Intranasal Delivery of RGD-Containing Osteopontin Heptamer Peptide Confers Neuroprotection in the Ischemic Brain and Augments Microglia M2 Polarization.

Osteopontin (OPN), a phosphorylated glycoprotein, is induced in response to tissue damage and inflammation in various organs, including the brain. In our previous studies, we reported the robust neuroprotective effects of the icosamer OPN peptide OPNpt20, containing arginine-glycine-aspartic acid (RGD) and serine-leucine-alanine-tyrosine (SLAY) motifs, in an animal model of transient focal ischemia and demonstrated that its anti-inflammatory, pro-angiogenic, and phagocytosis inducing functions are responsible for the neuroprotective effects. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motifs (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S), and their neuroprotective efficacy was examined in a rat middle cerebral artery occlusion (MCAO) model. Intranasal administration of all four peptides significantly reduced infarct volume; OPNpt7R (VPNGRGD), the 7-amino-acid peptide containing an RGD motif, was determined to be the most potent, with efficacy comparable to that of OPNpt20. Additionally, sensory-motor functional deficits of OPNpt7R-administered MCAO animals were significantly improved, as indicated by the modified neurological severity scores and rotarod test. Notably, the expression of M1 markers was suppressed, whereas that of M2 markers (Arginase 1, CD206, and VEGF) was significantly enhanced in OPNpt7R-treated primary microglia cultures. Inflammation resolution by OPNpt7R was further confirmed in MCAO animals, in which upregulation of anti-inflammatory cytokines (Arg1, IL-10, IL-4, and CD36) and enhanced efferocytosis were detected. Moreover, studies using three mutant peptides (OPNpt7R-RAA or OPNpt7R-RAD, where RGD was replaced with RAA or RAD, respectively, and OPNpt7R-sc containing scrambled sequences) revealed that the RGD motif plays a vital role in conferring neuroprotection. In conclusion, the RGD-containing OPN heptamer OPNpt7R exhibits neuroprotective effects in the post-ischemic brain by suppressing M1 markers and augmenting M2 polarization of microglia and the RGD motif plays a critical role in these activities.

Osteopontin heptamer peptide containing the RGD motif enhances the phagocytic function of microglia.

Osteopontin (OPN) is a phosphorylated glycoprotein expressed in various tissues, including brain, and mediates a wide range of cellular activities. In our previous studies, we reported recombinant OPN and RGD and SLAY-containing OPN-peptide icosamer (OPNpt20) exhibited robust neuroprotective activities in an animal model of transient focal ischemia, and attributed these effects to the anti-inflammatory, pro-angiogenic, and phagocytic functions of OPNpt20. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motif (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S) and their cell motility and migration inducing activities were examined in BV2 cells (a microglia cell line). All four peptides significantly enhanced BV2 cell motility and migration, but OPNpt7R, an RGD-containing 7-amino-acid OPN peptide (VPNGRGD), was found to be most potent and its potency was comparable to OPNpt20. Phagocytic activity and F-actin polymerization were also significantly enhanced in OPNpt7R-treated BV2 cells. Importantly, studies using two mutant peptides (OPNpt7R-RAA and OPNpt7R-RAD, wherein RGD in OPNpt7R was replaced with RAA or RAD, respectively) revealed that all these effects of OPNpt7R, motility, migration, F-actin polymerization, and phagocytosis induction, were RGD-dependent. Furthermore, the Erk, Fak, and Akt signaling pathways appeared to be involved in the induction of phagocytic activity by OPNpt7R. Co-treating cells with OPNpt7R and D98059 or wortmannin (pharmacological inhibitors of Erk and Akt, respectively) significantly suppressed OPNpt7R-mediated phagocytosis induction. These results indicate the RGD-containing OPN heptamer OPNpt7R triggers microglial motility, migration, and phagocytic activity and that the RGD motif plays a critical role in these activities.

Adenosine Triphosphate Accumulated Following Cerebral Ischemia Induces Neutrophil Extracellular Trap Formation.

