Effect Of Gpx1 On Selenium Antagonism Of DON On The Apoptosis And Methylation-related Enzymes In Spleen Lymphocytes Of Pigs

Deoxynivalenol（DON）is a mycotoxin commonly found in feeds in recent years and has the highest detection rate.DON can promote lymphocyte apoptosis through oxidative damage,and it can also have some influence on cell methylation.Selenium is one of the essential trace elements in the body,which acts as a selenoprotein to exert antioxidant properties and enhance the biological characteristics of the animal’s body immunity.In this study,primary porcine spleen lymphocytes,GPx1 knockdown porcine spleen lymphocytes and GPx1 overexpressing porcine spleen lymphocytes were used as experimental models.The deoxygenated snow rot was added separately/in combination.DON and sodium selenite（Na₂SeO₃）were used to investigate the effect of GPx1 on selenium antagonism of DON on apoptosis and methylation-related enzymes in spleen lymphocytes of pigs.Method:The experiment was divided into three models,10 groups of normal model,divided into blank control group（P group）,selenium alone group（Se group,Na₂SeO₃concentration was 2μmol/L）,DON alone group(D1,D2,D3,D4,DON concentrations were 100 ng/mL,200 ng/mL,400 ng/mL,800 ng/mL,respectively.Selenium and DON co-action groups（SD1,SD2,SD3,SD4,Na2SeO3 and DON concentrations were 2μmol/L and 100 ng/mL,2μmol/L and 200 ng/mL,2μmol/L and 400 ng/mL,2μmol/L and 800ng/mL）.GPx1 gene knockdown model 11 group,increased knockdown group（M group）;GPx1 gene overexpression model 12 groups,increased empty group（K group）and overexpression group（C group）.The remaining 10 groups of the two models are grouped the same as the normal cell model.After 48 h of cell culture,The apoptosis rate of spleen lymphocytes was detected by flow cytometry.The expression of Bax,Bcl-2,DNMT1,DNMT3a,DNMT3b and MBD2 mRNA was detected by qRT-PCR.Finally,the differences between the Se+DON combination group and the DON alone treatment group of each model of the three models were compared.Results:（1）In the normal model,compared with the blank control group,the apoptosis rate of the Se alone group was smaller than that of the blank group,the Bax mRNA expression level was decreased,and the Bcl-2 mRNA expression level was increased.When different concentrations of DON were added alone,the apoptotic rate increased and the apoptosis rate increased with the concentration of DON.The expression level of Bax mRNA was significantly increased（P<0.05）,and the expression level of Bcl-2mRNA was significantly decreased（P<0.05）.The apoptotic rate was decreased in the combination of selenium and different concentrations of DON compared with the corresponding DON alone group.the expression level of Bax mRNA was relatively decreased and the expression level of Bcl-2 mRNA was increased（P<0.05）.In the GPx1gene knockdown model,the apoptotic rate was increased（P<0.05）,the Bax mRNA expression level was increased,and the Bcl-2 mRNA expression level was significantly decreased in the knockdown group（M group）compared with the blank control group（P group）.The other groups of the GPx1 gene knockdown model showed the same trend as the normal model.In the GPx1 over-the-counter model,the apoptotic rate was increased（P<0.05）and the Bax mRNA expression level was significantly increased（P<0.05）,Bcl-2mRNA in the empty group（K group）compared with the control group（P group）.The level of expression is reduced.Compared with the control group（P group）,the overexpression group（group C）showed a decrease in apoptotic rate（P<0.05）,decreased Bax mRNA expression level,increased Bcl-2 mRNA expression level,and other groups of GPx1 gene overexpression model.The change trend of the test indicators is the same as the normal model.The apoptotic rate,the expression of Bax and Bcl-2 mRNA in the DON alone group and the corresponding Se+DON combination group were compared in three models.The rate of change in the GPx1 gene knockdown model is mostly lower than the normal model.It indicated that GPx1 was knocked down and Na₂SeO₃ was added.The antagonism of Na₂SeO₃ on DON-induced apoptosis was reduced.Compared with the normal model,the rate of apoptosis and the rate of change of Bcl-2 gene in the GPx1 gene overexpression model,except for the DON concentration of 800 ng/mL,the rate of change of the GPx1gene overexpression model did not increase compared with the normal model.And the rate of change of the Bax gene increased.It indicated that GPx1 was overexpressed and Na₂SeO₃ was added.Na₂SeO₃ enhanced the antagonism of DON-induced apoptosis-promoting gene Bax mRNA expression,but the increase of apoptosis rate and the decrease of apoptosis-inhibiting gene Bcl-2 mRNA were only in DON.The Na₂SeO₃antagonism was enhanced when the concentration of toxin was large.（2）In the normal model,compared with the blank control group,the expression levels of DNMT1,DNMT3a,and DNMT3b mRNA in the Se alone group increased（P<0.05）,and the MBD2 mRNA expression level decreased.After adding different concentrations of DON,the mRNA expression levels of DNMT1,DNMT3a and DNMT3b increased first and then decreased.The expression level of MBD2 mRNA is increased.Compared with the DON alone group,the expression levels of DNMT1,DNMT3a and DNMT3b mRNA and the expression level of MBD2 mRNA were decreased in selenium and different concentrations of DON.In the GPx1 gene knockdown model,the mRNA expression levels of DNMT1,DNMT3a,and DNMT3b were decreased and the expression level of MBD2mRNA was increased in the knockdown group（M group）compared with the blank control group（P group）.The trend of the other indicators in the GPx1 gene knockdown model was the same as the normal model.In the GPx1 gene overexpression model,the DNMT1,DNMT3a,and DNMT3b mRNA expression levels were decreased and the MBD2 mRNA expression level was increased in the empty group（K group）compared with the control group（P group）.Compared with the control group（P group）,the overexpression group（group C）increased the mRNA expression levels of DNMT1,DNMT3a and DNMT3b,and decreased the expression level of MBD2 mRNA.The other groups of the GPx1 gene overexpression model showed the same trend as the normal model.The rates of change of DNMT1,DNMT3a,DNMT3b mRNA and MBD2 mRNA were compared in three models.The rate of change in the GPx1 gene knockdown model was mostly lower than that of the normal model,indicating that the knockdown of GPx1followed by Na₂SeO₃,Na₂SeO₃ reduced the antagonism of DON-induced changes in DNA methylation-related enzymes.The rate of change in the GPx1 gene overexpression model was greater than that of the normal model in the rate of change in DNMT1 and DNMT3a in the GPx1 gene overexpression model.It was shown that GPx1 was overexpressed and Na₂SeO₃ was added.Na₂SeO₃ enhanced the antagonism of DON-induced changes in DNMT1 and DNMT3a mRNA expression.However,for the changes of DNMT3b and MBD2 mRNA,Na₂SeO₃ antagonism did not show enhanced.Conclusion:（1）In three cell models,DON caused apoptosis of porcine spleen lymphocytes,and the higher the DON concentration,the more severe the apoptosis,which was concentration-dependent.Selenium can exert its antagonism on DON-induced apoptosis of porcine spleen lymphocytes by selenoprotein GPx1.（2）In three cell models,800 ng/mL DON caused a decrease in the expression of lymphocyte methylase mRNA in pig spleen.Different concentrations of DON caused an increase in the mRNA expression of demethylase MBD2 gene in a concentration-dependent manner.The amount of selenium can exert its antagonism in the effect of DON on the methylation-related enzymes of pig spleen lymphocytes by selenoprotein GPx1.