State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun 130062, China.
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun 130062, China.
J Dairy Sci. 2024 Oct;107(10):8494-8507. doi: 10.3168/jds.2024-24800. Epub 2024 Jun 13.
Mitochondrial dysfunction has been reported to occur in the mammary gland of dairy cows suffering from ketosis. Prohibitin 2 (PHB2) plays a crucial role in regulating mitophagy, which clears impaired mitochondria to maintain normal mitochondrial function. Therefore, the current study aimed to investigate how PHB2 mediates mitophagy, thereby influencing mitochondrial function in the immortalized bovine mammary epithelial cell line (MAC-T cells). First, mammary gland tissue and blood samples were collected from healthy cows (n = 15, BHB <0.6 mM) and cows with clinical ketosis (n = 15, BHB >3.0 mM). Compared with healthy cows, cows with clinical ketosis exhibited lower DMI, milk production, milk protein, milk lactose, and serum glucose. In contrast, milk fat, serum nonesterified fatty acids (NEFA) and BHB were greater in cows with clinical ketosis. The protein abundance of PHB2, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), mitofusin 2 (MFN2) in whole cell lysates (WCL), as well as PHB2, sequestosome-1 (SQSTM1, also called p62), microtubule-associated protein 1 light chain 3-II (MAP1LC3-II, also called LC3-II), and ubiquitinated proteins in mitochondrial fraction were significantly lower in cows with clinical ketosis. The ATP content of mammary gland tissue in cows with clinical ketosis was lower than that of healthy cows. Second, MAC-T were cultured and treated with NEFA (0, 0.3, 0.6, 1.2 mM). The MAC-T treated with 1.2 mM NEFA displayed decreased protein abundance of PHB2, PGC-1α, and MFN2 in WCL, as well as protein abundance of PHB2, p62, LC3-II, and ubiquitinated proteins in mitochondrial fraction. The content of ATP and JC-1 aggregates in 1.2 mM NEFA group were lower than in the 0 mM NEFA group. Additionally, 1.2 mM NEFA disrupted the fusion between mitochondria and lysosomes. The MAC-T were then pretreated with 100 nM rapamycin, followed by treatment with or without NEFA. Rapamycin alleviated impaired mitophagy and mitochondria dysfunction induced by 1.2 mM NEFA. Third, MAC-T were transfected with small interfering RNA to silence PHB2 or a plasmid for overexpression of PHB2, followed by treatment with or without NEFA. The silencing of PHB2 aggravated 1.2 mM NEFA-induced impaired mitophagy and mitochondrial dysfunction, whereas the overexpression of PHB2 alleviated these effects. Overall, this study provides evidence that PHB2, in regulation of mitophagy, is a mechanism for bovine mammary epithelial cells to counteract NEFA-induced mitochondrial dysfunction.
线粒体功能障碍已被报道发生在患有酮病的奶牛的乳腺中。抑制素 2（PHB2）在调节线粒体自噬中起着至关重要的作用，线粒体自噬清除受损的线粒体以维持正常的线粒体功能。因此，本研究旨在探讨 PHB2 如何介导线粒体自噬，从而影响永生化牛乳腺上皮细胞系（MAC-T 细胞）中的线粒体功能。首先，从健康奶牛（n=15，BHB<0.6 mM）和患有临床酮病的奶牛（n=15，BHB>3.0 mM）中收集乳腺组织和血液样本。与健康奶牛相比，患有临床酮病的奶牛的 DMI、产奶量、乳蛋白、乳乳糖和血清葡萄糖较低。相反，患有临床酮病的奶牛的乳脂、血清非酯化脂肪酸（NEFA）和 BHB 较高。全细胞裂解物（WCL）中 PHB2、过氧化物酶体增殖物激活受体-γ共激活因子-1α（PGC-1α）、线粒体融合蛋白 2（MFN2）的蛋白丰度以及 PHB2、自噬相关蛋白 1 轻链 3-II（MAP1LC3-II，也称为 LC3-II）和线粒体部分的泛素化蛋白在患有临床酮病的奶牛中显著降低。患有临床酮病的奶牛的乳腺组织中的 ATP 含量低于健康奶牛。其次，培养 MAC-T 并分别用 0、0.3、0.6、1.2 mM 的 NEFA 处理。用 1.2 mM NEFA 处理的 MAC-T 显示 WCL 中 PHB2、PGC-1α 和 MFN2 的蛋白丰度降低，以及线粒体部分 PHB2、p62、LC3-II 和泛素化蛋白的蛋白丰度降低。1.2 mM NEFA 组的 ATP 含量和 JC-1 聚集物低于 0 mM NEFA 组。此外，1.2 mM NEFA 破坏了线粒体和溶酶体之间的融合。然后用 100 nM 雷帕霉素预处理 MAC-T，再用或不用 NEFA 处理。雷帕霉素缓解了 1.2 mM NEFA 诱导的受损的线粒体自噬和线粒体功能障碍。第三，用小干扰 RNA 沉默 PHB2 或过表达 PHB2 的质粒转染 MAC-T，然后用或不用 NEFA 处理。沉默 PHB2 加重了 1.2 mM NEFA 诱导的受损的线粒体自噬和线粒体功能障碍，而过表达 PHB2 则缓解了这些作用。总之，本研究提供的证据表明，PHB2 通过调节线粒体自噬，是牛乳腺上皮细胞抵抗 NEFA 诱导的线粒体功能障碍的一种机制。