In ischemic stroke, neutrophils infiltrate damaged brain tissue immediately following the ischemic insult and aggravate inflammation via various mechanisms which include neutrophil extracellular traps (NETs) formation. In the present study, we showed that adenosine triphosphate (ATP), a DAMP molecule, accumulates in the brain and induces NETosis in brain parenchyma and in circulating neutrophils (PMNs) isolated from a murine model of stroke induced by middle cerebral artery occlusion (MCAO). Expression of peptidylarginine deiminase-4 (PAD4), which induces citrullination of histones H3 (CitH3) and initiates NETosis, was significantly enhanced in brain parenchyma and blood PMNs following MCAO. ATP or BzATP (a prototypic P2X7R agonist) significantly enhanced the inductions of PAD4 and CitH3 in a P2X7R-dependent manner and intracellular Ca2+ influx, PKCα activation, and NADPH oxidase-dependent reactive oxygen species (ROS) production play critical roles in this ATP-P2X7R-mediated NETosis. In our MCAO animal model, NETosis was markedly suppressed by treatment with apyrase, an enzyme hydrolyzing ATP, but enhanced by co-treatment of BzATP, confirming ATP-P2X7R-mediated NETosis. Since ATP not only induced NETosis but was also extruded after NETosis, our results indicate that ATP accumulated in the ischemic brain induces NETosis, mediating a cross-talk linking NETosis with neuronal damage that might aggravate inflammation and brain damage.

Anti-Zn2+-Toxicity of 4-Hydroxybenzyl Alcohol in Astrocytes and Neurons Contribute to a Robust Neuroprotective Effects in the Postischemic Brain.

4-Hydroxybenzyl alcohol (4-HBA) is an important phenolic constituent of Gastrodia elata (GE) Blume, which is used as a traditional herbal medicine in East Asia. Many activities have been reported to underlie the beneficial effects of 4-HBA in brain, such as, anti-oxidative, anti-inflammatory, anti-excitotoxic, and anti-apoptotic effects in neurons and microglia. Here, the authors demonstrate the robust neuroprotective effects of 4-HBA in rat middle cerebral artery occlusion (MCAO) model of stroke, and showed anti-Zn2+ toxicity in neurons and astrocytes as a molecular mechanism contributing to these effects. Intraperitoneal administration of 4-HBA (20 mg/kg) in Sprague-Dawley rats 1 h after MCAO reduced infarct volumes to 27.1 ± 9.2% of that of MCAO controls and significantly ameliorated motor impairments and neurological deficits. Significant suppressions of Zn2+-induced cell death, ROS generation, and PARP-1 induction by 4-HBA were observed in primary cortical cultures. 4-HBA also protected astrocytes from Zn2+-induced toxicity and suppressing ROS generation by employing slightly different molecular mechanisms, i.e., suppressing PARP-1 induction and NAD depletion under acute Zn2+-treatment and suppressing p67 NADPH oxidase subunit induction under chronic Zn2+-treatment. Results indicate that the protective effects of 4-HBA against Zn2+-toxicity in neurons and astrocytes contribute to its robust neuroprotective effects in the postischemic brain. Considering the pleiotropic effects of 4-HBA, which have been reported in previous reports and added in the present study, it has therapeutic potential for the amelioration of ischemic brain damage.

Robust neuroprotective effects of intranasally delivered iNOS siRNA encapsulated in gelatin nanoparticles in the postischemic brain.

The therapeutic efficacy of intranasal iNOS siRNA delivery was investigated in the postischemic rat brain after encapsulating on in gelatin nanoparticles (GNPs; diameter 188.0 ± 60.9 nm) cross-linked with 0.0667% glutaraldehyde (GA). Intranasally delivered GNPs were found in extracellular and intracellular compartments of many brain regions, including the olfactory bulb, cerebral cortex, and striatum at 1 hour after infusion and continued to be detected for days. Infarct volumes were markedly suppressed (maximal reduction to 42.1 ± 2.6%) at 2 days after 60 minutes of middle cerebral artery occlusion (MCAO) when iNOS siRNA/GNPs were delivered at 6 hours post-MCAO. In addition, this protective effect was manifested by reductions in neurological and behavioral deficits that were sustained for 2 weeks. Therapeutic potency of iNOS siRNA/GNPs was significantly greater and sustained longer than that of bare siRNA and prolonged and efficient iNOS by iNOS siRNA/GNP is responsible for the robust neuroprotective effect.

Antidepressant effects of exercise are produced via suppression of hypocretin/orexin and melanin-concentrating hormone in the basolateral amygdala.

