Negative energy balance (NEB) typically occurs in dairy cows after delivery. Cows with a high yield are more likely to experience significant NEB. This type of metabolic imbalance could cause ketosis, which is often accompanied by a decline in reproductive performance. However, the molecular mechanisms underlying NEB have yet to be fully elucidated. During excessive NEB, the body fat is extensively broken down, resulting in the abnormal accumulation of non-esterified fatty acids (NEFAs), 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 remains poorly understood. Melatonin (MT) is recognized for its regulatory role in maintaining the homeostasis of mitochondrial reactive oxygen species (mitoROS). However, little is known as to whether MT could ameliorate the damage incurred by BEECs in response to PA and the molecular mechanism involved. Analysis showed that 0.2 mmol/L PA stress increased the level of cellular and mitochondrial oxidative stress, as indicated by increased reactive oxygen species (ROS) level. In addition, we observed mitochondrial dysfunction, including abnormal mitochondrial structure and respiratory function, along with a reduction in mitochondrial membrane potential and mitochondrial copy number, and the induction of apoptosis. Notably, we also observed the upregulation of autophagy proteins (PINK, Parkin, LC3B and Ubiquitin), however, the P62 protein was also increased. As we expected, 100 μmol/L of MT pre-treatment attenuated PA-induced mitochondrial ROS and restored mitochondrial respiratory function. Meanwhile, MT pretreatment reversed the upregulation of P62 induced by PA and activated the AMPK-mTOR-Beclin-1 pathway, contributing to an increase of autophagy and decline apoptosis. Our findings indicate that PA can induce mitochondrial dysfunction and enhance autophagy in BEECs. In addition, MT is proved to not only reduce mitochondrial oxidative stress but also facilitate the clearance of damaged mitochondria by upregulating autophagy pathways, thereby safeguarding the mitochondrial pool and promoting cellular viability. Our study provides a better understanding of the molecular mechanisms underlying the effect of an excess of NEB on the fertility outcomes of high yielding dairy cows.

中文翻译：

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褪黑激素通过减少牛子宫内膜上皮细胞的氧化应激和增强自噬来减轻棕榈酸诱导的线粒体功能障碍


负能量平衡（NEB）通常发生在奶牛产后。高产奶牛更有可能经历显着的 NEB。这种代谢失衡可能会导致酮症，而酮症通常伴随着生殖能力的下降。然而，NEB 的分子机制尚未完全阐明。在过度NEB期间，体内脂肪被广泛分解，导致以棕榈酸（PA）为代表的非酯化脂肪酸（NEFA）在子宫内异常积累。这种异常积累有可能损害牛子宫内膜上皮细胞 (BEEC)，但其参与 PA 诱导的 BEEC 损伤的分子机制仍知之甚少。褪黑激素 (MT) 因其在维持线粒体活性氧 (mitoROS) 稳态中的调节作用而受到认可。然而，MT是否可以改善BEECs对PA的反应及其分子机制却知之甚少。分析表明，0.2 mmol/L PA 应激会增加细胞和线粒体氧化应激水平，表现为活性氧 (ROS) 水平增加。此外，我们观察到线粒体功能障碍，包括线粒体结构和呼吸功能异常，以及线粒体膜电位和线粒体拷贝数的减少，以及细胞凋亡的诱导。值得注意的是，我们还观察到自噬蛋白（PINK、Parkin、LC3B 和泛素）的上调，但 P62 蛋白也有所增加。正如我们预期的那样，100 μmol/L MT 预处理减弱了 PA 诱导的线粒体 ROS，恢复了线粒体呼吸功能。 同时，MT预处理逆转了PA诱导的P62上调，并激活AMPK-mTOR-Beclin-1通路，有助于增加自噬并减少细胞凋亡。我们的研究结果表明，PA 可以诱导线粒体功能障碍并增强 BEEC 的自噬。此外，MT被证明不仅可以减少线粒体氧化应激，还可以通过上调自噬途径促进受损线粒体的清除，从而保护线粒体池并促进细胞活力。我们的研究让我们更好地了解过量 NEB 对高产奶牛生育力结果影响的分子机制。