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Abstract: Cerebral ischemia leads to brain injury via a complex series of pathophysiological events, and therefore, multi-drug treatments or multi-targeting drug treatments provide attractive options with respect to limiting brain damage. Previously, we reported that a novel multi-functional compound oxopropanoyloxy benzoic acid (OBA-09, a simple ester of pyruvate and salicylic acid) affords robust neuroprotective effects in the postischemic rat brain. OBA-09 exhibited anti-oxidative effects that appeared to be executed by OBA-09 and by the salicylic acid afforded by hydrolysis. Here, we report the anti-inflammatory effects of OBA-09. Microglial activation observed at 2 days post-middle cerebral artery occlusion (MCAO, 90min) and at 1 day after a LPS injection (0.5mg/kg, intravenously) in the brains of Sprague-Dawley rats were markedly suppressed by the administration of OBA-09 (10mg/kg). Inductions of proinflammatory markers (TNF-α, IL-1β, iNOS, and COX-2) were also suppressed by OBA-09 in both the LPS and MCAO models. Moreover, the anti-inflammatory effect of OBA-09 was accompanied by the suppression of infarct formation in the postischemic brain, but appeared to be independent of neuroprotection in LPS-treated rats. The inductions of proinflammatory markers were also inhibited by OBA-09 in LPS-treated BV2 cells (a microglia cell line) and in LPS-treated-primary neutrophils, possibly due to the suppression of NF-κB activity. Interestingly, the anti-inflammatory effect of OBA-09 was greater than that of equivalent co-treatment with pyruvate and salicylic acid. Together these results indicate that OBA-09 is a potent multi-modal neuroprotectant in the postischemic brain, and that its anti-inflammatory effect contributes to its neuroprotective function. [Copyright &y& Elsevier]

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca(2+) and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K(+)-induced transient [Ca(2+)]i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca(2+) as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca(2+) indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the A23187-induced intracellular Ca(2+) surge in BV2 cells and, under this condition, A23187-induced activations of Ca(2+)-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. (A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca(2+), and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.