Plasma High Mobility Group Box 1 Research Articles

Target Gene-Based Association Study of High Mobility Group Box Protein 1 in Intracranial Aneurysms in Koreans.

Objective: We investigated the effect of high mobility group box 1 (HMGB1) on intracranial aneurysms (IAs) by analyzing single-nucleotide polymorphisms (SNPs) based on genome-wide association study (GWAS) data. HMGB1 mRNA and protein expression levels in plasma were also analyzed. Methods: This study was a comprehensive analysis of a GWAS dataset, including 250 patients with IAs and 294 controls. The HMGB1 gene region was targeted within SNP rs3742305 ± 10 kbp. Multivariate logistic regression analysis determined its association with IAs after adjusting for relevant clinical factors. HMGB1 mRNA expression was analyzed in the plasma of 24 patients selected from the GWAS dataset. The HMGB1 protein was analyzed by Western blotting. Results: A total of seven polymorphisms, including rs1360485, rs185382445, rs2039338, rs1045411, rs3742305, rs2249825, and rs189034241, were observed. Two SNPs, including rs1045411 (UTR-3) and rs3742305 (intron), showed strong linkage disequilibrium (r2 = 0.99). However, none of the seven SNPs associated with IAs had an adjusted p-value of < 0.0016 on multiple comparison analysis. HMGB1 mRNA levels (2-ΔCt) did not differ significantly between patients with IAs and the control subjects [1.07 (1.00-1.15) in patients with IAs vs. 1.05 (0.94-1.12) in controls; p = 0.67)]. Also, no significant difference in the degree of plasma HMGB1 protein expression was seen between the two groups (p = 0.82). Conclusions: The number of SNPs associated with HMGB1 and the degree of HMGB1 mRNA and protein expression were not significantly different between patients diagnosed with IAs and the controls.

Assessment of the Role of Keratin 18 and High Mobility Group Box 1 Biomarkers for Early Prediction of Drugs and Chemicals Induced Hepatotoxicity A Prospective Study in the Poison Control Center of Ain Shams University Hospitals (PCC-ASUH)

Abstract Background Drug-induced hepatotoxicity represents a major clinical problem worldwide. Hepatotoxicity may be caused by drugs, chemicals used in laboratories and industries or natural products and rodenticides such as metal phosphides. Early identification of hepatotoxicity would facilitate patient- individualized treatment strategies. New biomarkers (HMGB1and K18) used to identify subsequent acute liver injury (ALI) in patients on admission to the hospital. Objectives This study aims to assess the role of keratin 18 and high mobility group box 1 biomarkers in early prediction of drugs and chemicals induced hepatotoxicity for better outcome and to compare these new liver biomarkers with the currently used parameters (ALT &amp; AST) in the early prediction of drugs and chemicals induced hepatotoxicity. Methods This prospective study was conducted on 50 adult patients of both sex presented to the PCC- ASUH with acute intoxication of hepatotoxic drugs or chemicals. For all patients, we measured high mobility group box-1 (HMGB1), keratin-18 (K18), AST and ALT on admission then patients were followed- up for development of liver injury . Subjects were classified into group I (control group), group II (no liver affection) and group III (with liver affection). Receiver operator characteristic (ROC) curve was used to compare sensitivity and specificity to report liver injury versus alanine transaminase (ALT) and aspartate transaminase (AST). Results This study showed that HMGB1 and K18 were much more elevated in patients who developed liver injury in the first hospital presentation when ALT and AST were within normal ranges with a highly significant difference between the hepatotoxic and non-hepatotoxic group. ROC analysis showed that HMGB1 and K18 were more sensitive than ALT and AST and more specific than AST in predicting ALI. Conclusion Elevations in plasma HMGB1, and K18 identified subsequent ALI development in patients on admission to the hospital, soon after drug and chemical overdose, and in patients with ALT and AST in the normal range.

Thrombomodulin Improves Cognitive Deficits in Heat-Stressed Mice.

