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NOD2 is involved in the inflammatory response after cerebral ischemia-reperfusion injury and triggers NADPH oxidase 2-derived reactive oxygen species.

Liu H, Wei X, Kong L, Liu X, Cheng L, Yan S, Zhang X, Chen L - Int. J. Biol. Sci. (2015)

Bottom Line: Our results showed that NOD2 expression was significantly increased in microglia and astrocytes in response to the I/R insult.Genetic ablation of the NOD2 gene significantly improved stroke outcomes and reduced inflammation, as evidenced by a lower expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNFα in conjunction with attenuated activation of nuclear factor κB (NF-κB), p38 mitogen activated protein kinases (MAPK) and JNK.Pharmacological targeting of NOD2-mediated inflammatory response at multiple levels may help design a new approach to develop therapeutic strategies for prevention of deterioration of cerebral function and for the treatment of stroke.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Pharmacology, School of Medicine, Shandong University, Wenhua West Road 44, Jinan, Shandong 250012, P.R. China.

ABSTRACT

Background: Increasing evidences suggest that innate immunity is involved in cerebral ischemia-reperfusion (I/R) injury, but the liable innate immune receptors have not been completely elucidated. Here, we explored the role of the nucleotide-binding oligomerization domain (NOD)2, a member of the cytosolic NOD-like receptor family, in acute focal cerebral I/R injury.

Methods: An in vivo middle cerebral artery occlusion (MCAO) model that in wild type (WT) and NOD2 deficient (NOD2(-/-)) mice and in vitro model of oxygen glucose deprivation and reoxygenation (OGD/R) in cultured primary microglia and astrocytes were used to investigate the expression of NOD2 and explore the roles of NOD2 in ischemic stroke.

Results: Our results showed that NOD2 expression was significantly increased in microglia and astrocytes in response to the I/R insult. Pretreatment with muramyl dipeptide, an extrinsic ligand of NOD2, significantly increased the infarct volume and neurological dysfunction in mice subjected to MCAO. Genetic ablation of the NOD2 gene significantly improved stroke outcomes and reduced inflammation, as evidenced by a lower expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNFα in conjunction with attenuated activation of nuclear factor κB (NF-κB), p38 mitogen activated protein kinases (MAPK) and JNK. Moreover, NOD2 deficiency prevented the upregulation of the NADPH oxidase (NOX) 2 and ROS generation induced by I/R. Mechanistically, NOD2-induced production of IL-6 in primary cultured microglia was mediated through activation of NOX2.

Conclusions: This study showed the contribution of NOD2 to inflammatory response and provided direct evidence that NOX2-mediated oxidative stress as an important target molecule linked NOD2 to inflammatory damage in ischemic stroke. Pharmacological targeting of NOD2-mediated inflammatory response at multiple levels may help design a new approach to develop therapeutic strategies for prevention of deterioration of cerebral function and for the treatment of stroke.

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NOD2 deficiency inhibited NF-κB and MAPK signaling activation after cerebral ischemia-reperfusion injury. (A and C) p-p65, IκBα, p-ERK , p-JNK and p-p38 MAPK protein expressions in the penumbral cortex from wild type (WT) mice at 2 h, 6 h, 12 h, 24 h and 48 h after reperfusion. (B and D) p-p65, IκBα, p-ERK , p-JNK and p-p38 MAPK protein expressions in the penumbral cortex from WT and NOD2-/- mice at 24 h after reperfusion. Statistical results from the densitometric measurements after normalization were calculated as the mean ± S.E.M. Results are representative of six independent experiments. * P<0.05, vs. sham-operated WT mice; # P<0.05, vs. sham-operated NOD2-/- mice; $ P<0.05, vs. I/R WT mice.
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Figure 5: NOD2 deficiency inhibited NF-κB and MAPK signaling activation after cerebral ischemia-reperfusion injury. (A and C) p-p65, IκBα, p-ERK , p-JNK and p-p38 MAPK protein expressions in the penumbral cortex from wild type (WT) mice at 2 h, 6 h, 12 h, 24 h and 48 h after reperfusion. (B and D) p-p65, IκBα, p-ERK , p-JNK and p-p38 MAPK protein expressions in the penumbral cortex from WT and NOD2-/- mice at 24 h after reperfusion. Statistical results from the densitometric measurements after normalization were calculated as the mean ± S.E.M. Results are representative of six independent experiments. * P<0.05, vs. sham-operated WT mice; # P<0.05, vs. sham-operated NOD2-/- mice; $ P<0.05, vs. I/R WT mice.

Mentions: The NF-κB signaling pathway plays an important role in the inflammatory process following the I/R injury 24. We assessed the I/R-induced activation of NF-κB in the absence of NOD2. As shown in Fig. 5A, the NF-κB pathway was markedly activated as evidenced by the phosphorylation of NF-κB subunit p65 and degradation in IκBα in the brain of WT mice in response to I/R insult. These changes in the NF-κB pathway were significantly inhibited in NOD2-/- mice subjected to 2 h of MCAO and 24 h of reperfusion (Fig. 5B). We also assessed I/R-injury induced changes in the MAPKs signaling pathway, including extracellular signal-regulated kinase (ERK) 1/2, Jun N-terminal kinase (JNK) and p38 MAPK. The results showed that cerebral I/R also resulted in strong phosphorylations of ERK1/2, JNK and p38 MAPK in the WT mice (Fig. 5C). However, I/R-induced activations of JNK and p38 MAPK were remarkably suppressed in NOD2-/- mice, and the activation of ERK was obviously unaffected (Fig. 5D).


