Limits...
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.

Show MeSH

Related in: MedlinePlus

NOD2 deficiency inhibited NADPH oxidase activity and NOX2 expression after cerebral ischemia-reperfusion injury. (A) Summarized data showing NADPH oxidase activity (reactive oxygen species production) in the penumbral cortex from WT mice after 2 h MCAO and 2 h, 6 h, 12 h, 24 h and 48 h reperfusion. (B) Western blot analysis of NOX2 protein levels in the penumbral cortex from WT mice after 2 h MCAO and 2 h, 6 h, 12 h, 24 h, 48 h reperfusion. (C) Summarized data showing NADPH oxidase activity in the penumbral cortex from NOD2 deficiency mice at 24 h after reperfusion. (D) Western blot analysis of NOX2 protein levels in the penumbral cortex from NOD2 deficiency mice at 24 h after reperfusion. 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4400384&req=5

Figure 6: NOD2 deficiency inhibited NADPH oxidase activity and NOX2 expression after cerebral ischemia-reperfusion injury. (A) Summarized data showing NADPH oxidase activity (reactive oxygen species production) in the penumbral cortex from WT mice after 2 h MCAO and 2 h, 6 h, 12 h, 24 h and 48 h reperfusion. (B) Western blot analysis of NOX2 protein levels in the penumbral cortex from WT mice after 2 h MCAO and 2 h, 6 h, 12 h, 24 h, 48 h reperfusion. (C) Summarized data showing NADPH oxidase activity in the penumbral cortex from NOD2 deficiency mice at 24 h after reperfusion. (D) Western blot analysis of NOX2 protein levels in the penumbral cortex from NOD2 deficiency mice at 24 h after reperfusion. 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: It has been demonstrated that intracellular ROS is required in TLRs - dependent inflammatory responses 25-27. However, it is unclear whether NOD2 signaling mediates ROS production and whether this mechanism contributes to cerebral I/R injury. In an in vivo study, consistent with the up-regulation of NOD2, the ROS production and expression of NADPH oxidase subunit NOX2 were dramatically increased after reperfusion (Fig. 6A and 6B). Interestingly, we found that the expression and activation of NOX2 were significantly inhibited in the NOD2-/- mice compared to WT mice (Fig. 6C and 6D). In an in vitro study, WT microglia was treated with MDP, a selective agonistic ligand of NOD2, and we found that MDP significantly induced NOX2 expression and ROS production in a time-dependent manner (Fig. 7A and 7B). To elucidate the role of NADPH oxidase on the NOD2 induced IL-6 production further, a well-known NADPH oxidase activity inhibitor, apocynin was used in this study. As shown in Fig. 7C, IL-6 production was markedly enhanced by OGD and MDP, and apocynin significantly inhibited the increase of IL-6 induced by OGD and MDP. The results suggest that NADPH oxidase activation is involved in the NOD2-mediated inflammatory response after cerebral I/R injury.


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 NADPH oxidase activity and NOX2 expression after cerebral ischemia-reperfusion injury. (A) Summarized data showing NADPH oxidase activity (reactive oxygen species production) in the penumbral cortex from WT mice after 2 h MCAO and 2 h, 6 h, 12 h, 24 h and 48 h reperfusion. (B) Western blot analysis of NOX2 protein levels in the penumbral cortex from WT mice after 2 h MCAO and 2 h, 6 h, 12 h, 24 h, 48 h reperfusion. (C) Summarized data showing NADPH oxidase activity in the penumbral cortex from NOD2 deficiency mice at 24 h after reperfusion. (D) Western blot analysis of NOX2 protein levels in the penumbral cortex from NOD2 deficiency mice at 24 h after reperfusion. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4400384&req=5

Figure 6: NOD2 deficiency inhibited NADPH oxidase activity and NOX2 expression after cerebral ischemia-reperfusion injury. (A) Summarized data showing NADPH oxidase activity (reactive oxygen species production) in the penumbral cortex from WT mice after 2 h MCAO and 2 h, 6 h, 12 h, 24 h and 48 h reperfusion. (B) Western blot analysis of NOX2 protein levels in the penumbral cortex from WT mice after 2 h MCAO and 2 h, 6 h, 12 h, 24 h, 48 h reperfusion. (C) Summarized data showing NADPH oxidase activity in the penumbral cortex from NOD2 deficiency mice at 24 h after reperfusion. (D) Western blot analysis of NOX2 protein levels in the penumbral cortex from NOD2 deficiency mice at 24 h after reperfusion. 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: It has been demonstrated that intracellular ROS is required in TLRs - dependent inflammatory responses 25-27. However, it is unclear whether NOD2 signaling mediates ROS production and whether this mechanism contributes to cerebral I/R injury. In an in vivo study, consistent with the up-regulation of NOD2, the ROS production and expression of NADPH oxidase subunit NOX2 were dramatically increased after reperfusion (Fig. 6A and 6B). Interestingly, we found that the expression and activation of NOX2 were significantly inhibited in the NOD2-/- mice compared to WT mice (Fig. 6C and 6D). In an in vitro study, WT microglia was treated with MDP, a selective agonistic ligand of NOD2, and we found that MDP significantly induced NOX2 expression and ROS production in a time-dependent manner (Fig. 7A and 7B). To elucidate the role of NADPH oxidase on the NOD2 induced IL-6 production further, a well-known NADPH oxidase activity inhibitor, apocynin was used in this study. As shown in Fig. 7C, IL-6 production was markedly enhanced by OGD and MDP, and apocynin significantly inhibited the increase of IL-6 induced by OGD and MDP. The results suggest that NADPH oxidase activation is involved in the NOD2-mediated inflammatory response after cerebral I/R injury.

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