High-mobility Group Box-1 Inhibitor Glycyrrhizin Research Articles

Co-treatment with natural HMGB1 inhibitor Glycyrrhizin exerts neuroprotection and reverses Parkinson’s disease like pathology in Zebrafish

Ethnopharmacological relevanceParkinson’s disease (PD) is the second most devastating age-related neurodegenerative diseases after Alzheimer diseases (AD) and is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN) and aggregation of α-synuclein (α-syn). The precise etiology of PD is not yet fully understood and lacks the disease-modifying therapeutic strategies that could reverse the ongoing neurodegeneration. In the quest of exploring novel disease modifying therapeutic strategies, natural compounds from plant sources have gained much attention in recent days. Glycyrrhizin (GL) is the main active ingredient of the roots and rhizomes of licorice (Glycyrrhiza glabra L), which are generally used in the treatment of inflammatory diseases or as a tonifying herbal medicine. In Persia, GL is a conventional neuroprotective agent that are used to treat neurological disorders. The traditional use of GL in Japan is to treat chronic hepatitis B. In addition, GL is a natural inhibitor of high mobility group box 1 (HMGB1) which has exerted neuroprotective effect against several HMGB1 mediated pathological conditions. Aim of the studyThe study is aimed to evaluate therapeutic effect of GL against PD in zebrafish. Material and methodsPD in zebrafish larvae is induced by administration of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Apoptosis was assessed with TUNEL assay. Gene expression was performed to assess the modulation in genes related to neuroinflammatory and autophagy. ResultsWe observed that GL co-treatment increased the length of DA neurons, decreased the number of apoptotic cells in zebrafish brain, and inhibited the loss of vasculature and disorganized vasculature induced by MPTP. GL co-treatment relieved the MPTP-induced locomotor impairment in zebrafish. GL co-treatment suppressed MPTP-induced upregulated mRNA expression of inflammatory markers such as hmgb1a, tlr4b, nfκb, il1β, and il6. GL co-treatment suppressed the autophagy related genes α-syn and atg5 whereas increased the mRNA expression level of parkin and pink1. In addition, molecular docking study reveals that GL has binding interaction with HMGB1, TLR4, and RAGE. ConclusionHence, the effect of GL co-treatment on MPTP-induced PD-like condition in zebrafish is to alleviate apoptosis and autophagy, as well as suppress inflammatory responses.

Autophagic secretion of HMGB1 from cancer-associated fibroblasts promotes metastatic potential of non-small cell lung cancer cells via NF\u03baB signaling

Tumor progression requires the communication between tumor cells and tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major components of stromal cells. CAFs contribute to metastasis process through direct or indirect interaction with tumor cells; however, the underlying mechanism is largely unknown. Here, we reported that autophagy was upregulated in lung cancer-associated CAFs compared to normal fibroblasts (NFs), and autophagy was responsible for the promoting effect of CAFs on non-small cell lung cancer (NSCLC) cell migration and invasion. Inhibition of CAFs autophagy attenuated their regulation on epithelial–mesenchymal transition (EMT) and metastasis-related genes of NSCLC cells. High mobility group box 1 (HMGB1) secreted by CAFs mediated CAFs’ effect on lung cancer cell invasion, demonstrated by using recombinant HMGB1, HMGB1 neutralizing antibody, and HMGB1 inhibitor glycyrrhizin (GA). Importantly, the autophagy blockade of CAFs revealed that HMGB1 release was dependent on autophagy. We also found HMGB1 was responsible, at least in part, for autophagy activation of CAFs, suggesting CAFs remain active through an autocrine HMGB1 loop. Further study demonstrated that HMGB1 facilitated lung cancer cell invasion by activating the NFκB pathway. In a mouse xenograft model, the autophagy specific inhibitor chloroquine abolished the stimulating effect of CAFs on tumor growth. These results elucidated an oncogenic function for secretory autophagy in lung cancer-associated CAFs that promotes metastasis potential, and suggested HMGB1 as a novel therapeutic target.

Argon Attenuates Multiorgan Failure in Relation with HMGB1 Inhibition.

