Synergistic Effects of Medium-chain and Omega-3 Triglycerides on Cellular Omega-3 Fatty Acid Enrichment: Characterization, Mechanisms, and Optimization

不良炎症反应导致细胞死亡和器官功能障碍。我们将研究含有富含二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）的中链甘油三酯（TG）（MCT）和omega-3（n-3）TG的脂肪乳剂的急性静脉（IV）治疗是否提供了一种新的治疗方法，通过靶向快速细胞n-3 FA膜富集来改善不良炎症反应。n-3脂肪酸（FA）由于其对细胞凋亡和自由基及促炎性类二十烷酸的产生的促愈合和抗炎作用而日益被认为是IV喂养中的有效补充剂。在实验动物模型中的研究表明，n-3富含TG的乳剂颗粒（TGRP）的推注在减少中风后的脑死亡和发病率方面具有治疗保护作用。我们已经发现，含有MCT和鱼油TG（8：2 wt/wt）的TGRP比其他TGRP更有效地富集具有n-3 FA的白色血细胞和内皮细胞的细胞膜。使用核磁共振（NMR），我们报告称，将MCT纳入模型TGRP可促进模型膜脂质紊乱，这可能会加速n-3递送速率，突出了推注用于n-3 FA富集的优势。然而，很少有研究涉及急性IV n-3给药的急性抗炎作用。目前，没有关于MCT与n-3 TG的最佳混合物以实现最快速和最高强度抗炎作用的数据。我们的中心假设是，包含MCT和n-3 TG的TGRP的推注将协同影响膜动力学，以加速n-3 FA掺入细胞膜，这将启动n-3介导的抗炎和抗凋亡作用。这些将在三个具体目标下进行研究。在目标1下，我们将使用生物物理方法结合基于质谱的脂质组学来表征具有不同n-3 TG含量的含MCT的TGRP的生物物理特性，以评估由含DHA、EPA或两者的特定MCT：n-3 TGRP介导的膜结构和活性的改变。在目标2中，我们将在培养的免疫效应巨噬细胞和小鼠模型中定义负责目标1中表征的特异性MCT：n-3 TGRP内化和代谢利用的细胞摄取途径。然后，我们将在目标3下研究MCT：n-3 TGRP细胞内化如何影响小鼠模型中单核细胞和巨噬细胞对急性免疫刺激（LPS）的功能活性。拟议的细胞和动物模型研究将建立创新方法，适用于将亲脂性和疏水性治疗剂靶向快速递送至特定器官或组织。我们将揭示MCT和n-3 TG之间的新型协同作用，调节膜脂质组成，细胞脂质代谢和免疫反应。我们的研究将对许多领域具有重要意义，包括感染和炎症相关的组织损伤和器官功能障碍的免疫反应。

Adverse inflammatory response leads to cell death and organ dysfunction. We will investigate whether acute intravenous (IV) treatments of lipid emulsions containing medium-chain triglycerides (TG) (MCT) and omega-3 (n-3) TG enriched in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) provide a new therapeutic approach to ameliorate adverse inflammatory responses by targeting rapid cellular n-3 FA membrane enrichment. n-3 fatty acids (FA) are increasingly recognized as a potent supplement in IV feeding because of their pro-healing and anti-inflammatory effects against apoptosis and production of free radicals and pro- inflammatory eicosanoids. Studies in experimental animal models demonstrate that bolus injections of n-3 TG- rich emulsion particles (TGRP) are therapeutically protective in decreasing brain death and morbidity after stroke. We have discovered that TGRP containing both MCT and fish oil TG (8:2 wt/wt) enriched cell membranes of white blood cells and endothelial cells with n-3 FA more efficiently than other TGRP. Using nuclear magnetic resonance (NMR), we reported that the inclusion of MCT into model TGRP promotes model membrane lipid disorder that may accelerate n-3 delivery rates, highlighting the advantage of bolus injection for n-3 FA enrichment. However, very few studies have addressed the acute anti-inflammatory effects of acute IV n-3 administration. Currently, there is no data on optimal mixtures of MCT with n-3 TG to achieve the most rapid and highest-magnitude anti-inflammatory action. Our central hypothesis is that bolus injections of TGRPs containing both MCT and n-3 TG will cooperatively affect membrane dynamics to accelerate n-3 FA incorporation into cell membranes, which will initiate n-3-mediated anti-inflammatory and anti-apoptotic actions. These will be studied under 3 Specific Aims. Under Aim 1, we will characterize biophysical properties of MCT-containing TGRP with different n-3 TG contents using biophysical approaches coupled with mass spectrometry-based lipidomics to assess alteration in membrane structure and activity mediated by specific MCT:n-3 TGRP containing DHA, EPA, or both. In Aim 2, we will define cellular uptake pathways responsible for internalization and metabolic utilization of specific MCT:n-3 TGRP characterized in Aim 1 in cultured immune-effector macrophages and in mouse models. We will then investigate under Aim 3 how MCT:n-3 TGRP cellular internalization affects functional activities of monocytes and macrophages in response to an acute immune stimulus (LPS) in mouse models. The proposed studies in cells and animal models will establish innovative methods that are adaptable for targeted, rapid delivery of lipophilic and hydrophobic therapeutic agents to specific organs or tissues. We will uncover novel synergies between MCT and n-3 TG that modulate membrane lipid composition, cell lipid metabolism, and immune responses. Our studies will be of high significance to many fields, including immune responses to infection and inflammation-related tissue injury and organ dysfunction.