Physical exercise is considered beneficial in the treatment of depression, but the underlying mechanism is not clearly understood. In the present study, we investigated the mechanism regulating antidepressant effects of exercise by focusing on the role of the amygdala using a well-defined animal model of depression. C57BL/6 mice treated with repeated restraint showed depression-like behaviors, which was counteracted by post-stress treatment with physical exercise. The two neuropeptides hypocretin/orexin (Hcrt/Orx) and melanin-concentrating hormone (MCH) were transcriptionally upregulated in the BLA after repeated stress, and their enhanced expression was downregulated by treatment with exercise, mirroring stress-induced depression-like behaviors and their reversal by exercise. Stereotaxic injection of either Hcrt/Orx peptide or MCH peptide within the BLA commonly increased phospho-CaMKIIα level and produced depression-like behaviors, mimicking the neural states in the BLA of mice subjected to repeated stress. In contrast, siRNA-mediated suppression of Hcrt/Orx or MCH in the BLA blocked stress-induced depression-like behaviors. Furthermore, siRNA-mediated inhibition of CaMKIIα in the BLA also counteracted stress-induced depression-like behaviors. Local injection of Hcrt/Orx peptide or MCH peptide within the BLA in exercise-treated animals blocked antidepressant-like effects of exercise. Together these results suggest that exercise produces antidepressant effects via suppression of Hcrt/Orx and MCH neural systems in the BLA.

Ethyl pyruvate inhibits HMGB1 phosphorylation and release by chelating calcium.

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca(2+) and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K(+)-induced transient [Ca(2+)]i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca(2+) as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca(2+) indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the A23187-induced intracellular Ca(2+) surge in BV2 cells and, under this condition, A23187-induced activations of Ca(2+)-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. (A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca(2+), and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.

Glycyrrhizin suppresses HMGB1 inductions in the hippocampus and subsequent accumulation in serum of a kainic acid-induced seizure mouse model.

Glycyrrhizin (GL), a triterpene present in the roots and rhizomes of licorice (Glycyrrhiza glabra), has been shown to have anti-inflammatory and anti-viral effects. In our previous reports, we demonstrated the neuroprotective effects of GL in the postischemic brain and in kainic acid (KA)-induced seizure animal model. In this KA-induced seizure model, the systemic administration of GL 30 min before KA administration significantly suppressed neuronal cell death and markedly suppressed gliosis and proinflammatory marker inductions. In the present study, we showed that high-mobility group box 1 (HMGB1), an endogenous danger signal, was induced in hippocampal CA1 and CA3 regions of the same KA-induced model, and peaked at ~3 h and at 6 days post-KA. HMGB1 was transiently induced in neurons and astrocyte at 3 h post-KA, and it was released from dying neurons and accumulated in serum at 12 h post-KA. Furthermore, after ~4 days of almost undetectable levels in the hippocampus, delayed and marked HMGB1 induction was detected at 6 days post-KA, mainly in astrocytes and endothelial cells, in which HMGB1 was localized in nuclei, and not secreted into serum. Interestingly, GL suppressed HMGB1 inductions in hippocampus and also suppressed its release into serum in KA-treated mice. Since we established previously that GL has anti-inflammatory and anti-excitotoxic effects in this KA-induced seizure model, these results indicate that the neuroprotective effect of GL in the KA-injected mouse brain might be attributable to the inhibitions of HMGB1 induction and release, which in turn, mitigates the inflammatory process.

Taurine Chloramine-Mediated Nrf2 Activation and HO-1 Induction Confer Protective Effects in Astrocytes.

Taurine is ubiquitously distributed in mammalian tissues, with the highest levels in the brain, heart, and leukocytes. Taurine reacts with hypochlorous acid (HOCl) to produce taurine chloramine (Tau-Cl) via the myeloperoxidase (MPO) system. In this study, we elucidated the antioxidative and protective effects of Tau-Cl in astrocytes. Tau-Cl increased the expression and nuclear translocation of nuclear factor E2-related factor (Nrf2) and the expression of Nrf2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Nrf2 activity is negatively regulated by Kelch-like ECH-associated protein 1 (Keap1). Tau-Cl decreased the level of the reduced thiol groups of Keap1, resulting in the disruption of the Keap1-Nrf2 complex. Consequently, Tau-Cl rescued the H2O2-induced cell death by enhancing HO-1 expression and suppressing reactive oxygen species. In conclusion, Tau-Cl confers protective effects in astrocytes by disrupting the Keap1-Nrf2 complex, thereby promoting Nrf2 translocation to the nucleus, wherein it binds to the antioxidant response element (ARE) and accelerates the transcription of antioxidant genes. Therefore, in astrocytes, the activation of the Keap1-Nrf2-ARE pathway by Tau-Cl may increase antioxidants and anti-inflammatory mediators as well as other cytoprotective proteins, conferring protection against brain infection and injury.