Argon inhalation attenuates multiorgan failure (MOF) after experimental ischemic injury. We hypothesized that this protection could involve decreased High Mobility Group Box 1 (HMGB1) systemic release. We investigated this issue in an animal model of MOF induced by aortic cross-clamping. Anesthetized rabbits were submitted to supra-coeliac aortic cross-clamping for 30 min, followed by 300 min of reperfusion. They were randomly divided into three groups (n = 7/group). The Control group inhaled nitrogen (70%) and oxygen (30%). The Argon group was exposed to a mixture of argon (70%) and oxygen (30%). The last group inhaled nitrogen/oxygen (70/30%) with an administration of the HMGB1 inhibitor glycyrrhizin (4 mg/kg i.v.) 5 min before aortic unclamping. At the end of follow-up, cardiac output was significantly higher in Argon and Glycyrrhizin vs. Control (60 ± 4 and 49 ± 4 vs. 33 ± 8 mL/kg/min, respectively). Metabolic acidosis was attenuated in Argon and Glycyrrhizin vs. Control, along with reduced amount of norepinephrine to reverse arterial hypotension. This was associated with reduced interleukin-6 and HMGB1 plasma concentration in Argon and Glycyrrhizin vs. Control. End-organ damages were also attenuated in the liver and kidney in Argon and Glycyrrhizin vs. Control, respectively. Argon inhalation reduced HMGB1 blood level after experimental aortic cross-clamping and provided similar benefits to direct HMGB1 inhibition.

Glycyrrhizin prevents SARS-CoV-2 S1 and Orf3a induced high mobility group box 1 (HMGB1) release and inhibits viral replication

Efforts to understand host factors critical for COVID-19 pathogenesis have identified high mobility group box 1 (HMGB1) to be crucial for regulating susceptibility to SARS-CoV-2. COVID-19 disease severity is correlated with heightened inflammatory responses, and HMGB1 is an important extracellular mediator in inflammation processes.In this study, we evaluated the effect of HMGB1 inhibitor Glycyrrhizin on the cellular perturbations in lung cells expressing SARS-CoV-2 viral proteins. Pyroptosis in lung cells transfected with SARS-CoV-2 S-RBD and Orf3a, was accompanied by elevation of IL-1β and extracellular HMGB1 levels. Glycyrrhizin mitigated viral proteins-induced lung cell pyroptosis and activation of macrophages. Heightened release of proinflammatory cytokines IL-1β, IL-6 and IL-8, as well as ferritin from macrophages cultured in conditioned media from lung cells expressing SARS-CoV-2 S-RBD and Orf3a was attenuated by glycyrrhizin. Importantly, Glycyrrhizin inhibited SARS-CoV-2 replication in Vero E6 cells without exhibiting cytotoxicity at high doses. The dual ability of Glycyrrhizin to concomitantly halt virus replication and dampen proinflammatory mediators might constitute a viable therapeutic option in patients with SARS-CoV-2 infection.

Cross-Talk between Sirtuin 1 and the Proinflammatory Mediator High-Mobility Group Box-1 in the Regulation of Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

ABSTRACTPurpose: High-mobility group box-1 (HMGB1) mediates inflammation and breakdown of blood-retinal barrier (BRB) in diabetic retina. Sirtuin-1 (SIRT1) has protective effects against inflammation and oxidative stress. The aim of this study was to investigate the interaction between HMGB1 and SIRT1 in regulating BRB breakdown in diabetic retina.Methods: BRB breakdown was assessed in vivo with fluorescein isothiocyanate-conjugated dextran. Vitreous samples from 47 proliferative diabetic retinopathy (PDR) and 19 nondiabetic patients, and epiretinal membranes from 13 patients with PDR were studied by enzyme-linked immunosorbent assay and immunohistochemistry. Retinas from 4-week diabetic rats and from normal rats intravitreally injected with HMGB1 were studied by spectrophotometric assay, Western blot analysis, and RT-PCR. We also studied the effect of the HMGB1 inhibitor glycyrrhizin and the SIRT1 activator resveratrol on diabetes-induced biochemical changes in the retina.Results: HMGB1 levels in vitreous samples from PDR patients were significantly higher than in nondiabetic controls, whereas SIRT1 levels were significantly lower in vitreous samples from patients with inactive PDR than those in patients with active PDR and nondiabetic controls. In epiretinal membranes, SIRT1 was expressed in vascular endothelial cells and stromal cells. Diabetes and intravitreal injection of HMGB1 in normal rats downregulated SIRT1expression, whereas glycyrrhizin and resveratrol normalized diabetes-induced downregulation of SIRT1. Resveratrol significantly attenuated diabetes-induced downregulation of occludin and upregulation of HMGB1 and receptor for advanced glycation end products in the retina and breakdown of BRB.Conclusions: Our findings suggest that a functional link between SIRT1 and HMGB1 is involved in regulating of BRB breakdown in diabetic retina.

