Thioredoxin-2 suppresses hydrogen peroxide-activated nuclear factor kappa B signaling via alleviating oxidative...

During the periparturient period, both oxidative stress, and inflammation of adipose tissue are considered high risk factors for metabolic disorder of dairy cows. Oxidative stress can activate transcription factor nuclear factor kappa B (NF-κB), which lead to the upregulation of genes involved in inflammatory pathways. Thioredoxin-2 (TXN2) is a mitochondrial protein that regulates cellular redox by suppressing mitochondrial reactive oxygen species (ROS) generation in nonruminant, whereas the function of TXN2 in bovine adipocytes was unclear. Thus, the objective of this study was to evaluate how or by which mechanisms TXN2 regulates oxidative stress and NF-κB signaling pathway in bovine adipocytes. Bovine pre-adipocytes isolated from 5 healthy Holstein cows were differentiated and used for (1) treatment with different concentrations of hydrogen peroxide (H₂O₂; 0, 25, 50, 100, 200, or 400 μM) for 2 h; (2) transfection with or without TXN2 small interfering RNA (si-TXN2) for 48 h and then treated with or without 200 μM H₂O₂ for 2 h; (3) transfection with scrambled negative control siRNA (si-control) or si-TXN2 for 48 h, and then treatment with or without 10 mM N-acetylcysteine (NAC) for 2 h; (4) transfection with or without TXN2-overexpressing plasmid for 48 h and then treatment with or without 200 μM H₂O₂ for 2 h. High concentrations of H₂O₂ (200 and 400 μM) decreased protein and mRNA abundance of TXN2, reduced total antioxidant capacity (T-AOC) and ATP content in adipocytes. Moreover, 200 and 400 μM H₂O₂ reduced protein abundance of inhibitor of kappa B α (IκBα), increased phosphorylation of NF-κB and upregulated mRNA abundance of tumor necrosis factor-α (TNFA) and interleukin-1B (IL-1B), suggesting that H₂O₂-induced oxidative stress and activated NF-κB signaling pathway. Silencing of TXN2 increased intracellular ROS content, phosphorylation of NF-κB and mRNA abundance of TNFA and IL-1B, decreased ATP content and protein abundance of IκBα in bovine adipocytes. Knockdown of TXN2 aggravated H₂O₂-induced oxidative stress and inflammation. In addition, treatment with antioxidant NAC ameliorated oxidative stress and inhibited NF-κB signaling pathway in adipocytes transfected with si-TXN2. In bovine adipocytes treated with H₂O₂, overexpression of TXN2 reduced the content of ROS and elevated the content of ATP and T-AOC. Overexpression of TXN2 alleviated H₂O₂-induced inflammatory response in adipocytes, as demonstrated by decreased expression of phosphorylated NF-κB, TNFA, IL-1B, as well as increased expression of IκBα. Furthermore, the protein and mRNA abundance of TXN2 was lower in adipose tissue of dairy cows with clinical ketosis. Overall, our studies contribute to the understanding of the role of TXN2 in adipocyte oxidative stress and inflammatory response.