Lipoteichoic acid (LTA), an organic acid of Gram-positive bacteria, is closely related to mastitis in dairy cows. This study evaluates the effect of LTA-induced endoplasmic reticulum stress (ER stress) in vitro using MAC-T (mammary epithelial cells) and in dairy cows with mastitis. LTA stimulation significantly increases ER stress and apoptosis-related factors in MAC-T. Further analysis suggests that the increase in ER stress may be associated with interactions involving PTGS2 and GRP78. Protein structural studies indicate a strong interaction between PTGS2 and GRP78. Lipidomics results further demonstrate that LTA disrupts lipid balance in MAC-T cells, affecting lipid metabolism in the endoplasmic reticulum, including PC, PE, TAG, and DAG, thereby exacerbating inflammation and ER stress. In dairy cows with mastitis caused by Gram-positive bacterial infection, damaged epithelial cells, inflammatory cell infiltration, and apoptotic vesicles are observed in affected tissues. In contrast, tissues from healthy cows exhibit regular epithelial cells without inflammatory cells or apoptotic vesicles. Furthermore, a significant ER stress and apoptosis increase is observed in mastitis tissues. This study demonstrates the close association between LTA-induced cell damage and ER stress, contributing to understanding the mechanisms underlying LTA-induced damage and supporting strategies for mastitis prevention and control in dairy cows.

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows. It mainly utilizes the properties of its pathogenic factor, lipoteichoic acid (LTA), to elicit a host-cell inflammatory response and evade the host-cell immune response. Arachidonic acid (AA) has a regulatory role in the inflammatory response, cell metabolism, and apoptosis. The study aimed to establish a cell model by determining the optimal concentration of LTA and AA for cell induction using the Cell Counting Kit−8 assay and the quantitative polymerase chain reaction of interleukin (IL)-1β, IL-2, and IL-6. MAC-T cells were planted in 36 10-cm² culture dishes at a density of 1 × 10⁷ cells per dish. They were treated with LTA for 24 h to constitute the LTA group and with AA for 12 h to constitute the AA group. The cells were pretreated with LTA for 24 h followed by treatment with AA for 12 h to constitute the LTA + AA group. Using proteomic, transcriptomic, and metabolomic analyses, this study determined that LTA can regulate the expression of Actin Related protein 2/3 complex (ARPC)3, ARPC4, Charged Multivesicular Body Protein 3, protein kinase cGMP-dependent, NF-κB Inhibitor Alpha,and other genes to affect cellular metabolism, immune regulation and promote apoptosis. In contrast, AA was observed to regulate the expression of genes such as ARPC3, ARPC4, Charged Multivesicular Body Protein 3, Laminin Gamma 1, Insulin Receptor, Filamin B, and Casein Kinase 1 Epsilon to inhibit cellular apoptosis and promote immune regulation, which provides a theoretical basis for future studies.

Mastitis typically arises from bacterial invasion, where host cell apoptosis significantly contributes to the inflammatory response. Gram-positive bacteria predominantly utilize the virulence factor lipoteichoic acid (LTA), which frequently leads to chronic breast infections, thereby impacting dairy production and animal husbandry adversely. This study employed LTA to develop models of mastitis in cow mammary gland cells and mice. Transcriptomic analysis identified 120 mRNAs associated with endocytosis and apoptosis pathways that were enriched in the LTA-induced inflammation of the Mammary Alveolar Cells-large T antigen (MAC-T), with numerous differential proteins also concentrated in the endocytosis pathway. Notably, actin-related protein 2/3 complex subunit 3 (ARPC3), actin-related protein 2/3 complex subunit 4 (ARPC4), and the heat shock protein 70 (HSP70) are closely related. STRING analysis revealed interactions among ARPC3, ARPC4, and HSP70 with components of the apoptosis pathway. Histological and molecular biological assessments confirmed that ARPC3, ARPC4, and HSP70 were mainly localized to the cell membrane of mammary epithelial cells. ARPC3 and ARPC4 are implicated in the mechanisms of bacterial invasion and the initiation of inflammation. Compared to the control group, the expression levels of these proteins were markedly increased, alongside the significant upregulation of apoptosis-related factors. While HSP70 appears to inhibit apoptosis and alleviate inflammation, its upregulation presents novel research opportunities. In conclusion, we deduced the development mechanism of ARPC3, ARPC4, and HSP70 in breast inflammation, laying the foundation for further exploring the interaction mechanism between the actin-related protein 2/3 (ARP2/3) complex and HSP70.

Cow mastitis is a major challenge in dairy farming, significantly affecting both milk quality and cow health. Cytochalasin B (CB) is a fungal toxin and an actin cytoskeleton depolymerizing agent that exhibits anti-inflammatory and antitumor properties; however, its mechanism in cow mastitis remains unclear. In this study, we systematically evaluated the effects of CB on mastitis using an LPS-induced inflammation model in bovine mammary epithelial cells (MAC-T) and a mouse mastitis model. The techniques employed included Real-time quantitative PCR detecting system (qPCR), Western blot, HE staining, immunofluorescence (IF), and immunohistochemistry (IHC). The results demonstrated that CB significantly alleviated LPS-induced mastitis by downregulating the expression of pro-inflammatory factors IL-1β, TNF-α, and the NLRP3 inflammasome while also reducing cell apoptosis. Further mechanistic investigations revealed that CB mitigates the inflammatory response by inhibiting the expression of ARPC3, ARPC4, and HSP70, thereby disrupting cytoskeletal rearrangement and the activation of the NLRP3 inflammasome. Overall, this study reveals the potential therapeutic role of CB in cow mastitis and provides a theoretical foundation for developing novel intervention strategies.