The Effect And Mechanism Of Zearalenone On The Activation Of Mouse B Lymphocyte In Vitro

Zearalenone(Zea)is one of those mycotoxins with estrogenic effect,which is widely distributed in cereal plants polluted by Fusarium fungi all over the world.Its toxin will harm the health of animals and human beings.In recent years,it has been found that zearalenone can cause damage to reproductive organs,endocrine system and immune organs.In order to study the humoral immune toxicity of Zea on splenic B lymphocyte,we used mouse splenic B lymphocytes to reveal the potential mechanism of Zea on humoral immune function,the effects of Zea on B cell activity,secretion function and signal pathway in vitro.1.Effects of Zea on the activity and proliferation of B lymphocytes in micePrimary spleen B lymphocytes were obtained from the spleen of 6-8 weeks old Balb/c mice by immunomagnetic beads negative sorting method.B lymphocytes were treated with 0(blank control),0.1,1.0,2.5,5.0,10.?g/mL lipopolysaccharide(LPS)for 24 h and 48 h.CCK-8 was used to detect the effect of LPS on spleen B lymphocytes,and to select the appropriate concentration of LPS.0(control group),5,10,20,40,80 ?mol/L ZEA were simultaneously treated with B lymphocytes without or with LPS(5 ?g/mL)for 24 h to find the effects of Zea on the activity and proliferation of spleen B lymphocytes detected by CCK-8.The results showed that,5 ?g/mL LPS stimulated mice spleen B lymphocyte activity increased significantly at 24 h(p<0.05).When the concentration of unactivated B lymphocytes exposed to ZEA reached 80 ?mol/L,the activity of B lymphocytes decreased significantly(p<0.05).In the tests,for the ZEA exposured cells with LPS,the cell activity of 20 ?mol/L Zea treatment group was significantly lower than that of LPS control group at 24 h(p<0.05).The 40,80 ?mol/L Zea treatment groups were significantly lower than that of LPS control group(p<0.01).The cell activity of 10 ?mol/L Zea treatment groups was significantly lower than the LPS control group at 48 h(p<0.05),and the 20,40 and 80?mol/L Zea treatment groups were significantly lower than the LPS control group at 48 h(p<0.01),which showed a dose-effect relationship.2.The effect of ZEA on the activation and secretion of B lymphocytes in miceMouse primary spleen lymphocytes were used as materials.The blank control group,LPS control group,20 and 40 ?mol/L ZEA with LPS 5?g/mL treatment group were set up.After exposure with ZEA for 24 h,flow cytometry was used to detect the expression levels of B lymphocyte surface antigens CD40,CD80,and CD86,the levels of cytokines IL-6,IL-10 and TNF-? were detected by Cytometric Beads Array.The expression levels of IgG and IgM were detected by ELISA kit.The results showed that the expression of CD40 and CD80 in LPS control group was significantly higher than that in blank control group(p<0.05).The expression of CD86 was significantly higher than that of blank control group(p<0.01).As the concentration of ZEA increased,the secretion of CD40 and CD80 in each exposure group began to show a downward trend.Compared with the LPS control group,the expression of CD40 and CD80 decreased significantly in 40 ?mol/L ZEA treatment group(p<0.05),the expression of CD86 did not change significantly(p>0.05).Compared with the blank control group,the secretion of IL-6,IL-10 and TNF-? in the LPS group all increased significantly(p<0.01).The levels of IL-6,IL-10 and TNF-? in the ZEA group were significantly lower than those in the LPS control group(p<0.01)which showed a dose-response relationship.Compared with the LPS control group,with the increase of the ZEA concentration,the IgG secretion of the exposure group showed a downward trend.The IgG secretion of the 20 ?mol/L ZEA treatment group was significantly lower than that of the LPS control group(p<0.05).The secretion of IgG in the 40 ?mol/L ZEA treatment group was significantly lower than that in the LPS control group(p<0.01).Compared with the blank control group,the antibody IgM secretion of the LPS control group was significantly increased(p<0.05).Compared with the LPS control group,the change trend of the IgM secretion of the ZEA treatment group was not significant(p>0.05).The results show that ZEA can reduce the expression of B cell surface antigens,inhibit the expression of related cytokines of B lymphocytes,and reduce the ability of B cells to secrete related antibodies.3.The effect of ZEA on signaling pathways related to the activation of mouse B lymphocytesUsing the sorted mouse spleen B lymphocytes as materials,a blank control group(without LPS),LPS(5 ?g/mL)control group,ZEA(20?mol/L)+LPS(5 ?g/mL)treatment group,ZEA(40 ?mol/L)+ LPS(5 ?g/mL)treatment group were established.After 24 hours,the expression levels of apoptosis signaling pathway related proteins JNK,ERK1/2,P38 and NF-?B were detected by western blot.The results showed that compared with the blank control group,the expression of JNK,ERK1/2,P38,and NF-?B protein in the LPS control group increased.The expression of NF-?B protein increased significantly(p<0.05).Compared with the LPS control group,the expression of JNK protein in the 20 ?mol/L ZEA treatment group increased significantly(p<0.05).The increase of it in the 40 ?mol/L ZEA treatment group was extremely significant(p<0.01).ERK1/2 Protein expression levels increased(p<0.05).P38 protein and NF-?B protein expression levels in the 40 ?mol/L ZEA treatment group decreased significantly(p<0.05).The results show that ZEA can activate the expression of JNK and ERK pathway proteins,and inhibit the expression of P38 and NF-?B pathway proteins.Conclusion:ZEA can inhibit the secretion of various cytokines,the secretion of antibodies and the expression of cell surface antigens.At the same time it can interfere with the expression of key proteins in related pathways which maintains the normal cell function.It can also affect the activation of B lymphocytes in mouse humoral immunity which affects the normal function of B cells and finally leads to the immunosuppression in animals.