Age-Dependent and Aß-Induced Dynamic Changes in the Subcellular Localization of HMGB1 in Neurons and Microglia in the Brains of an Animal Model of Alzheimer's Disease.

HMGB1 is a prototypical danger-associated molecular pattern (DAMP) molecule that co-localizes with amyloid beta (Aß) in the brains of patients with Alzheimer's disease. HMGB1 levels are significantly higher in the cerebrospinal fluid of patients. However, the cellular and subcellular distribution of HMGB1 in relation to the pathology of Alzheimer's disease has not yet been studied in detail. Here, we investigated whether HMGB1 protein levels in brain tissue homogenates (frontal cortex and striatum) and sera from Tg-APP/PS1 mice, along with its cellular and subcellular localization in those regions, differed. Total HMGB1 levels were increased in the frontal cortices of aged wildtype (7.5 M) mice compared to young (3.5 M) mice, whereas total HMGB1 levels in the frontal cortices of Tg-APP/PS1 mice (7.5 M) were significantly lower than those in age-matched wildtype mice. In contrast, total serum HMGB1 levels were enhanced in aged wildtype (7.5 M) mice and Tg-APP/PS1 mice (7.5 M). Further analysis indicated that nuclear HMGB1 levels in the frontal cortices of Tg-APP/PS1 mice were significantly reduced compared to those in age-matched wildtype controls, and cytosolic HMGB1 levels were also significantly decreased. Triple-fluorescence immunohistochemical analysis indicated that HMGB1 appeared as a ring shape in the cytoplasm of most neurons and microglia in the frontal cortices of 9.5 M Tg-APP/PS1 mice, indicating that nuclear HMGB1 is reduced by aging and in Tg-APP/PS1 mice. Consistent with these observations, Aß treatment of both primary cortical neuron and primary microglial cultures increased HMGB1 secretion in the media, in an Aß-dose-dependent manner. Our results indicate that nuclear HMGB1 might be translocated from the nucleus to the cytoplasm in both neurons and microglia in the brains of Tg-APP/PS1 mice, and that it may subsequently be secreted extracellularly.

NADPH oxidase mediates depressive behavior induced by chronic stress in mice.

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

Intranasal delivery of HMGB1 siRNA confers target gene knockdown and robust neuroprotection in the postischemic brain.

Noninvasive intranasal drug administration has been noted to allow direct delivery of drugs to the brain. In the present study, the therapeutic efficacy of intranasal small interfering RNA (siRNA) delivery was investigated in the postischemic rat brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA was delivered intranasally in normal adult rats using e-PAM-R, a biodegradable PAMAM dendrimer, as gene carrier. Florescence-tagged siRNA was found in the cytoplasm and processes of neurons and of glial cells in many brain regions, including the hypothalamus, amygdala, cerebral cortex, and striatum, in 1 hour after infusion, and the FITC-fluorescence was continuously detected for at least 12 hours. When siRNA for high mobility group box 1 (HMGB1), which functions as an endogenous danger molecule and aggravates inflammation, was delivered intranasally, the target gene was significantly depleted in many brain regions, including the prefrontal cortex and striatum. More importantly, intranasal delivery of HMGB1 siRNA markedly suppressed infarct volume in the postischemic rat brain (maximal reduction to 42.8 ± 5.6% at 48 hours after 60 minutes middle cerebral artery occlusion (MCAO)) and this protective effect was manifested by recoveries from neurological and behavioral deficits. These results indicate that the intranasal delivery of HMGB1 siRNA offers an efficient means of gene knockdown-mediated therapy in the ischemic brain.

HMGB1 induces hepcidin upregulation in astrocytes and causes an acute iron surge and subsequent ferroptosis in the postischemic brain.

