Abstract WP110: TLR5 Is Related To Neuroprotective Mechanism Of Postconditioning Through NF-κB-Akt Survival Pathway In Cerebral Ischemia

Abstract

Background and Purpose: Although postconditioning following stroke has indicated neuroprotective mechanisms, little is known about the mechanisms that lead to neuroprotection. Using flagellin, NF-kB activators and agonists of TLR5, we tested whether TLR5 plays an integral role in NF-kB activation and postconditioning-induced neuroprotection. Method: For the postconditioning group, 30 min of MCAO was induced, then 5 min of reperfusion was performed, after which the MCA was again occluded for 5 min. Using an anaerobic chamber, bEnd.3 and BV-2 cells in the postconditioning group were subjected to 180 min OGD (oxygen and glucose deprivation) and were allowed to recover for 10 min before being subjected to 10 more min of OGD. The control group was subjected to 180 min of OGD. Flagellin, an agonist of TLR5, was used to clarify the association between TLR5 and postconditioning in vivo (ICV; 50, 100 ng/mouse) and in vitro (0.1, 0.25, 0.5, 1 ng/ ml) experiments. Immunoprecipitation and immunoblotting were used to determine the interactions of TLR5, MyD88, p-Akt, and NF-κB. Results: Animals treated with postconditioning showed significant differences in infarct volumes compared with the MCAO group up to 60.51%. Postconditioning elevated NF-κB translocation to the nuclei (p<0.001) as early as 1 hour after postconditioning following tFCI and OGD. The protein interactions and expressions of TLR5, MyD88 and p-Akt (p<0.05) were increased in in vivo and in vitro postconditioning. Additionally, inhibition of Akt phosphorylation by Akt inhibitor IV decreased NF-κB translocation after postconditioning. To clarify the role of NF-κB in postconditioning, flagellin was used as a postconditioning mimicking agent. The treatment of flagellin reduced hypoxic-ischemic injury and increased nuclear accumulation of NF-κB and activation of Akt up to 4 hr. Conclusions: Postconditioning has neuroprotective effects by activating NF-κB and Akt survival pathway under TLR5 after stroke. Agonist of TLR5, flagellin simulated NF-κB and Akt survival pathways in cerebral ischemia. Our results suggested new mechanisms of postconditioning through TLR-5 and NF-κB, which leads to the possibility of development of a postconditioning mimicking agent using NF-κB activators such as flagellin.