The Effect Of Zearalenone On Immune Function Of B Lymphocytes In Mice And The Role Of TLR4/MAPK In It

Zearalenone(ZEA)is a nonsteroidal hormone mainly produced by Fusarium with estrogenic effects.It is contaminated all over the world and poses a great threat to human and animal health.ZEA can cause damage to animals’ reproductive system,immune system and endocrine system.In recent years,there have been many reports on the immunosuppression caused by ZEA in animals,but the specific mechanism of its action has not been fully explored,especially for B lymphocytes,which play an important role in humoral immune response.In order to explore the mechanism of immunotoxic action of ZEA,this study selected primary B lymphocytes of mouse as materials to explore the effects of ZEA on the related immune function of B lymphocytes and the mechanism of TLR4/MAPK in it.1.The effects of ZEA on the activity and activation-induced molecules of B lymphocytesPrimary B lymphocytes were isolated from the spleens of BALB/c mice aged 4-6 weeks.B lymphocytes with purity above 90%were selected by immunomagnetic bead method as material.Then B lymphocytes were treated with 0(Control group),5 μg/mL LPS(LPS group),5 μg/mL LPS+(10,20,30 μM)ZEA for 12 h,24 h,30 hand 72 h.①The effects of ZEA on the survival of mouse B lymphocytes in vitro were detected by CCK-8 assay.② The effects of ZEA on the activated aggregation of B lymphocytes was observed by the microscope.③Flow cytometry was used to detect the effects of ZEA on the activation-induced molecules of B lymphocytes at various stages.The results showed that:① At 24 h,different concentrations of ZEA significantly inhibited the LPS-induced increase in the activity of B lymphocytes(P<0.01).② Compared with the Control group,the volume of cells in the LPS group was significantly increased.The phenomenon of life clustering was complicated.After being cultured in 96-well U-shaped plate for 24 h,compared with the Control group,the cells in the LPS group were obviously aggregated,with a small amount of cell clones.Compared with the LPS group,cell clones disappeared after the treatment with 10 μM ZEA.Cell state was obviously dispersed after treatment with 20 and 30 μM ZEA.③ Being treated with ZEA for 12 h,30 h,72 h,the early,middle and late activated molecules of B lymphocytes was detected.It is found that LPS group compared with the Control,the amount of CD69,CD25,CD71 surface expression was significantly higher(P<0.01).All of the ZEA infected groups compared with LPS group,CD69,CD25,CD71 surface expression were significantly or extremely significantly decreased with dose-dependent effect(P<0.05 or P<0.01).These results indicate that ZEA can inhibit the activity and activation of B lymphocytes by LPS-induced.2.The effects of ZEA on proliferation,differentiation and phagocytosis of B lymphocytesAfter being treated with 0,5 μg/mL LPS and 5 μg/mL LPS+(20,30 μM)ZEA for 48 h and 72 h,①the proliferation of B lymphocytes was detected by CFSE method at 72 h.②The effect of ZEA on the differentiation of B lymphocytes to plasma cells was detected by flow cytometry.qRT-PCR was used to detect the effects of ZEA on B lymphocyte differentiation related regulatory genes in mice.③ The effect of ZEA on the phagocytosis of B lymphocytes was detected by flow cytometry.The results showed that:①At 72 h,different concentrations of ZEA significantly or extremely significantly inhibited the proliferation of LPS-induced primary B lymphocytes with dose-dependent(P<0.05 or P<0.01).② Different concentrations of ZEA were significantly inhibited by the B lymphocytes differentiate into plasma cell by LPS-induced(P<0.01).Different concentrations of ZEA can inhibit the genes promoting of differentiation and maturation of B lymphocytes in LPS-induced,IRF-4,XBP-1,and Blimp-1(P<0.05 or P<0.01).Different concentrations of ZEA can promote the genes suppression of differentiation of B lymphocytes by LPS-induced,BCL-6 and Pax-5,which could reach an extremely significant level at the 30 μM ZEA(P<0.01).③At 48 h,different concentrations of ZEA significantly inhibited LPS-induced phagocytosis of B lymphocytes(P<0.01).These results indicate that ZEA can inhibit the proliferation,differentiation and phagocytosis of B lymphocytes by LPS-induced.3.The role of TLR4/MAPK in the effect of ZEA on B lymphocyte-related immune functionThe 0,5 μg/mL LPS and 5 μg/mL LPS+(20,30 μM)ZEA treatment of cells for 24 h,20 μM ZEA and 10 μM of ERK inhibitor U0126 alone or combined treatment of the B lymphocytes induced by LPS for 24 h,20 μM ZEA and 10 μM SP600125 JNK inhibitor alone or combined treatment of B lymphocytes induced by LPS for 24 h were performed.① The expression levels of TLR4 signaling pathway and NFκB were detected by Western-blot.②The effect of ZEA on phosphorylation of ERK1/2 and JNK1/2 by LPS-induced in B lymphocytes,and the effect of ZEA alone or in combination with U0126 or SP600125 on phosphorylation of ERK1/2 and JNK1/2 in B lymphocytes were detected by Western-blot.③The effect of ZEA with U0126 or SP600125 alone or in combination,on the secretion of cytokines,IL-1β,IL-6 and TNF-α,which was induced by LPS,was detected by ELISA kit.The results showed that:①The expression of TLR4,My D88 and p-IκBa induced by LPS could be inhibited by different concentrations of ZEA.The expression of MyD88 protein was significantly decreased at 30 μM ZEA(P<0.05).The expression of p-IκBα was significantly or extremely significantly decreased(P<0.05 or P<0.01).Compared with LPS group,the total protein expression of NFκB showed a downward trend at different concentrations of ZEA group,and was significantly decreased at 30 μM ZEA(P<0.01).Cytoplasmic protein was increased at 30 μM ZEA and was significantly increased(P<0.01).The nucleoprotein expression was significantly decreased at 30 μM ZEA(P<0.05).② The phosphorylation of ERK1/2 and JNK1/2 were significantly or extremely significantly increased by different concentrations of ZEA(P<0.05 or P<0.01).Compared with the ZEA group,the phosphorylation of ERK1/2 in the ZEA and U0126 combined treatment group was significantly decreased(P<0.01).The phosphorylation of JNK1/2 in the ZEA and SP60015 combined treatment group was significantly decreased(P<0.01).③ Compared with the LPS group,the contents of IL-1β,IL-6 and TNF-α in cell supernatant were significantly decreased by 20 μM ZEA(P<0.05).Compared with single ZEA group,IL-1β,IL-6 and TNF-α in ZEA and U0126 combined treatment group,and ZEA and SP60015 combined treatment group had no significant changes(P>0.05).These results indicate that ZEA can inhibit the secretion of inflammatory cytokines in B lymphocytes by LPS-induced,which is mainly regulated by TLR4 signaling pathway.Conclusions:ZEA can inhibit NFκB entry into the nucleus by inhibiting TLR4 signaling pathway of B lymphocytes by LPS-induced.ZEA could also lead to the overactivation of MAPK signaling pathway.Therefore,it can inhibit the activity,activation,proliferation,differentiation and phagocytosis of B lymphocytes by LPS-induced,which leads to suppression of the immune response in animals.