添加胆碱、胆固醇对牛肝细胞脂质沉积调控机制的研究

Fatty liver of dairy cows during the transition period, which caused by the negative energy balance, was frequently showed the important pathological features, such as high blood nonesterified fatty acid (NEFA) and severely hepatic lipidosis, and a lower level of phospholipid, cholesterol and cholesterol ester secretion in liver. While the very low density lipoprotein (VLDL), which was synthesized by phospholipid, cholesterol, cholesterol ester and triglyceride, was the major way in liver to excrete triglyceride. It was considered the abnormal metabolism of phospholipid and cholesterol in dairy cows hepatocyte should play an important role in the hepatic lipidosis with negative energy balance. Choline is the important precursor of phospholipid. With used of rumen-protected choline, The morbidity of fatty liver in dairy cows was significantly decreased. While the effecting of choline and cholesterol on lipids metabolism pathway in hepatocyte lipidosis of dairy cows were incompletely understood. To explore the metabolism of phospholipid and cholesterol in liver with negative energy balance, in this research project, the negative energy balance bovine hepatocytes were cultured to establish the hepatocyte lipidosis, then treated with choline and cholesterol, respectively. The characteristics of hepatocyte lipid metabolism was measured and analyzed by proteomics and metabonomics to reveal the pathogenesis of fatty liver in dairy cows. This study will provide strong theoretical basis for preventing of the fatty liver in dairy cows.

能量负平衡导致的围产期奶牛脂肪肝以高非酯化脂肪酸（NEFA）血症、肝脏大量甘油三脂蓄积，但肝脏磷脂、胆固醇、胆固醇脂含量相对低下为病理特征。极低密度脂蛋白（VLDL）是反刍动物肝脏清除甘油三酯的主要形式，磷脂、胆固醇脂、胆固醇以及甘油三脂是合成VLDL的主要成分。因此，在奶牛处于能量负平衡时磷脂和胆固醇代谢异常对肝脏甘油三酯蓄积起重要作用。胆碱是肝脏合成磷脂的重要前体物，通过日粮添加过瘤胃胆碱可有效降低奶牛脂肪肝发病率。然而添加胆碱可影响肝脂代谢中哪些通路，而添加胆固醇是否能够有效减轻肝脂沉积及其调控机制，目前尚不明确。据此，本项目通过能量负平衡环境培养牛肝细胞，建立肝脂沉积病变，通过添加胆碱或胆固醇，利用代谢组学和蛋白组学技术对牛肝细胞脂质代谢特征进行系统全面分析，以阐明胆碱和胆固醇对能量负平衡导致的肝脂沉积的调控机制，进而明确其主要的代谢调控通路，为临床防治奶牛脂肪肝提供理论依据。

本项目旨在阐明添加胆碱和胆固醇对能量负平衡导致的肝脂沉积的调控机制。项目采用两步灌流法分离犊牛原代肝细胞，通过添加1.2 mM 非脂化脂肪酸（NEFA）培养12h成功复制肝细胞脂质沉积模型。在建立的肝细胞脂质沉积模型基础上添加10μm/dl氯化胆碱或50μm/dL胆固醇培养6h后收集样品，应用iTRAQ/MS蛋白组学技术及GC/MS代谢组学技术筛选差异物，以期阐明胆碱和胆固醇对能量负平衡导致的肝脂沉积的调控机制。结果显示，NEFA组与对照组相比共筛选出144个差异蛋白，22个差异代谢物。差异蛋白及代谢物分析显示NEFA诱导的犊牛肝细胞糖异生作用增强、线粒体内呼吸电子传递调控蛋白水平低下、柠檬酸循环代谢物水平低下导致线粒体完全氧化代谢障碍；NEFA诱导的犊牛肝细胞脂肪酸从头合成水平增强、肝细胞棕榈酸及硬脂酸代谢障碍、肝脂蓄积严重；此外NEFA介导的肝细胞存在ATF4通路内质网应激、NFKB炎性相关因子表达上调、细胞粘附相关蛋白水平升高、细胞生物氧化相关蛋白水平升高。NEFA+胆碱组与NEFA组相比共检测出74个差异蛋白，14个差异代谢物，表明添加胆碱促进了肝细胞磷脂合成以及VLDL的组装、降低花生四烯酸代谢进而降低肝脂蓄积；添加胆碱提高了肝细胞棕榈酸、丙酮酸线粒体转运以及精氨酸的转运调控蛋白水平；添加胆碱通过提高PTEN磷酸化降低PIP3调控相关蛋白水平。NEFA+胆固醇组和NEFA组相比共筛选了66个差异蛋白，17个差异代谢物。主要差异蛋白调控通路表明添加胆固醇降低了NEFA介导的PIP3及MAPK6水平、促进了硒代半胱氨酸代谢；添加胆固醇激活了SREBP通路的SCAP表达水平，提高了APOC3水平促进VLDL组装；此外添加胆固醇组细胞外葡萄糖和VC转运水平下降。本项目初步阐明添加胆碱和胆固醇对能量负平衡导致的肝脂沉积的调控机制，为临床防治奶牛脂肪肝提供理论支持。