The Cow Mammary Gland Epithelial Cells Induced By Selenium Of Peptidoglycan Inflammation Research Of Damage Effect And Related Signaling Pathway

Dairy mastitis is one of the most popular and complex diseases in the dairy industry and can cause huge economic losses.Selenium is an essential nutrient in a mammalian diet and has some antiinflammatory properties.Selenium in the form of selenium protein exists in the body,and mainly through its antioxidant participation in the body’s immune defense mechanism.A number of studies have confirmed that feeding an appropriate amount of selenium feed can minimize the incidence of mastitis,white myopathy and respiratory disease in animal populations.Peptidoglycan(PGN)is the main component of the cell wall structure of Staphylococcus aureus.It has been shown that the peptidoglycan of Staphylococcus aureus can stimulate the relevant pathogen recognition receptors on the host cells and activate the inflammatory signaling pathway.In the current experiment,we studied the the protective effect and associated mechanisms of Se against inflammation on mastitis to provide theoretical foundation and experimental date in the prevention and treatment of bovine mastitis.Staphylococcus aureus is one of the most important pathogens of dairy cow mastitis in clinical practice.By studying its pathogenic factors to explore its pathogenic mechanism and the mechanism of its reaction,it is important for the prevention and treatment of dairy cow mastitis Practical significance.We used a complete digestion of collagenase to culture a batch of primary mammary gland epithelial cells in vitro.PGN-infected cells were used to construct the mammary epithelial cells(bMEC)inflammatory model.and the changes of cytokine expression were detected under PGN stimulation.The results showed that mammary epithelial cells were typical island-like colonies before confluence,and the cell morphology after confluence was typical of paving stone and catheter-like structure,and the dome-like structure was formed.To explore the effect of Se on cell viability of bMECs,the cells were treated with Se(0,1,2,4,8,16,32,64 μM)for 12 h.The cell viabilities were measured by MTT assay.The result showed that when the concentration of Se is between 0～16 μM,the cell viabilities were not affected.With the increasing dosage of Se,the cell viabilities were decreasing,compared with control group the concentration of Se in 32 and 64 μM the cell viabilities decreased significantly(p<0.001),which suggested that high concentration of Se were toxic to bMECs.To further study the regulation effects of Se on TLR2 and Nod2 during inflammation,the cells were treated with Se(2,4,8 μM)for different time periods.Two signal pathway TLR2,Nod2/NF-KB,MAPK and cytokines which were closely relative with inflammation were tested.The dates showed that after 8 h,10 h,12 h infection,the concentration of Se in 2,4,8 μM could significantly inhibit the expression of TLR2,Myd88 on TLR2 pathways;Rip2 on Nod2 pathways and two pathways downstream of the NF-κB,TNF-α,IL-1β and IL-6 proinflammatory factor gene expression induced by PGN(p<0.01;p<0.001).At 8h,2 and 8 μM selenium did not significantly inhibit on Nod2.To explore the the regulation effect of Se on inflammation signal transduction pathway in protein level,the activation of NF-κB and MAPK were detected by using Western Blot.The cells were randomLy divided into 5 group,the Se groups were treated with three varying concentrations(2,4,8 μM)of Se for 12 h before infection with PGN for 0.5 h.While the model group was treated with PGN,the control group was left untreated.Then,the activation of TLR2,Myd88,Nod2,Rip2,Card9,IκBα,p65,p38,JNK and Erk were measured.The result showed compared with the control group the phosphorylation of TLR2,Myd88 in TLR2 pathway;the phosphorylation of Nod2,Rip2 Card9 in Nod2 pathway;the phosphorylation of IκBα,p65 in NF-κB pathway,the phosphorylation of p38,JNK and Erk in MAPK pathway were markedly increased after stimulation with PGN for 0.5 h(p<0.001).These data suggested that Se(4 μM)blocked TLR2 and Nod2 signal pathways to achieve the inhibition effects on NF-κB and MAPK,and then down-regulated the releasing of cytokines,ultimately decreasing the inflammation injury induced by PGN.