Polyunsaturated fatty acid (PUFA), inflammation and antioxidants

问题在上个世纪，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）。沿着这些变化，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，在加拿大人中激增。我们对PUFA代谢和炎症/氧化应激的理解存在基础知识差距。背景我们对n-6 PUFA驱动的炎症的大部分知识都集中在花生四烯酸（ARA）上，而花生四烯酸在慢性疾病中实际上较低。然而，最近的研究表明，其他n-6 PUFA如亚油酸（LA）和γ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/去饱和酶介导的PUFA生物转化仍然研究不足。然而，很难理解LA的具体作用，因为它在体内迅速生物转化为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 PUFA生物转化对炎症和氧化应激的作用。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。该计划的首要目标是了解LA和GLA对心肌细胞和巨噬细胞中炎症和氧化应激的氧化和代谢修饰的作用。我们的目标是在体外（使用H9 c2和RAW 264.7细胞系）和体内各种脂肪酸饮食（目标1）后，在高n-6 PUFA环境中氧化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，而不依赖于ARA（目标2）。在这方面，我们确实拥有Elovl 5基因缺失的独特敲除小鼠模型，其导致LA和ALA的体内积累，但减少ARA。因此，我们将在单不饱和（MUFA，作为对照）和富含n-6PUFA的饮食方案后从野生型同窝仔和Elovl 5 KO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花生酸，脂肪酸和氧化脂质代谢产物。细胞信号传导、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。意义本申请旨在了解单个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，但在功能上沿着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PUFA的冲击下，如果得到资助，这个项目可以导致PUFA代谢的基本知识合成，并确定哺乳动物细胞中n-6 PUFA介导的炎症的真正原因。

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.******