Effects Of ZEA,DON And Their Combined Exposure On Immune Function And Apoptosis Of T Lymphocytes In Vitro

Zearalenone(ZEA)and Deoxynivalenol(DON)are common mycotoxins in cereals and feeds in China,often coexisting naturally,and cause pollution in the production,storage and transportation of agricultural and sideline products.The main toxicity of ZEA is reproductive toxicity,while the main toxicity of DON is intestinal toxicity,which means the two toxins have different degrees of immunotoxicity.At present,there are few reports on the combined immunotoxicity of ZEA and DON.In view of the reality of the natural coexistence of the two mycotoxins,it is necessary to understand the combined immunotoxicity of the two mycotoxins.In this experiment,mouse primary spleen T lymphocytes were used as materials,and concanavalin A(Con A)was used as T cell activation stimulator.The effects of ZEA,DON and their combined exposure on immune function and apoptosis of T lymphocytes in vitro were explored.1.Effects of ZEA,DON and their combined exposure on T lymphocyte viability and ultrastructure in micePrimary T lymphocytes were isolated from the spleens of 4-5-week-old BALB/c mice by immunomagnetic beads negative sorting.The control group(excluding Con A),Con A(5 mg/L)group,Con A(5 mg/L)+ZEA(10,20,and 40?M)group,Con A(5 mg/L)+DON(0.5,1 and 2?M)group,Con A(5 mg/L)+(ZEA+DON group)(10?M ZEA+0.5?M DON,20?M ZEA+1?M DON,40?M ZEA+2?M DON)were established.After 24 h of exposure,the relative viability of T cells was detected by CCK-8 method.After 48 h of exposure,the ultrastructural damage of T cells was observed by transmission electron microscopy and scanning electron microscopy.The results showed that,after 24 h of exposure,compared with Control group,Con A group relative activity of T cells was significantly increased(p<0.01).Compared with Con A group,the T lymphocyte viability in different concentrations of ZEA and DON treated groups all showed significant or extremely significant decrease(p<0.05 or p<0.01)and showed a dose-effect relationship.Compared with the Con A group,the cell viability of the different concentrations of ZEA and DON treatment groups were significantly decreased(p<0.01).Compared with the different concentrations of ZEA groups,the relative viability of T cells in the corresponding concentration treatment groups were significant or extremely significant decreased(p<0.05 or p<0.01).Compared with different concentrations of DON groups,the relative viability of T cells in the 10?M ZEA+0.5?M DON combination treatment group was significantly decreased(p<0.05).The other combined treatment groups did not change significantly(p>0.05).Transmission electron microscopy showed that after 48 hours of Con A stimulation,the microvilli on the surface of T cell membrane increased,and the number of mitochondria increased.After adding 20?M ZEA and 1 ?M DON,the microvilli on the membrane surface disappeared.Moreover,the nuclear heterochromatin disappeared,the nuclear heterochromatin,mitochondrial swelling increased.After adding 20?M ZEA+1 ?M DON,the cell membrane was no longer intact.The vacuolization increased,and mitochondria and cell morphological damage were aggravated.After scanning the electron microscopy,it showed that after 48 hours of Con A stimulation,the volume of T cells.increased and the microvilli on the surface of the cell membrane increased.After adding 20?M ZEA and 1?M DON,the cell membrane was not completely destroyed.After adding 20?M ZEA+1 ?M DON,the cell membrane damage is intensified.The results showed that ZEA and DON could inhibit the cell viability of T cells and destroy the ultrastructure of cells,and the combined action of toxins had a synergistic effect.2.Effects of ZEA,DON and their combined exposure on T lymphocyte activation and cytotoxicity in micePrimary mouse T lymphocytes were used as materials.The Control group(excluding Con A),Con A(5 mg/L)group,Con A(5 mg/L)+ZEA(10,20,and 40?M)group,Con A(5 mg/L)+DON(0.5,1 and 2?M)group,Con A(5 mg/L)+(ZEA+DON)(10?M ZEA+0.5?M DON,20?M ZEA+1?M DON,40?M ZEA+2?M DON)group were established.After 24 h of exposure,the expression of CD25 and ICOS was detected by flow cytometry,after 48 h of exposure,the secretion of cytokines IL-2 and TNF-a was detected by CBA.The total amount of perforin(PFP)and granzyme A(GZMA)was detected by ELISA.The results showed that the expression of CD25 and ICOS,the secretion of IL-2 and TNF-a,and the total amount of PFP and GZMA were significantly increased in the Con A group compared with the control group after 24 h or 48 h(p<0.01).Compared with Con A group,CD25 and ICOS expression rates,IL-2 and TNF-a secretion,the total PFP and GZMA synthesis were significantly or significantly decreased(p<0.05 or p<0.01).After the treatment of T lymphocytes