Dysregulation of brain iron levels causes functional disturbances and damages neurons. Hepcidin (a peptide hormone) plays a principal role in regulating intracellular iron levels by modulating ferroportin (FPN, the only known iron exporter) through triggering its internalization and lysosomal degradation. We observed a significant and rapid iron surge in the cortices of ischemic hemispheres at 3 h after cerebral ischemia (middle cerebral artery occlusion, MCAO) that was maintained until 4 d post-MCAO. We showed upregulation of hepcidin expression in the brain as early as 3 h post-MCAO, mainly in astrocytes, and significant hepcidin accumulation in serum from 6 h post-MCAO, and these inductions were maintained for 1 day and 7 days, respectively. High mobility group box 1 (HMGB1), a prototypic danger-associated molecular pattern, accumulates markedly after transient MCAO and plays critical roles in damage aggravation via its proinflammatory effects. Here, we demonstrated that treatment with recombinant HMGB1 stimulated astrocytes to induce hepcidin expression in a TLR4- and CXCR4-dependent manner. Furthermore, hepcidin-mediated intracellular iron accumulation in neurons was confirmed by an experiment using N-methyl-D-aspartate (NMDA)-conditioned medium-treated primary astrocytes and fresh primary cortical neurons treated with hepcidin-containing astrocyte-conditioned medium. Moreover, HMGB1-mediated local hepcidin upregulation and subsequent local iron surge were found to cause ferroptosis in the postischemic brain, which was suppressed by the functional blocking of HMGB1 using intranasally administered HMGB1 A box or anti-HMGB1 antibody. These findings show that HMGB1 serves as a ferroptosis inducer by upregulating hepcidin in astrocytes and thus aggravates acute damage in the postischemic brain.

Glycyrrhizic acid affords robust neuroprotection in the postischemic brain via anti-inflammatory effect by inhibiting HMGB1 phosphorylation and secretion.

High mobility group box 1 (HMGB1) is an endogenous danger signal molecule. In a previous report, we showed that HMGB1 is massively released during NMDA-induced acute damaging process in the postischemic brain and triggers inflammatory processes, like microglial activation. siRNA-mediated HMGB1 knockdown markedly reduced infarct volumes, confirming the crucial role played by HMGB1 in the postischemic brain. In the present study, we showed neuroprotective effects of glycyrrhizin (GL) in the postischemic rat brain after middle cerebral artery occlusion (MCAO). GL, a triterpene present in the roots and rhizomes of licorice, Glycyrrhiza glabra, has been shown to have anti-inflammatory and anti-viral effects. It has been reported that GL binds directly to HMGB1, and inhibits its chemoattractant and mitogenic activities. The administration of GL (10mg/kg) intravenously at 3 or 6h after MCAO reduced infarct volumes to 12.9±4.2% and 46.2±9.9%, respectively, of untreated control. This neuroprotective effect was accompanied by improvements in motor impairment and neurological deficits and suppressions of microglia activation and proinflammatory cytokine induction. Interestingly, GL almost completely blocked HMGB1 secretion in the postischemic brain and in lipopolysaccharide (LPS)-treated microglia cells. Furthermore, HMGB1 phosphorylation, which is the initial step for HMGB1 secretion, and the interaction between HMGB1 and protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase IV (CaMKIV) were suppressed dose-dependently by GL. Here, we hypothesized that the blockage for the putative phosphorylation sites in HMGB1 by GL might be attributed to this suppression. In addition to the anti-inflammatory effects, we found that GL has anti-excitotoxic and anti-oxidative effects in neurons. Together these results indicate that GL has neuroprotective efficacy in the postischemic brain via its anti-inflammatory, anti-excitotoxic, and anti-oxidative effects and in particular, it exerts anti-inflammatory effect, at least in part, by inhibiting HMGB1 secretion.

Cytosolic HMGB1 Mediates LPS-Induced Autophagy in Microglia by Interacting with NOD2 and Suppresses Its Proinflammatory Function.

