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12/15-lipoxygenase (12/15-LO) enzyme and products have been associated with inflammation and atherosclerosis. However, the mechanism of effects of the 12/15-LO products has not been fully clarified. To study the role of 12/15-LO in cytokine expression, experiments with direct additions of the12/15-LO products, 12(S)-hydroxyeicosa tetraenoic acid or 12(S)-hydroperoxyeicosa-5Z, 8Z, 10E, or 14Z-tetraenoic acid to macrophages were first carried out, and results showed that the 12/15-LO products stimulated mRNA and protein expression of IL-6 and TNF-alpha in a dose-dependent manner. In contrast, an inactive analogue of 12(S)-hydroxyeicosa tetraenoic acid had no effect. To further explore the role of endogenous 12/15-LO in cytokine expression, we used an in vitro and in vivo model to test the effect of 12/15-LO overexpression. The models included Plox-86 cells, a J774A.1 cell line that stably overexpresses leukocyte-type 12/15-LO and primary mouse peritoneal macrophages (MPMs) from 12/15-LO transgenic mice. The results showed a clear increase in IL-6 and TNF-alpha expression in Plox-86 cells and MPMs from 12/15-LO transgenic mice, compared with mock-transfected J774A.1 cells and MPMs from control C57BL6 mice. IL-1beta, IL-12, and monocyte chemoattractant protein (MCP)-1 mRNA were also increased in Plox-86 cells. These data clearly suggest a clear role of 12/15-LO pathway in cytokine production. We also demonstrated that signaling pathways including protein kinase C, p38 MAPK (p38), c-jun NH(2)-terminal kinase as well as nicotinamide adenine dinucleotide phosphate oxidase are important for 12-(S)-hydroxyeicosatetraenoic acid-induced increases in IL-6 and TNF-alpha gene expression. These results suggest a potentially important mechanism linking 12/15-LO activation to chronic inflammation and atherosclerosis.

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.