Thrombomodulin (TM) exerts anticoagulant and anti-inflammatory effects to improve the survival of patients with septic shock. Heat stroke resembles septic shock in many aspects. We tested whether TM would improve cognitive deficits and related causative factors in heat-stressed (HS) mice. Adult male mice were exposed to HS (33°C for 2 hours daily for 7 consecutive days) to induce cognitive deficits. Recombinant human soluble TM (1mg/kg, i.p.) was administered immediately after the first HS trial and then once daily for 7 consecutive days. We performed the Y-maze, novel objective recognition, and passive avoidance tests to evaluate cognitive function. Plasma levels of lipopolysaccharide (LPS), high-mobility group box 1 (HMGB1), coagulation parameters, and both plasma and tissue levels of inflammatory and oxidative stress markers were biochemically measured. The duodenum and hippocampus sections were immunohistochemically stained. The intestinal and blood-brain barrier permeability were determined. Compared with controls, HS mice treated with TM had lesser extents of cognitive deficits, exacerbated stress reactions, gut barrier disruption, endotoxemia, blood-brain barrier disruption, and inflammatory, oxidative, and coagulatory injury to heart, duodenum, and hippocampal tissues, and increased plasma HMGB1. In addition to reducing cognitive deficits, TM therapy alleviated all the abovementioned complications in heat-stressed mice. The findings suggest that HS can lead to exacerbated stress reactions, endotoxemia, gut barrier disruption, blood-brain barrier disruption, hippocampal inflammation, coagulopathy, and oxidative stress, which may act as causative factors for cognitive deficits. TM, an anti-inflammatory, antioxidant, and anti-coagulatory agent, inhibited heat stress-induced cognitive deficits in mice.

The renoprotective effect of Tibolone in sepsis-induced acute kidney injury.

Sepsis-induced acute kidney injury (AKI) remains a major challenge in intensive care, contributing significantly to morbidity and mortality. Tibolone, known for its neuroprotective and hormonal properties, has not been explored for its potential in AKI management. This study investigates the protective effects of Tibolone and its underlying mechanisms involving Sirtuin-1 (SIRT1) and Yes-Associated Protein (YAP) in a rat sepsis model. Thirty-six female Wistar albino rats underwent cecal ligation and puncture (CLP) to induce sepsis. They were randomly assigned to control, CLP+Saline, and CLP+Tibolone groups. Tibolone was administered intraperitoneally. Biomarkers, including Sirtuin (SIRT1), Yes-associated protein (YAP), Tumor necrosis factor (TNF-α), High mobility group box 1 (HMGB1), malondialdehyde (MDA), creatinine, and urea, were assessed. Histopathological examination evaluated renal damage. Tibolone administration significantly reduced plasma TNF-α, HMGB1, MDA, creatinine, and urea levels compared to the CLP+Saline group. Moreover, Tibolone elevated SIRT1 and YAP levels in kidney tissues. Histopathological examination demonstrated a significant decrease in tubular epithelial necrosis, luminal debris, dilatation, hemorrhage, and interstitial inflammation in Tibolone-treated rats. This study unveils the protective role of Tibolone against sepsis-induced AKI in rats. The improvements in inflammatory and oxidative biomarkers and histological evidence suggest Tibolone's potential as a therapeutic intervention in sepsis-associated kidney injury. The upregulation of SIRT1 and YAP indicates their involvement in Tibolone's renoprotective mechanisms. Further investigations are warranted to explore Tibolone's translational potential in human sepsis-induced AKI.

Analysis of changes in high-mobility group box 1, receptor for advanced glycation endproducts, and T helper 17/regulatory T balance in severe preeclampsia with acute heart failure.

