Polyunsaturated fatty acid (PUFA), inflammation and antioxidants

在过去的一个世纪里，加拿大的饮食中充斥着n-6多不饱和脂肪酸(n-6 PUFA)，如亚油酸(LA)。伴随着这些变化，以异常炎症和氧化应激为特征的糖尿病和动脉粥样硬化等各种慢性疾病在加拿大人中激增。我们对多不饱和脂肪酸代谢和炎症/氧化应激的了解存在着基本的知识差距。 背景我们对n-6多不饱和脂肪酸引起的炎症的大部分知识都集中在花生四烯酸(ARA)，在慢性病中花生四烯酸(ARA)实际上较低。然而，最近的研究表明，其他n-6多不饱和脂肪酸，如亚油酸(LA)和伽马亚麻酸(GLA)是炎症过程中的主要参与者。在这方面，LA的氧化修饰和延伸酶/脱饱和酶介导的多不饱和脂肪酸的生物转化仍未得到充分研究。然而，很难理解LA的具体作用，因为它在体内被快速生物转化为GLA、DGLA，最后转化为ARA。 因此，总的来说，该计划的长期目标是阐明n-6多不饱和脂肪酸的生物转化在炎症和氧化应激中的作用。我的总体假设是，在LA的高可用性下，LA的生物转化在调节炎症/氧化应激方面至关重要。 该项目的主要目标是了解LA和GLA的氧化和代谢修饰对心肌细胞和巨噬细胞的炎症和氧化应激的作用。我们的目标是在体外(使用H9c2和RAW 264.7细胞株)和体内在不同脂肪酸饮食后(目标1)，在高n-6PUFA环境中靶向氧化LA代谢物和4-HNE的影响。接下来，我们的目标是确定LA和GLA在引起炎症/氧化应激中的作用，独立于ARA(目标2)。在这方面，我们确实拥有一种独特的Elov15基因缺失敲除小鼠模型，该模型导致LA和ALA在体内积累，但减少ARA。因此，我们将在单不饱和(MUFA)和n-6多不饱和脂肪酸(n-6)富含PUFA的饮食方案下，从野生型和Elov15KO中分离心肌细胞和巨噬细胞。质谱仪和气相色谱-将用于分析二十烷类化合物、脂肪酸和氧化脂质代谢物。细胞信号、炎症、氧化应激也将在这些细胞中进行评估，如早期研究所示。 意义这项应用是为了了解单个多不饱和脂肪酸在哺乳动物细胞(如巨噬细胞和心肌细胞)中的酶和非酶生物转化的基本作用，这些细胞在空间上相关，但功能不同，以及它们在炎症和氧化应激期间的反应。我相信，在每个加拿大人都受到高剂量膳食多不饱和脂肪酸的冲击下，如果得到资助，这个项目可以导致对多不饱和脂肪酸新陈代谢的基本知识的合成，并确定n-6多不饱和脂肪酸介导的哺乳动物细胞炎症的真正原因。

THE PROBLEM Over the last century, Canadian diets has been inundated with n-6 polyunsaturated fatty acids (n-6 PUFA), like linoleic acid (LA). Along with such changes, various chronic diseases like diabetes and atherosclerosis, characterized by aberrant inflammation and oxidative stress have soared among Canadians. Fundamental knowledge gaps exist in our understanding of PUFA metabolism and inflammation/oxidative stress. BACKGROUND Most of our knowledge of n-6 PUFA driven inflammation is centered around, arachidonic acid (ARA) which is actually lower in chronic diseases. However, recent research indicates that other n-6 PUFAs like linoleic (LA) and gamma linolenic acid (GLA) are major players during inflammation. In this regard, oxidative modifications of LA and elongase/desaturase mediated biotransformations of PUFA remain understudied. However, it is difficult to understand specific roles of LA as it is rapidly bioconverted sequentially to GLA, DGLA and finally to ARA in vivo. Thus overall, the LONG-TERM GOAL of this program is to elucidate the role of n-6 PUFA bio-transformation on inflammation and oxidative stress. My overall HYPOTHESIS is that under high LA availability, biological transformations of LA is vital in regulating inflammation/oxidative stress. THE PROGRAM The overarching aim of this program is to understand the roles of oxidative and metabolic modifications of LA and GLA towards inflammation and oxidative stress in cardiomyocytes and macrophages. We aim to target the impact of oxidized LA metabolites and 4-HNE in a high n-6 PUFA milieu both in vitro (using H9c2 and RAW 264.7 cell lines) and in vivo following various fatty acid diets (Objective 1). Next, we aim to identify the role of LA and GLA in causing inflammation/ oxidative stress independent of ARA (Objective 2). In this regard, we do possess a unique knockout mice model of Elovl5 gene deletion, which leads to in vivo accumulation of LA and ALA but reduces ARA. Thus, we will isolate cardiomyocytes and macrophages from wildtype littermates and Elovl5KO following monounsaturated (MUFA, as control) and n-6 PUFA-rich diet regimens. Mass Spectrometry and gas chromatography-will be used to analyze eicosanoids, fatty acids and oxidized lipid metabolites. Cell signaling, inflammation, oxidative stress will also be evaluated in these cells as shown in earlier studies. SIGNIFICANCE This application is to understand the fundamental role of enzymatic and non-enzymatic bio-transformations of individual PUFA in mammalian cells like macrophages and cardiomyocytes that are spatially related but functionally distinct along with their responses during inflammation and oxidative stress. I believe under the onslaught of high doses of dietary PUFAs, to which every Canadian is subjected to, this program if funded can lead to fundamental knowledge synthesis in PUFA metabolism and identification of true causes of n-6 PUFA mediated inflammation in mammalian cells.