β-arrestin 2 negatively regulates NOD2 signalling pathway through association with TRAF6 in microglia after cerebral ischaemia/reperfusion injury.

We previously reported that nucleotide-binding oligomerization domain-containing protein (NOD) 2 was involved in the inflammatory responses to cerebral ischaemia/reperfusion (I/R) insult. However, the mechanism by which NOD2 participates in brain ischaemic injury and the regulation of NOD2 in the process are still obscure. Increased β-arrestin 2 (ARRB2) expression was observed in microglia following cerebral I/R in wild-type mice besides the up-regulation of NOD2 and TRAF6. Stimulation of NOD2 by muramyl dipeptide (MDP) in BV2 cells induced the activation of NF-κB by the phosphorylation of p65 subunit and the degradation of IκBα. Meanwhile, the protein level of Cyclooxygenase-2 (COX-2), the protein expression and activity of MMP-9 were significantly increased in BV2 cells after administration of MDP. Furthermore, overexpression of ARRB2 significantly suppressed the inflammation induced by MDP, silence of ARRB2 significantly enhanced the inflammation induced by MDP in BV2 cells. In addition, we observed endogenous interaction of TRAF6 and ARRB2 after stimulation of MDP or cerebral I/R insult, indicating ARRB2 negatively regulates NOD2-triggered inflammatory signalling pathway by associating with TRAF6 in microglia after cerebral I/R injury. Finally, the in vivo study clearly confirmed that ARRB2 negatively regulated NOD2-induced inflammatory response, as ARRB2 deficiency exacerbated stroke outcomes and aggravated the NF-κB signalling pathway induced by NOD2 stimulation after cerebral I/R injury. These findings revealed ARRB2 negatively regulated NOD2 signalling pathway through the association with TRAF6 in cerebral I/R injury.