Hocheol Kim

Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia

Wogonin (5,7‐dihydroxy‐8‐methoxyflavone), a flavonoid originated from the root of a medicinal herb Scutellaria baicalensis Georgi, has been previously shown to have anti‐inflammatory activities in various cell types including macrophages. In this work, we have found that wogonin is a potent neuroprotector from natural source. Wogonin inhibited inflammatory activation of cultured brain microglia by diminishing lipopolysaccharide‐induced tumor necrosis factor‐α (TNF‐α), interleukin‐1β, and nitric oxide (NO) production. Wogonin inhibited NO production by suppressing inducible NO synthase (iNOS) induction and NF‐κB activation in microglia. Inhibition of inflammatory activation of microglia by wogonin led to the reduction in microglial cytotoxicity toward cocultured PC12 cells, supporting a neuroprotective role for wogonin in vitro. The neuroprotective effect of wogonin was further demonstrated in vivo using two experimental brain injury models; transient global ischemia by four‐vessel occlusion and excitotoxic injury by systemic kainate injection. In both animal models, wogonin conferred neuroprotection by attenuating the death of hippocampal neurons, and the neuroprotective effect was associated with inhibition of the inflammatory activation of microglia. Hippocampal induction of inflammatory mediators such as iNOS and TNF‐α was reduced by wogonin in the global ischemia model, and microglial activation was markedly down‐regulated by wogonin in the kainate injection model as judged by microglia‐specific isolectin B4 staining. Taken together, our results indicate that wogonin exerts its neuroprotective effect by inhibiting microglial activation, which is a critical component of pathogenic inflammatory responses in neurodegenerative diseases. The current study emphasizes the importance of medicinal herbs and their constituents as an invaluable source for the development of novel neuroprotective drugs.

Cultures of astrocytes and microglia express interleukin 18

Interleukin 18 (IL-18 or interferon-gamma inducing factor) is a recently discovered pro-inflammatory cytokine and powerful stimulator of the cell-mediated immune response. IL-18 is produced by several sources including monocytes/macrophages, keratinocytes and the zona reticularis and zona fasciculata of the adrenal cortex. IL-18 occurs in brain but its cellular source in the CNS has never been investigated. The presence of IL-18 and its response to stimulation in the brain was tested with primary cultures of microglia, astrocytes and hippocampal neurons. IL-18 mRNA was present in astrocytes and microglia, but not in neurons. The endotoxin lipopolysaccharide (LPS) did not affect IL-18 in astrocytes, but LPS robustly increased IL-18 mRNA in microglia. IL-18 protein was constitutively expressed in astrocytes and induced in microglia by LPS. The levels of interleukin-1beta converting enzyme (ICE), an activating enzyme, and caspase 3 (CPP32), an inactivating enzyme, were assessed to investigate the presence of the appropriate processing enzymes in the cultured cells. ICE was present at constitutive levels in microglia and astrocytes suggesting that these cell types may produce and secrete matured IL-18. Active forms of CPP32 were not detectable in either cell type indicating the absence of a degradative pathway of IL-18. The present results demonstrate that microglia and astrocytes are sources of brain IL-18 and add a new member to the family of cytokines produced in the brain.

Anti-inflammatory effects of Scutellaria baicalensis water extract on LPS-activated RAW 264.7 macrophages

AIM OF THE STUDY The root of Scutellaria baicalensis Georgi (Labiatae), also known as Scutellariae Radix, possesses anticancer, antiviral, and anti-inflammatory properties. And it is one of the most widespread herbal remedies used in Oriental medicine. In the present study, we investigated the effects of Scutellariae Radix water extract (SR) on proinflammatory mediators secreted from lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. MATERIAL AND METHODS Cell viability was assessed by MTT assay and nitric oxide (NO) concentration in the cultured medium was determined by the Griess reaction. Various Cytokines released from LPS-induced Raw 264.7 cells were measured in the cell culture supernatants using a multiplex bead array assay based on xMAP technology. RESULTS We found that SR significantly inhibited the production of NO, interleukin (IL)-3, IL-6, IL-10, IL-12p40, IL-17, interferon-inducible protein (IP)-10, keratinocyte-derived chemokine (KC), and vascular endothelial growth factor (VEGF) in LPS-induced RAW 264.7 cells at the concentrations of 25, 50, 100, 200 microg/ml (p<0.05). CONCLUSIONS These results suggest that SR has anti-inflammatory activity related with its inhibition of NO, cytokine, chemokine, and growth factor production in macrophages.