Spinal macrophage migration inhibitory factor and high mobility group box 1 mediate persistent bladder pain

Repeated intravesical PAR4 (protease activated receptor 4) activation elicits persistent bladder pain lasting 5 days after the last treatment. Persistent bladder pain was fully reversed by a systemic HMGB1 (high mobility group box 1) inhibitor while a MIF (macrophage migration inhibitory factor) antagonist partly reversed it. Since there is growing evidence that spinal MIF and HMGB1 mediate inflammatory and neuropathic pain we examined whether there were spinal changes occurring during persistent bladder pain that may be responsible for maintaining bladder pain. In addition, we tested whether we could modulate persistent bladder pain with spinal MIF or HMGB1 antagonists. Persistent bladder pain was elicited in female C57 mice by repeated (3x) intravesical instillation of PAR4-activating peptide while control animals received scramble peptide treatment. On day 4, spinal cord (L6-S1) changes in c-fos (non-specific marker of spinal activation) was assessed with immunofluorescence while MIF and HMGB1 were assessed with immunofluorescence, western blotting and real-time PCR. On day 7, mice received an intrathecal injection of a neutralizing MIF monoclonal antibody (15 μg in 5 μl PBS) or a HMGB1 inhibitor glycyrrhizin (25 μg in 5 μl of 5% alcohol in PBS) and abdominal mechanical threshold was tested. On day 9, mice were treated with vehicle or control and abdominal mechanical threshold was tested. Immunofluorescence showed that c-fos and MIF in the dorsal horn, dorsal grey commissure and intermediolateral areas significantly increased in PAR4-treated mice while HMGB1 was decreased. In addition, intrathecal treatment with MIF neutralizing mAb or glycyrrhizin significantly alleviated abdominal mechanical hypersensitivity at 1 and 2 h and the analgesic effect diminished at 6 h. Vehicle or control treatment had no effect. Persistent bladder pain is associated with spinal changes in MIF and HMGB1 levels. Furthermore, spinal treatment with MIF monoclonal antibody and HMGB1 inhibitor temporarily reversed bladder pain. Our findings suggest that spinal MIF and HMGB1 participate in persistent bladder pain induced by repeated intravesical PAR4 and may be potential therapeutic targets in chronic bladder pain conditions.

Inhibition of HMGB1 improves necrotizing enterocolitis by inhibiting NLRP3 via TLR4 and NF-κB signaling pathways.

To explore the relationship between high-mobility group box 1 (HMGB1) and NLR pyrin domain containing 3 (NLRP3) in the development of necrotizing enterocolitis (NEC). NEC rat models were constructed and treated with HMGB1 inhibitor glycyrrhizin (GL) with different concentration. An inflammatory condition of intestinal tissue in newborn NEC rats was observed by hematoxylin and eosin staining. The messenger RNA (mRNA) and protein expression of HMGB1, NLRP3, toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and caspase 1 were determined by real-time polymerase chain reaction and western blot analysis, respectively. The content of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay. Human intestinal epithelial cell lines were induced to NEC by lipopolysaccharides (LPSs). LPS-induced cells were transfected with small interfering RNA-HMGB1 and NLRP3 plasmid vector. The mRNA and protein expression of HMGB1, NLRP3, TLR4, NF-κB, caspase 1, IL-1β, and TNF-α were determined by real-time PCR and western blot analysis, respectively. The mRNA and protein expression of HMGB1 and NLRP3 in the NEC group was significantly higher than the control group. Inhibition of HMGB1 expression improved intestinal inflammation in newborn NEC rats. The expression of HMGB1, NLRP3, TLR4, NF-κB, and caspase 1 was upregulated in NEC and was weakened after treating with GL. LPS induction to intestinal epithelial cells markedly increased the expression of HMGB1, NLRP3, TLR4, NF-κB, caspase 1, IL-1β, and TNF-α. The knockdown of HMGB1 abolished the increase of expression, whereas further transfection with NLRP3 plasmid vector recovered the increase. HMGB1 and NLRP3 were all upregulated in the development of NEC. Inhibition on HMGB1 could improve the intestinal inflammation in NEC by inhibiting NLRP3 via TLR4 and NF-κB signaling pathways.