We measured the levels of High-Mobility Group Box 1 (HMGB1), Receptor for Advanced Glycation Endproducts (RAGE), T Helper 17 cells (Th17), Regulatory T cells (Treg), and related cytokines in the peripheral blood of patients with severe preeclampsia (SPE) complicated with acute heart failure (AHF) to explore the expression changes in these indicators. In total, 96 patients with SPE admitted to Gansu Provincial Maternity and Child-care Hospital between June 2020 and June 2022 were included in the study. The patients were divided into SPE+AHF (40 patients) and SPE (56 patients) groups based on whether they suffered from AHF. Additionally, 56 healthy pregnant women who either received prenatal examinations or were admitted to our hospital for delivery during the same period were selected as the healthy control group. An enzyme-linked immunosorbent assay was performed to detect the expression levels of HMGB1, RAGE, interleukin (IL)-17, IL-6, transforming growth factor β (TGF-β), IL-10, and NT-proBNP in plasma. Flow cytometry was employed to determine the percentages of Th17 and Treg cells. Compared to the healthy control group, the SPE+AHF and SPE groups had higher plasma levels of HMGB1 and RAGE expression, higher Th17 percentage and Th17/Treg ratio, and lower Treg percentage. Compared to the SPE group, the SPE+AHF group had higher plasma levels of HMGB1 and RAGE expression, higher Th17 percentage and Th17/Treg ratio, and lower Treg percentage (P<.05). In patients with SPE with AHF, plasma HMGB1 was positively correlated with RAGE, Th17, Th17/Treg, IL-17, and IL-6 and was negatively correlated with TGF-β and IL-10 (P<.05). Our findings revealed that patients with SPE with AHF had elevated levels of HMGB1 and RAGE while exhibiting Th17/Treg immune imbalance, suggesting that the abnormal expression of these indicators may be involved in the pathogenesis of SPE with AHF.

High-mobility group box 1 increases platelet surface P2Y12 and platelet activation in sickle cell disease.

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.

Plasma High-Mobility Group Box-1 and Galectin-9 in Patients with Trauma and Their Prognostic Potentials.

Plasma High-Mobility Group Box-1 and Galectin-9 in Patients with Trauma and Their Prognostic Potentials.

Toll-like receptor expression and functional behavior in platelets from patients with systemic lupus erythematosus

BackgroundMultiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. Materials and MethodsThirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. ResultsIn resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. ConclusionOur study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.

Effects of anterior approach to quadratus lumborum block on postoperative cognitive function following hip surgery in older people: a randomized controlled clinical trial

BackgroundPeripheral nerve block, including the quadratus lumborum block (QLB), has been used for postoperative analgesia in hip surgery. However, the effects of QLB on cognitive function after hip surgery remain unknown. This study aimed to assess the effects of the anterior approach to QLB on postoperative cognitive function in older people undergoing hip surgery.MethodsSixty older people who underwent hip surgery from May 2021 to May 2022 were randomly divided into the QLB (n = 30) and control groups (n = 30). The Montreal Cognitive Assessment (MoCA) score (mean ± SD) was measured one day preoperatively and seven and 30 days postoperatively. The frequency (%) of postoperative cognitive dysfunction (POCD) was examined seven and 30 days postoperatively. The visual analog scale (VAS) scores at rest and Bruggrmann comfort scale (BCS) scores [Median (IQR)] 6 h (t1), 12 h (t2), 24 h (t3), and 48 h (t4) after surgery were assessed. The plasma high mobility group box protein 1 (HMGB1) and levels of interleukin-6 (IL-6) (mean ± SD) were evaluated 1 h preoperatively (baseline) and 24 h postoperatively (day 1). The requirement for rescue analgesia [Median (IQR)], time to first off-bed activity (mean ± SD), and adverse effects after surgery were also recorded.ResultsCompared with the control group, the frequency of POCD was significantly lower in the QLB group seven days postoperatively (10.7% vs. 34.5%, P = 0.033), but no difference at 30 days postoperatively (3.6% vs. 10.3%, P = 0.319). There was no significant difference in MoCA scores between the two groups at one day preoperatively and 30 days postoperatively. However, the MoCA scores at seven days postoperatively were higher in the QLB group than in the control group (27.4 ± 1.81 vs. 26.4 ± 1.83, P = 0.043). In the QLB group, the VAS scores at t1, t2, and t3 were lower [3(2–4) vs. 4(3–4), P = 0.028; 3(2–3) vs. 4(3–5), P = 0.009; 2(1–3) vs. 2(2–3), P = 0.025], and the BCS scores at t1, t2, and t3 were higher than those in the control group [3(1–3) vs. 1(1–2), P = 0.006; 3(2–3) vs. 2(1–3), P = 0.011; 3(2–4) vs. 2(2–3), P = 0.041]. The patients in the QLB group reported significantly fewer requirements for rescue analgesia [0(0–1) vs. 1(0–2), P = 0.014]. The plasma levels of HMGB1 and IL-6 at 24 h postoperatively in the QLB group were significantly lower than in the control group (749.0 ± 185.7 vs. 842.1 ± 157.9, P = 0.046; 24.8 ± 8.1 vs. 31.9 ± 5.5, P < 0.001). The time to first off-bed activity from the end of surgery was shorter in the QLB group (25.3 ± 5.3 vs. 29.7 ± 6.9, P = 0.009). There was no significant difference in the incidence of postoperative complications between the two groups.ConclusionsAnterior QLB given to older people undergoing hip surgery could promote early postoperative cognitive function recovery, provide adequate postoperative analgesia, and inhibit the release of inflammatory factors.Trial registrationChictr.org.cn identifier ChiCTR2000040724 (Date of registry: 08/12/2020, prospectively registered).