Hypoxia induces nitric oxide production in mouse microglia via p38 mitogen-activated protein kinase pathway

In vitro exposure of microglial cells to hypoxia induces cellular activation. Also, in vivo studies of glial activation following ischemic hypoxia have shown that neuronal cell death is followed by microglial activation. Thus, it is likely that toxic inflammatory mediators produced by activated microglial cells under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. Nitric oxide (NO), which is known to be produced by activated microglia, may participate in this process. In the current work, we sought to determine whether and how the production of NO and the expression of inducible NO synthase (iNOS) are triggered by hypoxia in microglial cells. Exposure of established microglial cell lines as well as primary mouse microglial cultures to mild hypoxia (8 h) followed by reoxygenation (24 h) induced the production of NO and TNFalpha, indicating that hypoxia could lead to the inflammatory activation of microglia. Hypoxic induction of NO was accompanied by iNOS induction. Moreover, hypoxia induced the activation of p38 MAPK, but not ERK or JNK/SAPK, in BV-2 mouse microglial cells. SB203580, a specific inhibitor of p38 MAPK, blocked the hypoxic induction of NO and iNOS. Taken together, our results indicated that hypoxia could induce inflammatory activation of microglia, and the hypoxic induction of NO production in microglia is mediated through p38 MAPK pathway. Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but may also promote neuronal injury indirectly via microglial activation.

The plant flavonoid wogonin suppresses death of activated C6 rat glial cells by inhibiting nitric oxide production

Flavonoids are a group of low molecular weight polyphenolic compounds derived from plants. 5,7-dihydroxy-8-methoxyflavone (Wogonin), a flavonoid originated from the root of Scutellaria baicalensis Georgi, has been shown to exert various anti-inflammatory effects such as inhibition of nitric oxide (NO) and prostaglandin E2 production in macrophages. Because glial cells have been previously shown to undergo NO-dependent apoptosis upon inflammatory activation and this auto-regulatory process may be negatively affected by exogenous factors possessing anti-inflammatory activities, we examined the effects of wogonin on NO production and activation-induced cell death of C6 rat glial cells. Activation of C6 glial cells with lipopolysaccharide (LPS), interferon-gamma, and tumor necrosis factor-alpha induced NO production followed by cell death. Pretreatment of C6 cells with wogonin before LPS and cytokine treatment dose-dependently inhibited NO production as well as death of activated C6 cells. Wogonin-mediated inhibition of NO production was accompanied by suppression of inducible nitric oxide synthase (iNOS) protein induction and nuclear factor kappa B (NF-kappaB) reporter activity. Wogonin, however, did not affect a NO donor-induced cytotoxicity. Taken together, our results indicate that wogonin inhibits activation-induced death of C6 glial cells by suppressing NO production, and these inhibitory effects of wogonin on NO production are exerted through inhibition of NF-kappaB-mediated iNOS induction.

Cytoprotective effect of Scutellaria baicalensis in CA1 hippocampal neurons of rats after global cerebral ischemia

Based on the use of Scutellaria baicalensis for the treatment of stroke in traditional Oriental medicine, the current study was carried out to evaluate neuroprotective effects of S. baicalensis after transient global ischemia using rat 4-vessel occlusion model. Methanol extracts from the dried roots of S. baicalensis (0.1-10 mg/kg) administered intra-peritoneally significantly protected CA1 neurons against 10 min transient forebrain ischemia as demonstrated by measuring the density of neuronal cells stained with Cresyl violet. Methanol extract of S. baicalensis inhibited microglial tumor necrosis factor-alpha (TNF-alpha) and nitric oxide production, and protected PC12 cells from hydrogen peroxide-induced toxicity in vitro.

Neuroprotective herbs for stroke therapy in traditional eastern medicine

Abstract Traditional Eastern Medicine (TEM) has a long history in stroke therapy and its therapeutic efficacy has been confirmed by clinical studies. Extensive experience and abundant clinical data on TEM in stroke treatment have been accumulated over the past thousand years. Basic and clinical research in TEM constitutes a potentially rich source for new drug discovery and development with the integration of TEM and Western pharmacology. In recent years, many attempts have been made to document research data from extracts of composite formulas, single herbs, or single compounds from TEM herbs, according to orthodox pharmacological actions. This article reviews herbs and prescriptions that have been documented to have a neuroprotective effect in in vitro and in vivo ischemic model systems, and the neuroprotective compounds isolated from them. I also discuss the neuroprotective mechanisms of prescriptions, herbs, and single compounds relevant to the treatment of brain ischemia, including anti–oxidant, anti–excitotoxic, and anti–inflammatory effects.

