Effects And Mechanisms Of NDRG1 In The Proliferation And Milk Secretion Of Bovine Mammary Epithelial Cells

Bovine mastitis in one of the most important infectious diseases of dairy cattle,and it was usually caused by a variety of factors such as changes in body metabolism and physiology,mammary gland damage,and exposure to infectious or environmental pathogenic microorganisms.Currently,the most common cause of mammary infection is the invasion of pathogenic bacteria such as Escherichia coli(E.coli)and Staphylococcus aureus(S.aureus).When it occurs in the mammary gland,the proliferation and vitality of mammary epithelial cells and lactation of mammary glands will be affected,which could result in huge economic losses.Nowadays,the correlation between N-myc downstream regulated gene 1(NDRG1)and diverse tumor diseases has been proved by many researches.However,the reorganization about NDRG1 in bovine mastitis remains largely unknown.A high-throughput sequencing analysis of heat-inactivated E.coli and S.aureus-treated MAC-T cells showed that NDRG1 expression was evidently upregulated compared with the normal cells.To further validate the NDRG1 expression from the result of high-throughput sequencing,we examined NDRG1 expression in heat-inactivated E.coli and S.aureus-treated MAC-T cells by RT-q PCR and western blot.Results showed that E.coli and S.aureus treatment upregulated the gene and protein.In addition,we examined the expression level of NDRG1 in normal and mastitic mammary tissue of bovine,and results from RT-q PCR analysis showed that NDRG1 had an increased expression in inflammatory tissue compared to normal tissue.NDRG1-overexpressed and NDRG1-silenced MAC-T cell clones were establishment to investigate the effect of NDRG1 on mammary epithelial function in dairy cows.In this study,the inflammatory mediator and chemokine expressions in MAC-T cell clones overexpression or silenced for NDRG1 were determined by RT-q PCR.The effect of NDRG1 on epithelial cell proliferation was analyzed by using a CCK-8 kit.The effect of NDRG1 on MAC-T cells migration was analyzed with a scratch-recovery assay.?-casein protein secretion levels in the culture medium determined by ELISA.TGA secretion levels in the culture medium determined by enzymatic analysis.Moreover,the m TOR signaling pathway has been shown to be closely linked to mammary epithelial cell proliferation and milk protein synthesis.AKT1,p-AKT,m TOR and p-m TOR protein levels were determined by western blotting.In conclusion,results from this study showed that NDRG1 significantly inhibited cell proliferation and migration,?-casein and TG secretion,gene expressions of inflammatory cytokines and chemokines,and activation of m TOR signaling pathway of mammary epithelial cells.Increasing evidences indicate that n3 polyunsaturated fatty acids(n-3PUFA)exerts great influence in inflammation cascade and lipid-modulating,which similar with NDRG1.Therefore,this study continues to explore the effect of n-3PUFA and NDRG1 in inflammatory factors expression,?-casein and TG secretion on mammary epithelial cells.In conclusion,findings of this study uncover that NDRG1 was highly expressed in mammary epithelial cells under inflammation conditions and mastitic tissue,and inhibited the inflammatory mediator and chemokine expressions,cell proliferation and migration,?-casein and TG secretion by negatively regulates m TOR signaling pathway.In addition,n-3PUFA synergizes with NDRG1 gene to inhibit the secretion of inflammatory factors and TG in MAC-T cells,but n-3PUFA has negatively effect on the ?-casein secretion induced by NDRG1 gene in mammary epithelial cells.