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Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia¹

HEASUK LEE^*, YOUNG OK KIM, HOCHEOL KIM, SUN YEOU KIM, HAE SOOK NOH^*, SANG SOO KANG^*, GYEONG JAE CHO^*, WAN SUNG CHOI^* and KYOUNGHO SUK^*,2

^* Department of Anatomy and Neurobiology, Research Institute of Natural Science, and Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju, 660-751 Korea; and
Department of Herbal Pharmacology, Graduate School of East-West Medical Science, Kyunghee University, Seoul, 130-701 Korea

²Correspondence: Department of Anatomy and Neurobiology, Gyeongsang National University College of Medicine, 92 Chilam-dong, Jinju, Kyungnam 660-751, Korea. E-mail: ksuk{at}nongae.gsnu.ac.kr

SPECIFIC AIMS

Wogonin (5,7-dihydroxy-8-methoxyflavone) is a flavonoid derived from the root of Scutellaria baicalensis Georgi, a medicinal plant traditionally used in Oriental medicine. Based on the known anti-inflammatory activity of wogonin in macrophages and other cell types in periphery, we tested our hypothesis that wogonin may exert a similar anti-inflammatory effect in brain microglia and may be neuroprotective against brain injury where microglia-mediated inflammatory responses play an important pathogenic role.

PRINCIPAL FINDINGS

1. Flavonoid wogonin inhibited inflammatory activation of cultured brain microglia
Pretreatment of BV-2 mouse microglia cells or rat primary microglia cultures with wogonin (1–50 µM) diminished lipopolysaccharide (LPS) -induced TNF-, IL-1ß, and nitric oxide (NO) production in a dose-dependent manner (Fig. 1 ). Wogonin inhibition of LPS-induced NO production was accompanied by suppression of inducible NO synthase (iNOS) induction and NF-B activation in BV-2 microglia.

View larger version (47K): [in this window] [in a new window]   Figure 1. Wogonin inhibited inflammatory activation of cultured microglia. A) BV-2 cells were treated for 24 h with LPS (100 ng/mL) in the presence of NMMA (0.5 mM) or wogonin (1, 5, 25, 50 µM), then NO production was assessed by Griess reaction. NMMA (NOS inhibitor) was used for comparison. NO production by LPS alone was set to 100%. Results are mean ± SE of 4 independent experiments. Asterisks indicate significant differences from treatment with LPS alone (P<0.05). B, C) Rat primary microglia cultures (B) or BV-2 cells (C) were treated for 72 (B) or 24 h (C) with LPS (100 ng/mL) in the absence or presence of wogonin (50 µM), then production of NO or cytokines was evaluated by Griess reaction or specific ELISA. Cells were pretreated with wogonin for 1 h before LPS stimulation. Asterisks indicate significant differences (P<0.05).

2. Wogonin protected PC12 cells against microglial cytotoxicity
Coculture of microglia and neurons was used to determine whether wogonin inhibition of inflammatory activation of microglia could confer neuroprotection. Wogonin inhibition of microglial activation led to the reduction in microglial cytotoxicity toward cocultured PC12 cells, indicating a neuroprotective role for wogonin in vitro. Wogonin, however, did not influence NO donor-induced PC12 cell death.

3. Wogonin was protective against experimental brain injury in vivo
Wogonin (0.5–10 mg/kg) conferred neuroprotection against experimental brain injury by inhibiting inflammatory activation of microglia in vivo. In transient global ischemia (by 4-vessel occlusion), wogonin attenuated ischemic death of hippocampal neurons and reduced induction of inflammatory mediators such as iNOS and TNF- in hippocampus. Wogonin was also neuroprotective against kainate (30 mg/kg) -induced excitotoxic brain injury (Fig. 2 ). Neuroprotection was accompanied by inhibition of microglial activation as determined by microglia-specific isolectin B4 histochemistry.

View larger version (124K): [in this window] [in a new window]   Figure 2. Neuroprotection by wogonin against kainate-induced excitotoxic brain injury. Compared to extensive hippocampal cell death by administration of kainate (B, E), pretreatment of wogonin (10 mg/kg) prior to kainate injection (C, F) afforded neuroprotection by attenuating hippocampal cell death both in CA1 and CA3. Neuronal loss or damage is not visible in saline-administered control animals (A, D). Panels D, E, and F (x200) are CA3 regions of panels A, B, and C (x40) as indicated by arrowheads, respectively. Photomicrographs are representative results of Cresyl violet staining performed 2 days after kainate injection.

CONCLUSIONS AND SIGNIFICANCE

In this work, we found that wogonin is a potent neuroprotector from natural source. Wogonin inhibited inflammatory activation of cultured brain microglia in vitro and provided neuroprotection in microglia/PC12 coculture by mitigating microglia-induced PC12 cell death. The neuroprotective effect of wogonin was further demonstrated in vivo using two experimental brain injury models; transient global ischemia by 4-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. 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.

In neurodegenerative diseases, a pathogenic role of uncontrolled microglial activation is widely accepted. In search of neuroprotective agents, it is time to focus on killer cells (microglia) instead of killed cells (neurons); eliminating or at least suppressing killer microglial activation will provide a better chance for neuroprotection compared with just salvaging dying neurons. Now, our current work identified a potent neuroprotector from natural source that inhibits the killer cell activity. Our work will certainly instigate further investigations in the related areas, which will ultimately lead to the successful development of novel neuroprotective drugs based on wogonin or other constituents of the medicinal herb Scutellaria baicalensis.

View larger version (23K): [in this window] [in a new window]   Figure 3. Microglia as a target of wogonin action. Resting microglia (ramified type) can be activated by inflammatory stimuli such as LPS and IFN-. Activated microglia (amoeboid type) produce a variety of inflammatory mediators, including nitric oxide, TNF-, and IL-1ß, which cause neuronal injury (thereby resulting in neurodegeneration). Flavonoid wogonin may be neuroprotective by inhibiting the inflammatory activation of microglia.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0057fje; doi: 10.1096/fj.03-0057fje

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This Article Full Text (PDF) All Versions of this Article: 17/13/1943 03-0057fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by LEE, H. Articles by SUK, K. Search for Related Content PubMed PubMed Citation Articles by LEE, H. Articles by SUK, K.