High-Glucose and Free Fatty Acid-Induced Adipocytes Generate Increasing of HMGB1 and Reduced GLUT4 Expression

Background High-mobility group box 1 protein (HMGB1) is released from necrotic adipocytes into the extracellular milieu as an inflammatory alarmin in obesity. Although the impact of excess nutrient on adipocytes is well known, it is not clear how specific its component drive cell-size and damaged of adipocytes, and how this relates to the risk of insulin resistance. Objectives The aim of this study was to determine HMGB1 level in adipocytes cultures after high glucose and/or FFA exposures and to assess GLUT4 expression. We determined cellular features of adipocytes that correlates to HMGB1 released and insulin resistance. Methods Differentiated adipocytes were exposed to high glucose and/or FFAs for 7 days. ELISA was performed on supernatant to assess the HMGB1 level. Total GLUT4 expression were quantified by immunofluorescense. Results High glucose and FFA-exposed cells have significant increase of HMGB1 level with decreased of cell size and necrotic adipocytes features. The total GLUT4 were reduced in HG-cells (p <0,045), but not in FFA cells. Hypertrophic adipocytes (p <0.05) and slight decrease of GLUT4 expression were showed on HG+FFA exposures with no increase of HMGB1 level. There was a significant correlation between cell size and HMGB1 level (R -0,637, p < 0.026) Conclusion The expression level studies between high glucose, FFA, and a combination of both on adipocytes results strongly suggest that high glucose is more damaging to adipocyte compared to FFA. Nevertheless, the combination of the two causes adipocyte dysfunction with general features of adipose tissue in obesity, suggested it can be used as a hypertrophic adipocytes model to study obesity in vitro.

Pharmacological Inhibition of HMGB1 Prevents Muscle Wasting

Background: Cachexia is a multifactorial disorder characterized by weight loss and muscle wasting, making up for about 20% of cancer-related death. However, there are no effective drugs to combat cachexia at present.Methods: In this study, the effect of CT26 exosomes on C2C12 myotubes was observed. We compared serum HMGB1 level in cachexia and non-cachexia colon cancer patients. We further explored HMGB1 expression level in CT26 exosome. We added recombinant HMGB1 to C2C12 myotubes to observe the effects of HMGB1 on C2C12 myotubes and detected the expression level of the muscle atrophy-related proteins. Then, we used the HMGB1 inhibitor glycyrrhizin to reverse the effects of HMGB1 on C2C12 myotubes. Finally, HMGB1 inhibitor glycyrrhizin was utilized to relieve cachexia in CT26 cachexia mouse model.Results: Exosomes containing HMGB1 led to muscle atrophy with significantly decreased myotube diameter and increased expression of muscle atrophy-related proteins Atrogin1 and MuRF1. Further, we detected that HMGB1 induced the muscle atrophy mainly via TLR4/NF-κB pathway. Administration of the HMGB1 inhibitor glycyrrhizin could relieve muscle wasting in vitro and attenuate the progression of cachexia in vivo.Conclusion: These findings demonstrate the cachectic role of HMGB1, whether it is soluble form of HMGB1 or secreted from tumor cells as part of exosomes. HMGB1 inhibitor glycyrrhizin might be a promising drug in colon cancer cachexia.

LPS-induced macrophage HMGB1-loaded extracellular vesicles trigger hepatocyte pyroptosis by activating the NLRP3 inflammasome

Extracellular vesicles (EVs) have emerged as important vectors of intercellular dialogue. High mobility group box protein 1 (HMGB1) is a typical damage-associated molecular pattern (DAMP) molecule, which is cytotoxic and leads to cell death and tissue injury. Whether EVs are involved in the release of HMGB1 in lipopolysaccharide (LPS)-induced acute liver injuries need more investigation. EVs were identified by transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. The co-localization of HMGB1, RAGE (receptor for advanced glycation end-products), EEA1, Rab5, Rab7, Lamp1 and transferrin were detected by confocal microscopy. The interaction of HMGB1 and RAGE were investigated by co-immunoprecipitation. EVs were labeled with the PKH67 and used for uptake experiments. The pyroptotic cell death was determined by FLICA 660-YVAD-FMK. The expression of NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasomes were analyzed by western-blot or immunohistochemistry. Serum HMGB1, ALT (alanine aminotransferase), AST (aspartate aminotransferase), LDH (lactate dehydrogenase) and MPO (myeloperoxidase) were measured using a commercial kit. The extracellular vesicle HMGB1 was detected in the serums of sepsis patients. Macrophages were found to contribute to HMGB1 release through the EVs. HMGB1-RAGE interactions participated in the loading of HMGB1 into the EVs. These EVs shuttled HMGB1 to target cells by transferrin-mediated endocytosis leading to hepatocyte pyroptosis by the activation of NLRP3 inflammasomes. Moreover, a positive correlation was verified between the sepsis serum EVs-HMGB1 level and clinical liver damage. This finding provides insights for the development of novel diagnostic and therapeutic strategies for acute liver injuries.

