Excessive non-esterified fatty acids (NEFA) in the blood often lead to ketosis and fatty liver in peripartal cows. The integrity of mitochondrial structure and function plays a key role in the development of lipid metabolism imbalance and inflammation. However, the precise molecular mechanisms of NEFA's impact on these processes remain unclear. To explore how NEFA (0, 0.3, 0.6, 1.2, and 2.4mM) influence lipid metabolism, inflammation, and mitochondrial function in perinatal cow hepatocytes. Bovine primary hepatocytes were isolated from five 1-day-old Holstein calves using collagenase IV perfusion, hepatocytes were treated with various concentrations of NEFA for 12h to systematically simulate the metabolic changes in the negative energy balance (NEB) environment. Oxidative stress levels, lipid metabolism gene and protein expression, inflammatory pathway activation, and mitochondrial structure and function were assessed. Compared with the control group, 1.2 and 2.4mM NEFA significantly increased lipid accumulation (TAG, T-CHO, LDL-c) and oxidative stress (MDA), while reducing antioxidant levels (GSH, SOD, T-AOC). NEFA upregulated lipogenic genes (SREBP-1c, ACC, FASN) and downregulated lipid oxidation genes (PPARα, CPT1A). Lipid transport was impaired, with altered APOE, LDLR, CD36, and L-FABP expression. NEFA activated the TLR4/MyD88/IRAK2/NF-κB pathway, increasing pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Mitochondrial function was impaired, as evidenced by increased ROS, reduced mitochondrial membrane potential, decreased mtDNA levels, and downregulated expression of oxidative phosphorylation genes. NEFA enhanced IRAK2 mitochondrial translocation, inhibited the mitochondrial biogenesis process, and increased apoptosis. NEFA alters lipid metabolism, inflammation, and mitochondrial function in bovine hepatocytes via the TLR4/MyD88/IRAK2 pathway, providing a new target for preventing metabolic diseases in perinatal cows.

Keywords: Bovine hepatocytes; NEFA; lipid metabolism; metabolic inflammation; mitochondrial dysfunction.

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围产期奶牛血液中过多的非酯化脂肪酸（NEFA）常常导致酮病和脂肪肝。线粒体结构和功能的完整性在脂质代谢失衡和炎症的发展中起着关键作用。然而，NEFA对这些过程的确切分子机制仍不清楚。为了探索不同浓度的NEFA（0、0.3、0.6、1.2 和 2.4 mM）如何影响围产期奶牛肝细胞中的脂质代谢、炎症和线粒体功能。使用胶原酶IV灌注法从五头1日龄荷斯坦犊牛中分离出原代牛肝细胞，将肝细胞用不同浓度的NEFA处理12小时以系统地模拟负能量平衡（NEB）环境下的代谢变化。评估了氧化应激水平、脂质代谢基因和蛋白表达、炎症途径激活以及线粒体结构和功能。与对照组相比，1.2 mM 和 2.4 mM NEFA 显著增加了脂质积累（TG、TC、LDL-c），并提高了氧化应激（MDA）的水平，同时降低了抗氧化剂的水平（GSH、SOD、T-AOC）。NEFA上调了生脂基因（SREBP-1c、ACC、FASN），下调了脂质氧化基因（PPARα、CPT1A）。脂质转运受损，APOE、LDLR、CD36 和 L-FABP 的表达发生了改变。NEFA激活了TLR4/MyD88/IRAK2/NF-κB 通路，增加了促炎细胞因子的水平（IL-1β、IL-6、TNF-α）。线粒体功能受损，表现为ROS增加、线粒体膜电位降低、mtDNA 水平下降以及氧化磷酸化基因表达下调。NEFA促进了IRAK2向线粒体的转位，抑制了线粒体生物发生过程，并增加了细胞凋亡。NEFA通过TLR4/MyD88/IRAK2通路改变牛肝细胞中的脂质代谢、炎症和线粒体功能，为预防围产期奶牛代谢疾病提供了新的靶点。

关键词：牛肝细胞；NEFA；脂质代谢；代谢性炎症；线粒体功能障碍。

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