URB597 exerts neuroprotective effect against transient cerebral ischemic injury via restor⁃ing autophagy flux and inhibiting neuronal necroptosis in mice

OBJECTIVE URB597(KDS-4103)is a potent and selective inhibitor of the enzyme fatty acid amide hydrolase(FAAH)and can ele⁃vate the level of oleoylethanolamide(OEA),a naturally occurring endocannabinoid in the brain.However,the effect of URB597 on cerebral isch⁃emic injury in mice remains unclear.METHODS Focal cerebral ischemia was induced by middle cerebral artery occlusion for 30 min followed by reperfusion for 24 h in mice.To observe the dose-dependent effect,URB597(0.04-5.00 mg·kg-1,ip)was administered at the same time of reperfu⁃sion.To determine the time-dependent effect,URB597(1.00 mg·kg-1,ip)was administered as a single dose at 0,1,3 or 5 h after reperfusion.Twenty-four hours after brain ischemia,Beder⁃son scoring test and grip strength test were used to evaluate the neurological function;brain in⁃farct volume was assayed by 2,3,5-triphenyltetra⁃zolium chloride(TTC)staining or diffusion-weighted magnetic resonance imaging(MRI).Laser speckle imaging(LSI)technique was used to assay the regional cerebral blood flow(rCBF);NeuN immunofluorescence staining was used to observe the neuron survival in the penumbra.To further investigate the underlying mechanism,au⁃tophagy flux related proteins(LC3-Ⅱ,P62 and LAMP2)and necroptosis related proteins(pRIPK3 and pMLKL)were detected by Western blotting and immunofluorescence staining.RESULTS Twenty-four hours after brain ischemia,URB597 dose-dependently improved neurological func⁃tion and reduced brain infarct volume.The most effective dose was 1.00 mg·kg-1;the therapeutic time window was within 1 h after ischemic stroke.The protective effect is further confirmed by the results that post-ischemic treatment with URB597(1.00 mg·kg-1)significantly increased neurons survival,promoted autophagy flux and reduced cell necroptosis in cortical penumbra after cerebral I/R.CONCLUSION URB597 dose-and time-dependently exerts a neuroprotective effect against acute cerebral I/R injury.This neu⁃roprotective effect of URB597 may be associated with its restoration of autophagy flux and inhibi⁃tion of neuronal necroptosis in the cortical penumbra.