High Mobility Group Box 1: a Novel Host Restriction Factor in Zika Virus Replication

Neonatal microcephaly and adult Guillain-Barré syndrome are severe complications of Zika virus (ZIKV) infection. The robustly induced inflammatory cytokine expressions in ZIKV-infected patients may constitute a hallmark for severe disease. In the present study, the potential role of high mobility group box 1 protein (HMGB1) in ZIKV infection was investigated. HMGB1 protein expression was determined by the enzyme-linked immunosorbent assay (ELISA) and immunoblot assay. HMGB1’s role in ZIKV infection was also explored using treatment with dexamethasone, an immunomodulatory drug. Antiviral effects of dexamethasone treatment on both wild-typed (WT) and HMGB1-knockdown (shHMGB1) Huh7 cells were determined by the focus-forming assay. Results showed that the Huh7 cells were highly susceptible to ZIKV infection. The infection was found to induce HMGB1 nuclear-to-cytoplasmic translocation, resulting in a >99% increase in the cytosolic HMGB1 expression at 72h.p.i. The extracellular HMGB1 level was elevated in a time- and multiplicity of infection (MOI)- dependent manner. Dexamethasone 150 µM treatment of the ZIKV-infected cells reduced HMGB1 extracellular release in a dose-dependent manner, with a maximum reduction of 71 ± 5.84% (p < 0.01). The treatment also reduced virus titers by over 83 ± 0.50% (p < 0.01). The antiviral effects, however, was not observed in the dexamethasone-treated HMGB1-knockdown cells, suggesting the importance of the intracellular HMGB1 in ZIKV infection. Overall, these results suggest that translocation of HMGB1 occurred during ZIKV infection and inhibition of the translocation reduced ZIKV replication. These findings highlight the potential of developing therapeutics against ZIKV infection by affecting the translocation of HMGB1 from the nucleus to the cytoplasm.

Clinical Significance of Serum HMGB1 In COPD and Correlation with Severity of Airflow Restriction and Immune Function

BACKGROUND: We aimed to explore the serum HMGB1 levels in patients with smoking-induced chronic obstructive pulmonary disease (COPD) and the correlations with airflow restriction and immune function. METHODS: A total of 136 COPD patients were divided into mild, moderate and severe + extremely severe groups. Thirty-five healthy subjects were selected as control group. Serum HMGB1 levels were measured by ELISA, and the correlations with pulmonary and immune function indices were analyzed. Receiver operating characteristic (ROC) curve was plotted. RESULTS: Pa02, eosinophil count, FEV1/FVC, FEV1% pred, and IgA, IgM, IgG levels of COPD patients were lower than those of control group, and decreased with airflow restriction aggravation. PaC02, leukocyte count, neutrophil percentage, modified British Medical Research Council (mMRC) scale and COPD Assessment Test (CAT) scores, D-Dimer (D-D), PCT, CRP and HMGB1 levels, myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) counts, and mDCs/pDCs of COPD patients exceeded those of control group, and increased with airflow restriction aggravation (P<0.05). HMGB1 levels of COPD patients were negatively correlated with FEV1/FVC, FEV1% pred, IgA, IgM and IgG levels, and positively correlated with mDC count, pDC count and mDCs/pDCs (P<0.0001). The area under ROC curve was 0.883, the optimal cutoff value was 3.63 ng/mL, and sensitivity and specify were 86.7% and 85.9%, respectively. CONCLUSIONS: Serum HMGB1 level in patients with smoking-induced COPD rises with airflow restriction aggravation and has significant correlations with the decline of pulmonary and immune functions, with high predictive value for COPD. HMGB1 is a potential biomarker for evaluating COPD progression.

Abstract P336: Endogenous Alpha-1a Adrenergic Receptors Promote Cardiomyocyte Survival Through Suppression Of Rip Kinase-mediated Necroptosis

