| Zearalenone(Zearalenone,ZEA)is a kind of non-steroidal mycotoxin with estrogenic effect produced by many kinds of Fusarium,which has an influence all over the world.ZEA has a wide range of toxic effects on animals.In recent years,there has been a concern about the closed relationship between ZEA and male animal reproductive capacity.However,the related mechanism is not certain.Sertoli cells play an irreplaceable role in the process of spermatogenesis.Therefore,it is important to study the toxic effects of ZEA on Sertoli cells in order to provide a theoretical basis for the prevention and control of ZEA toxicity.In this study,we used the different concentrations of ZEA to cultured rat primary Sertoli cells to establish a vitro toxicity test model.The effects of ZEA on the structure,apoptosis rate and the apoptosis related proteins of Sertoli cells were studied by transmission electron microscopy,flow cytometry and western blotting,in order to explore the protection of N-acetyl-cysteine(NAC)on the pathological caused by ZEA.1.Effects of ZEA on the activity and ultrastructure of Sertoli cells.The primary Sertoli cells were treated with different concentrations of ZEA(0,5,10,20,40pmol/L)for 24 hours,then the CCK-8 method was used to estimate cells viability.The ultrastructural damage of Sertoli cells was observed by transmission electron microscopy.The results showed that the viability of the Sertoli cell in the exposed group was markedly or significantly inhibited(P<0.05 or P<0.01)with the increase of ZEA concentration compared with the control group.The shringkage of nuclear membrance,the shrinkage and even fragmentation of the chromatin,the swelling mitochondrial of the mitochondrial,the breakage of the mitochondrial cristae and the increase of the vacullization were observed in the exposed group by transmission electron microscopy.These results indicated that ZEA has a damage effect on primary Sertoli cells in a dose-dependent manner.2.Effect of ZEA on apoptosis of Sertoli cells.The primary Sertoli cells were treated with different concentrations of ZEA(0,5,10,20?mol/L)for 24 hours.AnnexinV-FITC/P1 double fluorescent was applied to dye the Sertoli cells which was detected by flow cytometry to access apoptosis.Western blot was used to detect the release of Cyt C and AIF and the expression of Bcl-2,Bax,cleaved-caspase-9 and cleaved-caspase-3.Flow cytometry showed that the apoptosis rate of each expose group was markedly or significantly higher than the control group(P<0.05 or P<0.01).Western blot analysis showed that the expression of Cyt C in mitochondria was markedly decreased in the 5 ?mol/L ZEA treatment group(P<0.05)and in the 10 or 20?mol/L ZEA treatment group significantly decreased(P<0.01),The expression of AIF in mitochondria was significantly decreased(P<0.01),The expression of Cyt C in cytoplasm was markedly increased in the 10?mol/L ZEA treatment group(P<0.05)and in the 20?mol/L ZEA treatment group significantly increased(P<0.01).The expression of AIF was significantly increased in 10 and 20p.mol/L ZEA treatment group(P<0.01).The expression of cleaved-caspase-9 and cleaved-caspase-3 were markedly or significantly incrased compared with the control group(P<0.05 or P<0.01),while the Bcl-2/Bax ration was markedly and significantly decreased(P<0.05 or P<0.01).The study indicated that ZEA could induce Sertoli cell apoptosis.The caspase-dependent and caspase-independent mitochondrial pathways were involved in this process.3.Effect of ZEA on oxidative damage of Sertoli cells.The primary Sertoli cells were treated with different concentrations of ZEA(0,5,10,20?mol/L)for 24 hours.The intracellular SOD activity and MDA content were measured by colorimetric method and the ROS level was detected by flow cytometry.The JC-1 probe was loaded,then the change of mitochondrial membrane potential(A??m)was detected by immunofluorescence and flow cytometry.The activity of SOD was markedly increased in the 10?mol/L ZEA treatment group(P<0.05)and significantly increased in the 20?mol/L ZEA treatment group(P<0.01).MDA content in the 10 and 20?mol/L ZEA group showed a significant increase(P<0.01).The level of ROS showed a markedly increased in 5?mol/L ZEA exposure group(P<0.05)and had a significantly increase in the 10,20?mol/L ZEA exposure group.The ??m showed a markedly decreased in 5?mol/L ZEA exposure group(P<0.05)and had a significantly decreased in the 10.20?mol/L ZEA exposure group.The results indicated that the oxidative stress mechanism of ZEA-induced mitochondrial damage is closely related to the toxicity of Sertoli cells.4.Protective effect of NAC on apoptosis induced by ZEA in Sertoli cells.We treated Sertoli used AnnexinV-FITC/PI double fluorescent to the apoptosis detection and the level of ROS by flow cytometry.Western blot was used to detect the expression of Bcl-2,Bax,cleaved-caspase-9 and cleaved-caspase-3.The intracellular SOD activity and the contenr of the MDA were measured by colorimetric method.The change of mitochondrial membrane potential(??m)was detected by immunofluorescence and flow cytometry through loading JC-1 probe.The results showed that the apoptotic rate,the expression of Bax,cleaved-caspase-9 and cleaved-caspase-3 in the Sertoli cells treated with 100?mol/L NAC and 20?mol/L ZEA were significantly decreased compared with 20p,mol/L ZEA(P<0.01).The activity of SOD and the content of MDA were decreased markedly in thel00?mol/L NAC and 20?mol/L ZEA combined group(P<0.05).The ??m and the expression of Bcl-2 in the combination of 100p.mol/L NAC and 20 ?mol/L ZEA group were significantly increased(P<0.01).These results indicated that NAC could reduce the oxidative damage of ZEA on Sertoli cells and inhibit the apoptosis induced by ZEA.It is concluded that ZEA can cause ultrastructural damage to the nucleus and mitochondria of Sertoli cells,induce oxidative stress and mediate the apoptosis of caspase-dependent and caspase-independent mitochondrial pathway.NAC can protect the Sertoli cells from apoptosis induced by ZEA through decreasing oxidative damage. |
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