Combination Treatment with Ethyl Pyruvate and Aspirin Enhances Neuroprotection in the Postischemic Brain

Abstract

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to act as an anti-inflammatory molecule in various pathological conditions, which include sepsis or hemorrhagic shock. Recently, we showed that ethyl pyruvate has a neuroprotective effect in the postischemic brain and also in KA-induced pathogenesis in the brain. In this study, we examined whether aspirin augments neuroprotective effect of ethyl pyruvate in transient focal ischemia model by complementing the neuroprotective effects of ethyl pyruvate. Although, most of neuroprotective effect of aspirin has been attributed to the anti-platelet action, aspirin also has direct neuroprotective effects, including NF-kappaB inhibition. Ethyl pyruvate dose-dependently suppressed infarct formation in the postischemic brain, wherein intravenous administration of 5 mg/kg ethyl pyruvate 30 min after the occlusion reduced infarct volume to 34.5 +/- 15.5% (n = 6, P < 0.01) of that of the untreated control. In combination with aspirin (5 mg/kg, i.v.), the neuroprotective effect was enhanced, resulting in 16.0 +/- 5.9% (n = 6, P < 0.01) infarct volume. The time window for synergistic neuroprotection by ethyl pyruvate and aspirin extended to 9 h post-MCAO. The synergistic reduction in infarct volume was accompanied by suppression of the clinical manifestations associated with cerebral ischemia including motor impairment and neurological deficits. Inflammatory processes including microglial activation and proinflammatory cytokine expression were notably suppressed by the combination treatment in the postischemic brain and in primary microglia cultures, wherein ethyl pyruvate and aspirin modulate NF-kappaB signaling differentially. Aspirin interferes with IkappaB phosphorylation and degradation in the cytoplasm, possibly by specifically inhibiting IkappaB kinase-beta, whereas, the effect of ethyl pyruvate seems to occur in the nucleus, where it may interfere with the binding of NF-kappaB to responsive promoter elements in the target genes. Similar enhancement in neuroprotective effect was also observed in primary cortical cultures after NMDA or Zn(2+) treatment or oxygen-glucose deprivation. Together, these results indicate that combination treatment of ethyl pyruvate and aspirin affords synergistic neuroprotection in the postischemic brain with a wide therapeutic window, in part via differential modulation of the NF-kappaB signaling pathway.

Robust Protective Effects of a Novel Multimodal Neuroprotectant Oxopropanoyloxy Benzoic Acid (a Salicylic Acid/Pyruvate Ester) in the Postischemic Brain

“…In our previous report, we showed that combination treatment with ethyl pyruvate and aspirin enhances neuroprotection in the postischemic brain (Kim et al, 2010). Here, we confirmed the complementary effects of pyruvate and salicylic acid, the metabolic products of ethyl pyruvate and aspirin, respectively.…”

“…It is beneficial for stroke patients in part due to its antiplatelet effect (Antithrombotic Trialists' Collaboration, 2002). In addition, aspirin also has anti-inflammatory (Pillinger et al, 1998;Feldman et al, 2001) and antiexcitotoxic effects (De Cristóbal et al, 2001;Castillo et al, 2003), which seem to complement the neuroprotective effects of ethyl pyruvate (Kim et al, 2010). In view of the multifactorial nature of cerebral ischemia, a multimechanism-based approach to drug development might be needed to maximize the efficacy of the drug treatment.…”

“…We showed recently that combination treatment of ethyl pyruvate and aspirin (acetylsalicylic acid) provides synergistic neuroprotection in the postischemic brain (Kim et al, 2010). Ethyl pyruvate has been reported to potently suppress infarct formation in the postischemic brain This work was supported by the Health Planning Technology and Evaluation Board [Translational Research Grant R05-003000-10562-0].…”

Simplified Heterocyclic Analogues of Fluoxetine Inhibit Inducible Nitric Oxide Production in Lipopolysaccharide-Induced BV2 Cells

“…The primer sequences of rat inducible NO synthase (iNOS) was described previously. 25) Immunoblotting BV2 cells were washed twice with cold phosphate buffered saline (PBS) and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodiumdeoxycholate, 150 mM NaCl, 1 mM Na 3 VO 4 , and 1 complete Mini protease inhibitor cocktail tablet (Roche Diagnostics, Switzerland)). The lysate was centrifuged for 15 min at 12000 rpm at 4°C and supernatants were loaded onto 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

“…The antigenantibody complex was detected by BM Chemiluminescence Blotting Substrate (Roche, Germany) using donkey antirabbit horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, U.S.A.). 25) …”