After parturition, bovine endometrial epithelial cells (BEECs) undergo serious inflammation and imbalance between oxidation and antioxidation, which is widely acknowledged as a primary contributor to the development of endometritis in dairy cows. Nevertheless, the mechanism of oxidative stress-mediated inflammation and damage in bovine endometrial epithelial cells remains inadequately defined, particularly the molecular pathways associated with mitochondria-dependent apoptosis. Hence, the present study was designed to explore the mechanism responsible for mitochondrial dysfunction-induced BEEC damage. In vivo, the expressions of proapoptotic protein caspase 3 and cytochrome C were increased significantly in dairy uteri with endometritis. Similarly, the levels of proapoptotic protein caspase 3, BAX, and cytochrome C were markedly increased in H₂O₂-treated BEECs. Our findings revealed pronounced BEEC damage in dairy cows with endometritis, accompanied by heightened expression of cyto-C and caspase-3 both in vivo and in vitro. The reduction in apoptosis-related protein of BEECs due to oxidant injury was notably mitigated following N-acetyl-L-cysteine (NAC) treatment. Furthermore, mitochondrial vacuolation was significantly alleviated, and mitochondrial membrane potential returned to normal levels after the removal of ROS. Excessive ROS may be the main cause of mitochondrial dysfunction. Mitochondrial permeability transition pore (mPTP) blockade by cyclophilin D (CypD) knockdown with CSA significantly blocked the flow of cytochrome C (cyto-C) and Ca²⁺ to the cytoplasm from the mitochondria. Our results indicate that elevated ROS and persistent opening of the mPTP are the main causes of oxidative damage in BEECs. Collectively our results reveal a new mechanism involving ROS-mPTP signaling in oxidative damage to BEECs, which may be a potential avenue for the clinical treatment of bovine endometritis.

Bovine endometritis is a reproductive disorder that is induced by mucus or purulent inflammation of the uterine mucosa. However, the intracellular control chain during inflammatory injury remains unclear. In the present study, we found that E. coli activated the inflammatory response through the assembly of the NLRP3 inflammasome and activation of the NF-κB p65 subunit in primary bovine endometrial epithelial cells (bEECs). Infection with E. coli also led to an abnormal increase in cytoplasmic calcium and mitochondrial dysfunction. Additionally, live-cell imaging of calcium reporters indicated that the increase in cytosolic calcium mainly was caused by the release of Ca²⁺ ions stored in the ER and mitochondria, which was independent of extracellular calcium. Cytoplasmic calcium regulates mitochondrial respiratory chain transmission, DNA replication, and biogenesis. Pretreatment with NAC, BAPTA-AM, or 2-APB reduced the expression of IL-1β and IL-18. Moreover, ERS was involved in the regulation of bovine endometritis and cytosolic calcium was an important factor for regulating ERS in E. coli-induced inflammation. Finally, activation of autophagy inhibited the release of IL-1β and IL-18, cytochrome c, ATP, ERS-related proteins, and cytoplasmic calcium. Collectively, our findings demonstrate that autophagy mediated E. coli-induced cellular inflammatory injury by regulating cytoplasmic calcium, mitochondrial dysfunction, and ERS.

Endometritis in high-yield dairy cows adversely affects lactation length, milk quality, and the economics of dairy products. Endoplasmic reticulum stress (ERS) in bovine endometrial epithelial cells (BEECs) occurs as a consequence of diverse post-natal stressors, and plays a key role in a variety of inflammatory diseases. Nuclear-factor-erythroid-2-related factor 2 (Nrf2) is an important protective regulatory factor in numerous inflammatory responses. However, the mechanism by which Nrf2 modulates inflammation by participating in ERS remains unclear. The objective of the present study was to explore the role of Nrf2 in lipopolysaccharide (LPS)-induced injury to BEECs and to decipher the underlying molecular mechanisms of this injury. The expression of Nrf2- and ERS-related genes increased significantly in bovine uteri with endometritis. Isolated BEECs were treated with LPS to stimulate the inflammatory response. The expression of Nrf2 was significantly higher in cells exposed to LPS, which also induced ERS in BEECs. Activation of Nrf2 led to enhanced expression of the genes for the inflammation markers TNF-α, p65, IL-6, and IL-8 in BEECs. Moreover, stimulation of Nrf2 was accompanied by activation of ERS. In contrast, Nrf2 knockdown reduced the expression of TNF-α, p65, IL-6, and IL-8. Additionally, Nrf2 knockdown decreased expression of ERS-related genes for the GRP78, PERK, eIF2α, ATF4, and CHOP proteins. Collectively, our findings demonstrate that Nrf2 and ERS are activated during inflammation in BEECs. Furthermore, Nrf2 promotes the inflammatory response by activating the PERK pathway in ERS and inducing apoptosis in BEECs.

In ruminants, the establishment of pregnancy requires a series of structural and functional changes in the endometrium under the action of hormones, thereby providing an optimal environment for the implantation of the embryo. In this study, we explored the molecular mechanism by which YPEL3 regulates endometrial function during gestation in goats. We found YPEL3 expression was significantly downregulated during early gestation and that YPEL3 overexpression inhibited the expression of ISG15, but had no significant effects on the expression of RSAD2 and CXCL10 in goat endometrial epithelial cells (gEECs). In addition, YPEL3 silencing significantly inhibited PGF_2α secretion and the expression of the prostaglandin synthesis-related rate-limiting enzyme-encoding genes PGFS and PTGES, with no significant effect on the expression of PTGS1 and PTGS2. Moreover, YPEL3 inhibited the expression of vimentin and β-catenin and pretreatment of gEECs with the β-catenin activator CHIR99021 prevented a YPEL3-induced decrease in vimentin expression. Collectively, our findings confirm that, as a hormone-regulated factor, YPEL3 regulates endometrial function by inhibiting the Wnt/β-catenin signaling pathway and provide new insights for further clarification of the mechanism by which YPEL3 functions during early pregnancy in ruminants.