Study On Expression Of Endogenous Antimicrobial Peptides In Bovine Mammary Epithelial Cells By Sodium Butyrate And Its Antimicrobial Activity Against Staphylococcus Aureus Infection

Staphylococcus aureus(S.aureus)is one of the major pathogens causing bovine mastitis,and the problem of antibiotic resistance and chronic inflammation are increasingly prominent.As a new strategy for disease control,antimicrobial peptides(AMPs)are usually produced naturally by animal cells,which are effective and extremely resistant to almost all types of pathogens.Sodium butyrate(Na B)is not only a short-chain fatty acid nutritional compound,but also a histone deacetylase inhibitor(HDACi).It has been reported that Na B can induce the expression of endogenous AMPs in animals through HDACi,and can also exert an anti-bacterial and anti-inflammatory effect,but the exact molecular mechanism is still unclear.Therefore,this study provides a theoretical basis in the molecular field for the effective prevention and control of bovine mastitis caused by S.aureus infection by comprehensively understanding the effect of Na B on gene expression of endogenous AMPs and clarifying the regulatory mechanism of Na B on inflammatory signaling pathways.In this study,bovine mammary epithelial cells(MAC-T)were used as cell models.Firstly,the effect of Na B on gene expression of endogenous AMPs was observed.Secondly,transcriptome identified and verified transcription factor,cell signal pathway and micro RNA on gene expression of endogenous AMPs regulated by Na B.Thirdly,the antibacterial activity of Na B on Staphylococcus aureus(S.aureus)infected MAC-T cells were observed.Finally,we explored the signal transduction mechanism of Na B in alleviating inflammatory response induced by S.aureus exotoxin lipoteichoic acid(LTA).The main results are list as follow:(1)Our results showed that Na B could up-regulate gene expression of endogenous AMPs in MAC-T cells,including β-defensin(DEFB1,DEFB3,DEFB4,DEFB5,DEFB6,DEFB8,DEFB9,DEFB10,DEFB11,DEFB12,DEFB13,LAP,TAP,BPTI,EBD)and cathelicidin(Bac1,Bac5,Indoliin,BMAP-28,BMAP-27,BMAP-34).AMPs were induced by Na B in a dose-and time-dependent manner,with DEFB1 and BMAP-34 being the most significant.Na B increased histone acetylation in MAC-T cells in a dose-dependent manner,and induced gene expression of DEFB5,Indolicidin and BMAP-34 was higher than trichostatin A(TSA).MAC-T cells expressed bovine homologous receptor protein and m RNA of GPR41 and GPR43,and Na B can up-regulated gene expression of GPR41 and GPR43.Silencing of GPR41 and GPR43 by specific si RNA attenuated the up-regulation of DEFB5,DEFB10 and BMAP-34 induced by Na B,and the effect of DEFB10 was most significant.(2)Transcriptome sequencing showed that EBD,TAP,DEFB5,DEFB1,DEFB4,LAP and BMAP-34 genes were up-regulated and differentially expressed.q PCR confirmed this result.Both AP-1 and DBP transcription binding sites were predicted in the upstream region of the promoter of the evaluated AMPs gene.Inhibition of p38 and NF-κB pathway down-regulated gene expression of BMAP-34 and DEFB1 induced by Na B,respectively.Inhibition of ERK pathway down-regulated the expression of BMAP-34 induced by Na B,but up-regulated the expression of DEFB1.In contrast,inhibition of JNK pathway up-regulated the expression of BMAP-34 induced by Na B and down-regulated the expression of DEFB1.Western blotting revealed that bta-mi R-677 had a significant positive regulatory effect on histone acetylation in MAC-T cells,whereas bta-mi R-2440 had no significant effect on it.bta-mi R-677 up-regulated gene expression of BMAP-34 and DEFB1 in MAC-T cells,while bta-mi R-2440 only inhibited BMAP-34 by Na B.(3)Our results showed that Na B inhibited S.aureus internalization into MAC-T cells in a dose-and time-dependent manner.Transmission electron microscopy showed that pretreatment with Na B ameliorated S.aureus induced disruption of MAC-T ultrastructure.S.aureus infection increased gene expression of TNF-α,IL-1β,IL-6,TLR2,NOD1 and NOD2 in MAC-T cells.S.aureus infection increased gene expression of BMAP-34 and TAP.After pretreatment with Na B,BMAP-34 and TAP were further up-regulated.Na B can significantly alleviate the mammary gland pathological damage and inflammation caused by S.aureus infection in mouse.(4)Our results showed that Na B significantly increased histone H3 acetylation level and BMAP-34,TAP,DEFB1,DEFB5 in MAC-T cells.LTA did not significantly increase histone acetylation level.After pretreatment Na B further increased histone acetylation level and AMPs expression.Pretreatment with Na B inhibited TNF-α,IL-1β and IL-6,and enhanced IL-10 in LTA-induced MAC-T cells.LTA significantly induced the activation of the subunit of NF-κB p65 and the degradation of IκBα.Pretreatment with Na B reduced the phosphorylation levels of NF-κB p65 and IκBα and reduced the nuclear translocation of NF-κB p65 in LTA-induced MAC-T cells.Na B significantly increased the phosphorylation protein expression levels of p38 and ERK,and inhibited the phosphorylated protein expression levels of JNK,AMPKα and ACCα in LTA-induced MAC-T cells.LTA significantly increased the protein expression levels of TLR2,NLRP3,ASC,and caspase-1,IL-1β in MAC-T cells,and this increase was attenuated by pretreatment with Na B.In conclusion,our study confirmed that Na B can induce the up-regulation gene expression of endogenous AMPs in bovine mammary epithelial cells(MAC-T),meanwhile,HDACi,short-chain fatty acid receptor GPR41/GPR43,NF-κB pathway,MAPKs pathway and micro RNA are involved in it.Na B plays an antibacterial and anti-inflammatory role in S.aureus infection and LTA stimulated MAC-T cells,and its effect is related to inhibition of TLR2/NF-κB/NLRP3 pathways and enhancement gene expression of endogenous AMPs.