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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 stimulation exacerbated stroke outcomes and NOD2 deficiency improved stroke outcomes. Wild type (WT) and NOD2 deficient (NOD2-/-) mice were suffered from 2 h occlusion of the middle cerebral artery (MCAO) and 24 h reperfusion. For muramyl dipeptide (MDP) group, MDP, an extrinsic ligand of NOD2 was intraventricularly administered to mice 30 min before MCAO. (A) Neurological deficit scores (B) Representative photographs of coronal brain sections following infarction, stained with 2, 3, 5-triphenyltetrazolium chloride. Red tissue is healthy; white tissue is infarcted. (C) Summary of cerebral infarct volume in brains. The infarct volume was expressed as the percentage of the contralateral hemispheric area. (D) Representative photomicrographs of H&E staining in the cortex and hippocampus. For magnification ×40, scale bars: 200 μm; for magnification ×400, scale bars: 20 μm. Results are representative of eight independent experiments. *P<0.05, compared with indicated groups.
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Figure 3: NOD2 stimulation exacerbated stroke outcomes and NOD2 deficiency improved stroke outcomes. Wild type (WT) and NOD2 deficient (NOD2-/-) mice were suffered from 2 h occlusion of the middle cerebral artery (MCAO) and 24 h reperfusion. For muramyl dipeptide (MDP) group, MDP, an extrinsic ligand of NOD2 was intraventricularly administered to mice 30 min before MCAO. (A) Neurological deficit scores (B) Representative photographs of coronal brain sections following infarction, stained with 2, 3, 5-triphenyltetrazolium chloride. Red tissue is healthy; white tissue is infarcted. (C) Summary of cerebral infarct volume in brains. The infarct volume was expressed as the percentage of the contralateral hemispheric area. (D) Representative photomicrographs of H&E staining in the cortex and hippocampus. For magnification ×40, scale bars: 200 μm; for magnification ×400, scale bars: 20 μm. Results are representative of eight independent experiments. *P<0.05, compared with indicated groups.

Mentions: Next, we determined if the NOD2 signaling pathway was involved in I/R insult. Pretreatment with MDP showed worse neurological dysfunctions (Fig. 3A) and larger infarction volume (Fig. 3B and 3C) as compared with WT mice at 24 h after reperfusion. To further assess the role of NOD2 stimulation in ischemia-evoked neuronal injury, H&E staining was used to show morphological features of injured neurons in hippocampus and cerebral cortex. It was found that the neuronal damage was exacerbated in mice pretreated with MDP (Fig. 3D). Collectively, these results implicate that the activation of NOD2 is detrimental to the brain during ischemia.


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 stimulation exacerbated stroke outcomes and NOD2 deficiency improved stroke outcomes. Wild type (WT) and NOD2 deficient (NOD2-/-) mice were suffered from 2 h occlusion of the middle cerebral artery (MCAO) and 24 h reperfusion. For muramyl dipeptide (MDP) group, MDP, an extrinsic ligand of NOD2 was intraventricularly administered to mice 30 min before MCAO. (A) Neurological deficit scores (B) Representative photographs of coronal brain sections following infarction, stained with 2, 3, 5-triphenyltetrazolium chloride. Red tissue is healthy; white tissue is infarcted. (C) Summary of cerebral infarct volume in brains. The infarct volume was expressed as the percentage of the contralateral hemispheric area. (D) Representative photomicrographs of H&E staining in the cortex and hippocampus. For magnification ×40, scale bars: 200 μm; for magnification ×400, scale bars: 20 μm. Results are representative of eight independent experiments. *P<0.05, compared with indicated groups.
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Figure 3: NOD2 stimulation exacerbated stroke outcomes and NOD2 deficiency improved stroke outcomes. Wild type (WT) and NOD2 deficient (NOD2-/-) mice were suffered from 2 h occlusion of the middle cerebral artery (MCAO) and 24 h reperfusion. For muramyl dipeptide (MDP) group, MDP, an extrinsic ligand of NOD2 was intraventricularly administered to mice 30 min before MCAO. (A) Neurological deficit scores (B) Representative photographs of coronal brain sections following infarction, stained with 2, 3, 5-triphenyltetrazolium chloride. Red tissue is healthy; white tissue is infarcted. (C) Summary of cerebral infarct volume in brains. The infarct volume was expressed as the percentage of the contralateral hemispheric area. (D) Representative photomicrographs of H&E staining in the cortex and hippocampus. For magnification ×40, scale bars: 200 μm; for magnification ×400, scale bars: 20 μm. Results are representative of eight independent experiments. *P<0.05, compared with indicated groups.
Mentions: Next, we determined if the NOD2 signaling pathway was involved in I/R insult. Pretreatment with MDP showed worse neurological dysfunctions (Fig. 3A) and larger infarction volume (Fig. 3B and 3C) as compared with WT mice at 24 h after reperfusion. To further assess the role of NOD2 stimulation in ischemia-evoked neuronal injury, H&E staining was used to show morphological features of injured neurons in hippocampus and cerebral cortex. It was found that the neuronal damage was exacerbated in mice pretreated with MDP (Fig. 3D). Collectively, these results implicate that the activation of NOD2 is detrimental to the brain during ischemia.

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