. In summary, this study demonstrates that resveratrol protects against lung endothelial barrier dysfunction initiated by HVT. Lung endothelial barrier protection by resveratrol involves inhibition of mechanical stretch-induced HMGB1 release and HMGB1-induced mitochondrial oxidative damage. These protective effects of resveratrol might be mediated through an Nrf2-dependent mechanism.

Tu1854 The Alarmin High Mobility Group Box 1 (HMGB1) Is Released From Enterocytes During Intestinal Inflammation and Causes Increased Interferon-γ and Decreased Na+/H+ Exchanger Isoform 3 Expression

Aims: The alarmin HMGB1 is released during cell damage and acts as an early or late stage inflammatory cytokine at multiple levels of target cells. The Na+/H+ exchanger Isoform 3 (NHE3) is the major intestinal salt and fluid absorptive transporter and its function is defective in intestinal inflammation. Aim of the study: We wanted to know if HMGB1 can affect NHE3 mRNA or protein expression as well as its localization in mucosal tissue form patients with ulcerative colitis and in NHE3-expressing Caco2bbe colonic cells. Methods and Results: We studied the mRNA and protein expression and localization of HMGB1 and NHE3 in rectal biopsies from the inflamed and noninflamed mucosa of patients with ulcerative colitis by quantitative qPCR and immunohistochemistry. TNF-α and IFN-γmRNA level was significantly elevated in lesions compared to that in its adjacent normal tissue samples of UC patient's rectal, suggesting a reliable endoscopic sampling of inflamed vs noninflamed tissue. HMGB1 mRNA and protein expression was increased in the inflamed as compared to adjacent normal tissues, and it was mostly distributed in cytoplasm and extracellular region. NHE3 protein was markedly reduced in inflamed compared to adjacent normal tissues, though its mRNA level was not significantly different between inflamed and noninflamed mucosa. When Caco2bbe cells overexpressing human NHE3 (Caco2bbe/ hNHE3) were incubated with high concentration of rh-HMGB1 (10μg/ml), NHE3 mRNA was found to be decreased at 1h (0.385±0.015 vs. 1.011±0.011, p 0.05). NHE3 protein, measured byWestern analysis, was not significantly changed when co-cultured with 10μg/ml rhHMGB1 for 1h or 6h, however, it was dramatically decreased at 24h of co-culture with 10μg/ml rhHMGB1. IFN-gamma levels were markedly increased when Caco2bbe/hNHE3 cells were incubated with 10 μg/ml rhHMGB1 for 1h as compared with normal group (29.855±0.055 vs. 29.525±0.050, p 0.05) and 24h (29.066±0.181 vs. 29.525±0.050, p>0.05) of co-culture with 10μg/ml rhHMGB1 as compared to normal group. This suggests that incubation with rhHMGB1 may down-regulate NHE3 mRNA expression via elevated IFN-gamma level at early stage (1h), resulting in a decrease in NHE3 protein expression at late stage (24h). Conclusion: Decreased NHE3 protein level was accompanied by elevated cytoplasm and extracellular HMGB1 in human ulcerative rectitis tissue. This may result from the regulatory effect of cytokines such as IFNgamma induced by exogenous HMGB1 at late stage of inflammation.

Inhibition effect of glycyrrhizin in lipopolysaccharide-induced high-mobility group box 1 releasing and expression from RAW264.7 cells.

High-mobility group box 1 (HMGB1) is a therapeutic target for sepsis. Glycyrrhizin (GL) is the aglycone of glycyrrhizin derived from licorice. We clarified the anti-inflammatory effects of GL. We explored the anti-HMGB1 effect of GL and elucidated its molecular mechanism, which will be of benefit for sepsis treatment. We stimulated murine macrophage-like RAW 264.7 cells with lipopolysaccharide (LPS) and LPS + GL, then measured the expression and release of HMGB1. The expression of related signal transduction factors was detected. High-mobility group box 1 was distributed mainly in the nucleus with lower cytoplasmic levels in RAW 264.7 cells before LPS stimulation. After stimulation, cytoplasmic HMGB1 levels increased gradually, whereas in nuclear fluctuation a trend of HMGB1 expression was observed. Significant upregulation of HMGB1 mRNA occurred 12 h after LPS stimulation. Glycyrrhizin prevented the transfer of HMGB1 from the nucleus to the cytoplasm and inhibited upregulation of HMGB1 mRNA induced by LPS. Phospho-p38 mitogen-activated protein kinase and activated activating protein 1 increased significantly 8 h after LPS stimulation. Tumor necrosis factor α and interleukin 6 increased 4 h after LPS stimulation and peaked at 48 h, and HMGB1 increased at 8 h. The Toll-like receptor 4/MD2/nuclear factor κB signaling pathway was activated 4 h after LPS stimulation. Glycyrrhizin inhibited this pathway. Glycyrrhizin inhibited the expression and release of HMGB1 through blocking the p38 mitogen-activated protein kinase/activating protein 1 signaling pathway then inhibited the massive release of tumor necrosis factor α and interleukin 6.