Evidence That Glutathione Depletion Is a Mechanism Responsible for the Anti-Inflammatory Effects of Ethyl Pyruvate in Cultured Lipopolysaccharide-Stimulated RAW 264.7 Cells

Abstract

Ethyl pyruvate (EP), an effective scavenger of reactive oxygen species, is also an anti-inflammatory agent in a variety of in vivo and in vitro model systems. To gain a better understanding of the molecular basis for the anti-inflammatory effects of EP, we compared the pharmacological properties of EP and N-acetyl-L-cysteine (NAC), a well studied scavenger of reactive oxygen species and a precursor for the endogenous antioxidant glutathione (GSH). The studies were performed using RAW 264.7 murine macrophage-like cells that were stimulated with lipopolysaccharide (LPS). Although EP and NAC both inhibited LPS-induced nitric oxide and interleukin (IL)-6 secretion, the former compound was considerably more potent than the latter. EP markedly inhibited inducible nitric-oxide synthase, IL-6, and IL-10 mRNA induction, whereas the effects of NAC were minimal. EP inhibited LPS-induced nuclear factor-B DNA binding to a much greater extent than did NAC. Both compounds inhibited LPS-induced lipid peroxidation, but the two compounds had qualitatively different effects on cellular levels of GSH. Although NAC increased GSH levels, EP had the opposite effect. The anti-inflammatory effects of EP were partially reversed when RAW 264.7 cells were treated with a cellpermeable GSH analog, glutathione ethyl ester. These data support the view that the anti-inflammatory effects of EP are mediated, at least in part, by the ability of EP to deplete cellular GSH stores. Moreover, the findings presented here suggest that an unusual combination of biochemical effects (inhibition of lipid peroxidation and GSH depletion) might account for the anti-inflammatory effects of EP.

Ethyl Pyruvate Inhibits Nuclear Factor-κB-Dependent Signaling by Directly Targeting p65

“…Results from in vivo and in vitro studies support the view that ethyl pyruvate inhibits NF-B DNA binding (Yang et al, , 2003(Yang et al, , 2004Uchiyama et al, 2003;Han et al, 2004;Song et al, 2004). Moreover, Song et al (2004) recently reported data suggesting that ethyl pyruvate might react with the sulfhydryl-containing compound, glutathione.…”

“…In many of these models of acute critical illness, treatment with ethyl pyruvate down-regulates the expression of various pro-inflammatory genes, including inducible nitric oxide synthase (iNOS), tumor necrosis factor, cyclooxygenase-2, and interleukin (IL)-6 (Yang et al, , 2003(Yang et al, , 2004Uchiyama et al, 2003). Similarly, ethyl pyruvate inhibits IL-6 and iNOS expression, nitric oxide production, and/or secretion of the pro-inflammatory protein HGMB1 by immunostimulated Caco-2 human enterocytelike cells or lipopolysaccharide-stimulated RAW 264.7 murine macrophage-like cells (Ulloa et al, 2002;Sappington et al, 2003a,b;Song et al, 2004). These latter findings support the view that ethyl pyruvate has activity as an anti-inflammatory agent; however, the molecular mechanisms underlying the anti-inflammatory effects of ethyl pyruvate remain to be elucidated.…”

“…Moreover, Song et al (2004) recently reported data suggesting that ethyl pyruvate might react with the sulfhydryl-containing compound, glutathione. Prompted by these findings, we carried out a series of in vitro experiments designed to better elucidate the mechanisms responsible for inhibition of NF-B-dependent signaling by ethyl pyruvate.…”

Glutathione-Redox Balance Regulates c-rel–Driven IL-12 Production in Macrophages: Possible Implications in Antituberculosis Immunotherapy

“…It is reported that 20 mM NAC downregulates basal NF-kB DNA binding in RAW 264.7 macrophages and also inhibits LPS-stimulated NF-kB DNA binding in these cells (54). In contrast, few studies indicate that NAC fails to inhibit LPS-induced activation of NF-kB in RAW 264.7 macrophages (55); yet in other studies, NAC has been shown to inhibit activation of NF-kB in alveolar macrophages induced by TNF-a (56).…”

Anti-HMGB1 Neutralizing Antibody Ameliorates Gut Barrier Dysfunction and Improves Survival after Hemorrhagic Shock

“…The serum was collected and stored frozen at -80 °C until assayed for IL-6 and IL-10 using ELISA kits from R&D Systems (Minneapolis, MN, USA) according to the manufacturer's instructions. Serum nitrite concentration was measured using the Greiss reaction, as described (32).…”