HMGB1 Is a Prognostic Factor for Mortality in Acute Kidney Injury Requiring Renal Replacement Therapy

Instruction: High mobility group box 1 (HMGB1) is a pro-inflammatory cytokine that reportedly causes kidney injury and other organ damage in rodent acute kidney injury (AKI) models. However, it remains unclear whether HMGB1 is associated with clinical AKI and related outcomes. This study aimed to evaluate the association with HMGB1 and prognosis of AKI requiring continuous renal replacement therapy (CRRT). Methods: AKI patients treated with CRRT in our intensive care unit were enrolled consecutively during 2013–2016. Plasma HMGB1 was measured on initiation. Classic initiation was defined as presenting at least one of the following conventional indications: hyperkalemia (K ≥6.5 mEq/L), severe acidosis (pH <7.15), uremia (UN >100 mg/dL), and diuretics-resistant pulmonary edema. Early initiation was defined as presenting no conventional indications. The primary outcome was defined as 90-day mortality. Results: A total of 177 AKI patients were enrolled in this study. HMGB1 was significantly associated with the primary outcome (hazard ratio, 1.06; 95% CI, 1.04–1.08). When the patients were divided into two-by-two groups by the timing of CRRT initiation and the HMBG1 cutoff value obtained by receiver operating curve (ROC) analysis, the high HMGB1 group (>10 ng/mL) with classic initiation was significantly associated with the primary outcome compared with the others, even after adjusting for other factors including the nonrenal serial organ failure assessment (SOFA) score. Conclusion: HMGB1 was associated with 90-day mortality in AKI patients requiring CRRT. Notably, the highest mortality was observed in the high HMGB1 group with classic initiation. These findings suggest that CRRT should be considered for AKI patients with high HMGB1, regardless of the conventional indications.

Chemotherapy induced peripheral neuropathy (CIPN) and biomarkers of gut microbial translocation in long-term survivors of testicular germ cell tumors.

e17032 Background: Survivors of testicular germ cell tumors (GCT) may suffer from long-term treatment induced peripheral neuropathy. We hypothesized markers of gut microbial translocation after chemotherapy and/or radiotherapy may be contributing factors of peripheral neuropathy. Methods: GCT survivors (N = 194) from National Cancer Institute of Slovakia completed the EORTC QLQ Chemotherapy-Induced Peripheral Neuropathy (CIPN20) questionnaires during their annual follow-up visit at 10-year median (range 2-25). Biomarkers of gut microbial translocaion and dysbiosis (GMT) lipopolysaccharide (LPS), high mobility group box-1 (HMGB-1), d-lactate and sCD14 were measured from peripheral blood obtained during the same visit. Upon obtaining the scores from questionnaires per recommended guidelines, each score was correlated with biomarkers of GMT scored as as high or low (above and below median). Survivors were treated with orchiectomy only (N = 30), cisplatin-based chemotherapy (N = 135), radiotherapy to the retroperitoneum (N = 14) or both chemotherapy and radiotherapy (N = 10). Results: GCT survivors with higher vs lower plasma levels of HMGB-1 had worse overall CIPN20 score (median ± SD = 18 229.0 ± 13 644.2 vs 17 387.0 ± 5106.6 p = 0.048). Patients with higher vs lower plasma HMGB-1 also had worse motor function (9.0 ± 1.8 vs 8.0 ± 2.3, p = 0.036). Sensory and autonomy subscales of CIPN20 qestionaire did not show significant association with HMGB-1 (both P &gt; 0.05). There was no significant difference in CIPN20 symptom burden associated with LPS, d-lactate and sCD14 (all P &gt; 0.05). Conclusions: HMGB-1 may serve as a promising biomarker of CIPN20 in long-term cancer survivors. Proinflammatory pathways are suggested as underlying mechanism of CIPN. Furhter research and larger patient cohorts are needed to explore the pathogenesis of CIPN in GCT survivors.

