Joo-Hyun Shin

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.

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.

Glucosamine exerts a neuroprotective effect via suppression of inflammation in rat brain ischemia/reperfusion injury.

We investigated the neuroprotective effect of glucosamine (GlcN) in a rat middle cerebral artery occlusion model. At the highest dose used, intraperitoneal GlcN reduced infarct volume to 14.3% ± 7.4% that of untreated controls and afforded a reduction in motor impairment and neurological deficits. Neuroprotective effects were not reproduced by other amine sugars or acetylated‐GlcN, and GlcN suppressed postischemic microglial activation. Moreover, GlcN suppressed lipopolysaccharide (LPS)‐induced upregulation of proinflammatory mediators both in vivo and in culture systems using microglial or macrophage cells. The anti‐inflammatory effects of GlcN were mainly attributable to its ability to inhibit nuclear factor kappaB (NF‐κB) activation. GlcN inhibited LPS‐induced nuclear translocation and DNA binding of p65 to both NF‐κB consensus sequence and NF‐κB binding sequence of inducible nitric oxide synthase promoter. In addition, we found that GlcN strongly repressed p65 transactivation in BV2 cells using Gal4‐p65 chimeras system. P65 displayed increased O‐GlcNAcylation in response to LPS; this effect was also reversed by GlcN. The LPS‐induced increase in p65 O‐GlcNAcylation was paralleled by an increase in interaction with O‐GlcNAc transferase, which was reversed by GlcN. Finally, our results suggest that GlcN or its derivatives may serve as novel neuroprotective or anti‐inflammatory agents.

Extracellular HMGB1 Released by NMDA Treatment Confers Neuronal Apoptosis via RAGE-p38 MAPK/ERK Signaling Pathway

High mobility group box 1 (HMGB1) was originally identified as ubiquitously expressed nonhistone DNA-binding protein, but recently, it was found to act as an endogenous danger molecule, which signals danger and traumatic cell death. Previously, the authors showed that HMGB1 is massively released immediately after an ischemic insult and that it subsequently activates microglia and induces inflammation in the postischemic brain. Here, we showed the endogenous danger molecule-like function of HMGB1 in primary cortical cultures. HMGB1 was found to be accumulated in NMDA-treated primary cortical culture media, and media collected from these cultures were able to induce neuronal cell death when added to fresh primary cortical cultures. However, HMGB1-depleted NMDA-conditioned media produced by HMGB1 siRNA transfection or by preincubation with anti-HMGB1 antibody or with HMGB1 A box failed to induce neuronal cell death. Furthermore, siRNA-mediated HMGB1 knockdown substantially suppressed NMDA- or Zn2+-induced cell death. It was interesting to find that extracellular HMGB1-induced neuronal apoptosis, as evidenced by TUNEL staining and caspase 3 assay in combination with double immunofluorescence staining. A series of RAGE and HMGB1 co-immunoprecipitation experiments in the presence of SB203580 and PD98059 (p38 MAPK and ERK inhibitors, respectively) demonstrated that RAGE-p38 MAPK and RAGE-ERK pathway might underlie extracellular HMGB1-mediated neuronal apoptosis. These results together with our previous reports regarding microglial activation by extracellular HMGB1 indicate that HMGB1 functions as a novel danger signal, which aggravates brain damage via autocrine and paracrine manners.

Up-down Regulation of HO-1 and iNOS Gene Expressions by Ethyl Pyruvate via Recruiting p300 to Nrf2 and Depriving It from p65

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to exert robust neuroprotection in various neuropathological conditions, such as, cerebral ischemia and KA-induced seizure animal models. The neuroprotective effect of EP is attributable to the anti-inflammatory, anti-oxidative, and anti-apoptotic effects. In the present study, we investigated convergence of anti-inflammatory and anti-oxidative functions of EP and present a novel molecular mechanism underlying anti-inflammatory effects of EP, which is conveyed by p300, a transcriptional co-activator for both Nuclear factor E2-related factor 2 (Nrf2) and p65. In BV2 cells, a microglia cell line, EP induced translocation of Nrf2 from the cytosol to the nucleus and enhanced the expression of hemeoxygenase 1 (HO-1) in a dose-dependent manner and 1h incubation with 10mM EP increased HO-1 to 4.9-fold. Nrf2 was found to translocate from the cytosol to the nucleus beginning 30 min after EP-treatment and binds to the antioxidant response element (ARE) located on HO-1 promoter. Interestingly, LPS-induced inducible NO synthase (iNOS) induction was substantially suppressed in EP-pre-treated BV2 cells and it was reverted by Nrf2 knockdown. We found that EP-induced Nrf2 accumulation in the nucleus recruits p300, a transcriptional co-activator of both Nrf2 and p65, inhibiting p65-p300 interaction. Competition between Nrf2 and p65 for p300 binding was confirmed by glutathione S-transferase (GST) pull down assay and reporter gene analysis. These results demonstrate that EP induced nuclear translocation of Nrf2 which binds to ARE along with p300 and hampers iNOS expression via depleting p300 from p65. This is a novel anti-inflammatory mechanism conveyed by EP, which enhances protective effect by converging anti-inflammatory and anti-oxidative effects and might be applicable to various Nrf2-activating agents, such as phytochemicals.

