The Effect And Mechanism Of Zearalenone On The Activation Of T Lymphocyte In Mice

Zearalenone(ZEA)is a nonsteroidal estrogenic mycotoxin produced by Fusarium and contaminated all over the world.The toxic effects of ZEA are found in the reproductive,endocrine and immune systems and so on.But the related mechanisms,especially molecular mechanisms,have not been fully explored.At present,there are few reports on the effects of the ZEA exposure on the activation of T lymphocytes in animal immune cells.In order to explore the mechanism of ZEA immunotoxicity,the primary T lymphocytes isolated from mice were used as materials.The effects of ZEA on the signal transduction pathway of T lymphocyte activation in vitro and its function after activation were studied.1.Effect of ZEA on T lymphocyte activity and activation-induced molecules in micePrimary T lymphocytes were isolated from the spleen of 6-8 week old Balb/c mice by immunomagnetic beads negative sorting technique.Treatment of primary mouse lymphocytes activated with/without Con A(5?g/mL)for 48 h,72 h,96 h with 0,2.5,5,10,20,40,80 ?M ZEA.The effect of ZEA on the activity of T lymphocytes before and after activation was detected by CCK-8.Cell agglomeration was observed in 96-well U cell culture plate.The expression of activation-induced molecules in T lymphocytes by ZEA at different stages was detected by flow cytometry.The results showed that there was no significant change in the activity of T cells in the groups without Con A compared with the control group(p>0.05);Compared with the blank control group,the cell activity of Con A control group increased significantly(p<0.01).The cell activity of 20 ?M ZEA treatment group was significantly lower than that of Con A group at 48 h(p<0.05).At 72h and 96h,the cell activity of 10?M ZEA treated group was significantly lower than that of Con A control group and showed a dose effect relationship(p<0.01).After 72 hours of cell grouping on the 96 well U substrate culture plate,we could see that the cell clone colony can hardly be seen in the control group without Con A;A large number of cell clones appeared in the Con A group;Cell aggregation was inhibited in 10?CM ZEA treatment group.20 ?M ZEA treatment group with 40 ?M ZEA group began to have shrinkage.Compared with the blank control group,the expression of the activation marker protein CD69,CD25,CD71 in the early,middle and late stage of T lymphocytes in the control group begin to express in large quantities(p<0.01)which was detected by double fluorescence staining flow cytometry.Compared with the control group of Con A,the positive expression rate of CD69?CD25?CD71 in T cells treated with 10 ?M ZEA or above decreased significantly(p<0.05 or p<0.01)and showed a dose-effect relationship.2.The effect of ZEA on TCR signaling pathwayMouse primary T lymphocytes were used as materials.The blank control group,Con A control group,20?40 ?M ZEA and Con A 5 ?g/mL treatment group were set up;The activity of LAT in the activation signal pathway of TCR and the activity and phosphorylation level of the activation initiation regulatory protein Lck,Zap-70 were detected by western blot method.Western blot method was also used to study the effect of ZEA exposure on T lymphocyte activated nuclear factor NF-AT,NF-?B pathway and the expression of two nuclear factors in the nucleus.The effect of ZEA on the costimulatory signal during TCR activation was analyzed by flow cytometry.The effect of ZEA on the downstream PI3K-Akt-mTOR signaling pathway mediated by CD28 was detected by western blot.The results show that,compared with the control group,the expression of LAT increased significantly(p<0.01).The activity of LAT was inhibited by different concentrations of ZEA,and the activity and phosphorylation of the activation initiation regulatory protein Lck,Zap-70 was inhibited by different concentrations of ZEA exposure.There was a dose-dependent relationship between the two levels(p<0.01).The exposure to different concentrations of ZEA inhibited the activity of key proteins in the activated nuclear factor NF-AT,NF-?B pathway(p<0.05)and inhibited the expression of two nuclear factors in the nucleus.In addition,ZEA interfered with the stability of TCR activation pathway and costimulated the signal CD28/CD152.10,20 ?M ZEA inhibited the expression of CD28 molecule and CD 152 molecule.Compared with Con A group,the 40 ?M ZEA exposure group showed no significant difference.The expression of key proteins in the downstream PI3k-Akt-mTOR signaling pathway mediated by CD28 in different concentrations of ZEA treatment group was significantly lower than that in Con A group(p<0.05 orp<0.01).3.Effects of ZEA on the expression of cytokines,chemokines and chemokine receptors in T cells after activationThe supernatant of mouse T lymphocytes was collected after 48h or/and 72h culture.The secretion of cytokine IL-2,IL-3,IL-5,IL-6,GM-CSF and chemokines MIP-1? and RANTES were detected by CBA.The expression of chemokine receptor CCR2 and CCR7 in T lymphocytes was detected by flow cytometry after 24 hours of cell culture.The results showed that the secretion levels of the cytokines IL-2?IL-3?IL5?IL6,GM-CSF,chemokines MIP-1?and RANTES were significantly higher than those of the control group(p<0.01).The secretion of five cytokines in the ZEA treatment group was significantly lower than that in the Con A group(p<0.01)and had a dose-effect relationship.With the increase of the concentration of ZEA,the secretion of MIP-1? and RANTES in the treatment group with 20?M ZEA and above decreased significantly compared with that in the Con A group.The RANTES secretion in the treatment group with 40 ?M ZEA was significantly lower than that in the Con A group.The results of flow double staining showed that compared with the unactivated T lymphocytes(blank control group),the T cells stimulated by Con A expressed a large amount of the CCR2 and CCR7(p<0.01).The cells were treated with different concentrations of ZEA.Compared with Con A group,20 ?M ZEA could significantly decrease the expression rate of CCR2 in T cells in a dose-dependent manner.The 40 ?M ZEA could significantly decrease the CCR7 expression(p<0.05)in a dose-dependent manner compared with Con A group.Conclusion:ZEA inhibited the activation of T lymphocytes mediated by Con A by interfering with TCR and costimulatory signaling pathway,which inhibited the synthesis and secretion of cytokines and chemokines after normal activation of T lymphocytes.The chemotactic effect of T cells was inhibited to some extent,which resulted in immunosuppression.