A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system

Takuya Iizumi, Shinichi Takahashi, Kyoko Mashima, Kazushi Minami, Yoshikane Izawa, Takato Abe, Takako Hishiki, Makoto Suematsu, Mayumi Kajimura, Norihiro Suzuki

Research output: Contribution to journalArticle

21 Citations (Scopus)

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 [1-14C]glucose and [6-14C]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.

Original languageEnglish
Article number99
JournalJournal of Neuroinflammation
Volume13
Issue number1
DOIs
Publication statusPublished - 2016 May 4

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Pentose Phosphate Pathway
Microglia
Lipopolysaccharides
Nitric Oxide
Toll-Like Receptor 4
Astrocytes
Reactive Oxygen Species
Oxidative Stress
Stroke
Glucose
Glucosephosphate Dehydrogenase
Glutathione Peroxidase
Mitogen-Activated Protein Kinases
Glutathione
Sprague Dawley Rats
Immunohistochemistry
Cytokines
Ligands
Inflammation

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)

ASJC Scopus subject areas

  • Neuroscience(all)
  • Cellular and Molecular Neuroscience
  • Neurology
  • Immunology

Cite this

A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system. / Iizumi, Takuya; Takahashi, Shinichi; Mashima, Kyoko; Minami, Kazushi; Izawa, Yoshikane; Abe, Takato; Hishiki, Takako; Suematsu, Makoto; Kajimura, Mayumi; Suzuki, Norihiro.

In: Journal of Neuroinflammation, Vol. 13, No. 1, 99, 04.05.2016.

Research output: Contribution to journalArticle

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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 [1-14C]glucose and [6-14C]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.",
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T1 - A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system

AU - Iizumi, Takuya

AU - Takahashi, Shinichi

AU - Mashima, Kyoko

AU - Minami, Kazushi

AU - Izawa, Yoshikane

AU - Abe, Takato

AU - Hishiki, Takako

AU - Suematsu, Makoto

AU - Kajimura, Mayumi

AU - Suzuki, Norihiro

PY - 2016/5/4

Y1 - 2016/5/4

N2 - 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 [1-14C]glucose and [6-14C]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.

AB - 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 [1-14C]glucose and [6-14C]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.

KW - Ara-C (cytosine β-d-arabinofuranoside hydrochloride)

KW - Astrocyte

KW - LME (l-leucine methyl ester)

KW - RNS (reactive nitrogen species)

KW - ROS (reactive oxygen species)

KW - TLR4 (Toll-like receptor 4)

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