Alterations in Histone Acetylation in Diabetic Painful Neuropathy

Peripheral sensory neuropathy is one of the most common complications of diabetes. Accumulating evidence suggests that chronic low-grade inflammation is involved in the pathogenesis of the disease. We hypothesize that hyperglycemia causes changes in histone acetylation and release of inflammatory mediators in the peripheral nervous system of diabetic animals with painful neuropathy; therefore blocking this increase will prevent or delay the development of neuropathy. High mobility group box 1 (HMGB1), a nuclear protein released by injured and severely stressed cells, promotes cytokine release via histone acetylation and its interaction with the Toll-like receptor (TLR). In this study we investigated the changes in inflammatory mediators and histone modifications in the dorsal root ganglia (DRG) and spinal cord dorsal horn neurons as well as compared the changes in behavior with treatment. Type 2 diabetic (T2D) animals with pain were treated with HMGB1 inhibitor Glycyrrhizin (GLC) for 2 days a week for 3 weeks at 50 mg/kg I.P. injection 6 weeks after diabetes. T2D animals demonstrated significant changes in thermal hyperalgesia manifested by a decrease in withdrawal latency to heat, mechanical hyperalgesia as measured by the Randall Sellito method of paw pressure at 6 weeks after diabetes and also exhibited marked increases in HMGB1, IL1β, TLR4 and H3K9 acetylation as determined by the Western blot analysis and immunohistochemistry. We analyzed the T2D animals with treatment or without treatment at 3 weeks after treatment. Our results show that animals treated with GLC had significant decrease in thermal hyperalgesia along with changes in histone acetylation and expression of inflammatory mediators. This preliminary study suggests that HMGB1 and histone acetylation play an important role in the inflammatory aspect of the painful neuropathy in T1D animals and may provide a novel treatment approach for this difficult-to-treat complication of diabetes. Disclosure M. Chattopadhyay: None. V. Thakur: None.

High-Mobility Group Box-1 Protein Mediates the Regulation of Signal Transducer and Activator of Transcription-3 in the Diabetic Retina and in Human Retinal Müller Cells

Purpose: The expression of high-mobility group box-1 (HMGB1) and signal transducer and activator of transcription-3 (STAT-3) is upregulated in the diabetic retina. We hypothesized that the activation of STAT-3 is under the control of HMGB1. Methods: Retinas from 1-month-old diabetic rats and from normal rats intravitreally injected with HMGB1 and human retinal Müller glial cells (MIO-M1) stimulated with HMGB1 or high glucose were studied by Western blot analysis and immunofluorescence. We also studied the effect of the HMGB1 inhibitor glycyrrhizin (GA) on high-glucose-induced pSTAT-3 nuclear translocation and upregulation in Müller cells and on pSTAT-3 expression in the retinas of diabetic rats (n = 7-10 in each group). In addition, we studied the effect of STAT-3 inhibitor on the HMGB1-induced induction of vascular endothelial growth factor (VEGF) by Müller cells and human retinal microvascular endothelial cell (HRMEC) migration. Results: Treatment of retinal Müller cells with recombinant HMGB1 induced nuclear translocation of pSTAT-3 but did not alter pSTAT-3 expression. High glucose induced a significant upregulation of HMGB1 and pSTAT-3 upregulation and nuclear translocation in retinal Müller cells. GA co-treatment normalized the high-glucose-induced upregulation of HMGB1 and pSTAT-3 upregulation and nuclear translocation in Müller cells. Intravitreal administration of HMGB1 in normal and diabetic rats upregulated pSTAT-3 expression in the retina. GA attenuated the diabetes-induced upregulation of pSTAT-3 in the retina. The STAT-3 inhibitor attenuated HMGB1-induced VEGF upregulation by Müller cells and HRMEC migration. Conclusions: The results suggest a role for HMGB1 in the modulation of STAT-3 expression in the diabetic retina.

High-mobility group box 1 is an important mediator of microglial activation induced by cortical spreading depression

Single episodes of cortical spreading depression (CSD) are believed to cause typical migraine aura, whereas clusters of spreading depolarizations have been observed in cerebral ischemia and subarachnoid hemorrhage. We recently demonstrated that the release of high-mobility group box 1 (HMGB1) from cortical neurons after CSD in a rodent model is dependent on the number of CSD episodes, such that only multiple CSD episodes can induce significant HMGB1 release. Here, we report that only multiple CSD inductions caused microglial hypertrophy (activation) accompanied by a greater impact on the transcription activity of the HMGB1 receptor genes, TLR2 and TLR4, while the total number of cortical microglia was not affected. Both an HMGB1-neurtalizing antibody and the HMGB1 inhibitor glycyrrhizin abrogated multiple CSD-induced microglial hypertrophy. Moreover, multiple CSD inductions failed to induce microglial hypertrophy in TLR2/4 double knockout mice. These results strongly implicate the HMGB1–TLR2/4 axis in the activation of microglia following multiple CSD inductions. Increased expression of the lysosomal acid hydrolase cathepsin D was detected in activated microglia by immunostaining, suggesting that lysosomal phagocytic activity may be enhanced in multiple CSD-activated microglia.

