The neuropeptide ␣-melanocyte-stimulating hormone (␣-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) in leukocytes via stimulation of ␣-MSH cell surface receptors. However, the signaling mechanism of ␣-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which ␣-MSH inhibits lipopolysaccharide (LPS)-induced TNF-␣ production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor B (NF B) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NF B to induce TNF-␣ production. We found that pretreatment of cells with ␣-MSH blocked LPS-induced p38 kinase and NF B activation as well as TNF-␣ production. This response was proportional to ␣-MSH receptor expression levels, and addition of an ␣-MSH receptor antagonist abolished the inhibitory effects. In addition, ␣-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of ␣-MSH on p38 kinase activation, NF B activation, and TNF-␣ production. Taken together, our results indicate that stimulation of PKA by ␣-MSH causes inhibition of LPS-induced activation of p38 kinase and NF B to block TNF-␣ production.

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Oxidative stress contributes to cellular injury following clinical and experimental ischemia/reperfusion episodes. Oxidative injury can induce cellular and subcellular damage that results in apoptotic cell death. We tested whether 2',4',7-trihydroxyflavanone, a synthetic flavonoid derivative, inhibits hydrogen peroxide (H(2)O(2))-induced toxicity in human umbilical endothelial cells (HUVECs). Cultured HUVECs were incubated for 30 minutes with 0.2 mM H(2)O(2) in the absence and presence of 2',4',7-trihydroxyflavanone at a non-toxic concentration of 50 microM. The effect of 2',4',7-trihydroxyflavanone on apoptosis parameters was compared with that of naringenin and two flavonol derivatives, 2',4',7-trihydroxyflavonol and 2',4',6-trihydroxyflavonol. H(2)O(2) induced cell death within 24 hours over the range of 0.05-1.0 mM, and decreased cell viability by approximately 30% at 0.2 mM. This cytotoxicity was associated with nuclear condensation and DNA fragmentation, indicating induction of apoptosis. 2',4',7-Trihydroxyflavanone, as well as naringenin, was effective as an inhibitor of oxidative stress, protecting cell viability with >/=85% viable cells compared with the control, and no apparent nuclear condensation or DNA fragmentation. In contrast, the flavonol derivatives had no such effect. In addition, immunocytochemical data and Western blot analysis revealed that, unlike flavonol derivatives, the expression of Bcl-2 was markedly up-regulated, and that the expression of Bax and activation of caspase-3 were strongly inhibited by this flavanone derivative, thereby implicating antioxidant activity-related anti-apoptotic mechanisms of 2',4',7-trihydroxyflavanone. These results indicate that the synthetic flavonoid derivative 2',4',7-trihydroxyflavanone is an effective antioxidant, preventing endothelial apoptosis induced by H(2)O(2).

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