Heilongjiang Provincial Key Laboratory of Prevention and Control of Bovine Diseases, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, No. 5 Xinyang Road, Daqing, Heilongjiang Province 163319, China.
College of Veterinary Medicine, Hunan Agricultural University, Changsha, 410128, China.
J Dairy Sci. 2021 Dec;104(12):12830-12844. doi: 10.3168/jds.2021-20732. Epub 2021 Sep 16.
Bovine mammary epithelial cells undergo an increase in metabolic rate, mitochondrial dysfunction, and oxidative stress after calving. Nuclear factor erythroid 2-related factor 2 (NFE2L2), a master regulator of cellular redox homeostasis, plays crucial roles in the regulation of mitochondrial function. The objective of this study was to investigate the role of NFE2L2 on mitochondrial function in bovine mammary epithelial cells under hyperlipidemic conditions. Three experiments were conducted as follows: (1) the immortalized bovine mammary epithelial cell line MAC-T was treated with various concentrations of free fatty acids (FFA; 0, 0.6, 1.2, or 2.4 mM) for 24 h to induce stress; (2) MAC-T cells were transfected with small interfering RNA targeting NFE2L2 (si-NFE2L2) and scrambled nontarget negative control (si-Control) for 48 h; and (3) MAC-T cells were pretreated with 10 μM sulforaphane (SFN), an activator of NFE2L2, for 24 h followed by treatment with 1.2 mM FFA for an additional 24 h. Results indicated that exogenous FFA challenge induced linear and quadratic increases in concentrations of mitochondrial reactive oxygen species (ROS). Compared with 0 mM FFA, mitochondrial membrane potential, mRNA abundance of oxidative phosphorylation complexes (CO I-V), protein abundance of nuclear respiratory factor 1 (NRF1), peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), mitochondrial transcription factor A (TFAM), and NFE2L2 along with the contents of ATP, mitochondrial DNA (mtDNA), and total mitochondria were greater in the MAC-T challenged with 0.6 mM FFA group, but lower in the 1.2 and 2.4 mM FFA cultures. Knockdown of NFE2L2 via small interfering RNA led to greater mitochondrial ROS content and lower mitochondrial membrane potential along with contents of ATP, mtDNA, and total mitochondria. The SFN pretreatment upregulated protein abundance of NFE2L2 and attenuated the downregulation of NFE2L2 induced by FFA. Pretreatment with SFN attenuated the downregulation induced by FFA of PGC-1α, NRF1, and TFAM protein abundance along with contents of mtDNA and total mitochondria. Furthermore, SFN pretreatment attenuated the upregulation of mitochondrial ROS content, the downregulation of mitochondrial membrane potential, and the decreases in ATP, mtDNA, and mitochondrial content induced by FFA. Overall, data indicated that FFA inhibit NFE2L2, resulting in mitochondrial dysfunction and ROS production in bovine mammary epithelial cells. Thus, NFE2L2 may be a promising therapeutic target against metabolic challenge-driven mitochondrial dysfunction and oxidative stress in bovine mammary epithelial cells.
奶牛乳腺上皮细胞在产后会经历代谢率增加、线粒体功能障碍和氧化应激。核因子红细胞 2 相关因子 2（NFE2L2）是细胞氧化还原平衡的主要调节剂，在调节线粒体功能方面发挥着关键作用。本研究旨在探讨 NFE2L2 在高脂条件下奶牛乳腺上皮细胞中线粒体功能中的作用。进行了以下三个实验：（1）用不同浓度的游离脂肪酸（FFA；0、0.6、1.2 或 2.4 mM）处理永生化奶牛乳腺上皮细胞系 MAC-T 24 h 以诱导应激；（2）用靶向 NFE2L2 的小干扰 RNA（si-NFE2L2）和无义对照（si-Control）转染 MAC-T 细胞 48 h；（3）用 10 μM 萝卜硫素（SFN）预处理 MAC-T 细胞 24 h，激活 NFE2L2，然后用 1.2 mM FFA 处理 24 h。结果表明，外源性 FFA 挑战诱导了线粒体活性氧（ROS）浓度的线性和二次增加。与 0 mM FFA 相比，线粒体膜电位、氧化磷酸化复合物（CO I-V）的 mRNA 丰度、核呼吸因子 1（NRF1）、过氧化物酶体增殖物激活受体 γ 共激活因子 1α（PGC-1α）、线粒体转录因子 A（TFAM）和 NFE2L2 的蛋白丰度以及 ATP、线粒体 DNA（mtDNA）和总线粒体的含量在 0.6 mM FFA 组中更高，但在 1.2 和 2.4 mM FFA 培养物中更低。通过小干扰 RNA 敲低 NFE2L2 导致线粒体 ROS 含量增加，线粒体膜电位降低，同时 ATP、mtDNA 和总线粒体含量降低。SFN 预处理上调了 NFE2L2 的蛋白丰度，并减轻了 FFA 诱导的 NFE2L2 下调。SFN 预处理减轻了 FFA 诱导的 PGC-1α、NRF1 和 TFAM 蛋白丰度以及 mtDNA 和总线粒体含量的下调。此外，SFN 预处理减轻了 FFA 诱导的线粒体 ROS 含量增加、线粒体膜电位降低以及 ATP、mtDNA 和线粒体含量减少。总体而言，数据表明 FFA 抑制 NFE2L2，导致奶牛乳腺上皮细胞中线粒体功能障碍和 ROS 产生。因此，NFE2L2 可能是一种有前途的治疗靶点，可用于治疗奶牛乳腺上皮细胞中由代谢应激引起的线粒体功能障碍和氧化应激。