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.

Promoting AMPK/SR-A1-mediated clearance of HMGB1 attenuates chemotherapy-induced peripheral neuropathy

BackgroundChemotherapy-induced peripheral neuropathy (CIPN) is a serious side effect of chemotherapy with poorly understood mechanisms and few treatments. High-mobility group box 1 (HMGB1)-induced neuroinflammation is the main cause of CIPN. Here, we aimed to illustrate the role of the macrophage scavenger receptor A1 (SR-A1) in HMGB1 clearance and CIPN resolution.MethodsOxaliplatin (L-OHP) was used to establish a CIPN model. Recombinant HMGB1 (rHMGB1) (his tag) was used to evaluate the phagocytosis of HMGB1 by macrophages.ResultsIn the clinic, HMGB1 expression and MMP-9 activity were increased in the plasma of patients with CIPN. Plasma HMGB1 expression was positively correlated with the cumulative dose of L-OHP and the visual analog scale. In vitro, engulfment and degradation of rHMGB1 increased and inflammatory factor expression decreased after AMP-activated protein kinase (AMPK) activation. Neutralizing antibodies, inhibitors, or knockout of SR-A1 abolished the effects of AMPK activation on rHMGB1 engulfment. In vivo, AMPK activation increased SR-A1 expression in the dorsal root ganglion, decreased plasma HMGB1 expression and MMP-9 activity, and attenuated CIPN, which was abolished by AMPK inhibition or SR-A1 knockout in the CIPN mice model.ConclusionActivation of the AMPK/SR-A1 axis alleviated CIPN by increasing macrophage-mediated HMGB1 engulfment and degradation. Therefore, promoting HMGB1 clearance may be a potential treatment strategy for CIPN.1JEzHJvY7WaTcecv7_-Z6vVideo abstract

Probiotic Treatment Ameliorates Exercise Intolerance, Deficits in Fear Learning and Systemic and Neuro-inflammation in a Mouse Model of Gulf War Illness

Veterans of the Persian Gulf War (GW) continue to suffer from Gulf War Illness (GWI) characterized by cognitive deficit and fatigue. Our group and others have suggested possible involvement of the microbiota-gut-brain axis in GWI pathology (Kozlova et a., 2021a; 2021b). The current study tested the hypothesis that probiotic treatment (P) prevents GWI symptoms, and systemic and neuro-inflammation. Adult male C57Bl/6N mice were separated into 4 groups (n=16/group): GW group was exposed to 8.7 mg/kg/d pyridostigmine bromide (PB) in saline (150uL/30g bw, oral gavage), 1.3 mg/kg PER in 100% DMSO (dermal), and 33% DEET in 70% EtOH (dermal) for 28 days (5 days/week). CON/S group was sham-treated. CON/S+P and GW+P groups were treated with a probiotic (P) cocktail of L. reuteri, L. rhamnosus, L. casei, B. longum (10 8 CFU/mL, oral gavage) 3 times/week for 2 weeks prior to and during sham/GW agent treatment and until sacrifice (post-treatment (PT) 150). All groups were subjected to restraint stress (5 min/d for 28 d). RT-qPCR on fecal pellets showed bacteria colonization of all strains except L. rhamnosus and B. longum after administration of 6-7 doses. At PT1, all strains showed increased colonization relative to baseline, which persisted to PT150, except B. longum. Body weight, fat, lean mass at PT150 showed no changes among groups. An exercise endurance (EE) test was performed to measure differences in fatigue. GW mice tired faster relative to CON/S at PT56 which persisted at PT150. Probiotic normalized exercise endurance to CON/S levels at PT56. On the passive avoidance test all groups behaved normally at PT50 but GW showed deficits on day 1 acquisition trial of fear learning when tested again 14 weeks later at PT150. GW+P behaved similarly to CON/S. Strength and depression-like behavior was examined before and after exercise stress to simulate post-exertional malaise. Mean percent time spent mobile on tail suspension test (TST) were significantly lower for GW but not GW+P at PT50. All groups were normal at PT150. There was no effect of GW agents or probiotics on the hanging wire test. Systemic and neuroinflammatory markers: C-reactive protein (CRP), high mobility group box 1 (HMGB1), and interleukin-6 (IL-6) were examined using ELISA. Liver and plasma CRP was increased in GW group but not GW+P in liver (p=0.06 in plasma). Plasma HMGB1 was reduced and brain IL-6 was elevated in GW mice but not GW+P. Immunofluorescence analysis in fixed hippocampal sections showed more Iba-1 positive cells in CA1 and DG in GW vs CON/S, while GW+P showed no significant changes vs CON/S. These results show that GW mice can be used to model long-lasting fatigue and other GWI symptoms as well as systemic and neuro-inflammation concurrently. After exercise stress GW agents produced depression. Importantly, probiotic treatment improved exercise endurance, depression, systemic and neuro-inflammation in GW mice, indicating its potential therapeutic use in GWI. Clara Berdasco and Elena Kozlova are co-first authors. Supported by DoD #W81XWH-19-1-0802 (MCC). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protective effects of an anti-4-HNE monoclonal antibody against liver injury and lethality of endotoxemia in mice