The high mobility group box 1 (HMGB1), a well-known danger-associated molecule pattern (DAMP) molecule, is a non-histone chromosomal protein localized in the nucleus under normal physiological conditions. HMGB1 exhibits diverse functions depending on its subcellular location. In the present study, we investigated the role of HMGB1-induced autophagy in the lipopolysaccharide (LPS)-treated BV2 microglial cell line in mediating the transition between the inflammatory and autophagic function of the nucleotide-binding oligomerization domain-containing 2 (NOD2), a cytoplasmic pattern-recognition receptor. The induction of the microtubule-associated protein 1 light chain 3 (LC3), an autophagy biomarker, was detected slowly in BV2 cells after the LPS treatment, and peak induction was detected at 12 h. Under these conditions, NOD2 level was significantly increased and the binding between HMGB1 and NOD2 and between HMGB1 and ATG16L1 was markedly enhanced and the temporal profiles of the LC3II induction and HMGB1-NOD2 and HMGB1-ATG16L1 complex formation coincided with the cytosolic accumulation of HMGB1. The LPS-mediated autophagy induction was significantly suppressed in BV2 cells after HMGB1 or NOD2 knock-down (KD), indicating that HMGB1 contributes to NOD2-mediated autophagy induction in microglia. Moreover, NOD2-RIP2 interaction-mediated pro-inflammatory cytokine induction and NF-κB activity were significantly enhanced in BV2 cells after HMGB1 KD, indicating that HMGB1 plays a critical role in the modulation of NOD2 function between pro-inflammation and pro-autophagy in microglia. The effects of the cell-autonomous pro-autophagic pathway operated by cytoplasmic HMGB1 may be beneficial, whereas those from the paracrine pro-inflammatory pathway executed by extracellularly secreted HMGB1 can be detrimental. Thus, the overall functional significance of HMGB1-induced autophagy is different, depending on its temporal activity.

Robust protective effects of a novel multimodal neuroprotectant oxopropanoyloxy benzoic acid (a salicylic acid/pyruvate ester) in the postischemic brain.

Cerebral ischemia leads to brain injury via a complex series of pathophysiological events. Therefore, multidrug treatments or multitargeting drug treatments are attractive options in efficiently limiting brain damage. Here, we report a novel multifunctional compound oxopropanoyloxy benzoic acid (OBA-09), a simple ester of pyruvate and salicylic acid. This protective effect was manifested by recoveries from neurological and behavioral deficits. OBA-09 exhibited antioxidative effects in the postischemic brain, which was evidenced by remarkable reduction of lipid peroxidation and 4-hydroxy-2-nonenal staining in OBA-09-administered animals. Reactive oxygen species generation was markedly suppressed in primary cortical cultures under oxygen-glucose deprivation. More interestingly, OBA-09 was capable of scavenging hydroxyl radical in cell-free assays. High-performance liquid chromatography results demonstrated that OBA-09 was hydrolyzed to salicylic acid and pyruvate with t(1/2) = 43 min in serum and 4.2 h in brain parenchyma, indicating that antioxidative function of OBA-09 is executed by itself and also by salicylic acid after the hydrolysis. In addition to antioxidative function, OBA-09 exerts anti-excitotoxic and anti-Zn(2+)-toxic functions, which might be attributed to attenuation of ATP and nicotinamide adenine dinucleotide depletion and to the suppression of nuclear factor-κB activity induction. Together these results indicate that OBA-09 has a potent therapeutic potential as a multimodal neuroprotectant in the postischemic brain and these effects were conferred by OBA-09 itself and subsequently its hydrolyzed products.

Simplified heterocyclic analogues of fluoxetine inhibit inducible nitric oxide production in lipopolysaccharide-induced BV2 cells.

A series of fluoxetine, where the N-methylamino group was replaced and then simplified, were synthesized and their inhibitory effect was tested for nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-induced BV2 cells. Although the synthesized compounds generally revealed weaker activity or greater cytotoxicity than fluoxetine, compound 10a, in which the N-methylamino group in fluoxetine was replaced by morpholine, and the trifluoromethylphenyl ring was substituted with simple oxo group, suppressed NO production dose-dependently at 10, 20 and 40 µM concentrations with less cytotoxicity than fluoxetine, and inhibited iNOS mRNA and protein expression at the same concentrations in LPS-induced BV2 cells. The results suggested that the trifluoromethylphenyl ring moiety in fluoxetine is not necessary for the suppression of NO production and that 10a has the potential as a potent inhibitor of NO production.

Taurine Protects against Postischemic Brain Injury via the Antioxidant Activity of Taurine Chloramine.

