Yinchuan Jin

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.

Fluoxetine attenuates kainic acid-induced neuronal cell death in the mouse hippocampus

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) and one of the commonly prescribed antidepressants. Numerous clinical observations and animal studies indicate that fluoxetine enhances the anticonvulsant potencies of several antiepileptic drugs. In the previous report, we showed that fluoxetine strongly protects against delayed cerebral ischemic injury. In the present study, the authors investigated whether fluoxetine has a beneficial effect on KA-induced neuronal cell death. An intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 microg) of KA produced typical neuronal cell death both in CA1 and CA3 regions of the hippocampus. Although, there was no significant difference in the time course or severity of epileptic behavior, the systemic administration of fluoxetine 30 min before KA administration significantly attenuated this neuronal cell death. Fluoxetine was found to suppress neuronal cell loss when injected at 10 mg/kg and the effect was enhanced at 50 mg/kg. Furthermore, this fluoxetine-induced neuroprotection was accompanied by marked improvements in memory impairment, as determined by passive avoidance tests. KA-induced gliosis and proinflammatory marker (COX-2, IL-1beta, and TNF-alpha) inductions were also suppressed by fluoxetine administration. It is interesting to note here that fluoxetine treatment suppressed NF-kappaB activity dose-dependently in KA-treated mouse brains, suggesting that this explains in part its anti-inflammatory effect. Together, these results suggest that fluoxetine has therapeutic potential in terms of suppressing KA-induced pathogenesis in the brain, and that these neuroprotective effects are associated with its anti-inflammatory effects.

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.

Glycyrrhizin Attenuates Kainic Acid-Induced Neuronal Cell Death in the Mouse Hippocampus

Glycyrrhizin (GL), a triterpene that is present in the roots and rhizomes of licorice (Glycyrrhiza glabra), has been reported to have anti-inflammatory and anti-viral effects. Recently, we demonstrated that GL produced the neuroprotective effects with the suppression of microglia activation and proinflammatory cytokine induction in the postischemic brain with middle cerebral artery occlusion (MCAO) in rats and improved motor impairment and neurological deficits. In the present study, we investigated whether GL has a beneficial effect in kainic acid (KA)-induced neuronal death model. Intracerebroventricular (i.c.v.) injection of 0.94 nmole (0.2 µg) of KA produced typical neuronal death in both CA1 and CA3 regions of the hippocampus. In contrast, administration of GL (10 mg/kg, i.p.) 30 min before KA administration significantly suppressed the neuronal death, and this protective effect was more stronger at 50 mg/kg. Moreover, the GL-mediated neuroprotection was accompanied with the suppression of gliosis and induction of proinflammatory markers (COX-2, iNOS, and TNF-α). The anti-inflammatory and anti-excitotoxic effects of GL were verified in LPS-treated primary microglial cultures and in NMDA- or KA-treated primary cortical cultures. Together these results suggest that GL confers the neuroprotection through the mechanism of anti-inflammatory and anti-excitotoxic effects in KA-treated 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.

Biodegradable gelatin microspheres enhance the neuroprotective potency of osteopontin via quick and sustained release in the post-ischemic brain.

Gelatin microspheres (GMSs) are widely used as drug carriers owing to their excellent biocompatibilities and toxicologically safe degradation products. The drug release profile is easily tailored by controlling the cross-linking density and surface-to-volume ratio, i.e. size, of the GMS. In this study, we employed GMSs which are 25 μm in diameter and cross-linked with 0.03125% glutaraldehyde, to enable rapid initial and a subsequent sustained release. Therapeutic potency of human recombinant osteopontin (rhOPN) with or without encapsulation into GMSs was investigated after administrating them to rat stroke model (Sprague-Dawley; middle cerebral artery occlusion, MCAO). The administration of rhOPN/GMS (100 ng/100 μg) at 1h post-MCAO reduced the mean infarct volume by 81.8% of that of the untreated MCAO control and extended the therapeutic window at least to 12h post-MCAO, demonstrating a markedly enhanced therapeutic potency for the use of OPN in the post-ischemic brain. Scanning electron microscopy micrographs revealed that GMSs maintained the three-dimensional shape for more than 5 days in normal brain but were degraded rapidly in the post-ischemic brain, presumably due to high levels of gelatinase induction. After encapsulation with GMS, the duration of OPN release was markedly extended; from the period of 2 days to 5 days in normal brain, and from 2 days to 4 days in the post-ischemic brain; these encompass the critical period for recovery processes, such as vascularization, and controlling inflammation. Together, these results indicate that GMS-mediated drug delivery has huge potential when it was used in the hyperacute period in the post-ischemic brain.

