Won-Ki Kim

Inhibition of MMP-3 or -9 suppresses lipopolysaccharide-induced expression of proinflammatory cytokines and iNOS in microglia

Recently, matrix metalloproteinases (MMPs) are emerging as important molecules in neuroinflammation as well as neuronal cell death. However, the role of MMPs in activated microglia remains unclear. In the present study, we found that expressions of MMP‐1, ‐3, ‐8 and ‐9 were significantly induced by single or combined treatment of immunostimulants lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) in primary cultured microglia and BV2 microglial cells. Inhibition of MMP‐3 or ‐9 significantly suppressed the expression of iNOS and pro‐inflammatory cytokines and the activities of NF‐κB, AP‐1, and MAPK in LPS‐stimulated microglia. The results suggest that MMP‐3 and ‐9 both mediate LPS‐induced inflammatory reactions. Inhibition of reactive oxygen species (ROS) by N‐acetyl‐cysteine or diphenylene iodonium significantly suppressed the expression of MMP‐3, MMP‐9, NO and TNF‐α in LPS‐stimulated microglia, suggesting that ROS is an early signaling inducer in LPS‐stimulated microglial cells. MMP inhibitors also suppressed ROS production, suggesting a cross‐talk between ROS and MMPs. Collectively, the present study demonstrates that MMP‐3 and MMP‐9 play a role as inflammatory mediators in activated microglia. Pharmacological intervention of MMPs especially MMP‐3 and ‐9 would be a therapeutic strategy for the treatment of inflammatory diseases in the CNS caused by over‐activation of microglial cells.

Role of glutathione peroxidase in the ontogeny of hippocampal oxidative stress and kainate seizure sensitivity in the genetically epilepsy-prone rats.

Oxidative stress may contribute to epileptogenicity in genetic models of epilepsy. To address this, we examined the enzymatic activity of cytosolic Cu/Zn superoxide dismutase (SOD-1), mitochondrial Mn superoxide dismutase (SOD-2), and glutathione peroxidase (GPx) in the developing hippocampus of genetically epilepsy-prone rats (GEPR-9s). We also measured changes in the GSH/GSSG ratio, lipid peroxidation, and protein oxidation at post-natal days (PD) 7, 30, and 90, respectively. Compared with control Sprague-Dawley (SD) rats, GEPR-9s showed similar SOD-1 and SOD-2 activity but lower GPx activity. Epilepsy-prone rats also showed lower GSH/GSSG ratios than controls, and more lipid peroxidation (as measured by malondialdehyde levels) and protein oxidation (as measured by carbonyl levels). Treatment with kainic acid (KA) resulted in more pronounced seizures, less GPx activity, and lower GSH/GSSG ratios in GEPR-9s than in controls, but KA did not significantly affect SOD-1 or SOD-2 activity, suggesting that GEPR-9s do not compensate for reduced GPx activity by increasing SOD. Moreover, KA treatment resulted in significantly a lower GSH/GSSG ratio and GPx-like immunoreactivity and higher malondialdehyde and carbonyl levels in GEPR-9s than in controls. These findings were more evident in GEPR-9s at PD 90 than at PD 30, indicating that oxidative stress is age-dependent. Double-labeling immunocytochemical analysis demonstrated co-localization of GPx-immunoreactive glia-like cells and reactive astrocytes, as labeled by glial fibrillary acidic protein (GFAP). This suggests that mobilization of astroglial cells for synthesis of GPx protein is a response to KA insult, intended to decrease the neurotoxicity induced by peroxides. These responses were more pronounced in control SD rats than in GEPR-9s. Our results suggest that impairment of the GPx (including glutathione)-mediated antioxidant system contributed to epileptogenesis in GEPR-9s.

Protection against kainate neurotoxicity by ginsenosides: Attenuation of convulsive behavior, mitochondrial dysfunction, and oxidative stress

We previously demonstrated that kainic acid (KA)‐mediated mitochondrial oxidative stress contributed to hippocampal degeneration and that ginsenosides attenuated KA‐induced neurotoxicity and neuronal degeneration. Here, we examined whether ginsenosides affected KA‐induced mitochondrial dysfunction and oxidative stress in the rat hippocampus. Treatment with ginsenosides attenuated KA‐induced convulsive behavior dose‐dependently. KA treatment increased lipid peroxidation and protein oxidation and decreased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio to a greater degree in the mitochondrial fraction than in the hippocampal homogenate. KA treatment resulted in decreased Mn‐superoxide dismutase expression anddiminished the mitochondrial membrane potential. Furthermore, KA treatment increased intramitochondrial Ca2+ and promoted ultrastructural degeneration in hippocampal mitochondria. Treatment with ginsenosides dose‐dependently attenuated convulsive behavior and the KA‐induced mitochondrial effects. Protection appeared to be more evident in mitochondria than in tissue homogenates. Collectively, the results suggest that ginsenosides prevent KA‐induced neurotoxicity by attenuating mitochondrial oxidative stress and mitochondrial dysfunction.

Resveratrol, purified from the stem of Vitis coignetiae Pulliat, inhibits food intake in C57BL/6J Mice.

