| As one of the harmful gases in the livestock house,NH3 affects the health and growth of livestock and poultry and reduces production performance.Apoptosis and autophagy play an important role in the maintenance of the number and function of mammary cells,and they are the key factors affecting the lactation performance of dairy cows.SIRT5 has a variety of enzyme activities and can regulate the autophagy of tumor cells induced by ammonia.Our previous research found that NH3 regulates the apoptosis and autophagy of cow mammary epithelial cells through PI3K/Akt/mTOR signal.Whether SIRT5 regulates NH3-induced apoptosis and autophagy in dairy cow mammary epithelial cells remains unclear.In this study,MAC-T cells were used to construct SIRT5 overexpression and knockout MAC-T cells,combined with SIRT5 inhibitors to treat MAC-T cells,and detect the contents of NH3,GLS,glutamate,and ADP/ATP in the cells.To understand the effect of SIRT5 on MAC-T cell NH3 release;use RT-PCR,Western blot,Annexin Ⅴ FITC/PI,transmission electron microscopy and other methods to evaluate the effect of SIRT5 on MAC-T cell apoptosis and autophagy;use NH4Cl treatment SIRT5 overexpression and knockout MAC-T cells and cells in the inhibitor group,combined with PI3K or mTOR inhibitor treatment,clarify the effect of SIRT5 on NH3 inducing MAC-T cell apoptosis and autophagy;aim to reveal SIRT5 on NH3 Induced MAC-T cell apoptosis and regulation of autophagy activity.The results are as follows:SIRT5 overexpression and knockout MAC-T cell line establishment:The SIRT5 gene fragment was cloned from MAC-T cells with a length of 933 bp;SIRT5 overexpression vector pEGFP-SIRT5 and SIRT5 knockout vector pX330-sgRNA-SIRT5 were constructed;The results of RT-PCR and Western blot showed that a stable MAC-T cell line overexpressing or knocking out SIRT5 was successfully obtained.SIRT5 reduces the release of NH3 in cells:Compared with the control group,the SIRT5 overexpression group reduces the content of NH3,GLS and glutamate in the cells,and reduces the ratio of ADP/ATP,the results of the SIRT5 knockout group and the inhibitor group to the opposite.It is suggested that SIRT5 can inhibit glutamine metabolism and reduce the release of NH3.SIRT5 inhibits MAC-T cell apoptosis and autophagy:Compared with the control group,the mRNA and protein levels of Bax,Cspase-3,LC3,and Becline-1 decreased in the SIRT5 overexpression group,and the mRNA and protein levels of Bcl-2 and p62 increased.High,the results of SIRT5 knockout group and inhibitor group are opposite;suggesting that SIRT5 inhibits MAC-T cell apoptosis and autophagy.The Annexin Ⅴ FITC/PI results further prove that SIRT5 can inhibit the apoptotic response of MAC-T cells.SIRT5 inhibits MAC-T cell apoptosis and autophagy:Compared with the control group,the mRNA and protein levels of Bax,Caspase-3,LC3,and Becline-1 decreased in the SIRT5 overexpression group,and the mRNA and protein levels of Bcl-2 and p62 increased.High,the results of SIRT5 knockout group and inhibitor group are opposite;suggesting that SIRT5 inhibits MAC-T cell apoptosis and autophagy.The Annexin Ⅴ FITC/PI results further prove that SIRT5 can inhibit the apoptotic response of MAC-T cells.SIRT5 inhibits NH3-induced apoptosis and autophagy of MAC-T cells:After co-treatment with NH4Cl,SIRT5 reduces the content of NH3,GLS and glutamate in cells;Bax,Caspase-3,LC3,Beclinel mRNA and protein levels Decrease,Bcl-2,p62 mRNA and protein levels increase;Annexin Ⅴ FITC/PI results show that SIRT5 reduces the apoptotic response of cells.It is suggested that SIRT5 can inhibit the apoptosis and autophagy of MAC-T cells induced by NH3.PI3K/Akt/mTOR signal is involved in the regulation of NH3-induced autophagy and apoptosis by SIRT5:After PI3K or mTOR inhibitor treatment,the number of NH4Cl-induced apoptosis cells induced by SIRT5 overexpression was significantly reduced;The mRNA and protein expression levels of pro-apoptotic factor Bax and Caspase-3 were significantly decreased,while the mRNA and protein expression levels of anti-apoptotic factor Bcl-2 were significantly increased.The expression of autophagy factor LC3B and Becline-1 mRNA decreased,the expression of p62 mRNA increased,the ratio of LC3Ⅱ/Ⅰ protein and the expression of p62 protein increased significantly,and the expression of Becline-1 protein decreased significantly.The results were reversed in the SIRT5 knockout group and the inhibitor group.It is suggested that PI3K/Akt/mTOR signal is involved in the regulation of NH3-induced autophagy and apoptosis by SIRT5. |
