Polyunsaturated fatty acid (PUFA), inflammation and antioxidants

在上个世纪，加拿大人的饮食中充斥着n-6多不饱和脂肪酸（n-6 PUFA），比如亚油酸（LA）。随着这些变化，以异常炎症和氧化应激为特征的各种慢性疾病，如糖尿病和动脉粥样硬化，在加拿大人中急剧增加。我们对PUFA代谢和炎症/氧化应激的理解存在基础知识缺口。我们对n-6 PUFA驱动炎症的大部分知识都集中在花生四烯酸（ARA）上，而ARA在慢性疾病中实际上含量较低。然而，最近的研究表明，其他n-6 PUFAs，如亚油酸（LA）和γ亚麻酸（GLA）是炎症的主要参与者。在这方面，LA的氧化修饰和延长酶/去饱和酶介导的PUFA生物转化仍有待研究。然而，由于LA在体内快速依次转化为GLA、DGLA，最后转化为ARA，因此很难理解其具体作用。因此，总的来说，这个项目的长期目标是阐明n-6 PUFA生物转化在炎症和氧化应激中的作用。我的总体假设是，在高LA可用性下，LA的生物转化在调节炎症/氧化应激中至关重要。该计划的总体目标是了解LA和GLA在心肌细胞和巨噬细胞炎症和氧化应激中的氧化和代谢修饰的作用。我们的目标是在体外（使用H9c2和RAW 264.7细胞系）和体内不同脂肪酸饮食下，在高n-6 PUFA环境中氧化LA代谢物和4-HNE的影响（目标1）。接下来，我们的目标是确定LA和GLA在独立于ARA引起炎症/氧化应激中的作用（目标2）。在这方面，我们确实拥有一个独特的Elovl5基因缺失敲除小鼠模型，该模型会导致LA和ALA在体内的积累，但会减少ARA。因此，我们将在单不饱和（MUFA，作为对照）和富含n-6 pufa的饮食方案下，从野生型幼崽和Elovl5KO中分离心肌细胞和巨噬细胞。质谱法和气相色谱法将用于分析类二十烷酸、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。该应用旨在了解单个PUFA在哺乳动物细胞（如巨噬细胞和心肌细胞）中的酶促和非酶促生物转化的基本作用，这些细胞在空间上相关，但在功能上不同，以及它们在炎症和氧化应激中的反应。我相信，在每个加拿大人都要承受的高剂量膳食PUFA的冲击下，如果得到资助，这个项目可以导致PUFA代谢的基础知识合成，并确定哺乳动物细胞中n-6 PUFA介导炎症的真正原因。

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.