Neuropeptide Y (NPY) and agouti-related protein (AgRP) have powerful stimulatory effects on food intake, which suggests that the downregulation of brain NPY or AgRP may help reduce obesity and diabetes by inhibiting food intake. To search for active compounds that inhibit NPY and AgRP expression, we made two luciferase reporter assay systems consisting of NPY and AgRP promoter-driven luciferase genes, together with the puromycin resistance gene, in a plasmid vector. Each plasmid was permanently transfected into N29-4 neuronal cells. Using the systems, resveratrol was purified from the stem of Vitis coignetiae Pulliat by activityguided fractionation. Resveratrol downregulated NPY and AgRP promoter-driven luciferase activity in a dose-dependent manner. The inhibitory concentrations (IC50, 50% inhibition) of resveratrol against pNPY-luc and pAgRP-luc activities were 8.9 μM and 8.0 μM, respectively. Furthermore, one-time intraperitoneal injection of resveratrol (100 mg/kg) suppressed 20.0% and 17.2% of food intake during 24 and 48 h, respectively. These results indicated that resveratrol inhibited food intake, which may be related to the downregulation of NPY and AgRP gene expression.

Ginsenosides attenuate kainic acid-induced synaptosomal oxidative stress via stimulation of adenosine A2A receptors in rat hippocampus

Treatment with ginsenosides attenuated KA-induced seizures and oxidative stress in the synaptosome, and reduced synaptic vesicles at the presynaptic terminals dose-dependently. The adenosine A(2A) receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine reversed the ginsenoside-mediated pharmacological actions. Neither the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine nor the adenosine A(2B) receptor antagonist alloxazine affected the ginsenoside-mediated pharmacological actions. Our results suggest that ginsenosides block KA-induced synaptosomal oxidative stress, associated with hippocampal degeneration, through activation of adenosine A(2A) receptors.

Role of TGF-β in Survival of Phagocytizing Microglia: Autocrine Suppression of TNF-α Production and Oxidative Stress

Microglia are recognized as residential macrophageal cells in the brain. Activated microglia play a critical role in removal of dead or damaged cells through phagocytosis activity. During phagocytosis, however, microglia should survive under the harmful condition of self-producing ROS and pro-inflammatory mediators. TGF-β has been known as a classic anti-inflammatory cytokine and controls both initiation and resolution of inflammation by counter-acting inflammatory cytokines. In the present study, to understand the self-protective mechanism, we studied time-dependent change of TNF-α and TGF-β production in microglia phagocytizing opsonized-beads (i.e., polystyrene microspheres). We found that microglia phagocytized opsonized-bead in a time-dependent manner and simultaneously produced both TNF-α and TGF-β. However, while TNF-α production gradually decreased after 6 h, TGF-β production remained at increased level. Microglial cells pre-treated with lipopolysaccharides (a strong immunostimulant, LPS) synergistically increased the production of TNF-α and TGF-β both. However, LPS-pretreated microglia produced TNF-α in a more sustained manner and became more vulnerable, probably due to the marked and sustained production of TNF-α and reduced TGF-β. Intracellular oxidative stress appears to change in parallel with the microglial production of TNF-α. These results indicate TGF-β contributes for the survival of phagocytizing microglia through autocrine suppression of TNF-α production and oxidative stress.

4-hydroxy-2(E)-Nonenal facilitates NMDA-Induced Neurotoxicity via Triggering Mitochondrial Permeability Transition Pore Opening and Mitochondrial Calcium Overload

N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is one of the major causes for neuronal cell death during cerebral ischemic insult. Previously, we reported that the final product of lipid membrane peroxidation 4-hydroxy-2E-nonenal (HNE) synergistically increased NMDA receptor-mediated excitotoxicity (J Neurochem., 2006). In this study, we investigated the mechanism involved in the synergistic neuronal cell death induced by co-treatment with HNE and NMDA. Although neither HNE (1 µM) nor NMDA (2 µM) alone induced the death of cortical neurons, simultaneous treatment of neuronal cells with HNE and NMDA synergistically evoked the death of the cells. However, the synergistic effect on neuronal death was observed only in the presence of calcium. HNE neither increased the cytosolic calcium level ([Ca2+]i) nor altered the NMDA-induced intracellular calcium influx. However, HNE together with NMDA elevated the mitochondrial calcium level and depolarized the mitochondrial transmembrane potential. Furthermore, HNE evoked damage of isolated mitochondria at the cytosolic calcium level (200 nM), which is maximally induced by 2 µM NMDA. Consistently, ATP was depleted in neurons when treated with both HNE and NMDA together. Ciclopirox, a potent inhibitor of mitochondrial permeability transition pore opening (Br. J. Pharmacol., 2005), largely prevented the synergistic damage of mitochondria and death of cortical neurons. Therefore, although low concentrations of HNE and NMDA cannot individually induce neuronal cell death, they can evoke the neuronal cell death by synergistically accelerating mitochondrial dysfunction.

Etoposide Reduces Peroxynitrite-Induced Cytotoxicity via Direct Scavenging Effect

Previously, we reported that glucose-deprived astrocytes are more vulnerable to the cytotoxicity of peroxynitrite, the reaction product of nitric oxide and superoxide anion. The augmented vulnerability of glucose-deprived astrocytes to peroxynitrite cytotoxicity was dependent on their proliferation rate. Inhibition of cell cycle progression has been shown to inhibit the apoptotic cell death occurring in cerebral ischemia-reperfusion. In the present study, we demonstrate that the increased death of glucose-deprived astrocytes by peroxynitrte was largely blocked by the cell cycle phase G2/M transition blocker etoposide. However, the cytoprotective effect of etoposide was not associated with its inhibition of cell cycle progression. Instead, etoposide effectively scavenged peroxynitrite. However, etoposide did not scavenge individual nitric oxide and superoxide anion and it did not prevent the hydrogen peroxide-induced cytotoxicity. The present results indicate that etoposide prevents the toxicity of peroxynitrite in astrocytes by directly scavenging peroxynitrite, not by inhibiting cell cycle progression.