能量负平衡内生信号NEFA和BHBA介导的UPR在奶牛酮病发生发展中的作用机制

Negative energy balance (NEB) is considered as the pathological basis of ketosis in dairy cows, and non-esterified fatty acids (NEFA) and β-hydroxybutyric acid (BHBA) are the important endogenous signal molecules produced by the body under the condition of NEB. Studies have shown that livers of early lactation cows emerge the obvious unfolded protein response (UPR), which may participate in the pathological process of energy metabolism disorders. On the basis of the facts that NEFA could mediate the hepatocyte UPR of dairy cows and BHBA involves in the metabolic adaptive adjustment through reducing UPR, we hypothesize that NEFA mediates hepatocyte UPR thus contributing to the ketosis in dairy cows, while BHBA could influence the occurrence and development of ketosis in dairy cows via participating in the UPR process. In vivo, dairy cows with ketosis were used as animal model. In vitro, the bovine hepatocytes were cultured and treated with varied concentrations of NEFA and/or BHBA, and transfected with overexpression or silencing of PERK, IRE1 and ATF6 adenovirus, respectively. The key molecules in the hepatic lipid metabolism and UPR signaling pathway were measured to make clear the NEFA- and BHBA-mediated the regulation of UPR on lipid metabolism of dairy cows, which will explore the signaling mechanism of the occurrence and development of ketosis in dairy cows affected by BHBA participating in the NEFA-mediated UPR process. The present study will provide theoretical basis for searching new approach for preventing of ketosis in dairy cows.

能量负平衡是奶牛酮病的病理学基础，非酯化脂肪酸（NEFA）和β-羟丁酸（BHBA）是能量负平衡条件下机体产生的重要内生性信号分子；泌乳初期奶牛肝脏存在明显的未折叠蛋白应答（UPR），其有可能参与能量代谢障碍性疾病的病理学过程。本项目以NEFA可介导奶牛肝细胞UPR，且BHBA能通过缓解UPR参与机体代谢适应性调节的事实为依据，提出NEFA介导肝细胞UPR促发奶牛酮病，而BHBA通过参与UPR过程影响奶牛酮病发生发展的理论假设。通过不同类型奶牛酮病的体内实验，结合体外培养奶牛肝细胞添加NEFA和BHBA实验，分别构建重组腺病毒载体过表达和沉默PERK、IRE1和ATF6，并分别转染上述腺病毒，检测肝脂代谢和UPR关键分子变化，明确NEFA和BHBA可介导UPR调控奶牛肝脂代谢，探究BHBA通过参与NEFA介导的UPR过程影响奶牛酮病发生发展的作用机制，为探索奶牛酮病防治的新途径提供理论支持。

围产期奶牛能量需求增加与干物质摄入量减少导致的能量负平衡是诱发酮病和脂肪肝等能量代谢障碍性疾病的病理学基础。能量负平衡条件下，机体通过增加储备的体脂动员和分解以改善能量缺乏，致使围产期奶牛血液循环中非酯化脂肪酸（NEFA）浓度升高。近年来，越来越多的研究表明肝脏内质网应激（ERS）与肝脂代谢紊乱性疾病的发生有关，尽管有研究显示高NEFA可诱导肝细胞内质网应激的发生，但NEFA能否介导ERS信号通路调节肝脏脂质代谢？且其作用机制尚不清楚。本项目以分离培养的奶牛原代肝细胞为模型，通过外源添加NEFA模拟NEB条件下奶牛肝脏代谢环境，检测信号通路关键蛋白表达变化以及脂质转运、合成和氧化关键因子表达水平的变化以及自噬状态，旨在明确NEFA介导ERS信号通路调控奶牛肝脂代谢和自噬的分子机制。结果显示，NEFA可介导PERK-eIF2α信号通路促进细胞内脂质的从头生成，促进奶牛肝细胞脂质氧化。PERK-eIF2α信号通路的激活可降低VLDL组装成分APOB的表达和VLDL的分泌，从而在一定程度上抑制了NEFA诱导的VLDL的组装与分泌。NEFA可上调自噬相关基因的蛋白水平以及奶牛肝细胞中自噬体数量。总之，NEFA可介导PERK-eIF2α信号通路促进肝细胞脂质合成和脂质氧化，同时促进自噬体形成，但抑制了VLDL的装配与分泌，最终在一定程度上降低了NEFA诱导的肝细胞中脂质蓄积。上述结果有利于进一步探明ERS信号通路在脂肪肝发生发展中的作用，并为探寻肝脏脂质代谢紊乱性疾病防治的新靶标和途径提供了理论依据。