NOD2 is involved in the inflammatory response after cerebral ischemia-reperfusion injury and triggers NADPH oxidase 2-derived reactive oxygen species.

Liu H, Wei X, Kong L, Liu X, Cheng L, Yan S, Zhang X, Chen L - Int. J. Biol. Sci. (2015)

NOD2 deficiency inhibited NF-κB and MAPK signaling activation after cerebral ischemia-reperfusion injury. (A and C) p-p65, IκBα, p-ERK , p-JNK and p-p38 MAPK protein expressions in the penumbral cortex from wild type (WT) mice at 2 h, 6 h, 12 h, 24 h and 48 h after reperfusion. (B and D) p-p65, IκBα, p-ERK , p-JNK and p-p38 MAPK protein expressions in the penumbral cortex from WT and NOD2-/- mice at 24 h after reperfusion. Statistical results from the densitometric measurements after normalization were calculated as the mean ± S.E.M. Results are representative of six independent experiments. * P<0.05, vs. sham-operated WT mice; # P<0.05, vs. sham-operated NOD2-/- mice; $ P<0.05, vs. I/R WT mice.
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Figure 5: NOD2 deficiency inhibited NF-κB and MAPK signaling activation after cerebral ischemia-reperfusion injury. (A and C) p-p65, IκBα, p-ERK , p-JNK and p-p38 MAPK protein expressions in the penumbral cortex from wild type (WT) mice at 2 h, 6 h, 12 h, 24 h and 48 h after reperfusion. (B and D) p-p65, IκBα, p-ERK , p-JNK and p-p38 MAPK protein expressions in the penumbral cortex from WT and NOD2-/- mice at 24 h after reperfusion. Statistical results from the densitometric measurements after normalization were calculated as the mean ± S.E.M. Results are representative of six independent experiments. * P<0.05, vs. sham-operated WT mice; # P<0.05, vs. sham-operated NOD2-/- mice; $ P<0.05, vs. I/R WT mice.
Mentions: The NF-κB signaling pathway plays an important role in the inflammatory process following the I/R injury 24. We assessed the I/R-induced activation of NF-κB in the absence of NOD2. As shown in Fig. 5A, the NF-κB pathway was markedly activated as evidenced by the phosphorylation of NF-κB subunit p65 and degradation in IκBα in the brain of WT mice in response to I/R insult. These changes in the NF-κB pathway were significantly inhibited in NOD2-/- mice subjected to 2 h of MCAO and 24 h of reperfusion (Fig. 5B). We also assessed I/R-injury induced changes in the MAPKs signaling pathway, including extracellular signal-regulated kinase (ERK) 1/2, Jun N-terminal kinase (JNK) and p38 MAPK. The results showed that cerebral I/R also resulted in strong phosphorylations of ERK1/2, JNK and p38 MAPK in the WT mice (Fig. 5C). However, I/R-induced activations of JNK and p38 MAPK were remarkably suppressed in NOD2-/- mice, and the activation of ERK was obviously unaffected (Fig. 5D).

Bottom Line: Our results showed that NOD2 expression was significantly increased in microglia and astrocytes in response to the I/R insult.Genetic ablation of the NOD2 gene significantly improved stroke outcomes and reduced inflammation, as evidenced by a lower expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNFα in conjunction with attenuated activation of nuclear factor κB (NF-κB), p38 mitogen activated protein kinases (MAPK) and JNK.Pharmacological targeting of NOD2-mediated inflammatory response at multiple levels may help design a new approach to develop therapeutic strategies for prevention of deterioration of cerebral function and for the treatment of stroke.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Pharmacology, School of Medicine, Shandong University, Wenhua West Road 44, Jinan, Shandong 250012, P.R. China.

ABSTRACT

Background: Increasing evidences suggest that innate immunity is involved in cerebral ischemia-reperfusion (I/R) injury, but the liable innate immune receptors have not been completely elucidated. Here, we explored the role of the nucleotide-binding oligomerization domain (NOD)2, a member of the cytosolic NOD-like receptor family, in acute focal cerebral I/R injury.

Methods: An in vivo middle cerebral artery occlusion (MCAO) model that in wild type (WT) and NOD2 deficient (NOD2(-/-)) mice and in vitro model of oxygen glucose deprivation and reoxygenation (OGD/R) in cultured primary microglia and astrocytes were used to investigate the expression of NOD2 and explore the roles of NOD2 in ischemic stroke.

Results: Our results showed that NOD2 expression was significantly increased in microglia and astrocytes in response to the I/R insult. Pretreatment with muramyl dipeptide, an extrinsic ligand of NOD2, significantly increased the infarct volume and neurological dysfunction in mice subjected to MCAO. Genetic ablation of the NOD2 gene significantly improved stroke outcomes and reduced inflammation, as evidenced by a lower expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNFα in conjunction with attenuated activation of nuclear factor κB (NF-κB), p38 mitogen activated protein kinases (MAPK) and JNK. Moreover, NOD2 deficiency prevented the upregulation of the NADPH oxidase (NOX) 2 and ROS generation induced by I/R. Mechanistically, NOD2-induced production of IL-6 in primary cultured microglia was mediated through activation of NOX2.

Conclusions: This study showed the contribution of NOD2 to inflammatory response and provided direct evidence that NOX2-mediated oxidative stress as an important target molecule linked NOD2 to inflammatory damage in ischemic stroke. Pharmacological targeting of NOD2-mediated inflammatory response at multiple levels may help design a new approach to develop therapeutic strategies for prevention of deterioration of cerebral function and for the treatment of stroke.

Show MeSH
Related in: MedlinePlus