Gao Yujin, Li Yina, Wang Jiamian, Zhang Xijun, Yao Dan, Ding Xuanpan, Zhao Xingxu, Zhang Yong
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China.
Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China.
Animals (Basel). 2023 Jul 28;13(15):2449. doi: 10.3390/ani13152449.
Bovine endometritis is characterized by reduced milk production and high rates of infertility. Prior research has indicated that melatonin may possess anti-inflammatory and antioxidant properties that can counteract the progression of inflammatory diseases. In this research, we attempted to elucidate the protective effects of melatonin on LPS-induced endometritis. The results obtained from enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR) revealed that melatonin effectively reduced the production and release of pro-inflammatory cytokines in an LPS-induced bovine endometrial epithelial cell line (BEND cells). Furthermore, western blotting demonstrated that melatonin treatment reduced the expression levels of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-related proteins, including NLRP3, activated caspase-1, and cleaved IL-1β. Importantly, we further demonstrated that the anti-inflammatory effect of melatonin on BEND cells was related to autophagy by western blotting. Moreover, we used western blotting to detect autophagy-related proteins, MitoSOX to detect mitochondrial reactive oxygen species production (mtROS), and mitochondrial membrane potential (MMP) assay to detect mitochondrial membrane potential. The administration of melatonin demonstrated a significant enhancement in autophagy within BEND cells, leading to the effective elimination of impaired mitochondria. This process resulted in a reduction in the generation of reactive oxygen species within the mitochondria, restoration of mitochondrial membrane potential, and inhibition of the NLRP3 inflammasome activation. Moreover, in a mouse model of LPS-induced endometritis, melatonin treatment repressed the expression of pro-inflammatory cytokines by ELISA and qRT-PCR, alleviated pathological changes by hematoxylin-eosin staining (H&E), and inhibited myeloperoxidase (MPO) activity. In conclusion, our study showed that melatonin inhibited the activation of the NLRP3 inflammasome in BEND cells through autophagy, which may provide a novel therapeutic strategy for bovine endometritis.
牛子宫内膜炎的特征是产奶量降低和不育率高。先前的研究表明，褪黑素可能具有抗炎和抗氧化特性，能够对抗炎症性疾病的进展。在本研究中，我们试图阐明褪黑素对脂多糖（LPS）诱导的子宫内膜炎的保护作用。酶联免疫吸附测定（ELISA）和定量实时聚合酶链反应（qRT-PCR）的结果显示，褪黑素有效地降低了LPS诱导的牛子宫内膜上皮细胞系（BEND细胞）中促炎细胞因子的产生和释放。此外，蛋白质印迹法表明，褪黑素处理降低了含NOD样受体家族吡啉结构域3（NLRP3）炎性小体相关蛋白的表达水平，包括NLRP3、活化的半胱天冬酶-1和裂解的白细胞介素-1β。重要的是，我们通过蛋白质印迹法进一步证明，褪黑素对BEND细胞的抗炎作用与自噬有关。此外，我们使用蛋白质印迹法检测自噬相关蛋白，用MitoSOX检测线粒体活性氧生成（mtROS），并用线粒体膜电位（MMP）测定法检测线粒体膜电位。褪黑素的给药显示BEND细胞内自噬显著增强，导致受损线粒体的有效清除。这一过程导致线粒体内活性氧生成减少、线粒体膜电位恢复以及NLRP3炎性小体激活受到抑制。此外，在LPS诱导的子宫内膜炎小鼠模型中，褪黑素处理通过ELISA和qRT-PCR抑制促炎细胞因子的表达，通过苏木精-伊红染色（H&E）减轻病理变化，并抑制髓过氧化物酶（MPO）活性。总之，我们的研究表明，褪黑素通过自噬抑制BEND细胞中NLRP3炎性小体的激活，这可能为牛子宫内膜炎提供一种新的治疗策略。