Oleic acid reduces lipopolysaccharide-induced expression of iNOS and COX-2 in BV2 murine microglial cells: Possible involvement of reactive oxygen species, p38 MAPK, and IKK/NF-κB signaling pathways

Microglia are the major cells involved in neuroinflammation resulting in brain tissue damage during infection and neurodegenerative diseases. In this study, we examined the effects of the monounsaturated fatty acid oleic acid (OA) on LPS-induced proinflammatory mediators production and the mechanisms involved in BV2 microglia. OA inhibited LPS-induced expression of iNOS and COX-2 as well as production of NO and prostaglandin E2. We showed that OA blocked LPS-induced NF-kappaB activation and phosphorylation of inhibitor kappaB kinase (IKK). We also showed that OA inhibited LPS-induced phosphorylation of Akt and p38 MAPK, but not that of ERK. Finally, we showed that OA reduced reactive oxygen species (ROS) accumulation and an anti-oxidant N-acetylcysteine inhibited NF-kappaB transactivation and phosphorylation of IKK and Akt in LPS-stimulated BV2 cells. Taken together, our results suggest that OA shows an anti-inflammatory effect by inhibiting ROS, p38 MAPK, and Akt/IKK/NF-kappaB signaling pathways in LPS-stimulated BV2 microglia.

Activation-induced cell death of rat astrocytes

Inflammatory activation of astrocytes has been implicated in various neurodegenerative diseases. The elimination of activated astrocytes by apoptosis or the deactivation may be the mechanisms for auto-regulation of activated astrocytes. To test the possibility of apoptotic elimination of activated astrocytes, we examined a potential correlation between activation state of astrocytes and their viability using C6 rat glial cells and rat primary astrocyte cultures exposed to a variety of inflammatory stimuli such as lipopolysaccharide, interferon-gamma, and tumor necrosis factor-alpha. Nitric oxide production was measured to evaluate inflammatory activation of astrocytes. We found that: (i) the activation of astrocytes by the combination of lipopolysaccharide and inflammatory cytokines, but not by either alone, led to nitric oxide production followed by apoptotic cell death; (ii) the amount of nitric oxide produced by activated astrocytes was inversely proportional to the viability of the cells; (iii) inhibition of nitric oxide synthase by N-monomethyl L-arginine blocked death of activated astrocytes; and (iv) nitric oxide donors induced apoptosis of astrocytes in a caspase-dependent manner. Taken collectively, our results suggest that activated astrocytes produce nitric oxide as an autocrine mediator of caspase-dependent apoptosis, and this type of programmed cell death of astrocytes may be the underlying mechanism for the auto-regulation of inflammatory activation of astrocytes.

Neuroprotection by methanol extract of Uncaria rhynchophylla against global cerebral ischemia in rats

In traditional Oriental medicine, Uncaria rhynchophylla has been used to lower blood pressure and to relieve various neurological symptoms. However, scientific evidence related to its effectiveness or precise modes of action has not been available. Thus, in the current study, we evaluated neuroprotective effects of U. rhynchophylla after transient global ischemia using 4-vessel occlusion model in rats. Methanol extract of U. rhynchophylla administered intraperitoneally (100-1000 mg/kg at 0 and 90 min after reperfusion) significantly protected hippocampal CA1 neurons against 10 min transient forebrain ischemia. Measurement of neuronal cell density in CA1 region at 7 days after ischemia by Nissl staining revealed more than 70% protection in U. rhynchophylla-treated rats compared to saline-treated animals. In U. rhynchophylla-treated animals, induction of cyclooxygenase-2 in hippocampus at 24 hr after ischemia was significantly inhibited at both mRNA and protein levels. Furthermore, U. rhynchophylla extract inhibited TNF-alpha and nitric oxide production in BV-2 mouse microglial cells in vitro. These anti-inflammatory actions of U. rhynchophylla extract may contribute to its neuroprotective effects.