4-hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product that is known to be elevated during oxidative stress. During systemic inflammation and endotoxemia, plasma levels of 4-HNE are elevated in response to lipopolysaccharide (LPS) stimulation. 4-HNE is a highly reactive molecule due to its generation of both Schiff bases and Michael adducts with proteins, which may result in modulation of inflammatory signaling pathways. In this study, we report the production of a 4-HNE adduct-specific monoclonal antibody (mAb) and the effectiveness of the intravenous injection of this mAb (1 mg/kg) in ameliorating LPS (10 mg/kg, i.v.)-induced endotoxemia and liver injury in mice. Endotoxic lethality in control mAb-treated group was suppressed by the administration of anti-4-HNE mAb (75 vs. 27%). After LPS injection, we observed a significant increase in the plasma levels of AST, ALT, IL-6, TNF-α and MCP-1, and elevated expressions of IL-6, IL-10 and TNF-α in the liver. All these elevations were inhibited by anti-4-HNE mAb treatment. As to the underlining mechanism, anti-4-HNE mAb inhibited the elevation of plasma high mobility group box-1 (HMGB1) levels, the translocation and release of HMGB1 in the liver and the formation of 4-HNE adducts themselves, suggesting a functional role of extracellular 4-HNE adducts in hypercytokinemia and liver injury associated with HMGB1 mobilization. In summary, this study reveals a novel therapeutic application of anti-4-HNE mAb for endotoxemia.

Anti-high-mobility group box-1 treatment strategies improve trauma-induced coagulopathy in a mouse model of trauma and shock

BackgroundTrauma-induced coagulopathy is associated with platelet dysfunction and contributes to early mortality after traumatic injury. Plasma concentrations of the damage molecule high-mobility group box-1 (HMGB-1) increase after trauma, which may contribute to platelet dysfunction. We hypothesised that inhibition of HMGB-1 with a monoclonal antibody (mAb) or with recombinant thrombomodulin (rTM) improves trauma-induced coagulopathy in a murine model of trauma and shock. MethodsMale 129S2/SvPasOrlRJ mice were anaesthetised, mechanically ventilated, and randomised into five groups: (i) ventilation control (VENT), (ii) trauma/shock (TS), (iii) TS+anti-HMGB-1 mAb (TS+AB), (iv) TS+rTM (TS+TM), and (v) TS+anti-HMGB-1 mAb+rTM (TS+COMBI). Primary outcome was rotational thromboelastometry EXTEM. Secondary outcomes included tail bleeding time, platelet count, plasma HMGB-1 concentration, and platelet activation. ResultsTrauma and shock resulted in a hypocoagulable thromboelastometry profile, increased plasma HMGB-1, and increased platelet activation markers. TS+AB was associated with improved clot firmness after 5 min compared with TS (34 [33–37] vs 32 [29–34] mm; P=0.043). TS+COMBI was associated with decreased clot formation time (98 [92–125] vs 122 [111–148] s; P=0.018) and increased alpha angle (77 [72–78] vs 69 [64–71] degrees; P=0.003) compared with TS. TS+COMBI also reduced tail bleeding time compared with TS (P=0.007). The TS+TM and TS+COMBI groups had higher platelet counts compared with TS (P=0.044 and P=0.041, respectively). ConclusionsInhibition of HMGB-1 early after trauma in a mouse model improves clot formation and strength, preserves platelet count, and decreases bleeding time.