MP28-14 PROTEASE-ACTIVATED RECEPTOR 4 INDUCES BLADDER PAIN THROUGH DISULFIDE HIGH MOBILITY GROUP BOX-1 ACTING ON RECEPTORS FOR ADVANCED GLYCATION ENDPRODUCTS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2016MP28-14 PROTEASE-ACTIVATED RECEPTOR 4 INDUCES BLADDER PAIN THROUGH DISULFIDE HIGH MOBILITY GROUP BOX-1 ACTING ON RECEPTORS FOR ADVANCED GLYCATION ENDPRODUCTS Dimitrios Kouzoukas, Fei Ma, Katherine Meyer-Siegler, Karin Westlund, David Hunt, and Pedro Vera Dimitrios KouzoukasDimitrios Kouzoukas More articles by this author , Fei MaFei Ma More articles by this author , Katherine Meyer-SieglerKatherine Meyer-Siegler More articles by this author , Karin WestlundKarin Westlund More articles by this author , David HuntDavid Hunt More articles by this author , and Pedro VeraPedro Vera More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1064AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Pain without obvious bladder pathology is a hallmark of Interstitial Cystitis / Painful Bladder Syndrome (IC/PBS). In a mouse model of bladder pain, urothelial protease activated receptor 4 (PAR4) stimulation causes pain without inflammation through macrophage migration inhibitory factor (MIF) release (Kouzoukas et al 2014). High mobility group box-1 (HMGB1), a ubiquitous nuclear non-histone DNA-binding protein, also mediates bladder pain, but not inflammation (Tanaka et al, 2014). Two redox states of all-thiol and disulfide HGMB1 respectively act through receptors for advanced glycation endproducts (RAGE) and toll-like receptor 4 (TLR4). Which HMGB1 form(s) contribute to bladder pain is unknown. We investigated whether: 1) bladder PAR4 stimulation causes urothelial HMGB1 release; 2) blocking MIF prevents urothelial HMGB1 release; 3) blocking HMGB1 prevents PAR4-induced hypersensitivity; 4) specific HMGB1 redox form causes pain, changes in micturition parameters and/or bladder inflammation; and 5) antagonizing specific HMGB1 receptors affect HMGB1-induced hypersensitivity. METHODS Female C57BL/6 mice were given intravesical PAR4 peptides (100 μM; 1 hr). Intraluminal fluid was collected and assayed for HMGB1, fluid parameters, abdominal hypersensitivity and histology were determined 24 hrs after pretreatment with HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; i.p.), MIF blocker (ISO-1: 20 mg/kg; i.p.), specific HMGB1 redox forms (all-thiol or disulfide; 1, 2, 5, 10, 20, μg/150 μl; 1 hr), RAGE antagonist (FPS-ZM1; 10 mg/kg, i.p.), TLR4 antagonist (TAK-242; 3 mg/kg, i.p.), or vehicle. RESULTS PAR4-AP triggered urothelial HMGB1 release and Intravesical PAR4 induced abdominal hypersensitivity was prevented by blocking HMGB1. MIF inhibition blocked PAR4-mediated urothelial HMGB1 release. Intravesical disulfide HMGB1 (≥ 10 μg) instillation induced abdominal hypersensitivity, decreased micturition volume and increased voiding frequency. Thiol HMGB1 was ineffective. Systemic FPS-ZM1 fully and TAK-242 partially prevented disulfide HMGB1 (10 μg)-induced hypersensitivity. All doses of HMGB1 produced minimal to mild bladder edema and reactive changes. CONCLUSIONS Bladder PAR4 receptor stimulation induced urothelial HMGB1 release, mediated by MIF. Abdominal hypersensitivity, mediated by disulfide HGMB1 (not all-thiol), acted mainly through RAGE receptors. Urothelial MIF and HGMB1 receptors represent novel therapeutic targets for bladder pain conditions. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e376 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Dimitrios Kouzoukas More articles by this author Fei Ma More articles by this author Katherine Meyer-Siegler More articles by this author Karin Westlund More articles by this author David Hunt More articles by this author Pedro Vera More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1.