Our previous work has demonstrated essential protective roles for the endogenous cardiomyocyte alpha-1A adrenergic receptor (α1A-AR) subtype in mouse models of heart failure. However, the underlying mechanism of this protective phenotype is unclear. To address this gap in knowledge, we bred a mouse line lacking α1A-ARs on cardiomyocytes by crossing αMHC-cre mice with floxed α1A mice (CMKO= cre+ fl/fl, CMWT= cre- fl/fl), and subjected males to permanent LAD ligation. CMKO mice had increased serum HMGB1 level, larger infarcts and higher mortality. We found that RIP1/3-mediated programmed necrosis (necroptosis), but not apoptosis was exaggerated in CMKO mice 3 days after ligation. We then tested whether RIP1 inhibition with Nec-1s could mitigate this injury. Mice were given Nec-1s (1.65 mg/kg) or vehicle 10 mins prior to LAD ligation, followed by daily IV injection. Nec-1s treatment diminished post-ligation RIP1 (0.62±0.02 vs. 0.78±0.23 A.U., p=NS) and RIP3 expression (0.33±0.1 vs. 0.26±0.10 A.U., p=NS) in CMWT and CMKO mice respectively. Serum level of HMGB1 on D3 was markedly reduced in both CMWT (45.1%) and CMKO (61.1 %) after Nec-1s treatment. There was no difference between Nec-1s treated CMWT and CMKO mice (147±53 vs. 174±37 pg/mL, p=NS), indicating that blocking the RIP kinase pathway abrogates the exaggerated cell death in CMKO mice after ligation. Likewise, Nec-1s-treated CMKO mice had similar infarct areas to CMWT controls (16.2±4.5 vs. 19.9±4.6%, p=NS), further confirming that targeting necroptosis abrogates pathological damage. Collectively these Nec-1s data suggest that RIP-mediated necroptosis may account for larger infarcts in CMKO mice. Interestingly, expression of the apoptosis markers c-caspase-3 and PARP was similar between CMWT and CMKO mice, suggesting that the α1A-AR specifically regulates necroptosis. In sum, our data demonstrate that RIP kinase-mediated necroptosis contributes to susceptibility to injury in mice lacking cardiomyocyte α1A-ARs.

Serum HMGB1 level is correlated with serum I-FABP level in neonatal patients with necrotizing enterocolitis

Background This study aims to investigate clinical significance of HMGB1 in neonatal patients with necrotizing enterocolitis (NEC). Methods This observational study enrolled a total of 106 stage II-III NEC neonatal patients, who were admitted in our hospital from March 2014 to March 2019. In addition, 99 suspected NEC patients and 200 healthy controls were included. The serum levels of HMGB1, I-FABP, and inflammatory factors CRP, IL-1β, IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA). Then, the demographic data and clinical characteristics of all patients were collected. Statistical analysis was conducted to determine the correlation between HMGB1 and the clinical characteristics. Results No significant difference was found in the basic characteristics of NEC patients and healthy controls, except for birth weight and gestational age. The expression levels of HMGB1, I-FABP, and inflammatory factors IL-1β, IL-6 and TNF-α were significantly higher in NEC patients, when compared to healthy controls. The serum levels of HMGB1, I-FABP, IL-1β and IL-6 markedly increased in stage II-III NEC patients, when compared to stage I NEC patients. The Pearson’s analysis revealed a positive correlation between HMGB1 and I-FABP, HMGB1 and IL-1β, and HMGB1 and IL-6. The ROC curve revealed that both HMGB1 and I-FABP can potentially be used as diagnostic factors for NEC. The logistic multivariate regression revealed that I-FABP, IL-1β and IL-6 are independent risk factors for mortality in neonatal NEC patients. Conclusions Serum HMGB1 levels are upregulated in neonatal NEC patients, and these are correlated with the patient’s prognosis.

Modulation of HMGB1 Release in APAP-Induced Liver Injury: A Possible Strategy of Chikusetsusaponin V Targeting NETs Formation

Acetaminophen (APAP), one of the most common antipyretic analgesics, which is safe at therapeutic dose, cause acute liver injury and even death at overdose. However, the mechanism of APAP-induced inflammation in liver injury is still controversial. Therefore, effective drug intervention is urgently needed. The aim of this study was to explore the inflammatory exact mechanism of APAP, especially on neutrophils, and to study the intervention effect of Chikusetsusaponin V (CKV) derived from Panax japonicus. Establishment of hepatotoxicity model of APAP in vitro and in vivo. In vitro, HepG2 cells, AML12 cells, primary mouse hepatocytes and neutrophils were used to mimic APAP-affected hepatocytes and neutrophil. In vivo, C57BL/6 mice were administrated overdose of APAP with or without neutrophil depletion or abolishing neutrophil extracellular traps (NETs) formation. In this study, APAP stimulation increased the level of HMGB1, IL-1β and Caspase-1 in mouse liver, especially hepatocytes, which had a synergistic effect with LPS/ATP combination. NETs were formatted at early stage of APAP or HMGB1-stimulated neutrophils’ damage. Conditioned mediums from APAP-treated hepatocytes induced more significant NETs than direct APAP stimulation. Neutrophil depletion or abolishing NETs formation decreased HMGB1 level, eventually blocked hepatocytes necrosis. CKV pretreatment interfered Caspase-1 activation and HMGB1 release in APAP-damaged hepatocytes. CKV also prevented NETs formation. These results indicate that the production of HMGB1 may depend on the activation of Caspase-1 and play a key role in liver inflammation caused by APAP. The cross-dialogue between hepatocytes and neutrophils can be mediated by HMGB1. Therefore, CKV has a positive intervention effect on NETs-related inflammation in APAP-damaged liver, targeting Caspase-1-HMGB1.