High mobility group box 1 derived mainly from platelet microparticles exacerbates microvascular obstruction in no reflow

IntroductionNo reflow manifests coronary microvascular injury caused by continuous severe myocardial ischemia and reperfusion. Microvascular obstruction (MVO) has emerged as one fundamental mechanism of no reflow. However, the underlying pathophysiology remains incompletely defined. Herein, we explore the contribution of high mobility group box 1 (HMGB1), derived mainly from platelet microparticles exacerbating MVO in no reflow. Materials and methods44 STEMI patients undergoing successful primary percutaneous coronary intervention (PCI) were included in our study. Plasma HMGB1 levels in both the peripheral artery (PA) and infarct-related coronary artery (IRA) were measured by ELISA. Flow cytometry and confocal microscopy assessed the level of HMGB1+ platelet derived microparticles (PMPs) and platelet activation. Flow cytometry and western blot evaluated the procoagulant activity (PCA) and the release of inflammatory factors of human microvascular endothelial cells (HCEMCs). ResultsHMGB1 levels were significantly higher in the IRA in no-reflow patients. The levels of HMGB1+ PMPs were considerably higher in the IRA of patients with no reflow and were strongly associated with platelet activation. Moreover, our results show that HMGB1 interacts with human microvascular endothelial cells primarily through TLR4, inducing HCMEC proinflammatory, procoagulant phenotype, and monocyte recruitment, accelerating microvascular obstruction and facilitating the development of no reflow. ConclusionOur results illustrate a novel mechanism by which HMGB1, derived mainly from PMPs, plays a crucial role in the pathogenesis of no-reflow, revealing a novel therapeutic target.

Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is the most common acquired autoimmune bleeding disorder. Abnormally increased levels of High Mobility Group Box 1 (HMGB1) protein associate with thrombocytopenia and therapeutic outcome in ITP. Previous studies proposed that a natural inhibitor of HMGB1, 18β-glycyrrhetinic acid (18β-GA), could be used for its anti-inflammatory and immune-modulatory effects, although its ability to correct immune balance in ITP is unclear. In this study, we showed that plasma HMGB1 correlated negatively with platelet counts in ITP patients, and confirmed that 18β-GA stimulated the production of regulatory T cells (Treg), restored the balance of CD4+ T-cell subsets and enhanced the suppressive function of Treg through blocking the effect on HMGB1 in patients with ITP. HMGB1 short hairpin RNA interference masked the effect of 18β-GA in Treg of ITP patients. Furthermore, we found that 18β-GA alleviated thrombocytopenia in mice with ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The proportion of circulating Treg increased significantly, while the level of plasma HMGB1 and serum antiplatelet antibodies decreased significantly in ITP mice along 18β-GA treatment. In addition, 18β-GA reduced phagocytic activity of macrophages towards platelets both in ITP patients and ITP mice. These results indicate that 18β-GA has the potential to restore immune balance in ITP via inhibition of HMGB1 signaling. In short, this study reveals the role of HMGB1 in ITP, which may serve as a potential target for thrombocytopenia therapy.

Glycyrrhizin through liquorice intake modulates ACE2 and HMGB1 levels-A pilot study in healthy individuals with implications for COVID-19 and ARDS.