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.

HMGB1 Promotes Mitochondrial Dysfunction-Triggered Striatal Neurodegeneration via Autophagy and Apoptosis Activation.

Impairments in mitochondrial energy metabolism are thought to be involved in many neurodegenerative diseases. The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces striatal pathology mimicking neurodegeneration in vivo. Previous studies showed that 3-NP also triggered autophagy activation and apoptosis. In this study, we focused on the high-mobility group box 1 (HMGB1) protein, which is important in oxidative stress signaling as well as in autophagy and apoptosis, to explore whether the mechanisms of autophagy and apoptosis in neurodegenerative diseases are associated with metabolic impairment. To elucidate the role of HMGB1 in striatal degeneration, we investigated the impact of HMGB1 on autophagy activation and cell death induced by 3-NP. We intoxicated rat striata with 3-NP by stereotaxic injection and analyzed changes in expression HMGB1, proapoptotic proteins caspase-3 and phospho-c-Jun amino-terminal kinases (p-JNK). 3-NP–induced elevations in p-JNK, cleaved caspase-3, and autophagic marker LC3-II as well as reduction in SQSTM1 (p62), were significantly reduced by the HMGB1 inhibitor glycyrrhizin. Glycyrrhizin also significantly inhibited 3-NP–induced striatal damage. Neuronal death was replicated by exposing primary striatal neurons in culture to 3-NP. It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1. These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.

Mutual enhancement between high-mobility group box-1 and NADPH oxidase-derived reactive oxygen species mediates diabetes-induced upregulation of retinal apoptotic markers.

The expression of the proinflammatory cytokine high-mobility group box-1 (HMGB1) is upregulated in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and in the diabetic retina. We hypothesized that a novel mechanism exists where HMGB1 and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are mutually enhanced in the diabetic retina, which may be a novel mechanism for promoting upregulation of retinal apoptotic markers induced by diabetes. Vitreous samples from 48 PDR and 34 nondiabetic patients, retinas from 1-month diabetic rats and from normal rats intravitreally injected with HMGB1 and human retinal microvascular endothelial cells (HRMEC) stimulated with HMGB1 were studied by enzyme-linked immunosorbent and spectrophotometric assays, Western blot analysis, RT-PCR, and immunofluorescence. We also studied the effect of the HMGB1 inhibitor glycyrrhizin and apocynin on diabetes-induced biochemical changes in the retinas of rats (n = 5-7 in each groups). HMGB1 and the oxidative stress marker protein carbonyl content levels in the vitreous fluid from PDR patients were significantly higher than in controls (p = 0.021; p = 0.005, respectively). There was a significant positive correlation between vitreous fluid levels of HMGB1 and the levels of protein carbonyl content (r = 0.62, p = 0.001). HMGB1 enhanced interleukin-1β, ROS, Nox2, poly (ADP-ribose) polymerase (PARP)-1, and cleaved caspase-3 production by HRMEC. Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of ROS, Nox2, PARP-1, and cleaved caspase-3 in the retina. Constant glycyrrhizin and apocynin intake from onset of diabetes did not affect the metabolic status of the diabetic rats, but restored these increased mediators to control values. The results of this study suggest that there is a mutual enhancement between HMGB1 and Nox-derived ROS in the diabetic retina, which may promote diabetes-induced upregulation of retinal apoptotic markers.

Glutamate/NMDA excitotoxicity and HMGB1/TLR4 neuroimmune toxicity converge as components of neurodegeneration

Neurodegeneration in brain is linked to both excitotoxicity and neuroimmune gene induction,although the mechanisms are poorly understood. High-mobility group box 1 (HMGB1) is a cytokine like molecule released in brain by glutamate that has been found to enhance neuronal excitability through Toll-like receptor 4 (TLR4). To explore the role of HMGB1 in glutamate/NMDA excitotoxicity or neuroimmune-induced neurodegeneration we used an <em>ex vivo</em> model of organotypic hippocampal-entorhinal cortex (HEC) slice culture. Concentration response and time course studies find release of HMGB1 precedes neuronal death induced by glutamate,NMDA,TNFα and LPS. Blockade of glutamate receptors with antagonist MK-801 prevents glutamate/NMDA stimulation release of HMGB1 and neuronal death as well as blocking neuroimmune (LPS and TNFα) induced neuronal death. Similarly,HMGB1 neutralizing antibodies or inhibitor glycyrrhizin block glutamate/NMDA as well as neuroimmune (LPS and TNFα) induced neuronal death. Further,delayed neuronal cell death mediated by LPS and TNFα was rescued by NR2B inhibitor ifenprodil. Together,these findings suggest HMGB1 contributes a critical element of both glutamate/NMDA as well as neuroimmune induced neurodegeneration indicating HMGB1 may be a novel target crossing multiple neurodegeneration pathologies.

