Mechanism Of Staphylococcus Aureus Affecting Milk Protein Synthesis And The Protective Effect Of Chlorogenic Acid

Dairy cow mastitis is a series of inflammatory reactions caused by breast tissue infection,which lead to decreased milk production and changes in milk composition.Staphylococcus aureus(S.aureus)is one of the most serious pathogens causing mastitis.The invasion of S.aureus into bovine mammary epithelial cells(BMECs)affect the expression of inflammatory cytokines and synthesis of milk protein.Chlorogenic acid(CGA)is a kind of natural product widely existing in many plants,which exhibit anti-oxidation,anti-inflammatory and other biological activities,and has been applied in the prevention and treatment of dairy cow mastitis.At present,the roles and mechanisms of CGA in alleviating the reduction of milk protein synthesis induced by S.aureus infection of BMECs are still unclear.The main contents of this study are as follows:(1)The in vitro mastitis model of BMECs was firstly established,and the internalization of S.aureus into BMECs was confirmed by colony counting method,transmission electron microscope and laser confocal microscope.(2)The expression of pro-inflammatory factors(TNF-α,IL-1β,IL-6,IL-8),absorption of amino acid(Met and Lys)and synthesis of α-Lactalbumin(α-LA),β-Lactoglobulin(β-LG),β-Casein(CSN2)were detected after S.aureus invasion.(3)The expression of inflammatory factors and milk protein were checked by ELISA and RT-q PCR after CGA treatment prior to S.aureus infection to explore the protective effect of CGA against S.aureus infection.(4)The RNA sequencing was performed to analyze and verified the differentially expressed genes and predict the potential target of CGA against S.aureus infection;(5)The roles and mechanisms of CGA in alleviating the reduction of milk protein synthesis caused by S.aureus infection on BMECs were confirmed by ELISA,RT-q PCR,Western blot based on the transcriptome analysis results.The results showed that :(1)S.aureus could internalized into the BMECs and the number of intracellular S.aureus was increasing along with the prolongation of infection time,which results indicating that S.aureus can proliferate within cells;(2)The expression of inflammatory factors were induced(p<0.05)after S.aureus infection,while the absorption of Met and Lys(p<0.05),synthesis and secretion of milk proteins(p<0.05)were inhibitied;(3)When S.aureus invading BMECs,the optimal treatment concentration of CGA was 40 μg/m L,and the optimal treatment time was 48 h.CGA under this treatment condition exhibit less toxicity on the proliferation of BMECs and S.aureus,which treatment also reduce the expression of infiammatory factors induced by S.aureus infection;(4)CGA treatment can reduce the number of intracellular S.aureus after infection(p<0.01);(5)CGA treatment could alleviate the decrease of Met and Lys absorption and milk protein synthesis caused by S.aureus infection of BMECs(p<0.05);(6)The transcriptome sequencing results showed that the number of differentially expressed genes detected in the CGA vs.Control,S.aureus vs.Control,and CGA + S.aureus vs.S.aureus were 94,111 and 77,respectively;(7)CGA could promote the expression of IL-10 and IL-10 RA in BMECs,which alleviate the decrease of milk protein synthesis(p<0.05)and inhibit the activation of NF-κB induced by S.aureus infection;(8)CGA can relieve the inhibition effect of the phosphorylation of m TOR,S6 and STAT5 after S.aureus infection;(9)The expression of SF-1(Steroidogenic Factor 1)were suppressed by S.aureus infection,and sh SF-1 inhibit the synthesis of milk protein,while CGA can alleviate the decrease of α-LA,β-LG and CSN2 synthesis caused by SF-1 gene sliencing;(10)The CGA may target NF-κB to regulate anti-inflammatory effect,and may target SF-1 to increase the synthesis of milk protein.In conclusion,S.aureus can invade BMECs and proliferate within cells,resulting in reduced absorption of amino acids and inhibit synthesis of milk protein.However,CGA had a mitigating effect on this phenomenon.In this study,we established an in vitro mastitis model to determine the protective effect of CGA against S.aureus infection of BMECs,and preliminarily discussed the possible mechanism of CGA based on the analysis and screening of transcriptome results and the prediction of bioinformatics,which will provide a theoretical basis for the treatment of cow mastitis and the clinical application of CGA.