The Mechanism Of Taurine Activates Mammary Epithelial Cells Autophagy And Alleviates The Infection Of Streptococcus Uberis

This study focused on bovine mammary epithelial cells,taurine and Streptococcus uberis 0140J to explore the effect of taurine on mammary epithelial cells autophagy and the role of cell autophagy in mammary epithelial cells infected by Streptococcus uberis.In the study,the mechanism of taurine in alleviating the damage caused by uberis infection was clarified from the perspective of the occurrence and regulation of autophagy,by observing the cell autophagy and related signaling pathways,providing new ideas and methods for the prevention and control of bovine mastitis.The results are as follows:1.The effects of taurine on mammary epithelial cells autophagyIn order to investigate the effects of taurine on autophagy of mammary epithelial cells,the cells(MAC-T)were treated with different concentrations of taurine,then the autophagy marker proteins,autophagosomes,and autophagy flow were observed or measured by western blot,immunofluorescence and transmission electron microscopy.The results showed that the safety concentration of taurine is 0-90 mmol·L-1.The MAC-T was incubated with 50 nmmol·L-1 Baf-Al before treated with 70 mmol·L-1 taurine for 4 h,which could significantly increase the expression level of autophagy marker proteins such as LC3B-Ⅱ,Beclin-1,p62.The results of immunofluorescence and transmission electron microscopy showed that taurine treatment could significantly increase the number of autophagosomes.The cells transfection test of mCherry-EGFP-LC3B showed that taurine treatment could significantly increase the number of autophagosomes.With the increase of treatment time,the color of autophagosomes migrated from yellow(the superposition of red fluorescence and green fluorescence at 4h)to red(at 24h),indicating that autophagosomes and lysosome were fused to form autolysosomes,which showed a complete autophagy process was formed.The above results showed that treatment with 70 mmo·L-1 taurine for 4 h could significantly increase the expression of autophagy marker proteins and the number of autophagosomes,trigger complete autophagy flow and activate cells autophagy.2.Taurine activates mammary epithelial cells autophagy in an mTOR-dependent mannerAutophagy,as an important physiological phenomenon,is regulated by mTOR-dependent and independent signaling pathways.In order to investigate whether taurine regulates autophagy through mTOR-dependent signaling pathways in MAC-T cells,the cells were transfected with siRNA to interfere with the gene expression of taurine transport vectors(TauT,PAT1)and a specific inhibitor of PTEN(VOTH)to inhibit PTEN activity.Then the Western Blot,immunofluorescence and ELISA techniques were used to detect the expression of key note protein in the mTOR pathway and autophagy-associated proteins,the number of autophagosomes,and the content of inositol phospholipid(PIP2,PIP3).The results showed that taurine could significantly increase the phosphorylation level of PTEN and decrease the phosphorylation level of Akt and mTOR.The decrease of mTOR phosphorylation reduced the phosphorylation level of autophagy-associated proteins ATG13 and ULK1,which in turn activated autophagy.The addition of taurine transporter interfering RNA(siTauT,siPAT1)or PTEN-specific inhibitor(VOTH)alone or simultaneously could reduce the inhibitory effect of taurine on mTOR and its related signaling pathway and the activation effect of taurine on cells autophagy.All these results suggested that taurine could activate autophagy by mTOR-dependent mechanism in bovine mammary epithelial cells.3.The role of autophagy in mammary epithelial cells infected by Streptococcus uberisTo investigate the role of autophagy in mammary epithelial cells infected by Streptococcus uberis,the bovine mammary epithelial cells(MAC-T)and Streptococcus uberis(S.uberis 0140J)were used as the research objects,and the cells autophagy was activated by taurine,Rapa or silenced by siATG5,a specific inhibitor of ATG 5,and was inhibited by a specific inhibitor of 3-MA.Next,biochemical kit,inverted microscope,transmission electron microscope,Western Blot,ELISA kits and CCK-8 kits were used to detect the activity of NAGase and LDH,the contents of cytokines such as TNF-α,IL-1 β,and IL-6 in cellular supernatant,the amount of intracellular bacteria load,cell morphology,intracellular bacterial morphology,and the key proteins expression of inflammatory signaling pathway such as IKK,IκB,NF-κB and the expression of autophagy-associated proteins such as LC3B,ATG5,p62,and so on.The above results indicated that activation of cell autophagy by taurine or Rapa could significantly reduce the increase of the NAGase,LDH activity and the secretion of TNF-α,IL-1β and IL-6 in the cell supernatant caused by Streptococcus uberis infection.At the same time,it could also significantly reduce the intracellular bacteria load and inhibit the activation of IKK,IκB and NF-κB caused by Streptococcus uberis infection.The inhibition of cell autophagy with siATG5 or 3-MA could further increase the activity of NAGase,LDH and the secretion of TNF-α,IL-1 β and IL-6 in the cell supernatant，intracellular bacteria load and the activation of IKK,IκB,NF-κB caused by Streptococcus uberis infection.In addition,the inhibition of cell autophagy could decrease the alleviated effects of taurine or Rapa on Streptococcus uberis infection.It is suggested that cell autophagy plays an important role in Streptococcus uberis infection,and taurine might alleviate the damage of Streptococcus uberis infection by activating cell autophagy.In conclusion,we could infer that taurine might inhibit mTOR and the related signaling pathway by activating PTEN to induce autophagy in bovine mammary epithelial cells,speed up the process of eliminating intracellular Streptococcus uberis,inhibit the over-activation of inflammatory signaling pathway,which alleviate the inflammation and cell damage caused by Streptococcus uberis infection.This study will provide a theoretical basis for the application of nutritional elements to regulate the body’s innate immunity to resist the infection of Streptococcus uberis and provide new ideas and methods for the prevention and control of bovine mastitis.