线粒体功能紊乱与炎症在奶牛脂肪肝肝脂代谢紊乱中的作用机制

Dairy cows with fatty liver display high blood concentration of nonesterified fatty acid (NEFA) and hepatic lipid metabolic disorder. High blood concentration of NEFA exists lipotoxicity. Mitochondria are the main organelles that metabolize NEFA in hepatocytes. Therefore, high level of NEFA may impair the mitochondrial function. Furthermore, dairy cows with fatty liver display different levels of inflammation, which may cooperate with mitochondrial dysfunction influences the hepatic lipid metabolism. Accordingly, we hypothesized that high blood concentration of NEFA could influence the mitochondrial function and inflammatory pathway in bovine hepatocytes. In vivo, dairy cows with fatty liver were used as animal model. In vitro, the bovine hepatocytes were cultured and treated with varied levels of NEFA and antioxidant N-acetyl-L-cysteine (NAC), and transfected with PGC-1α, Mfn2, NRF1, TLR2 and TLR4 overexpression adenovirus and TLR2 and TLR4-silencing adenovirus, respectively. The key molecules in the hepatic lipid metabolism, mitochondrial function and inflammatory pathway were detected to evaluate the changes of mitochondrial function and inflammatory pathway, to investigate the effect of NEFA on the lipid metabolism, mitochondrial function and inflammatory pathway, and to reveal the mechanism of cooperationof effects of mitochondrial dysfunction and inflammatory pathway on the hepatic lipid metabolism in dairy cows with fatty liver. This study will provide valuable information to reveal the pathogenesis of fatty liver in dairy cows.

脂肪肝奶牛存在高非酯化脂肪酸（NEFA）血症和肝脂肪代谢紊乱。高NEFA具有脂毒性，线粒体是NEFA的主要代谢场所，所以高NEFA可能影响线粒体功能。此外，脂肪肝奶牛还表现出不同程度的炎性反应，有可能与线粒体功能紊乱一起扰乱脂代谢。据此，本项目提出高NEFA血症可影响奶牛肝细胞的线粒体代谢功能和活化炎性通路的科学假设。通过不同程度脂肪肝奶牛体内实验，及体外培养奶牛肝细胞添加NEFA和抗氧化剂（NAC）实验，构建重组腺病毒载体过表达PGC-1α、Mfn2、NRF1、TLR2和TLR4及沉默TLR2和TLR4，并分别转染上述腺病毒，检测肝细胞脂代谢、线粒体功能及炎性信号通路关键分子变化，以明确不同程度脂肪肝奶牛线粒体功能和炎性通路活化水平，明确NEFA对肝细胞脂代谢、线粒体功能和炎性信号通路的影响及线粒体功能紊乱与炎性通路协同影响肝脂代谢的机制，为阐明奶牛脂肪肝的发病机制提供理论依据。

围产期能量负平衡（NEB）引起奶牛脂肪组织动员，由此产生的高浓度非酯化脂肪酸（NEFA）可导致脂肪肝和酮病，二者的发生可引起奶牛肝脏和乳腺组织代谢变化。因此，本项目的主要研究内容为探究围产期NEB奶牛高NEFA血症引起的肝脏和乳腺组织的代谢紊乱。本项目通过围产期奶牛体内实验发现，脂肪肝奶牛肝脏存在内质网应激（ERS），脂合成升高，脂氧化降低，炎性信号通路激活，MFN2低表达，由此引起肝脏脂蓄积和炎性反应；酮病奶牛存在系统性炎症，肝脏存在脂蓄积及肝损伤，炎性信号通路激活；临床酮病奶牛乳腺组织存在线粒体功能障碍和氧化应激，酮病奶牛乳腺组织自噬增强。通过体外奶牛肝细胞实验发现，高浓度的NEFA可以抑制奶牛肝细胞自噬激活、脂氧化和MFN2表达，可诱导ERS，促进脂合成和氧化应激，激活炎性信号通路；丙酸可通过促进自噬缓解PA引起的肝细胞自噬活性降低及ERS，增加细胞活力。体外肝细胞沉默MFN2可促进高浓度NEFA诱导的奶牛肝细胞脂蓄积和炎性信号通路的激活；过表达MFN2可缓解高浓度NEFA诱导的奶牛肝细胞脂蓄积和炎性信号通路激活。本项目研究结果阐明了围产期NEB奶牛肝脏和乳腺线粒体功能、脂代谢信号通路、炎性信号通路、氧化应激、ERS及自噬信号通路的变化，明确了高浓度的NEFA引起奶牛肝脏和乳腺代谢紊乱的具体作用机制，可为揭示围产期奶牛营养代谢病的发病机制及科学防控提供实验和理论依据。