Melatonin alleviates palmitic acid–induced mitochondrial dysfunction by decreasing oxidative stress and enhancing autophagy combined in bovine endometrial epithelial cells

Abstract Background: Negative energy balance (NEB) typically occurs in dairy cows after delivery, with those in high yielding being more likely to experience significant NEB. Such metabolic imbalance could cause ketosis, which is often accompanied by a decline in reproductive performance, but the molecular mechanism has not been fully elucidated. During excessive NEB, the body fat is extensively broken down, resulting in abnormal accumulation of non-esterified fatty acids (NEFA), represented by palmitic acid (PA), within the uterus. Such an abnormal accumulation has the potential to damage bovine endometrial epithelial cells (BEECs), while the molecular mechanisms underlying its involvement in the PA-induced injury of BEECs is poorly understood. Melatonin (MT) is recognized for its regulatory role in maintaining the homeostasis of mitochondrial reactive oxygen species (mitoROS). However, less is known about whether MT could ameliorate the damage of PA on BEECs and its molecular mechanism. Results: Our results demonstrated that 0.2 mM PA stress increased the level of cellular and mitochondrial oxidative stress. Further, we observed mitochondrial dysfunction, including an abnormal incidence of mitochondrial structure and respiratory function, reduction in mitochondrial membrane potential and mitochondrial copy number, which in turn induced apoptosis. Notably, we also found an upregulation of autophagy protein (PINK, Parkin, LC3B et al), however, the P62 protein was also increased. As we expected, 100uM MT pre-treatment attenuated PA-induced mitochondrial ROS and restored mitochondrial respiratory function. Meanwhile, pre-treated MT reversed the upregulation of P62 induced by PA and activate the AMPK-mTOR-Beclin-1 pathway, contributing to increase of autophagy and decline apoptosis. Conclusions: These findings indicate that PA could induce mitochondrial dysfunction and also enhance autophagy in BEECs. In addition, MT is hypothesized to not only reduce mitochondrial oxidative stress but also facilitate the clearance of damaged mitochondria through the regulation of autophagy pathways, thereby safeguarding the mitochondrial pool and promoting cellular viability. Our study helps better understand the molecular mechanism involved in excessive NEB's impact on the fertility outcomes of high yielding dairy cows.