Anti-inflammatory And Neuroprotective Effects Of Combined NADPH And The NOX Inhibitor Apocynin In A Mouse Model Of Stroke

Aim: To investigate the effects of co-treatment with NADPH and NOX inhibitor apocynin on ischemia/reperfusion-induced brain inflammation and neuronal injury.Methods: Transient middle cerebral artery occlusion(tMCAO)was produced by using an intraluminal filament technique in mice.The effects of co-treatment with NADPH and NOX inhibitor apocynin were analyzed by scoring neurological deficits,assessing brain infarction volume with 2,3,5-triphenyltetrazolium chloride(TTC),determining the brain water content 24 h after reperfusion,recovering of long-term neurological functions and performing histopathological examination.The changes of ROS in ischemic cerebral cortex were detected by DHE probe and the levels of GSH and NADPH were measured by the corresponding detection kits.Western blot analysis was employed to determine alterations in NOX2,NOX4,p-IKK?,I?B?,p-I?B?,p-NF-?B p65,NF-?B p65,COX2 and iNOS in cerebral cortex at the indicated time.Brain sections were immune stained for NF-?B p65 nuclear translocation and inflammasome proteins expression in cerebral cortex.Results: Both NADPH and apocynin markedly attenuated ischemia/reperfusioninduced increases in the levels of NOX2,NOX4 and ROS.NADPH and apocynin significantly inhibited the phosphorylation and degradation of I?B?,NF-?B p65 nuclear localization,and the expression of NF-?B target gene cyclooxygenase(COX2)and inducible nitric oxide synthase(iNOS).Furthermore,both NADPH and apocynin suppressed the expression of inflamma some proteins including NLRP3 ASC,caspase-1,interleukin(IL)-1? and IL-18 in the ischemic cortex as revealed by Western blot analysis and immunofluorescence.Moreover,all these effects were greatly amplified by combination of NADPH and apocynin.Both NADPH and apocynin significantly reduced infarct volume,improved post-stroke survival,and recovery of neurological functions in mouse model of stroke.Consistently,the combination of NADPH and apocynin produced greater beneficial effects in against ischemic brain damage.Conclusion: Beyond anti-oxidative effects,NADPH may also have anti-inflammatoryeffects and combination of NADPH and NOX inhibitors could produce a greater neuroprotective effect in ischemic stroke.