Study On The Combined Toxicity Of ZEA And Don On Sertoli Cells In Piglets And The Protective Effect Of NAC

Zearalenone(ZEA)and deoxynivalenol(DON)are two mycotoxins with high detection rate and quantity in agricultural products and feed,and often coexist in contaminated diets.Studies have shown that both ZEA and DON can cause reproductive toxicity in male animals,reduce the number of germ cells in the testis of male animals,reduce the concentration of testosterone in serum,and affect fertility.Therefore,we take the Sertoli cells of piglets as the test object to study the combined toxicity of ZEA and DON on Sertoli cells of piglets,and to evaluate the alleviative effect of N-acetyl-l-cystein(NAC)on the combined toxicity of ZEA and DON.The Sertoli cells of piglets were treated with ZEA(10?g/m L),DON(0.2?g/m L)and NAC(0.6mg/m L)for 24 hours.CCK-8method was used to determine the optimal toxin concentration and protective agent concentration.The morphological and ultrastructural changes of Sertoli cells were observed by inverted microscope and transmission electron microscope;The expression and distribution of mitochondrial membrane potential and tight junction protein were detected by laser confocal microscopy;the apoptosis rate,mitochondrial membrane potential,ROS expression and cell cycle were detected by flow cytometry;q RT-PCR was used to determine the m RNA expression of Bcl-2,Bax,Bid,Caspase-3 and Caspase-9.Western-blot was used to determine the expression levels of apoptosis related proteins Bcl-2,Bax,Bid,Caspase-3 and Caspase-9,and to evaluate the combined toxic effect of ZEA and DON on Sertoli cells and the protective effect of NAC.These results showed that:1.10?g/m L ZEA and 0.2?g/m L DON could significantly reduce the cell viability(P<0.05),10?g/m L ZEA combined with 0.2?g/ml DON could significantly reduce the testicular sertoli cell viability(P<0.01).The results of inverted microscope showed that the combination of ZEA and DON could cause the growth of Sertoli cells to become worse,the cells to arrange sparsely,and the cells to be suspended or not tightly attached to the wall to increase.The results of electron microscopy showed that the combination of ZEA and DON resulted in obvious cell shrinkage,cell membrane damage,increase of autophagic vesicles in the cytoplasm,rupture of endoplasmic reticulum and mitochondrial cristae,fuzzy border of nuclear membrane attached by nuclear chromatin,deepening and concentration of nuclear chromatin.The results of immunofluorescence showed that ZEA and DON could significantly affect the expression and distribution of ZO-1,and reduce the expression level of ZO-1.2.ZEA and DON can cause oxidative damage to Sertoli cells and disturb cell cycle.ZEA and DON significantly increased ROS expression(P<0.01),SOD,CAT,GSH-Px activity(P<0.05 or P<0.01)and MDA content(P<0.05 or P<0.01)in Sertoli cells of testis.ZEA and DON significantly decreased mitochondrial membrane potential(P<0.01)and disturbed cell cycle,resulting in G2 phase arrest.3.The results showed that ZEA and DON could significantly increase the apoptosis rate of Sertoli cells(P<0.05 or P<0.01).Moreover,ZEA and DON significantly or extremely increased the expression of Bax,Bid,Caspase-3 and Caspase-9 protein and m RNA(P<0.05 or P<0.01),and significantly or extremely decreased the expression of Bcl-2 protein and m RNA(P<0.05 or P<0.01).4.The Sertoli cells of testis were cultured with NAC,ZEA and DON.The protective effects of NAC on Sertoli cells were evaluated by cell morphology,ultrastructure,oxidation and antioxidant factors,cell cycle and apoptosis.The results showed that NAC could significantly reduce the cell damage and apoptosis induced by ZEA and DON.Compared with the combined toxin group,NAC could significantly alleviate the damage of ZEA and DON to the organelle and cell structure of Sertoli cells,significantly reduce the expression of ROS and MDA(P<0.01),and significantly or extremely increase the activity of SOD,CAT and GSH-P_X(P<0.05 or P<0.01).NAC could alleviate the cell cycle arrest caused by ZEA and DON,and significantly reduce the apoptosis rate of Sertoli cells(P<0.01).NAC can significantly or extremely significantly reduce the expression level of Bax,Bid,Caspase-3,Caspase-9 protein and m RNA(P<0.05 or P<0.01),and significantly increase the expression level of Bcl-2 protein and m RNA(P<0.05).In conclusion,ZEA combined with DON can cause oxidative damage to Sertoli cells,destroy organelles and ultrastructure,block cell cycle,and induce apoptosis.Its action pathway is related to mitochondrial mediated apoptosis pathway.The oxidative damage effect of ZEA and DON aggravates the apoptosis of cells.NAC can alleviate the damage of Sertoli cells caused by the combination of ZEA and DON.It can reduce the oxidative damage of ZEA and DON by antioxidation...