SREBP-1c/Cidea信号通路在能量负平衡奶牛肝脂沉积中的作用机制

The pathologic basis of fatty liver in dairy cows is negative energy balance. Dairy cows with negative energy balance could result in a high blood concentration of nonesterified fatty acid (NEFA) and/or β-hydroxybutyric acid (BHBA). The above metabolic signals are associated with the development of fatty liver in dairy cows. Sterol regulatory element-binding protein 1c (SREBP-1c) and cell death-inducing DFF45-like effectors a (Cidea) are the pivotal regulators for the hepatic lipid metabolism. Therefore, we hypothesized that high blood concentration of NEFA and BHBA activate SREBP-1c/Cidea signaling pathway to induce hepatic lipidosis in dairy cows. In vivo, dairy cows with fatty liver were used as animal model. In vitro, the bovine hepatocytes were cultured and treated with NEFA and BHBA, respectively. Furthermore, the hepatocytes were transfected with SREBP-1c and Cidea overexpression adenovirus and silencing Cidea expression adenovirus, respectively. The changes of key molecules in SREBP-1c/Cidea signaling pathway and the expression and activity of lipid metabolism enzymes were detected. Therefore, the regulation mechanism of SREBP-1c/Cidea on hepatic lipid metabolism in dairy cows could be revealed. In addition, the effects of NEFA and BHBA on the SREBP-1c/Cidea signaling pathway were evaluated. This study will provide valuable information to reveal the pathogenesis of fatty liver in dairy cows.

奶牛脂肪肝的病理学基础是能量负平衡，能量负平衡可导致高非酯化脂肪酸（NEFA）和/或高β-羟丁酸（BHBA）血症，而这与奶牛脂肪肝发生发展密切相关。固醇调节元件结合蛋白1c（SREBP-1c）和DNA断裂因子相似蛋白a (Cidea)是肝脂代谢的决定性调节因子。据此，本项目提出高NEFA和高BHBA血症可能通过激活SREBP-1c/Cidea信号通路促进肝脂沉积的科学假设。本项目通过脂肪肝奶牛体内实验，及体外培养奶牛肝细胞添加NEFA和BHBA实验，并分别转染过表达SREBP-1c和Cidea腺病毒和沉默Cidea表达的腺病毒，观测SREBP-1c/Cidea通路关键分子变化，及下游脂代谢相关酶表达水平和活性，以明确SREBP-1c/Cidea调节奶牛肝细胞脂代谢的作用机制，明确高NEFA和高BHBA血症对SREBP-1c/Cidea通路的影响，为阐明奶牛脂肪肝的发病机制提供理论依据。

奶牛脂肪肝是围产期奶牛常发生的以能量负平衡为病理基础的营养代谢病。能量负平衡，动员脂肪，造成高非酯化脂肪酸血症（NEFA）。固醇调节元件结合蛋白(SREBP-1c)是调节肝脂代谢的重要脂质转录因子。诱导细胞死亡的DNA断裂因子45样效应因子α (Cidea)作为新发现的SREBP-1c通路下游分子的一员，与人和小鼠脂肪肝的发生发展有密切关系。体内实验表明，围产期脂肪肝奶牛存在严重的能量负平衡，SREBP-1c和Cidea分子在脂肪肝奶牛肝脏中高表达。脂肪酸从头合成关键酶：脂肪酸合成酶 ( FAS)， 乙酰辅酶A羧化酶1 (ACC1)在脂肪肝奶牛肝脏中表达显著升高；而参与极低密度脂蛋白 VLDL 组装相关脂蛋白：微粒体甘油三脂转运蛋白 (MTP)、载脂蛋白B100 (ApoB100) 和载脂蛋白E (ApoE) 的表达显著下降。体外培养奶牛肝细胞，结果表明，SREBP-1c能直接上调Cidea的基因mRNA和蛋白表达；过表达Cidea能显著促进奶牛肝细胞脂肪酸合成关键酶FAS和ACC1的表达，抑制脂肪转运相关蛋白MTP、ApoB100、ApoE的表达，导致肝细胞TAG积累和脂滴的变大；过表达Cidea可反向促进SREBP-1c的表达、转录活性和成熟。混合转染过表达SREBP-1c和沉默Cidea表达腺病毒，发现沉默Cidea能够抑制奶牛肝细胞脂肪酸从头合成，缓解SREBP-1c诱导的肝细胞TAG积累。转染沉默SREBP-1c腺病毒，混合添加不同浓度的NEFA，能够上调SREBP-1c和Cidea表达，1.2 mM时达到峰值；1.2 mM NEFA能显著促进SREBP-1c的入核和转录活性；SREBP-1c被沉默后，NEFA对SREBP-1c的激活作用被阻断。转染沉默Cidea腺病毒，混合添加1.2 mM的NEFA，结果表明，沉默Cidea能缓解NEFA诱导的奶牛肝细胞TAG积累。以上体内实验结果表明，脂肪肝奶牛肝脏SREBP-1c/Cidea通路被激活。体外实验表明，Cidea促进奶牛肝细胞TAG沉积和脂滴的变大；NEFA能够激活SREBP-1c/Cidea通路促进奶牛肝细胞TAG沉积。