Elsevier

Neurochemistry International

Volume 52, Issues 4–5, March–April 2008, Pages 878-886
Neurochemistry International

Apigenin inhibits the production of NO and PGE2 in microglia and inhibits neuronal cell death in a middle cerebral artery occlusion-induced focal ischemia mice model

https://doi.org/10.1016/j.neuint.2007.10.005Get rights and content

Abstract

Flavonoids have been intensively studied on their pharmacological activities such as anti-cancer, anti-oxidant and anti-inflammation. However, little is known about their neuroprotective effects. Recent studies suggest that inflammation mediated by microglia may play a role in neurodegenerative diseases. In this study, we evaluated the anti-inflammatory effect of various flavonoid compounds by using BV-2, a murine microglia cell line. Of the compounds that were evaluated, apigenin inhibited the production of nitric oxide and prostaglandin E2 by suppressing the expression of inducible nitric oxide synthase and cyclooxygenase-2 protein, respectively. Moreover, apigenin suppressed p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) phosphorylation without affecting the activity of extracellular signal-regulated kinase (ERK). Apigenin was also found to protect neuronal cells from injury in middle cerebral artery occlusion.

Introduction

Although there are various inducing factors in diverse neurodegenerative diseases, neuronal cell death is the final result in the diseases. Recent studies demonstrate that neuroinflammation mediated by microglia contributes to neuronal cell death, which is not restricted to a specific disease but implicated in various diseases such as ischemia, Parkinson's disease and Alzheimer's disease (Gehrmann et al., 1992, Le et al., 2001, Ryu et al., 2002, Stoll and Jander, 1999). It has been proposed that microglia, a resident macrophage-like population of brain cells, plays a role in host defense and tissue repair in the central nervous system (CNS) (Perry and Gordon, 1988). However, overactivated microglia cells produce excessive inflammatory substances such as nitric oxide, various cytokines, and prostaglandins. Accumulation of these proinflammatory and cytotoxic factors is deleterious to neurons (Chao et al., 1992).

In brain hypoxia/ischemia, expression of inducible nitric oxide synthase (iNOS) increases in infiltrating macrophages and activated microglia (Giulian and Lachman, 1985, Nakashima et al., 1995). Some researchers report that iNOS is in part responsible for ischemic injury (del Zoppo et al., 2000, Eliasson et al., 1999), and inhibition of nitric oxide (NO) production protects neuronal cells from damage caused by oxygen and glucose deficiency (Cardenas et al., 1998, Jiang et al., 2005). Therefore, anti-inflammatory agents are thought to be potentially potent neuroprotective drugs.

Flavonoids are abundant in vegetables and fruits and have been extensively studied for their biological activities. It has been reported that flavonoids have anti-oxidant, anti-inflammation and anti-cancer activities (Ishige et al., 2001, Middleton et al., 2000). Furthermore, baicalein was recently found to inhibit LPS and cytokine-stimulated microglia cells and wogonin was reported to protect neuronal cells from ischemic damage by blocking microglial activation (Chen et al., 2004, Lee et al., 2003), thus, suggesting that flavonoids may be developed as neuroprotective agents. However, each compound included in flavonoid family shows different biological activity.

Therefore, it is vital to study the connection between structural features of the flavonoids and the corresponding biological activities. In this study, we investigated the relationship between flavonoid structure and inhibitory activity on NO production in microglia. It has been shown that apigenin (5,7,4′-trihydroxyflavone) possesses anti-inflammatory, anti-carcinogen and free radical scavenging effect (Kim et al., 1998), but little is known about any neuroprotective effects that it may possess. We investigated the neuroprotective effect of apigenin using a murine microglia cell line and middle cerebral arterial occlusion (MCAO).

Section snippets

Cell culture

BV-2 cells, a murine microglia cell line, were maintained in 10 ml of Dulbecco's Modified Eagle's Medium (DMEM, Gibco BRL) supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin (Gibco BRL). Primary microglia cells were cultured from the whole brains of neonatal Sprague–Dawley rats (1 day old). The whole brains were triturated into single cells in DMEM containing 10% FBS and plated into a 75 cm2 T-flask for 2 weeks. The microglia were detached by mild shaking and applied to a

Effect of flavonoids on nitric oxide production in LPS-activated BV-2 cells

To assess the effect of 9 flavonoids (Table 1) on LPS-activated NO production, BV-2 cells were treated with flavonoids with 100 ng/ml LPS. Three groups of flavonoids including flavones (chrysin, apigenin, and luteolin), flavonols (galangin, kaempferol, quercetin, and myricetin) and flavanones (pinocembrin and naringenin) were tested. Five flavonoids (chrysin, apigenin, luteolin, kaempferol, and quercetin) showed inhibitory effects on NO production in dose-dependent manners (Fig. 1). Among tested

Discussion

Microglia are believed to play a major role in host defense and tissue repair in the central nervous system (Kreutzberg, 1996). Nevertheless, activation of microglia is observed in brain injuries (Giulian et al., 1994) and is also induced after the exposure of lipopolysaccharide, interferon-γ or β-amyloid (Zielasek and Hartung, 1996). Microglia activation has been considered as a result of neuronal damage. Also, recently it becomes to recognize as a possible cause of the damage in various

Acknowledgements

This work was supported by the grants (PF0320601-00) from Plant Diversity Research Center of 21st Century Frontier Research Program funded by the Ministry of Science and Technology of The Korean government and by the SRC/ERC program of MOST/KOSEF (R11-2002-103).

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