Allicin Alleviates The Inflammatory Response Of Mammary Epithelial Cells In Dairy Cows And Its Mechanism Of Action

Mastitis is one of the most frequent diseases in dairy cattle production,and the occurrence of mastitis in dairy cattle seriously affects milk quality and milk production.The causes of mastitis include pathogenic infections,environmental management and individual cow causes,etc.Pathogenic infections are usually the most likely causative agent,with a variety of Gram-negative bacteria,led by Escherichia coli,being the causative agent of mastitis,and the lipopolysaccharide(LPS)of the outer wall of Gram-negative bacteria is an endocytic toxin that causes inflammation.A sustained and prolonged inflammatory response results in damage to the lobules of the breast,inflammatory cell infiltration,mammary epithelial cell hyperplasia and enlarged mammary lymph nodes,which in severe cases can lead to necrosis and atrophy of the breast tissue until lactation is lost.At the same time,flocculent precipitation and offensive odors appear in the milk,accompanied by increased somatic cell content in the milk,resulting in lower milk production,poorer milk quality and higher treatment costs for cows,and even premature culling of cows,resulting in huge economic losses.The effective means of treating mastitis in dairy cows is the use of antibiotics,but due to the massive negative effects of the overuse of antibiotics in recent years,the use of natural and harmless antibiotic alternatives has become a hot topic of research and public concern.The natural extract allicin,a sulfur-containing organic compound in garlic,has various pharmacological effects,such as antioxidant,antibacterial and anticancer,and has also been demonstrated to have anti-inflammatory effects.Therefore,the aim of this study was to investigate the effect and mechanism of allicin on LPS-induced inflammation in mammary epithelial cells of dairy cows.In this study,the mammary epithelial cell line(MAC-T)was treated with LPS to construct an in vitro inflammation model of mastitis in cows,and the protective effect and mechanism of action of allicin on LPS-induced cell inflammation was investigated by exogenous addition of allicin.In addition,an in vivo mastitis model was constructed using mice to investigate the effect of allicin on mastitis in mice.The effects of allicin on mammary gland inflammatory response were investigated by realtime fluorescent quantitative PCR(RT-q PCR),Western blot and hematoxylin & eosin staining(H&E).The results showed that.1.Treatment of mammary epithelial cells in cows with 10 μg/m L of LPS did not change cell viability,and 10 μg/m L of LPS increased the levels of inflammatory cytokines in the cells.RT-q PCR assays showed significant increases in the m RNA expression of tumor necrosis factor α(TNF-α),interleukin 1β(IL-1β),interleukin 6(IL-6)and interleukin 8(IL-8),indicating that a model of inflammation in mammary epithelial cells in cows was successfully constructed.2.The addition of 2.5 μM allicin to a mammary epithelial cell inflammation model in dairy cows significantly suppressed inflammatory cytokine levels that were elevated due to LPS induction.RT-q PCR results showed that the m RNA expression of TNF-α,IL-1β,IL-6 and IL-8 and the activation of Nod-like receptor protein 3(NLRP3)inflammatory vesicles were significantly reduced by the addition of allicin.3.To further investigate the mechanism by which allicin exerts its antiinflammatory effects,this experiment investigated the effect of allicin on the LPSactivated TLR4-NF-κB signaling pathway.Allicin decreased the m RNA expression and protein level of Toll-like receptor 4(TLR4)and inhibited the phosphorylation of nuclear factor kappa B p65(NF-κB p65),nuclear factor kappa B inhibitor protein α(IκB-α).It indicates that allicin reduces the production of pro-inflammatory cytokines by inhibiting LPS-induced activation of TLR4-NF-κB signaling pathway and NLRP3 inflammatory vesicle activation.4.In a mouse mastitis model,allicin alleviated histopathological damage to the mammary gland caused by LPS,suggesting that allicin has a mitigating effect on inflammation in vivo,while allicin exerts anti-inflammatory effects by reducing myeloperoxidase(MPO)activity and production of inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-8 in the mammary gland.In summary,this study found that allicin protected MAC-T cells from LPSinduced inflammatory responses by inhibiting the activation of TLR4-NF-κB pathway and NLRP3 inflammatory vesicles,and that allicin also had alleviating effects on LPS-induced mastitis in mice.The combined in vitro and in vivo results suggest that allicin is effective against the damage caused by LPS to the mammary gland and could provide theoretical support for allicin as a potential alternative to antibiotics in the treatment of mastitis in dairy cows.