Effects Of LTA-sa On Milk Protein Synthesis And Related Signaling Pathways In Bovine Mammary Epithelial Cells

Staphylococcus aureus can induce mastitis in dairy cows,is the most difficult to treat clinically and causes the most economic loss,and seriously hinders the healthy development of the dairy industry.When Staphylococcus aureus infection occurs in the mammary glands of dairy cows,Staphylococcus aureus lipoteichoic acid(LTA-sa),an inherent component on the surface,can be recognized as a ligand,causing an inflammatory reaction in dairy mammary gland tissues and changing milk proteins in milk and milk fat and other milk ingredients.But the specific mechanism is not very clear.In this experiment,bovine mammary epithelial cells(BMECs)cultured in vitro were used as lactation models to study the effects of LTA-sa on milk protein synthesis and related signaling pathways in BMECs.To further study the regulatory mechanism of milk protein synthesis,Improving the nutritional quality of milk provides a scientific basis.To determine the optimal concentration of LTA-sa for the construction of inflammatory cell models and the nuclear factor-κB(Nuclear factor-κB)inhibitor PDTC and Signal transducer and activator of transcription 3,(STAT3)The optimal concentration of inhibitor stattic.BMECs were treated with LTA-sa,PDTC and stattic at different concentrations.The m RNA expression of related inflammatory cytokines and the phosphorylation of NF-κB and STAT3 proteins were detected.The cytotoxicity test was performed to determine the optimal treatment concentration of LTA-sa,PDTC and stattic as 20 μg / m L,10 μM and 10 μM.The experiment was divided into three parts:(1)20 μg / m L LTA-sa alone was used to treat BMECs at different times(0 h,1 h,3 h,6 h,12 h,and 24 h),and detection of inflammation and lactation-related NF-κB,STAT3,Suppressor of cytokine signaling 3(SOCS3),Janus kinase 2(JAK2),STAT5,and β-casein(CSN2)Protein expression level and phosphorylated protein expression level,detect the m RNA expression levels of SOCS3 and CSN2,and detect the nuclear localization of p-STAT3,SOCS3,p-STAT5 and CSN2;(2)BMECs first use 10 μM PDTC Treat 3 h,then use 20 μg / m L of LTA-sa to treat BMECs at different times(0 h,1 h,3 h,6 h,12 h,24 h),and detect the above-mentioned related indicators;(3)BMECs first use 10 μM stattic was pretreated for 3 h,and then BMECs were treated with LTA-sa at 20 μg / m L for different time(0 h,1 h,3 h,6 h,12 h,24 h),and the above related indicators were also detected.The results showed that LTA-sa treatment of BMECs alone could significantly increase the protein expression levels of NF-κB p-p65,p-STAT3 and SOCS3,significantly reduce the protein expression levels of p-JAK2,p-STAT5 and CSN2.The expression level increased while CSN2 decreased.These results indicate that LTA-sa can activate the NF-κB inflammatory pathway and reduce CSN2 expression by inhibiting JAK2-STAT5 pathway conduction;in the subsequent PDTC and LTA-sa treatment groups,the results were similar to the LTA-sa treatment group,and we found that inhibition of NF-κB does not resist the reduction of milk protein synthesis by LTAsa,which indicates that activation of the NF-κB pathway is not the key to inhibit JAK2-STAT5;in the final statistical and LTA-sa treatment groups,the phosphorylation level of STAT3 After being inhibited,LTA-sa can still activate the NF-κB pathway,but the expression of SOCS3 cannot be activated,and it can no longer inhibit the conduction of JAK2-STAT5 pathway,nor can it reduce the expression of CSN2.This shows that STAT3 can activate SOCS3 and is a key factor in the reduction of CSN2 synthesis induced by LTA-sa.STAT3 and SOCS3 can inhibit the activation of JAK2-STAT5 pathway and reduce the function of CSN2 synthesis.In summary,LTA-sa can activate the NF-κB inflammatory pathway,and by activating STAT3,activating SOCS3 expression,inhibiting JAK2-STAT5 pathway conduction,and ultimately affecting CSN2 synthesis and expression.