Modulation of the arachidonic acid cascade by food polyphenols - A comprehensive analysis of their effects on 5-Lipoxygenase, Cytochrome-P450 and autoxidation pathways

二十烷基类化合物和其他多不饱和脂肪酸(PUFAs)的氧化代谢产物参与细胞内信号转导和疼痛调节，是最有效的内源性炎症介质之一。由于它们的重要生理作用，世界上超过一半的药物直接影响这些氧化脂质的形成。虽然一些研究表明营养会改变内源性氧脂模式，但大多数食物成分，如多酚，对氧脂形成的影响还没有被调查。此外，多酚对花生四烯酸(AA)级联反应和炎症的显著影响的研究集中在分析单酶的表达水平或单二十烷类化合物的水平。这些方法忽视了对生物过程的调节依赖于大量的氧化脂质。因此，对单个氧化脂质的监测描绘了一幅不完整的图景。因此，在这个项目中，我们将从机械上研究多酚对AA级联反应的影响。为此，我们将使用液-质联用(LC-MS)对AA级联的所有分支的一套全面的氧脂进行定量，作为靶向代谢组学方法。我们将分析在不同的细胞培养方法和无细胞方法中，多酚对AA级联的环氧合酶、脂氧合酶、细胞色素P450和环氧化物水解酶途径活性的影响。此外，我们将利用氧化脂质模式来监测炎症过程中发生的氧化应激以及食物中的抗氧化剂对其的改善。有了这些高含量的筛选工具，我们不仅可以发现潜在的活性多酚，还可以获得关于其作用模式的信息，关于其效力的定量数据，以及关于其影响广度的定性数据。随后，将在脂多糖诱导的脓毒症和葡聚糖硫酸钠诱导的急性和慢性结肠炎的啮齿动物模型中研究最有效的化合物。氧脂模式的改变将与炎症的生理、组织病理学和分子生物学参数相关。总体而言，该项目将使人们更深入地了解营养导致的氧化脂质模式的变化及其对炎症的影响。我们将确定那些能够有效改变调节血脂水平的多酚类物质，阐明它们的作用机制，并评估它们在体内的生物学效应。这些数据将有助于了解多酚对炎症的一般影响，并评估它们在调节急性和慢性炎症过程中的潜在治疗价值。

Eicosanoids and oxidative metabolites of other poly-unsaturated fatty acids (PUFAs) are involved in intracellular signaling as well as the regulation of pain, and represent one of the most potent classes of endogenous mediators of inflammation. Due to their crucial physiological role more than half of the world pharmaceuticals directly influence the formation of these oxylipins. Although several studies demonstrate that nutrition alters the endogenous oxylipin pattern, the influence of most food ingredients, such as polyphenols, on the formation of oxylipins has not been investigated yet. Moreover, the studies demonstrating a significant impact of polyphenols on the arachidonic acid (AA) cascade and inflammation focused on the analysis of expression levels of single enzymes or the levels of single eicosanoids. These approaches disregard that the regulation of biological processes relies on a multitude of oxylipins. Thus, monitoring of single individual oxylipins draws an incomplete picture.Therefore, in this project, we will mechanistically investigate the impact of polyphenols on the AA cascade. For this purpose we will employ liquid chromatography mass spectrometry (LC-MS) for the quantification of a comprehensive set of oxylipins of all branches of the AA cascade as targeted metabolomics approach. We will analyze the effect of polyphenols on the activity of cyclooxgenase, lipoxygenase, cytochrome P450 and epoxide hydrolase pathways of the AA cascade in different cell culture assays as well as in cell free approaches. Moreover, we will utilize the oxylipin pattern to monitor oxidative stress occurring during inflammation and its amelioration by food-born antioxidants. With these high-content screening tools, we will not only discover potentially active polyphenols, but also gain information on their mode of action, quantitative data on their potency and qualitative data on the breadth of their effects. Subsequently, the most potent compounds will be investigated in rodent models of lipopolysaccharid induced sepsis and dextran sulfate sodium induced acute and chronic colitis. Changes in the oxylipin pattern will be correlated to physiological, histopathological and molecular biological parameters of inflammation. Overall, this project will lead to a deeper understanding of the nutritionally induced alterations in oxylipin patterns and their impact on inflammation. We will identify those polyphenols that potently alter regulatory lipid levels, elucidate their mechanistic mode of action and evaluate their biological effects in vivo. The obtained data will help to understand the general influence of polyphenols on inflammation and to evaluate their potential therapeutic value in modulating acute and chronic inflammatory processes.