左旋肉碱对冷应激断奶羔羊棕色化脂肪线粒体抗氧化功能的作用研究

Cold stress is a common stressor of weaning lambs, accompanying high incidence of oxidative stress diseases due to short of nutrition, resulting in lamb slow growth or even death. Therefore, an effective way of improving lamb cold resistance is to clarify the causes of cold stress by regulating nutritional factors. Brown adipose tissue is an beneficial soft gold, also called “heat producer” based on many previous studies ."Green nutrition"-L-carnitine is the only vehicle during fat burning, as a way of replenishing energy, which can alleviate the body stress status. Here we hypothesize that L-carnitine could induce white fat brownization of cold-stressed weaning lambs, which inhibits the excessive production of reactive oxygen species (ROS), subsequently maintaining the body homeostasis, and which could be the main mechanism of lamb cold resistance development. Lambs and primary white adipose cell browning model will be needed. Effects of L-carnitine on the brown fat formation in cold-stimulated lambs will be evaluated, and the corresponding influence on major antioxidative molecules of Nrf2-ARE signaling pathway in lamb brown adipose cell mitochondria will be investigated in order to further clarify the cold tolerance mechanism of L-cartine in brown fat. Therefore, this study could provide a feasible way for weaning lambs to resist cold stress and lay the foundation for prevention of oxidative stress diseases in lambs.

冷应激是断奶羔羊常见应激原，同时会伴随营养因素缺乏引起氧化应激性疾病高发，导致羔羊生长缓慢甚至死亡。因此通过调控营养因素揭示抗寒的成因，是提高羔羊抗冷应激反应有效途径。目前大量研究表明，棕色脂肪组织作为有益软黄金，被认为是 “产热器”。“绿色营养素”左旋肉碱是脂肪燃烧过程中唯一的运载工具，其作为补充能量的手段，可缓解机体应激状态。基于此，项目组推测左旋肉碱可能会促使冷刺激断奶羔羊白色脂肪棕色化的形成，通过棕色化脂肪细胞抑制过量活性氧（ROS）的产生维持机体稳态平衡，这可能是羔羊抵抗冷刺激的重要机制。拟采用断奶肉羔羊在体和原代白色脂肪细胞棕色化模型，评价左旋肉碱对冷刺激羔羊棕色化脂肪形成的影响，检测左旋肉碱对羔羊棕色化脂肪线粒体在Nrf2-ARE信号转导通路中抗氧化关键分子变化，以明确左旋肉碱和棕色化脂肪之间抗寒机制，为断奶羔羊抵抗冷应激提供可行途径，预防羔羊氧化应激性疾病奠定基础。

左旋肉碱是脂肪燃烧过程中唯一的运载工具，其作为补充能量的手段，可缓解冷应激时羔羊断奶损伤状态。基于此，项目组推测左旋肉碱可能会促使冷刺激断奶羔羊白色脂肪棕色化的形成，通过棕色化脂肪细胞抑制过量活性氧（ROS）的产生维持机体稳态平衡，这可能是羔羊抵抗冷刺激的重要机制。项目采用断奶肉羔羊在体实验，检测了左旋肉碱、CPT-Ⅰ、CPT-Ⅱ、ROS、SOD、GSH-Px、H2O2等含量，结果显示左旋肉碱在冷刺激环境下对断奶羔羊血液中抗氧化指标具有保护作用，L-Carnitine对UCP1和Elovl3mRNA冷刺激-高水平左旋肉碱组影响最显著。同时检测结果显示L-Carnitine能降低羔羊脂肪组织脂肪酸含量，可预防因脂肪酸过多沉积引发的机体损伤，且脂肪组织中较血液中长链脂肪酸种类增多，特别是二十烷酸以后的脂肪酸或其衍生物，CPT1位于线粒体外膜，可以催化长链脂肪酸CoA与肉碱结合，穿过线粒体外膜，到达线粒体内膜，使脂酰CoA从细胞质进入线粒体，是脂肪氧化分解至关重要的一步，说明L-carnitine可以分解脂肪酸代谢。采用体外培养羔羊原代皮下白色脂肪细胞棕色化模型，结果显示L-carnitine冷处理组可以显著提高PUFA的含量，提示我们绿色饲料添加剂L-carnitine在冷刺激条件下可以增强脂肪酸的代谢途径，降低白色脂肪大量蓄积，为脂肪代谢稳态平衡提供了理论依据。采用qRT-PCR 和Western-Blotting技术，结果显示添加L-carnitine冷刺激组NQO1、HO-1、SOD、Nrf2、Keap1等蛋白表达水平显著高于对照组，提示我们L-Carnitine可以激活棕色脂肪。蛋白质组学结果研究表明，L-Carnitine对冷应激断奶羔羊脂肪棕色化形成时，NADPH氧化酶复合体是脂肪细胞呼吸爆发产生ROS的最为关键的酶系，调控NAPDH的表达和活化，促进ROS的产生，基于Nrf2/ARE通路L-Carnitine对棕色化脂肪抗氧化的必要性。目前结果表明L-carnitine能加强分解脂肪代谢，参与抗氧化作用，L-carnitine可以激活棕色化脂肪的形成，其抗冷应激原理是通过捕获过氧自由基来抑制过氧化链式反应的进行，阐明他们之间的关联对防治断奶羔羊氧化应激损伤疾病具有重要意义。