Non-esterified fatty acids disrupt hepatic lipid metabolism and mitochondrial function via TLR4/MyD88/IRAK2 signaling in bovine hepatocytes

Abstract

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 2.4 mM) 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 12 h 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 2.4 mM 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.