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.

Staphylococcus pseudintermedius (S. pseudintermedius) is a common pathogen that causes canine corneal ulcers. However, the pathogenesis remained unclear. In this study, it has been demonstrated that S. pseudintermedius invaded canine corneal epithelial cells (CCECs) intracellularly, mediating oxidative damage and pyroptosis by promoting the accumulation of intracellular reactive oxygen species (ROS) and activating the NLRP3 inflammasome. The canine corneal stroma was infected with S. pseudintermedius to establish the canine corneal ulcer model in vivo. The intracellular infectious model in CCECs was established in vitro to explore the mechanism of the ROS - NLRP3 signalling pathway during the S. pseudintermedius infection by adding NAC or MCC950. Results showed that the expression of NLRP3 and gasdermin D (GSDMD) proteins increased significantly in the infected corneas (p < 0.01). The intracellular infection of S. pseudintermedius was confirmed by transmission electron microscopy and immunofluorescent 3D imaging. Flow cytometry analysis revealed that ROS and pyroptosis rates increased in the experimental group in contrast to the control group (p < 0.01). Furthermore, NAC or MCC950 inhibited activation of the ROS - NLRP3 signalling pathway and pyroptosis rate significantly, by suppressing pro-IL-1β, cleaved-IL-1β, pro-caspase-1, cleaved-caspase-1, NLRP3, GSDMD, GSDMD-N, and HMGB1 proteins. Thus, the research confirmed that oxidative damage and pyroptosis were involved in the process of CCECs infected with S. pseudintermedius intracellularly by the ROS - NLRP3 signalling pathway. The results enrich the understanding of the mechanisms of canine corneal ulcers and facilitate the development of new medicines and prevention measures.

Klebsiella pneumoniae (K. pneumoniae), a prominent causative agent of mastitis in dairy cattle, remains enigmatic in its pathogenic mechanisms. This study aimed to reveal the effects of K. pneumoniae on mammary glands via the induction of ferroptosis, as well as the protective role of Ferrostatin-1 (Fer-1) against this pathogen-mediated damage in bovine mammary epithelial cells (BMECs). Holstein cows were used to establish an intramammary infection model of K. pneumoniae. In vitro, primary BMECs were treated with 10 μM Fer-1 and K. pneumoniae alone or in combination. The results showed that mammary glands infected with K. pneumoniae exhibited increased transcriptional levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Concurrently, significant elevations in iron, 4-hydroxynonenal, and reactive oxygen species (ROS) levels were observed. Conversely, K. pneumoniae infection downregulated nuclear factor erythroid 2-related factor 2 (Nrf2), cystine/glutamate antiporter (xCT), glutathione peroxidase 4 (GPX4), and glutathione levels. Following K. pneumoniae invasion, intracellular Fe²⁺ and lipid ROS accumulated in the BMECs, impeding activation of Nrf2/xCT/GPX4 signal transduction. Additionally, Fer-1 facilitated the nuclear translocation of Nrf2 protein, upregulating the protein levels of Nrf2/xCT/GPX4 while downregulation transcriptional levels of IL-1β, IL-6, IL-8, and TNF-α. In conclusion, Fer-1 alleviates K. pneumoniae-induced inflammatory factor activation and ferroptosis in BMECs via upregulation of the Nrf2/xCT/GPX4 pathway, supporting ferroptosis inhibition holds promise as a feasible therapeutic agent for the control of mastitis.

Radiation-induced intestinal injury (RIII) remains a major clinical challenge, partly due to the lack of local delivery systems capable of concurrently coordinating oxidative stress and immune responses at the lesion site. In this study, we identify the transmembrane mucin MUC13 as a key regulator of intestinal epithelial homeostasis. MUC13 deficiency aggravates oxidative stress, epithelial apoptosis, and inflammatory responses, whereas supplementation with recombinant human MUC13 (rhMUC13) markedly attenuates epithelial injury. To achieve site-specific and durable delivery to inflamed tissue, we developed an inflammation-responsive polydopamine–gelatin methacryloyl (PDA–GelMA) hydrogel microsphere system that integrates targeted local delivery with intrinsic microenvironment modulation. The therapeutic efficacy arises from complementary, component-specific functions. The PDA shell mediates inflammation-associated adhesion and prolonged mucosal retention, rapidly scavenges reactive oxygen species (ROS), suppresses early inflammatory amplification, and promotes macrophage polarization toward a reparative M2 phenotype. Within this favorable redox–immune milieu, the GelMA core enables sustained rhMUC13 release, enhancing epithelial survival and barrier reconstruction by inhibiting NF-κB–associated pro-apoptotic signaling (the Bax/Bcl-2 axis) and restoring tight-junction proteins (ZO-1, Occludin, and Claudin-1). This cooperative mechanism likely accounts for the superior efficacy of the composite microspheres compared with single-component controls. Moreover, 16S rRNA gene sequencing revealed that irradiation induces pronounced gut microbiota dysbiosis, characterized by disrupted microbial diversity and community structure, increased inflammation-associated opportunistic taxa, and reduced beneficial commensal/metabolism-related bacteria; notably, PDA–GelMA@rhMUC13 partially ameliorated irradiation-induced dysbiosis, showing an overall remodeling trend toward reduced inflammation-associated taxa and increased putatively beneficial and metabolism-related bacteria. Collectively, these findings establish MUC13 as a critical mediator of epithelial–immune crosstalk and introduce a multifunctional hydrogel microsphere platform that combines targeted protein delivery with endogenous antioxidative and immunomodulatory capacities. This strategy may additionally promote restoration of intestinal homeostasis through microbiota remodeling, offering translational potential for RIII and other inflammatory intestinal disorders.