Toll-Like Receptor 4/Spleen Tyrosine Kinase Complex in High Glucose Signal Transduction of Proximal Tubular Epithelial Cells

Background/Aims: High glucose activates spleen tyrosine kinase (Syk) in human proximal tubular epithelial cells (HK-2 cells), which leads to NF-κB activation and transforming growth factor-ß1 (TGF-ß1) production. We explored the signal transduction pathway from high glucose to Syk activation. Methods: The pathway was evaluated by siRNA transfection, immunoprecipitation and Western blot. Results: High glucose stimulated Syk activation within 10 min. Depletion of toll-like receptor 4 (TLR4) attenuated high glucose-induced Syk activation, NF-κB p65 nuclear translocation, and TGF-ß1 production. In addition, TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody, and depletion of myeloid differentiation factor 88 (MyD88) all attenuated high glucose-induced Syk activation. As an evidence of TLR4 activation, interleukin-1 receptor-associated kinase 1 was recruited to MyD88 and TLR4 upon exposure to high glucose. Syk was co-immunoprecipitated with TLR4, and Syk bound to TLR4 was activated by high glucose. High-mobility group box-1 (HMGB-1), an endogenous activator of TLR4, rapidly increased in TLR4 immunoprecipitates upon high glucose stimulation, and this association was reduced by N-acetylcysteine, an antioxidant. An HMGB-1 inhibitor glycyrrhizin suppressed high glucose-induced Syk activation. Conclusion: Syk is constitutively associated with TLR4. High glucose induces an immediate, reactive oxygen species-dependent, extracellular release of HMGB-1 which binds to TLR4 and activates it, leading to Syk activation.

High-Mobility Group Box-1 Modulates the Expression of Inflammatory and Angiogenic Signaling Pathways in Diabetic Retina

Purpose: The expression of high-mobility group box-1 (HMGB1) is upregulated in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy and in the diabetic retina. HMGB1 mediates inflammation, breakdown of the blood–retinal barrier and apoptosis in the diabetic retina. Here, we investigated inflammatory and angiogenic signaling pathways activated by HMGB1 in diabetic retina.Methods: Human retinal microvascular endothelial cells (HRMEC) and retinas from 1-month diabetic rats and normal rats intravitreally injected with HMGB1 were studied using RT-PCR, Western blot analysis and co-immunoprecipitation. We also studied the effect of the HMGB1 inhibitor glycyrrhizin on diabetes-induced biochemical changes in the retina.Results: Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of the mRNA levels of the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) receptor CXCR4 and protein levels of hypoxia-inducible factor-1α, early growth response-1, tyrosine kinase 2 and the CXCL12/CXCR4 chemokine axis. Constant glycyrrhizin intake from onset of diabetes did not affect the metabolic status of the diabetic rats, but it restored these increased mediators to control values. Stimulation of HRMEC with HMGB1 and intraviteral injection of HMGB1 significantly increased the expression of vascular endothelial growth factor (VEGF) and VEGF receptor-2. Co-immunoprecipitation studies showed that diabetes increased the interaction between CXCL12 and CXCR4 and between HMGB1 and receptor for advanced glycation end products (RAGE), but not between HMGB1 and the CXCL12/CXCR4 chemokine axis.Conclusions: Our findings suggest that HMGB1 activates inflammatory and angiogenic signaling pathways in diabetic retina mediated by RAGE.

