Background: Primary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells for in vitro analyses. Results: In this study, CCECs were separated and purified by trypsin and dispase II enzymatic analysis. Next, the cells were immortalized by transfection with a lentiviral vector expressing Simian vacuolating virus 40 large T (SV40T). The immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passages and monoclonal selection. The biological characteristics of CCEC-SV40T cells were evaluated based on the cell proliferation rate, cell cycle pattern, serum dependence, karyotype, and cytokeratin 12 immunofluorescence detection. In addition, we infected CCEC-SV40T cells with Staphylococcus pseudintermedius (S. pseudintermedius) and detected the inflammatory response of the cells. After the CCEC-SV40T cells were passaged continuously for 40 generations, the cells grew in a cobblestone pattern, which was similar to CCECs. The SV40T gene and cytokeratin 12 can be detected in each generation. CCEC-SV40T cells were observed to have a stronger proliferation capacity than CCECs. CCEC-SV40T cells maintained the same diploid karyotype and serum-dependent ability as CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression levels of NLRP3, Caspase-1 and proinflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α, were upregulated, and the protein levels of MyD88, NLRP3 and the phosphorylation of Iκbα and p65 were upregulated. Conclusions: In conclusion, the CCEC-SV40T line was successfully established and can be used for in vitro studies, such as research on corneal diseases or drug screening.

Endometritis represents a prevalent condition in perinatal dairy cows. Bovine endometrial epithelial cells (BEECs), as the primary interface between cavity and the external environment, are particularly vulnerable to infection by pathogenic bacteria following parturition. A20 is essential for regulating inflammation and modulating immune responses. Nevertheless, the exact role of A20 in the BEECs in response to inflammatory response is not fully understood. An endometritis model infected by Escherichia coli (E. coli) in vivo and a BEECs inflammation model induced with lipopolysaccharide (LPS) in vitro were built to investigate the function and governing mechanisms of A20 in endometritis. The results showed that infection with E. coli resulted in endometrial damage, inflammatory cell infiltration, and upregulation of inflammatory factors in dairy cows. Furthermore, A20 expression was upregulated in the endometrium of cows with endometritis and in BEECs following LPS stimulation. A20 overexpression attenuated the level of proinflammatory cytokines in LPS-stimulated BEECs; conversely, A20 knockdown lead to an exacerbated response to LPS stimulation. The overexpression of A20 was shown to activate autophagy and suppress the NF-κB signaling pathway in LPS-stimulated BEECs. However, blocking autophagy with chloroquine notably attenuated the anti-inflammatory effect of A20, leading to the activation of the NF-κB signaling pathway. In summary, the study demonstrated that A20’s suppression of inflammation in LPS-stimulated BEECs is associated with the activation of autophagy. Therefore, the A20 protein showed potential as a novel treatment focus for managing endometritis in dairy cows.

During the postpartum period, domestic ruminants suffer elevated endogenous cortisol levels, which are associated with an increased risk of uterine infections. Selenium is a trace mineral nutrient with beneficial impacts on animals. The study aimed to investigate whether selenium yeast (SeY) could attenuate Escherichia coli (E. coli)-induced endometrial injury in goats with high cortisol background. Goats were examined after oral SeY administration for 21 days and were treated with glacial acetic acid, E. coli, and hydrocortisone to establish an endometritis model with high cortisol background. The results showed that endometrial injury caused by E. coli was aggravated under high cortisol background. Supplementation with SeY alleviated endometrial inflammation and serum LDH content. The mRNA expression of pro-inflammatory cytokines and defensin beta 2 and the phosphorylation level of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-b (NF-κB) signaling pathways were decreased by SeY supplementation. Total antioxidant capacity and antioxidant enzymes activities were increased by SeY supplementation, but malondialdehyde and 4-hydroxynonenal content were decreased. Moreover, nuclear factor erythroid-2 related factor 2 (NRF2) in the nucleus, heme oxygenase-1, and NAD(P)H quinone dehydrogenase 1 were increased by SeY supplementation. So, supplementation with SeY alleviated E. coli-induced endometritis in goats by activating the NRF2 pathway and inhibiting the activation of the MAPK and NF-κB pathways under postpartum stress.