Ibuprofen attenuates oxidative damage through NOX2 inhibition in Alzheimer's disease

Abstract

Considerable evidence points to important roles for inflammation in Alzheimer's disease (AD) pathophysiology. Epidemiological studies have suggested that long-term nonsteroidal anti-inflammatory drug (NSAID) therapy reduces the risk for Alzheimer's disease; however, the mechanism remains unknown. We report that a 9-month treatment of aged R1.40 mice resulted in 90% decrease in plaque burden and a similar reduction in microglial activation. Ibuprofen treatment reduced levels of lipid peroxidation, tyrosine nitration, and protein oxidation, demonstrating a dramatic effect on oxidative damage in vivo. Fibrillar β-amyloid (Aβ) stimulation has previously been demonstrated to induce the assembly and activation of the microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leading to superoxide production through a tyrosine kinase-based signaling cascade. Ibuprofen treatment of microglia or monocytes with racemic or S-ibuprofen inhibited Aβ-stimulated Vav tyrosine phosphorylation, NADPH oxidase assembly, and superoxide production. Interestingly, Aβ-stimulated Vav phosphorylation was not inhibited by COX inhibitors. These findings suggest that ibuprofen acts independently of cyclooxygenase COX inhibition to disrupt signaling cascades leading to microglial NADPH oxidase (NOX2) activation, preventing oxidative damage and enhancing plaque clearance in the brain.

Figure 1. Chronic ibuprofen treatment reduces AD-related plaque pathology in B6-R1.40 mice

(A) Sagittal sections from age-matched non-treated (−IBU) and ibuprofen-treated (+IBU) 24-month-old B6-R1.40 mice were immunstained with anti-human Aβ monoclonal antibody 6E10. (B) Average plaque number/section in the parenchyma was reduced by 90% (n=5/group, ***p<0.001) in +IBU animals. Dense core plaques were identified in (C) −IBU and (D) +IBU mice by Thioflavin-S positive staining (green). Nuclei were visualized with propidium iodide (red). White arrows indicate blood vessels with amyloid deposition.

Figure 2. Chronic ibuprofen treatment reduces microglial activation in B6-R1.40 mice

(A) Representative photomicrograph depicting phenotypically activated microglia stained with Iba1 (green) adjacent to a 6E10+ plaque (red) in the non-treated (−IBU) but not the ibuprofen-treated (+IBU) B6-R1.40 mouse. Nuclei are stained with DAPI (blue); scale bar=50 μm. (B) Sagittal sections from −IBU and +IBU-treated B6-R1.40 mice were stained for anti-CD45; scale bar=200μm.

Figure 3. Ibuprofen treatment reduces AD-related oxidative damage

(A) Effect of the hAPP transgene on oxidative damage as measured by 4-HNE levels in brain homogenates. Samples from 24-month-old age-matched C57BL/6 (B6) and B6-R1.40 mice are shown. (B) Representative immunoblot from individual non-treated (−IBU) and ibuprofen-treated (+IBU) brain homogenates analyzed for lipid peroxidation measured by 4-HNE protein adduct levels (n=5, **p<0.01). (C) Representative immunoblot from −IBU and +IBU-treated animals analyzed for 3-nitrotyrosine (3-NT) levels (n=5, *p<0.05). Blots were stripped and reprobed with GAPDH as a protein loading control.

Figure 4. Chronic ibuprofen treatment reduced protein oxidation in aged B6-R1.40 mice

Protein oxidation was measured using an Oxyblot kit. Representative blot from non-treated (−IBU) and ibuprofen-treated (+IBU) brain homogenates analyzed by immunoblot analysis with an anti-DNP antibody (n=5, *p< 0.05). Blots were stripped and reprobed with GAPDH as a protein loading control.

Figure 5. Fibrillar Aβ-stimulated Vav phosphorylation is inhibited by ibuprofen pretreatment

(A) C57Bl/6 microglia were pretreated with ibuprofen (600 μM) for 1hr followed by incubation in serum-free DMEM-F12 containing NBT +/− fAβ_25–35 (60 μM) for 30 min. PMA (390 nM) was a positive control. Superoxide generation was monitored by the presence of insoluble formazan and visualized on a Leica DMIRB inverted microscope. Three random fields of cells (>100 cells) were counted (n=6, ***p< 0.001). (B) THP-1 cells (5 × 10⁶ cells) were pretreated 1 hr +/− ibuprofen and then stimulated with fAβ_25-35 for 3 min. Vav immunoprecipitates were analyzed by Western blot analysis using a phospho-Tyr antibody (4G10). Blots were stripped and reprobed with Vav as a protein-loading control. Band intensity was analyzed as the level of phophorylated Vav normalized to total Vav protein levels and expressed as relative density (n=3, *p<0.05). (C) Dose response of S-ibuprofen pretreatment on Vav protein-Tyr phosphorylation in THP-1 cells treated with fAβ_25-35 (60 μM) for 3 min. (*p<0.05 at 200 μM and **p<0.01 at 300 μM). R-ibuprofen (200 μM) pretreatment had no effect (n=4). (D) THP-1 cells pretreated 1 hr with either a COX1 (sc560) or a COX2 (CAY10404) inhibitor were stimulated with fAβ_25-35 for 3 min. Vav immunoprecipitates were analyzed by Western blot using a phospho-Tyr antibody (4G10). Blots were stripped and reprobed with Vav as a protein-loading control.

Figure 6. S-ibuprofen disrupts NOX2 complex assembly

(A) Dose response for THP-1 cells pretreated with S-ibuprofen for 1 hr followed by stimulation with fAβ_25-35 (60 μM) for 10 min. Lysates were subjected to differential centrifugation and membrane fractions were immunoblotted for Rac. Cell fractions were also immunoblotted with a flotillin antibody to assess the efficacy of the fractionation procedure. (B) THP-1 cells pretreated with either S- or R-ibuprofen for 1 hr were stimulated with fAβ_25-35. Lysates were immunoblotted for phosphorylated p38. Blots were stripped and reprobed with p38 as a protein loading control. Band intensity was analyzed as the level of phophorylated p38 normalized to total p38 protein levels and expressed as relative density (n=4, **p<0.01).

Figure 7. S-ibuprofen inhibits the generation of NOX2-derived radicals in microglia stimulated with fAβ

Primary C57Bl/6 microglia were preincubated with S-ibuprofen (200 μM) for 1 hr in serum-free DMEM-F12. NBT was added to the media and microglia were stimulated with fAβ_25-35 (60 μM) or PMA (390 nM) for 30 min. Superoxide anion generation was monitored by the presence of insoluble formazan. Three random fields of cells (>100 cells) were counted (n=3, **p < 0.01).