As a critical disease usually infected by Staphylococcus aureus, with a worldwide effect on dairy animals, subclinical mastitis is characterized by persistence and treatment resistance. During mastitis, the blood–milk barrier (BMB)’s integrity is impaired, resulting in pathogen invasion and milk quality decline. In this study, it was found that ginsenoside Rg1 (Rg1), a natural anti-inflammatory and antioxidant compound derived from ginseng, inhibited the onset of tight junction (TJ) dysfunction and ameliorated lipoteichoic acid (LTA)-induced BMB disruption inside and outside the organisms. According to subsequent mechanistic studies, Rg1 inhibited excessive autophagy and inactivated the NLRP3 inflammasome by blockading ROS generation, thereby alleviating TJ dysfunction. Peroxisome proliferator-activated receptor gamma (PPARγ) was identified as a potential target of Rg1 by means of molecular docking plus network pharmacology analysis. Furthermore, it was demonstrated that Rg1 inhibited the oxidative stress levels by activating PPARγ, and regulating the upstream autophagy-related AMPK/mTOR signaling pathway, thus decreasing excessive in vivo and in vitro autophagy. The ROS/autophagy/NLRP3 inflammasome axis was identified as a promising target for treating subclinical bovine mastitis in this study. In conclusion, Rg1 is proven to alleviate BMB disruption by activating PPARγ to inhibit oxidative stress and subsequent excessive autophagy in the case of subclinical bovine mastitis.

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.

Mastitis in dairy cows, typically caused by bacterial infection, is a common inflammatory condition of the mammary tissue that leads to fibrosis, adversely affecting cow health, milk production, and dairy product quality. Astragalus polysaccharide (APS) has shown effectiveness in alleviating inflammation and fibrosis in various organs. The study employed lipopolysaccharide (LPS) to induce fibrotic conditions in two experimental systems: MAC-T bovine mammary epithelial cells and Kunming mouse models. Key parameters, including relative gene mRNA expression, protein levels, and reactive oxygen species (ROS) levels, were assessed using RT-qPCR, Western blotting (WB), and 2’,7’-Dichlorofluorescin diacetate (DCFH-DA) techniques, while histological analysis of mammary tissue was performed using H&E and Masson trichrome staining. Measuring malondialdehyde (MDA) levels, assessing the enzyme activities of catalase (CAT), and superoxide dismutase (SOD) were two methods of assessing oxidative stress. These methods were also tested in mouse mammary glands. APS significantly decreased ROS concentrations (p < 0.01), restored oxidative stress balance in mice (p < 0.05), and reduced fibrosis and inflammation, as demonstrated by histological observations and analysis. It also exerted regulatory effects on fibrosis markers (E-cadherin, N-cadherin, α-SMA) and inflammation markers (NLRP3, ASC, Caspase-1, IL-1β), as demonstrated by changes in their mRNA and protein expression. These findings endorse APS’s viability as an alternative therapeutic agent for mammary fibrosis therapy by demonstrating its ability to inhibit epithelial-mesenchymal transition (EMT) in vitro and mammary fibrosis in vivo, while also mitigating ROS production and reducing inflammation.

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.

Mastitis in dairy cows, typically caused by bacterial infection, is a common inflammatory condition of the mammary tissue that leads to fibrosis, adversely affecting cow health, milk production, and dairy product quality. Astragalus polysaccharide (APS) has shown effectiveness in alleviating inflammation and fibrosis in various organs. In this study, a lipopolysaccharide (LPS)-induced fibrosis model was established using a bovine mammary epithelial cell line (MAC-T) and Kunming rats. Key parameters, including relative gene mRNA expression, protein levels, and reactive oxygen species (ROS) content, were assessed using RT-qPCR, Western blotting (WB), and DCFH-DA techniques, while histological analysis of breast tissue was performed using H&E and Masson trichrome staining. Oxidative stress markers, such as malondialdehyde (MDA) concentration and enzymatic activities of superoxide dismutase (SOD) and catalase (CAT), were also evaluated in mouse mammary glands. APS treatment modulated fibrosis markers (E-cadherin, N-cadherin, and α-SMA) and inflammation markers (NLRP3, ASC, Caspase-1, and IL-1β) at both mRNA and protein levels, significantly reduced ROS concentrations (P<0.01), restored oxidative stress balance in mice (P<0.05), and mitigated fibrosis and inflammation, as revealed by histological observations. These findings demonstrate that APS can mitigate ROS accumulation, reduce inflammation, and inhibit epithelial-mesenchymal transition (EMT) in vitro and mammary fibrosis in vivo, supporting its potential as an alternative therapeutic agent for mammary fibrosis treatment.

Canine endometritis is commonly associated with bacterial infections caused by Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Streptococcus canis (S. canis), leading to reproductive disorders in dogs. With increasing concern regarding antimicrobial resistance, alternative therapeutic strategies are needed. This study evaluated the in vitro antibacterial activity and underlying mechanisms of protocatechuic acid (PCA) against clinical isolates of these pathogens obtained from dogs diagnosed with endometritis. The antibacterial efficacy of PCA was assessed by determining minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and bacterial growth curves. PCA inhibited the growth of all three pathogens, with MIC values of 4 mg/mL for E. coli and S. aureus and 2 mg/mL for S. canis. The MBCs for E. coli and S. aureus were equal to their MICs, while the MBC for S. canis was twice the MIC, indicating bactericidal activity. Mechanistic analyses demonstrated that PCA disrupted bacterial membrane integrity, induced membrane depolarization, reduced intracellular ATP levels, and increased reactive oxygen species (ROS) accumulation. These effects were supported by SYTO9/PI fluorescence staining and scanning electron microscopy. In conclusion, PCA exhibits notable in vitro antibacterial activity against key pathogens associated with canine endometritis and represents a promising natural antimicrobial candidate.