BackgroundGlycyrrhizin, an active component of liquorice root extract, exhibits antiviral and immunomodulatory properties by direct inhibition of the pro-inflammatory alarmin HMGB1 (High-mobility group box 1).ObjectiveThe aim of this study was to explore the role of liquorice intake on the viral entry receptor ACE2 (angiotensin-converting enzyme 2) and the immunoregulatory HMGB1 in healthy individuals and to explore HMGB1 expression in coronavirus disease 2019 (COVID-19) or non-COVID-19 in ARDS (acute respiratory distress syndrome patients).Material and methodsThis study enrolled 43 individuals, including hospitalised patients with i) acute respiratory distress syndrome (ARDS) due to COVID-19 (n = 7) or other underlying causes (n = 12), ii) mild COVID-19 (n = 4) and iii) healthy volunteers (n = 20). Healthy individuals took 50 g of liquorice (containing 3% liquorice root extract) daily for 7 days, while blood samples were collected at baseline and on day 3 and 7. Changes in ACE2 and HMGB1 levels were determined by Western blot analysis and enzyme-linked immunosorbent assay, respectively. Additionally, HMGB1 levels were measured in hospitalised COVID-19 patients with mild disease or COVID-19 associated acute respiratory distress syndrome (ARDS) and compared with a non-COVID-19-ARDS group.ResultsLiquorice intake significantly reduced after 7 days both cellular membranous ACE2 expression (-51% compared to baseline levels, p = 0.008) and plasma HMGB1 levels (-17% compared to baseline levels, p<0.001) in healthy individuals. Half of the individuals had a reduction in ACE2 levels of at least 30%. HMGB1 levels in patients with mild COVID-19 and ARDS patients with and without COVID-19 were significantly higher compared with those of healthy individuals (+317%, p = 0.002), but they were not different between COVID-19 and non-COVID-19 ARDS.ConclusionsLiquorice intake modulates ACE2 and HMGB1 levels in healthy individuals. HMGB1 is enhanced in mild COVID-19 and in ARDS with and without COVID-19, warranting evaluation of HMGB1 as a potential treatment target and glycyrrhizin, which is an active component of liquorice root extract, as a potential treatment in COVID-19 and non-COVID-19 respiratory disease.

Histamine induced high mobility group box-1 release from vascular endothelial cells through H1 receptor.

BackgroundSystemic allergic reaction is characterized by vasodilation and vascular leakage, which causes a rapid, precipitous and sustained decrease in arterial blood pressure with a concomitant decrease of cardiac output. Histamine is a major mediator released by mast cells in allergic inflammation and response. It causes a cascade of inflammation and strongly increases vascular permeability within minutes through its four G-protein-coupled receptors (GPCRs) on endothelial cells. High mobility group box-1 (HMGB1), a nonhistone chromatin-binding nuclear protein, can be actively secreted into the extracellular space by endothelial cells. HMGB1 has been reported to exert pro-inflammatory effects on endothelial cells and to increase vascular endothelial permeability. However, the relationship between histamine and HMGB1-mediated signaling in vascular endothelial cells and the role of HMGB1 in anaphylactic-induced hypotension have never been studied.Methods and resultsEA.hy 926 cells were treated with different concentrations of histamine for the indicated periods. The results showed that histamine induced HMGB1 translocation and release from the endothelial cells in a concentration- and time-dependent manner. These effects of histamine were concentration-dependently inhibited by d-chlorpheniramine, a specific H1 receptor antagonist, but not by H2 or H3/4 receptor antagonists. Moreover, an H1-specific agonist, 2-pyridylethylamine, mimicked the effects of histamine, whereas an H2-receptor agonist, 4-methylhistamine, did not. Adrenaline and noradrenaline, which are commonly used in the clinical treatment of anaphylactic shock, also inhibited the histamine-induced HMGB1 translocation in endothelial cells. We therefore established a rat model of allergic shock by i.v. injection of compound 48/80, a potent histamine-releasing agent. The plasma HMGB1 levels in compound 48/80-injected rats were higher than those in controls. Moreover, the treatment with anti-HMGB1 antibody successfully facilitated the recovery from compound 48/80-induced hypotension.ConclusionHistamine induces HMGB1 release from vascular endothelial cells solely through H1 receptor stimulation. Anti-HMGB1 therapy may provide a novel treatment for life-threatening systemic anaphylaxis.