Abstract:

The aim was to investigate the effect and mechanisms of melatonin on the mitochondrial dynamics of bovine endometrial epithelial cells (BEECs) induced by palmitic acid (PA). BEECs were used as the research subjects and divided into 3 groups: control group, PA group, and melatonin group. The control group was supplemented with 0.2 mmol·L^-1 BSA as the solvent, the PA group was supplemented with 0.2 mmol·L^-1 PA, and the melatonin group was supplemented with both 0.2 mmol·L^-1 PA and 100 nmol·L^-1 melatonin. The experiments included: assessing cell viability and apoptosis using the CCK8 method (n=4) and Annexin V-FITC/PI double staining method (n=3); detecting the ROS levels in mitochondria using flow cytometry(n=3); further analyzing mitochondrial respiration-related indicators such as oxygen consumption rate, basal respiration, ATP production capacity using Seahorse XFe96 cellular metabolism analysis system (n=4); measuring the mRNA expression changes of mitochondrial dynamics-related genes MFN1, DRP1 and PGC1-α were detected by qRT-PCR (n=3); finally, the protein expression changes of mitochondrial dynamics-related proteins MFN1, p-DRP1, DRP1 and PGC1-α were detected by Western-Blot (n=3). The results showed that: compared with the PA group, 1) the melatonin significantly reduced the mitochondrial ROS level of BEECs, increased cell viability, and decreased apoptosis rate significantly (P<0.05); 2) Seahorse results indicated that melatonin significantly increased mitochondrial basal respiration, ATP production, and maximum oxygen consumption (P<0.05), suggesting that melatonin could improve mitochondrial function, promote aerobic respiration, and increase ATP production; 3) qRT-PCR results showed that the melatonin significantly reduced the mRNA expression of MFN1 and DRP1, while significantly increasing the mRNA expression of PGC1-α(P<0.05); 4) Western-Blot analysis revealed a significant decrease in MFN1 protein expression and a significant increase in p-DRP1/DRP1 and PGC1-α protein expression in the melatonin group (P<0.05). In conclusion, the study suggests that melatonin may alleviate PA-induced mitochondrial oxidative stress and improve mitochondrial respiration by improving mitochondrial dynamics of BEECs, which ultimately enhances the viability of BEECs and reduces apoptosis. This study preliminarily explored the mechanism by which melatonin mitigates PA-induced injury in BEECs, offering a theoretical foundation for improving the low reproductive capacity of severe NEB cows.

Key words: melatonin, palmitic acid, bovine endometrial epithelial cells, mitochondrial dynamics, apoptosis