Effects of miR-1305 on Proliferation and Apoptosis of Non-Small Cell Lung Cancer Cells via Regulating High Mobility Group Box-1 Level

Abnormal expression of HMGB1 is closely related to non-small cell lung cancer (NSCLC). miR-1305 regulates HMGB1 level by MiRDB analysis. Therefore, we investigated whether miR-1305 affects NSCLC cell proliferation and apoptosis by regulating HMGB1. The control group (NC group), miR-1305 Mimics group and miR-1305 Mimics+pcDNA-HMGB1 group were set followed by analysis of miR-1305 and HMGB1 mRNA level real time-PCR, relationship between miR-1305 and HMGB1 by dual fluorescein reporter assay, HMGB1 and Tubulin level by Western blot, cell proliferation by clone formation assay, cell apoptosis by Annexin V-FITC/PI staining. Compared with normal tissues, miR-1305 was significantly downregulated in NSCLC tissues (P <0.01), while HMGB1 mRNA was upregulated (P <0.01). HMGB1 was the target gene of miR-1305. Compared to NC group, HMGB1 level in miR-1305 Mimics group was significantly reduced (P <0.01). Compared with miR-1305 Mimics group, HMGB1 level was significantly increased in miR-1305 Mimics+pcDNA-HMGB1group (P <0.05). HMGB1 mRNA level was not significantly changed. In addition, the number of cell clones and proliferation ability was decreased in miR-1305 Mimics group, which were reversed in miR-1305 Mimics+pcDNA-HMGB1 group. miR-1305 can bind HMGB1 3′-UTR, reduce its protein level, thereby inhibiting NSCLC cell proliferation and promoting cell apoptosis. HMGB1 overexpression can prevent the effect of miR-1305.

Neuroprotective Effects of Intraoperative Dexmedetomidine Infusion Combined With Goal-directed Hemodynamic Therapy for Patients Undergoing Cranial Surgery: A Double-blinded Randomized Controlled Trial

BackgroundDespite dexmedetomidine may be neuroprotective in patients undergoing cranial surgery by inhibiting neuroinflammation; however, it reduces cardiac output and cerebral blood flow. We proposed that dexmedetomidine infusion combined with goal-directed hemodynamic therapy (GDHT) could improve cranial surgery neurological outcomes without hemodynamic perturbation.MethodsA randomized, double-blind trial was conducted. One hundred sixty adult patients undergoing elective cranial surgery received either dexmedetomidine (0.5 μg kg−1 h−1) or saline during surgery. The goal-directed hemodynamic therapy was used for stroke volume optimization. The proportion of patients who developed postoperative new neurological deficits was compared. The severities of new neurological deficit were assessed by using in-hospital Barthel index changes and the 30-day modified Rankin Scale (mRS). Postoperative delirium was identified using the Intensive Care Delirium Screening Checklist (ICSDC) criteria. The level of a perioperative serum neuroinflammatory mediator, high motility group box 1 protein (HMGB1), was compared.ResultsThe dexmedetomidine group exhibited a lower cardiac index than did the control group (3.0 ± 0.8 vs. 3.4 ± 1.8 L min−1 m−2; p = 0.0482) without lactate accumulation. Fewer patients in the dexmedetomidine group developed new postoperative deficits (26.3% versus 43.8%; p = 0.031) but numbers of patients remained symptomatic neurological deficit before discharge were comparable between the two groups (23.8% vs. 38.8%; p= 0.060). In addition, an attenuated Barthel index decline (−6.3 ± 20.4 vs. −13.6 ± 24.8; p = 0.043), a more favorable 30-day mRS profile (p = 0.013), and a higher incidence of postoperative delirium-free (ICDSC scored 0: 84.6% versus 64.2%; p = 0.012) were observed in the dexmedetomidine group. Furthermore, dexmedetomidine induced a significant decline in perioperative serum HMGB1 level (222.5 ± 408.3 vs. 152.2 ± 280.0 ng mL−1; p = 0.0033).ConclusionsDexmedetomidine infusion combined with GDHT mitigates neuroinflammation during cranial surgery without hemodynamic perturbation, thus achieving neuroprotective effects.Clinical Trial RegistrationProspectively registered at clinicaltrials.gov. (identifier NCT02878707, date of registration: August 25, 2016)