p38β MAPK affords cytoprotection against oxidative stress-induced astrocyte apoptosis via induction of αB-crystallin and its anti-apoptotic function

The activation of p38 mitogen-activated protein kinases (MAPKs) has been implicated in many cellular processes, such as, inflammation, cell death, and survival. In mammals, four distinct genes encode the four known members of p38 MAPKs, p38α, p38β, p38γ, and p38δ. Despite the fact that p38α and p38β MAPKs share over 75% homology sequences, they have distinct, perhaps even opposite roles under stress conditions. In our previous report, we showed that p38β MAPK is induced in activated astrocytes in the penumbra of the postischemic brain, wherein it was co-localized with αB-crystallin and MAPKAPK-2. To investigate the functional significance of p38β MAPK in astrocytes, a C6 astroglioma cell line stably over-expressing p38β MAPK was generated. In these cells, hydrogen peroxide-induced apoptosis was reduced to 44.3% of that obtained from normal C6 cells. Interestingly, we found that expression of a small heat shock protein, αB-crystallin, was significantly increased in these cells, but that the expressions of HSP27 and HSP70 were not. Repression of αB-crystallin expression by αB-crystallin siRNA transfection suppressed the protective effect and recovered caspase 3 activity, indicating that αB-crystallin induction plays a crucial role in the protection against H₂O₂-induced apoptosis observed in p38β-overexpressing C6 astroglioma cells. We found that the binding between αB-crystallin and partially processed caspase-3 (a p24 intermediate) was significantly increased in p38β-overexpressing cells, which might result in suppression of caspase 3 activity in these cells. These results indicate that p38β confers protection against H₂O₂-induced astrocytes apoptosis by inducing a small heat shock protein, αB-crystallin, which inhibits caspase-3 activation.

Anti-inflammatory effects of OBA-09, a salicylic acid/pyruvate ester, in the postischemic brain.

Cerebral ischemia leads to brain injury via a complex series of pathophysiological events, and therefore, multi-drug treatments or multi-targeting drug treatments provide attractive options with respect to limiting brain damage. Previously, we reported that a novel multi-functional compound oxopropanoyloxy benzoic acid (OBA-09, a simple ester of pyruvate and salicylic acid) affords robust neuroprotective effects in the postischemic rat brain. OBA-09 exhibited anti-oxidative effects that appeared to be executed by OBA-09 and by the salicylic acid afforded by hydrolysis. Here, we report the anti-inflammatory effects of OBA-09. Microglial activation observed at 2 days post-middle cerebral artery occlusion (MCAO, 90 min) and at 1 day after a LPS injection (0.5 mg/kg, intravenously) in the brains of Sprague-Dawley rats were markedly suppressed by the administration of OBA-09 (10 mg/kg). Inductions of proinflammatory markers (TNF-α, IL-1β, iNOS, and COX-2) were also suppressed by OBA-09 in both the LPS and MCAO models. Moreover, the anti-inflammatory effect of OBA-09 was accompanied by the suppression of infarct formation in the postischemic brain, but appeared to be independent of neuroprotection in LPS-treated rats. The inductions of proinflammatory markers were also inhibited by OBA-09 in LPS-treated BV2 cells (a microglia cell line) and in LPS-treated-primary neutrophils, possibly due to the suppression of NF-κB activity. Interestingly, the anti-inflammatory effect of OBA-09 was greater than that of equivalent co-treatment with pyruvate and salicylic acid. Together these results indicate that OBA-09 is a potent multi-modal neuroprotectant in the postischemic brain, and that its anti-inflammatory effect contributes to its neuroprotective function.