title: A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system.
abstract: BACKGROUND
  Toll-like receptor 4 (TLR4) plays a pivotal role in the pathophysiology of stroke-induced inflammation. Both astroglia and microglia express TLR4, and endogenous ligands produced in the ischemic brain induce inflammatory responses. Reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines produced by TLR4 activation play harmful roles in neuronal damage after stroke. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose phosphate pathway (PPP), reducing oxidative stress by glutathione peroxidase. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP, using TLR4 stimulation and hypoxia in concert with microglia.
  
  
  METHODS
  In vitro experiments were performed using cells prepared from Sprague-Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using L-leucine methyl ester (LME). Cells were exposed to LPS (0.01 μg/mL) or hypoxia (1 % O2) for 12-15 h. PPP activity was measured using [1-(14)C]glucose and [6-(14)C]glucose. ROS and NO production were measured using 2',7'-dichlorodihydrofluorescein diacetate and diaminofluorescein-FM diacetate, respectively. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2), a cardinal transcriptional factor under stress conditions that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP, was evaluated using immunohistochemistry.
  
  
  RESULTS
  Cultured astroglia exposed to LPS elicited 20 % increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. LPS enhanced astroglial ROS production after glutathione depletion. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial-microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. Hypoxia induced astroglial PPP activation independent of the microglia-NO pathway. Elimination of ROS and NO production by sulforaphane, a natural Nrf2 activator, confirmed the astroglial protective mechanism.
  
  
  CONCLUSIONS
  Astroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke.
authors.affiliation: Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582, Tokyo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Iizumi
authors.affiliation: Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582, Tokyo, Japan. takashin@tka.att.ne.jp.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582, Tokyo, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Mashima
authors.affiliation: Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582, Tokyo, Japan.
authors.forename: Kazushi
authors.initials: K
authors.lastname: Minami
authors.affiliation: Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582, Tokyo, Japan.
authors.forename: Yoshikane
authors.initials: Y
authors.lastname: Izawa
authors.affiliation: Department of Neurology, Osaka City University Graduate School of Medicine, Osaka-shi, 545-8585, Osaka , Japan.
authors.forename: Takato
authors.initials: T
authors.lastname: Abe
authors.affiliation: Department of Biochemistry, Keio University School of Medicine, Shinjuku-ku, 160-8582, Tokyo, Japan.|Clinical and Translational Research Center, Keio University School of Medicine, Shinjuku-ku, 160-8582, Tokyo, Japan.|JST Exploratory Research for Advanced Technology (ERATO) Suematsu Gas Biology Project, Shinjuku-ku, 160-8582, Tokyo , Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Hishiki
authors.affiliation: Department of Biochemistry, Keio University School of Medicine, Shinjuku-ku, 160-8582, Tokyo, Japan.|JST Exploratory Research for Advanced Technology (ERATO) Suematsu Gas Biology Project, Shinjuku-ku, 160-8582, Tokyo , Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Suematsu
authors.affiliation: Department of Biochemistry, Keio University School of Medicine, Shinjuku-ku, 160-8582, Tokyo, Japan.|JST Exploratory Research for Advanced Technology (ERATO) Suematsu Gas Biology Project, Shinjuku-ku, 160-8582, Tokyo , Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Kajimura
authors.affiliation: Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582, Tokyo, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D000072019:Kelch-Like ECH-Associated Protein 1 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D017628:Microglia / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D010427:Pentose Phosphate Pathway / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D021241:Spectrometry, Mass, Electrospray Ionization; D020521:Stroke / Q000378:metabolism*
chemical_list: C486610:KEAP1 protein, rat; D000072019:Kelch-Like ECH-Associated Protein 1; D008070:Lipopolysaccharides; D051267:NF-E2-Related Factor 2; C495637:Nfe2l2 protein, rat; D017382:Reactive Oxygen Species; D009569:Nitric Oxide
keywords: Ara-C (cytosine β-D-arabinofuranoside hydrochloride); Astrocyte; LME (L-leucine methyl ester); RNS (reactive nitrogen species); ROS (reactive oxygen species); TLR4 (Toll-like receptor 4)