with different concentrations of ZEA and DON,the result showed a dose-effect relationship.Compared with the Con A group,CD25 and ICOS expression rates,IL-2 and TNF-a secretion,total PFP and GZMA synthesis were significantly or highly significantly decreased(p<0.05 or p<0.01)in different concentrations of ZEA and DON.Compared with different concentrations of ZEA and DON groups,CD25 and ICOS expression rates,IL-2 and TNF-a secretion,the total PFP and GZMA synthesis were significantly or significantly decreased(p<0.05 or p<0.01).The results indicated that ZEA and DON could inhibit the expression of CD25,ICOS,PFP and GZMA in T lymphocytes.The secretion of IL-2 and TNF-a could affect the autocrine effect,co-stimulatory effect,the cytotoxic function of the granzyme pathway of T lymphocytes.The combined effect of toxins,has varying degrees of synergistic or antagonistic effects.3.Effects of ZEA,DON and their combined exposure on T lymphocyte apoptosis rate,Fas/FasL pathway,mitochondrial pathway and MAPK pathway-related proteins in micePrimary mouse T lymphocytes were used as materials.The control group(excluding Con A),Con A(5 mg/L)group,Con A(5 mg/L)+ZEA(10,20,and 40?M)group,Con A(5 mg/L)+DON(0.5,1 and 2?M)group,Con A+ZEA+DON(5 mg/L ConA+10?M ZEA+0.5?M DON,5 mg/L ConA+20?M ZEA+1?M DON,5 mg/L ConA+40?M ZEA+2?M DON)group were established.After 24 h of exposure,T cell apoptosis rate was detected by flow cytometry.The control group(excluding Con A),Con A group,Con A+ZEA co-treatment group(Con A+10,20 or 40?M ZEA),Con A+DON co-treatment group(ConA+0.5,1 or 2?M DON),Con A+ZEA+DON co-processing group(ConA+20?M ZEA+1?M DON)were established.After 24 h of exposure,Fas/FasL pathway-related proteins(Fas,FasL,Cleved Caspase-8),mitochondrial pathway-associated proteins(Cleved Caspase-9,Cleved Caspase-3),and Bax/Bcl-2 ratio,phosphorylation levels of proteins associated with the MAPK pathway(ERK1/2,JNK1/2,p38)were detected by Western Blot.The results showed that the apoptosis rate of Con A group increased but the difference was not significant compared with the control group after 24 h exposure.Compared with Con A group,the apoptosis rate of ZEA and DON groups was significantly or extremely significantly increased(p<0.05 or p<0.01),which showed a dose-effect relationship.Compared with the Con A group,the apoptotic rate of the different concentrations of ZEA and DON treatment group was significantly increased(p<0.01).Compared with the different concentrations of ZEA group,the apoptosis rate of the corresponding combination treatment group was significantly increased(p<0.01).Compared with different concentrations of DON group,the apoptosis rate of the corresponding combined treatment group increased,but the difference was not significant(p>0.05).Compared with the Con A group,the expression levels of Fas,FasL,and Cleved Caspase-8 were significantly or significantly increased(p<0.05 or p<0.01).The mitochondrial pathway Bax/Bc1-2 ratio,the expression level of Cleved Caspase-9,-3 were significantly or significantly increased(p<0.05 or p<0.01).The phosphorylation levels of MAPK pathway-related proteins MEK1/2,ERK1/2,and JNK1/2 were significantly or significantly increased(p<0.05 or p<0.01).The results showed that ZEA and DON could induce T cell apoptosis.The combination of toxins had a synergistic effect.Besides,the ZEA and DON alone or combined exposure activated T cell Fas/FasL pathway,mitochondrial pathway and MAPK pathway.To explore the regulation of JNK in the mitochondrial pathway,20?M ZEA and 1?M DON were used alone or in combination with 10?M SP600125 for Con A-stimulated T lymphocytes for 24 h.Western Blot was used to detect JNK 1/2 phosphorylation,and Bax and Cleved Caspase-9,-3 protein expression levels.The results showed that,compared with the ZEA group,the phosphorylation level of JNK1/2,the protein expression of Bax,Cleved Caspase-9,and Cleved Caspase-3 were significantly decreased in the combination of ZEA and SP600125(p<0.05).Compared with the DON group,the phosphorylation level of JNK1/2 and the protein expression of Bax and Cleved Caspase-9 were significantly decreased in the combination of DON and SP600125(p<0.05).The results indicate that JNK is involved in the regulation of the mitochondrial pathway.In conclusion,ZEA and DON can cause the decrease of T lymphocyte activity and ultrastructural damage,inhibit the expression of T cell costimulatory molecules and cytokines,as well as inhibit the activation and killing efficacy of T cells.ZEA and DON can induce T cell apoptosis,ZEA and DON can participate in the regulation of T lymphocyte apoptosis through Fas/FasL pathway and mitochondrial pathway,while JNK participates in the regulation of mitochondrial pathway.The combined exposure of ZEA and DON has synergistic effect.