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.

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.

Mastitis is a common inflammatory disease that harms mammary gland health. Its development is closely linked to dysregulated inflammatory signaling. Eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, has potential anti-inflammatory effects. However, its molecular mechanism in mastitis prevention remains unclear. In this study, we used both in vivo and in vitro models to evaluate how EPA pretreatment regulates mastitis-related inflammatory signaling. Transcriptome analysis showed that differentially expressed genes after EPA treatment were mainly enriched in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. In an LPS-induced mastitis model, EPA restored the LPS-reduced PPARγ protein level and suppressed NF-κB p65 activation, consistent with reduced nuclear translocation of p65. Similar effects were observed in mammary epithelial cells, where EPA inhibited NF-κB activation at 50 and 100 μM. Functional experiments further showed that a PPARγ agonist mimicked the inhibitory effect of EPA on p65, whereas PPARγ antagonist partially abrogated EPA-mediated inhibition of p65. Collectively, these data indicate that EPA attenuates mastitis-associated inflammation at least in part through the PPARγ–NF-κB axis.

Bovine mastitis is a common inflammatory disease that can progress to mammary fibrosis, thereby impairing udder health, milk yield, and milk quality. This study investigated the protective effects of palmatine on lipopolysaccharide (LPS)-induced epithelial–mesenchymal transition (EMT) in bovine mammary epithelial cells and mammary fibrosis in mice, as well as the underlying mechanisms. In vitro, palmatine markedly reversed LPS-induced EMT by increasing E-cadherin expression and decreasing N-cadherin and α-SMA expression. In vivo, palmatine alleviated inflammatory cell infiltration and collagen deposition in mammary tissue and reduced the expression of TGF-β1, p-Smad2, p-Smad3, p-p65, TNF-α, IL-1β, and IL-6. These findings suggest that palmatine alleviates LPS-induced mammary fibrosis, possibly through inhibition of the NF-κB/TGF-β1/Smad signaling pathway, and may represent a potential therapeutic strategy for the prevention and treatment of mammary fibrosis.

Cytochalasin B (CB) inhibits actin polymerization and modulates cytoskeletal dynamics, but its role in mastitis remains unclear. This study investigated the protective effects of CB against mastitis and its association with PI3K/N-WASP/ARP2/3-related molecular alterations. Transcriptomic analysis of mammary tissues from healthy cows and cows with subclinical or clinical mastitis revealed extensive transcriptional remodeling during mastitis progression. Validation showed enhanced inflammatory responses accompanied by increased expression of PI3K, N-WASP, CLTC, ARP2, and ARP3 in mastitic tissues. In LPS-stimulated bovine mammary epithelial cells (MAC-T), CB reduced pro-inflammatory cytokine expression, increased IL-10 expression, and attenuated the abnormal elevation of PI3K/N-WASP/ARP2/3-related molecules. Consistently, in an LPS-induced mouse mastitis model, CB alleviated mammary tissue injury and inflammatory cell infiltration, improved inflammatory cytokine profiles, and reduced the expression of PI3K, N-WASP, CLTC, ARP2, and ARP3. Collectively, CB attenuates LPS-induced mammary inflammation and tissue injury, and these effects are associated with alterations in PI3K/N-WASP/ARP2/3-related cytoskeletal molecules. These findings provide new evidence linking cytoskeleton-related molecular alterations to mastitis inflammation and the anti-inflammatory effects of CB.