NEFA Promotes Bovine Granulosa Cell Apoptosis via Activation of the PERK/eIF2α/ATF4/CHOP Pathway

Previous studies have identified oxidative stress and inflammatory responses in granulosa cells (GCs) of periparturient dairy cows. However, whether non-esterified fatty acids (NEFA)-induced endoplasmic reticulum (ER) stress is involved in GC apoptosis remains unclear. In this study, treatment with NEFA (0.9 mM, 24 h) activated the ER stress pathway. This was evidenced by increased expression of both CHOP and GRP78. Furthermore, upregulation of pro-apoptotic factors BAX and Caspase-3 and downregulation of the anti-apoptotic factor Bcl-2 were observed. Pretreatment of GCs with 4-phenylbutyric acid (4-PBA, 2.5 mM, 2 h) reversed the ER stress and apoptotic effects. This suggests that NEFA-induced apoptosis is mediated through activation of the PERK pathway of ER stress, and that 4-PBA alleviates this effect. Furthermore, targeted metabolomics revealed disruptions in lipid and hormone metabolism in GCs following NEFA treatment. Analysis revealed an increase in the levels of 26 types of fatty acids and 8 types of hormones, while a decrease was detected in the levels of 3 types of fatty acids and 4 types of hormones. In summary, NEFA induces ER stress and disrupts intracellular fatty acid and hormone metabolism, ultimately leading to cell apoptosis. Our findings offer valuable insights for developing strategies to regulate follicular development in dairy cows and mitigate the impacts of postpartum negative energy balance (NEB).