NEFA介导内质网应激对奶牛肝细胞自噬和脂质代谢的调控机制

Negative energy balance (NEB) is considered as the pathological basis of energy metabolic disorders in periparturient dairy cows. Under the condition of NEB, excessive fat mobilization results in a marked influx of non-esterified fatty acids (NEFA) in blood, which is crucial to cause energy metabolic disorders, like fatty liver and ketosis etc. Preliminary experiment data showed that high NEFA concentrations increased the expression of endoplasmic reticulum stress (ERS) marker molecules in hepatocytes of dairy cows. Studies have shown that ERS occurs under various pathological conditions, and plays a vital role in pathologic processes of numerous diseases. However, molecular mechanisms of how NEFA-induced ERS is involved in the regulation of hepatocyte autophagy and lipid metabolism in dairy cows remain poorly understood. Therefore, we hypothesize that NEFA-induced ERS regulates hepatocyte autophagy and lipid accumulation, thereby promoting the occurrence of energy metabolic disorders in periparturient dairy cows. In order to make clear the involvement and regulation of NEFA-induced ERS in hepatocyte autophagy, expressions of several key indicators of ERS and autophagy are measured, and their correlation with blood NEFA concentration and liver TG content are as well analyzed in both healthy periparturient dairy cows and cows with high blood NEFA. Primary cultured cow hepatocytes with/without high NEFA are established as a experiment model in vitro. The expression of the key signal molecules of ERS, autophagy and lipid metabolism and their relationships are determined in cultured hepatocytes, which will explore the regulatory mechanism of hepatocyte autophagy and lipid metabolism by NEFA-induced ERS in dairy cows. The present project will further reveal the pathogenesis of energy metabolism disorders in dairy cows, and establish the theoretical basis for searching new targets and methods for prevention and treatment of those diseases.

脂肪过度动员导致的高非酯化脂肪酸（NEFA）血症是诱发围产期奶牛能量代谢障碍性疾病的前提。申请者前期研究显示，高浓度NEFA可显著上调奶牛肝细胞中内质网应激（ERS）标志性分子表达。研究表明ERS与多种疾病发生有关，但NEFA介导的ERS是否参与奶牛肝细胞自噬和脂质代谢调节还不明确。本项目以“NEFA介导的ERS调控肝细胞自噬并促进肝脂沉积，进而促发奶牛能量代谢障碍性疾病”为理论假设，通过测定健康与高NEFA血症奶牛肝脏中ERS和自噬关键因子表达水平，并分析其与血液NEFA浓度和肝脂沉积的相关性，明确NEFA介导ERS并参与奶牛肝细胞自噬调控；通过体外培养奶牛肝细胞并模拟高NEFA血症，测定ERS、自噬和脂质代谢关键信号分子表达水平及其相互关系，探究NEFA介导ERS对奶牛肝细胞自噬和脂质代谢的调控机制，为进一步揭示奶牛能量代谢障碍性疾病的发病机理并探寻其防治的新靶标和途径奠定理论基础。

产后能量摄入无法满足机体代谢需求导致的能量负平衡（NEB）是引发奶牛脂肪肝和酮病的共同病理学基础；NEB条件下，储备的体脂被大量动员而引起的高非酯化脂肪酸（NEFA）血症是引发奶牛能量代谢障碍性疾病的前提。内质网应激（ERS）与多种疾病的发生密切相关，本项目通过测定血液中ERS下游调控脂代谢关键肝脏因子血管生成素样蛋白4（ANGPTL4）和成纤维细胞生长因子21（FGF21）的浓度，明确两种肝脏因子在围产期健康奶牛血液中的变动范围和规律，以及发生能量代谢障碍性疾病时两种肝脏因子的变化；在建立奶牛肝细胞体外培养体系的基础上，添加NEFA模拟NEB条件下奶牛肝脏代谢环境，测定不同浓度的NEFA对ANGPTL4和FGF21表达和分泌的影响；同时，通过比较围产期健康奶牛与高NEFA血症奶牛肝脏中ERS标志性分子的表达水平，明确罹患能量代谢障碍性疾病奶牛肝细胞ERS状态的变化。利用奶牛原代肝细胞中添加NEFA处理不同时间和浓度，评价NEFA对肝细胞内质网应激和自噬相关标志性分子及下游基因表达的影响，以期探究围产期奶牛肝脂代谢紊乱性疾病的分子机制。结果表明，血清中AGNPTL4和FGF21的变化受机体能量状态的影响，能量负平衡可上调两种肝脏因子；酮病和脂肪肝奶牛血清中两种肝脏因子的浓度显著高于同时期健康对照组奶牛；高浓度NEFA可显著促进体外培养的奶牛肝细胞中ANGPTL4和FGF21的表达和分泌，根据ANGPTL4和FGF21可诱导胰岛素抵抗的前提，推测两种肝脏因子可能参与围产期奶牛能量代谢障碍性疾病的病理学过程。NEFA可诱导奶牛原代肝细胞的ERS及自噬，并且ERS在一定程度上介导NEFA诱导的自噬，NEFA介导PERK-eIF2α信号通路可诱导奶牛原代肝细胞自噬并促进肝脂沉积。以上结果为揭示奶牛能量代谢障碍性疾病的发病机理并探寻其防治的新靶标和途径奠定了理论基础。