PHB2-mediated inhibition of OPA1 cleavage alleviates nonesterified fatty acid–induced apoptosis in bovine mammary epithelial cells

ABSTRACT Excessive nonesterified fatty acids (NEFA) during the transition period impose metabolic stress on the bovine mammary gland. High NEFA levels induce oxidative stress and trigger apoptosis in bovine mammary epithelial cells. Prohibitin 2 (PHB2) is a mitochondrial inner-membrane scaffolding protein known for its role in maintaining mitochondrial cristae integrity and regulating apoptosis. However, the specific effect of PHB2 on apoptosis in response to NEFA-induced stress in bovine mammary epithelial cells remains unclear. Therefore, this study aimed to investigate the regulatory role of PHB2 in high-NEFA-induced bovine mammary epithelial cells apoptosis, as well as the underlying mechanisms involved. First, mammary gland tissue from healthy (n = 15, BHB < 0.6 mM) and ketotic cows (n = 15, BHB > 3.0 mM) were collected. Compared with healthy cows, ketotic cows exhibited higher protein abundances of cleaved caspase-3 and cleaved PARP, lower protein abundances of PHB2 and long-form OPA1 (L-OPA1) in mammary glands, increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling in mammary tissues, and elevated lactate dehydrogenase activity in milk. Second, MAC-T cells were treated with NEFA (0, 0.3, 0.6, or 1.2 mM). MAC-T treated with 1.2 mM NEFA displayed increased protein abundance of cleaved caspase-3, cleaved PARP and cytoplasmic cytochrome c (Cyt C), and decreased protein abundance of PHB2, L-OPA1, and mitochondrial Cyt C. Additionally, MAC-T treated with 1.2 mM NEFA had increased apoptosis rate and TUNEL labeling. Third, MAC-T cells were transfected with small interfering RNA to silence PHB2 or plasmid for overexpression of PHB2, followed by treatment with or without NEFA. PHB2 silencing aggravated NEFA-induced apoptosis in MAC-T cells, and PHB2 overexpression alleviated this effect. Fourth, MAC-T cells were transfected with noncleavable OPA1 mutant overexpression adenovirus. Overexpression of noncleavable OPA1 mutant ameliorates NEFA-induced mitochondrial dysfunction and apoptosis. In conclusion, PHB2 alleviates NEFA-induced mitochondrial dysfunction and apoptosis by preventing OPA1 cleavage.