Taurine is ubiquitously distributed in mammalian tissues and is highly concentrated in the heart, brain, and leukocytes. Taurine exerts neuroprotective effects in various central nervous system diseases and can suppress infarct formation in stroke. Taurine reacts with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) to produce taurine chloramine (Tau-Cl). We investigated the neuroprotective effects of taurine using a rat middle cerebral artery occlusion (MCAO) model and BV2 microglial cells. Although intranasal administration of taurine (0.5 mg/kg) had no protective effects, the same dose of Tau-Cl significantly reduced infarct volume and ameliorated neurological deficits and promoted motor function, indicating a robust neuroprotective effect of Tau-Cl. There was neutrophil infiltration in the post-MCAO brains, and the MPO produced by infiltrating neutrophils might be involved in the taurine to Tau-Cl conversion. Tau-Cl significantly increased the levels of antioxidant enzymes glutamate-cysteine ligase, heme oxygenase-1, NADPH:quinone oxidoreductase 1, and peroxiredoxin-1 in BV2 cells, whereas taurine slightly increased some of them. Antioxidant enzyme levels were increased in the post-MCAO brains, and Tau-Cl further increased the level of MCAO-induced antioxidant enzymes. These results suggest that the neutrophils infiltrate the area of ischemic injury area, where taurine is converted to Tau-Cl, thus protecting from brain injury by scavenging toxic HOCl and increasing antioxidant enzyme expression.

Role of HMGB1 in the Interplay between NETosis and Thrombosis in Ischemic Stroke: A Review.

Neutrophil extracellular traps (NETs) comprise decondensed chromatin, histones and neutrophil granular proteins and are involved in the response to infectious as well as non-infectious diseases. The prothrombotic activity of NETs has been reported in various thrombus-related diseases; this activity can be attributed to the fact that the NETs serve as a scaffold for cells and numerous coagulation factors and stimulate fibrin deposition. A crosstalk between NETs and thrombosis has been indicated to play a role in numerous thrombosis-related conditions including stroke. In cerebral ischemia, neutrophils are the first group of cells to infiltrate the damaged brain tissue, where they produce NETs in the brain parenchyma and within blood vessels, thereby aggravating inflammation. Increasing evidences suggest the connection between NETosis and thrombosis as a possible cause of "tPA resistance", a problem encountered during the treatment of stroke patients. Several damage-associated molecular pattern molecules have been proven to induce NETosis and thrombosis, with high mobility group box 1 (HMGB1) playing a critical role. This review discusses NETosis and thrombosis and their crosstalk in various thrombosis-related diseases, focusing on the role of HMGB1 as a mediator in stroke. We also addresses the function of peptidylarginine deiminase 4 with respect to the interplay with HMGB1 in NET-induced thrombosis.

Ninjurin 1 dodecamer peptide containing the N-terminal adhesion motif (N-NAM) exerts proangiogenic effects in HUVECs and in the postischemic brain.

Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is a cell adhesion molecule responsible for cell-to-cell interactions between immune cells and endothelial cells. In our previous paper, we have shown that Ninj1 plays an important role in the infiltration of neutrophils in the postischemic brain and that the dodecamer peptide harboring the Ninj1 N-terminal adhesion motif (N-NAM, Pro26-Asn37) inhibits infiltration of neutrophils in the postischemic brain and confers robust neuroprotective and anti-inflammatory effects. In the present study, we examinedt the pro-angiogenic effect of N-NAM using human umbilical vein endothelial cells (HUVECs) and rat MCAO (middle cerebral artery occlusion) model of stroke. We found that N-NAM promotes proliferation, migration, and tube formation of HUVECs and demonstrate that the suppression of endogenous Ninj1 is responsible for the N-NAM-mediated pro-angiogenic effects. Importantly, a pull-down assay revealed a direct binding between exogenously delivered N-NAM and endogenous Ninj1 and it is N-terminal adhesion motif dependent. In addition, N-NAM activated the Ang1-Tie2 and AKT signaling pathways in HUVECs, and blocking those signaling pathways with specific inhibitors suppressed N-NAM-induced tube formation, indicating critical roles of those signaling pathways in N-NAM-induced angiogenesis. Moreover, in a rat MCAO model, intranasal administration of N-NAM beginning 4 days post-MCAO (1.5 µg daily for 3 days) augmented angiogenesis in the penumbra of the ipsilateral hemisphere of the brain and significantly enhanced total vessel lengths, vessel densities, and pro-angiogenic marker expression. These results demonstrate that the 12-amino acid Ninj1 peptide, which contains the N-terminal adhesion motif of Ninj1, confers pro-angiogenic effects and suggest that those effects might contribute to its neuroprotective effects in the postischemic brain.