Oxidative Stress Induces Bovine Endometrial Epithelial Cell Damage through Mitochondria-Dependent Pathways

SIMPLE SUMMARY: Polymorphonuclear neutrophil (PMN) count is the main diagnostic method of bovine endometritis. High neutrophil PMN counts in the endometrium of cows affected by endometritis suggest the involvement of oxidative stress among the causes of impaired fertility. The damage mechanism of oxidative stress on bovine endometrial epithelial cells (BEECs) is still unelucidated. The objective of this experiment was to investigate the relationship between oxidative stress and graded endometritis in dairy uteri and the molecular mechanism of oxidative stress injury to BEECs. Our research showed that there was an imbalance of antioxidant stress in dairy cow uterine with endometritis, oxidative stress damaged dairy cow endometrial epithelial cells through mitochondria-dependent pathways. These findings may provide new insight into the therapeutic target of bovine endometrial cell injury. ABSTRACT: Bovine endometritis is a mucosal inflammation that is characterized by sustained polymorphonuclear neutrophil (PMN) infiltration. Elevated PMN counts in the uterine discharge of dairy cows affected by endometritis suggest that oxidative stress may be among the causes of impaired fertility due to the condition. Nevertheless, the effects of oxidative stress-mediated endometritis in dairy cows largely remain uninvestigated. Therefore, fresh uterine tissue and uterine discharge samples were collected to diagnose the severity of endometritis according to the numbers of inflammatory cells in the samples. Twenty-six fresh uteri were classified into healthy, mild, moderate, and severe endometritis groups based on hematoxylin and eosin stain characteristics and the percentage of PMNs in discharge. BEECs were treated with graded concentrations of H(2)O(2) from 50 μM to 200 μM in vitro as a model to explore the mechanism of oxidative stress during bovine graded endometritis. The expressions of antioxidant stress kinases were detected by quantitative fluorescence PCR to verify the oxidative stress level in uteri with endometritis. Reactive oxygen species were detected by fluorescence microscope, and inflammation-related mRNA expression increased significantly after H(2)O(2) stimulation. Moreover, mRNA expression levels of antioxidant oxidative stress-related enzymes (glutathione peroxidase, superoxide dismutase, and catalase) and mitochondrial membrane potential both decreased. Further investigation revealed that expression of the apoptosis regulator Bcl-2/Bax decreased, whereas expression of the mitochondrial apoptosis-related proteins cytochrome c and caspase-3 increased in response to oxidative stress. Our results indicate that an imbalance exists between oxidation and antioxidation during bovine endometritis. Moreover, apoptosis induced in vitro by oxidative stress was characterized by mitochondrial damage in BEECs.