Ethyl pyruvate-mediated Nrf2 activation and hemeoxygenase 1 induction in astrocytes confer protective effects via autocrine and paracrine mechanisms

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to act as an anti-inflammatory molecule under various pathological conditions, such as, during cerebral ischemia and sepsis in animal models. Here, the authors investigated the novel molecular mechanism underlying the anti-oxidative effect of EP in primary astrocyte cultures, particularly with respect to nuclear factor E2-related factor 2 (Nrf2) activation and hemeoxygenase 1 (HO-1) induction. EP was found to induce Nrf2 translocation and the inductions of various genes downstream of Nrf2 and these resulted in the amelioration of the oxidative damage of H(2)O(2). Furthermore, EP dose-dependently suppressed H(2)O(2)-induced astrocyte cell death (12h preincubation with 5mM EP increased cell survival after 1h exposure to 100 μM H(2)O(2) from 32.6±0.7% to 63±1.8%). HO-1 was markedly induced (4.9-fold) in EP-treated primary astrocyte cultures and Nrf2 was found to translocate from the cytosol to the nucleus and bind to the antioxidant response element (ARE) located on HO-1 promoter after EP treatment. siRNA-mediated HO-1 or Nrf2 knockdown and zinc protoporphyrin (ZnPP)-mediated inhibition of HO-1 activity showed that Nrf2 activation and HO-1 induction were responsible for the observed cytoprotective effect of EP, which was found to involve the ERK and Akt signaling pathways. Furthermore, EP-conditioned astrocyte culture media was found to have neuroprotective effects on primary neuronal cultures exposed to oxidative or excitotoxic stress, and this seemed to be mediated by glial cell line-derived neurotrophic factor (GDNF) and glutathione (GSH), which accumulated in EP-treated astrocyte culture media. Interestingly, we also found that in addition to HO-1, EP-induced Nrf2 activation increased the expressions of various anti-oxidant genes, including GST, NQO1, and GCLM. The study shows that EP-mediated Nrf2 activation and HO-1 induction in astrocytes act via autocrine and paracrine mechanisms to confer protective effects.

Ethyl pyruvate inhibits HMGB1 phosphorylation and secretion in activated microglia and in the postischemic brain.

Ethyl pyruvate (EP) has been shown to have anti-inflammatory effects and confer protective effects in various pathological conditions. For example, EP inhibits secretion of high mobility group box 1 (HMGB1), which is known to be released from activated or dying cells and aggravate inflammatory pathways. In the present study, we investigated whether EP reduces HMGB1 phosphorylation and release in ischemic brain and in cultured microglia. In the postischemic brains (60 min middle cerebral artery occlusion (MCAO)), HMGB1 was released extracellularly, generating dual peaks in cerebrospinal fluid (CSF) around 1 and 7 days after ischemic insult, which were probably generated from damaged neurons and activated inflammatory cells, respectively. We showed that treatment with EP 30 min post-MCAO (5 mg/kg, i.v.), which has been shown to confer a robust neuroprotective effect in the postischemic brain, reduced both peaks. In addition, delayed EP treatment from 4 days post-MCAO reduced HMGB1 accumulation in CSF at 7 day post-MCAO in the absence of accompanying amelioration of ischemic brain damage, indicating that the suppression of HMGB1 release is a direct effect. We also found that EP markedly suppressed the LPS-induced nuclear translocations of protein kinase C alpha and calcium/calmodulin-dependent protein kinase IV, HMGB1 phosphorylation, and subsequent secretion of HMGB1 induced by LPS in BV2 cells and EP-mediated above-mentioned effects were also independent of cell death or survival. These results indicate that EP inhibits HMGB1 phosphorylation and release in activated microglia, which might be responsible for EP-mediated suppression of HMGB1 release in the postischemic brain.

Intranasal delivery of HMGB1-binding heptamer peptide confers a robust neuroprotection in the postischemic brain.

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 and induces neuronal apoptosis. We have also reported a robust neuroprotection of intranasally delivered HMGB1 siRNA in the postischemic rat brain (middle cerebral artery occlusion (MCAO), 60 min). In the present study, we investigated the therapeutic efficacy of intranasally delivered HMGB1 binding heptamer peptide (HBHP; HMSKPVQ), which was selected using a phage display approach, in the same stroke animal model. A pull-down assay using biotin-labeled HBHP showed that HBHP binds directly to HMGB1, specifically to HMGB1 A box, confirming HMGB1/HBHP interaction. HBHP significantly suppressed HMGB1-mediated neuronal cell death in primary cortical cultures and HMGB1/HBHP binding was detected in NMDA-conditioned culture media. However, a heptamer peptide composed of a scrambled sequence of the seven amino acids in HBHP failed to bind HMGB1 and had no protective effect. Furthermore, HBHP (300 ng) delivered intranasally at 30 min before MCAO significantly suppressed infarct volume in the postischemic rat brain (maximal reduction by 41.8±5.4%) and ameliorated neurological and behavioral deficits. In contrast, scrambled heptamer peptide had no protective effect at the same dose. Together these results suggest that intranasal HBHP ameliorates neuronal damage in the ischemic brain by binding HMGB1, which might inhibit the function of HMGB1 as an endogenous danger signal molecule.

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 Ca2+ 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 [Ca2+]i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca2+ as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca2+ indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the A23187-induced intracellular Ca2+ surge in BV2 cells and, under this condition, A23187-induced activations of Ca2+-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 Ca2+, and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.