The proinflammatory cytokine high‐mobility group box‐1 mediates retinal neuropathy induced by diabetes

AbstractPurpose To test the hypothesis that increased expression of proinflammatory cytokine high‐mobility group box‐1 (HMGB1) in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy and in retinas of diabetic rats plays a pathogenetic role in mediating diabetes‐induced retinal neuropathy.Methods Retinas of 1‐month diabetic rats and HMGB1 intravitreally injected normal rats were studied using Western blot analysis, RT‐PCR and glutamate assay. In addition, we studied the effect of the HMGB1 inhibitor glycyrrhizin on diabetes‐induced biochemical changes in the retina.Results Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of HMGB1 protein and mRNA, activated extracellular signal‐regulated kinase 1 and 2 (ERK1/2) cleaved caspase‐3 and glutamate and significant downregulation of synaptophysin, tyrosine hydroxylase, glutamine synthetase and glyoxalase 1. Constant glycyrrhizin intake from the onset of diabetes did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes‐induced upregulation of HMGB1 protein and mRNA, activated ERK1/2 , cleaved caspase‐3 and glutamate. In the glycyrrhizin‐fed diabetic rats, the decrease in synaptophysin, tyrosine hydoxylase and glyoxalase 1 caused by diabetes was significantly attenuated.Conclusion These findings suggest that early retinal neuropathy of diabetes involves upregulated expression of HMGB1 and can be ameliorated by inhibition of HMGB1.

Release of Neuronal HMGB1 by Ethanol through Decreased HDAC Activity Activates Brain Neuroimmune Signaling

Neuroimmune gene induction is involved in many brain pathologies including addiction. Although increased expression of proinflammatory cytokines has been found in ethanol-treated mouse brain and rat brain slice cultures as well as in post-mortem human alcoholic brain, the mechanisms remain elusive. High-mobility group box 1 (HMGB1) protein is a nuclear protein that has endogenous cytokine-like activity. We previously found increased HMGB1 in post-mortem alcoholic human brain as well as in ethanol treated mice and rat brain slice cultures. The present study investigated the mechanisms for ethanol-induced release of HMGB1 and neuroimmune activation in a model of rat hippocampal-entorhinal cortex (HEC) brain slice cultures. Ethanol exposure triggered dose-dependent HMGB1 release, predominantly from neuronal cells. Inhibitors of histone deacetylases (HDACs) promoted nucleocytoplasmic mobilization of HDAC1/4 and HMGB1 resulting in increased total HMGB1 and acetylated HMGB1 release. Similarly, ethanol treatment was found to induce the translocation of HDAC1/4 and HMGB1 proteins from nuclear to cytosolic fractions. Furthermore, ethanol treatment reduced HDAC1/4 mRNA and increased acetylated HMGB1 release into the media. These results suggest decreased HDAC activity may be critical in regulating acetylated HMGB1 release from neurons in response to ethanol. Ethanol and HMGB1 treatment increased mRNA expression of proinflammatory cytokines TNFα and IL-1β as well as toll-like receptor 4 (TLR4). Targeting HMGB1 or microglial TLR4 by using siRNAs to HMGB1 and TLR4, HMGB1 neutralizing antibody, HMGB1 inhibitor glycyrrhizin and TLR4 antagonist as well as inhibitor of microglial activation all blocked ethanol-induced expression of proinflammatory cytokines TNFα and IL-1β. These results support the hypothesis that ethanol alters HDACs that regulate HMGB1 release and that danger signal HMGB1 as endogenous ligand for TLR4 mediates ethanol-induced brain neuroimmune signaling through activation of microglial TLR4. These findings provide new therapeutic targets for brain neuroimmune activation and alcoholism.

The proinflammatory cytokine high-mobility group box-1 mediates retinal neuropathy induced by diabetes.

To test the hypothesis that increased expression of proinflammatory cytokine high-mobility group box-1 (HMGB1) in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy and in retinas of diabetic rats plays a pathogenetic role in mediating diabetes-induced retinal neuropathy. Retinas of 1-month diabetic rats and HMGB1 intravitreally injected normal rats were studied using Western blot analysis, RT-PCR and glutamate assay. In addition, we studied the effect of the HMGB1 inhibitor glycyrrhizin on diabetes-induced biochemical changes in the retina. Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of HMGB1 protein and mRNA, activated extracellular signal-regulated kinase 1 and 2 (ERK1/2), cleaved caspase-3 and glutamate; and significant downregulation of synaptophysin, tyrosine hydroxylase, glutamine synthetase, and glyoxalase 1. Constant glycyrrhizin intake from the onset of diabetes did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of HMGB1 protein and mRNA, activated ERK1/2, cleaved caspase-3, and glutamate. In the glycyrrhizin-fed diabetic rats, the decrease in synaptophysin, tyrosine hydroxylase, and glyoxalase 1 caused by diabetes was significantly attenuated. These findings suggest that early retinal neuropathy of diabetes involves upregulated expression of HMGB1 and can be ameliorated by inhibition of HMGB1.