The Molecular Mechanisms Of ZEA Induced-cell Apoptosis And Destroyed Cytoskeleton In TM4 Sertoli Cells

Zearalenone(ZEA),a non-steroidal estrogen mycotoxin produced by several species of Fusarium fungi,can be metabolized into many other derivatives by microorganisms,plants,animals and humans.Several studies have suggested that the exposure to ZEA can reduce the number of germ cells,alter the morphology of testis,cause testicular cells to differentiate abnormally and affect fertility.ZEA can disrupt puberty and the estrous cycle,and affect early pregnancy events including fertilization,embryo development,and embryo implantation.ZEA and its derivatives can not only stimulate the cell growth but also inhibit the cell viability and cause cell death including apoptosis and necrosis.Sertoli cells around germ cells are considered a barrier that protects spermatogenesis from harmful influences.One of the major roles of Sertoli cells is to establish the blood-testis barrier(BTB)which provides an exclusive and stable environment for germ cell development.Thus,any agent that can impair the stability or function of Sertoli cells may profoundly affect spermatogenesis.The detailed molecular mechanisms of ZEA-induced cytotoxicity have not yet been completely detected.Thus,the purpose of this study is to reveal the molecular mechanisms of ZEA-induced cytotoxicity in Sertoli cells.In current study,the TM4 cell line was selected as the experimental subject for exploring the reproductive toxicity of ZEA.1.Effects of ZEA on the cell proliferation and cell cycle distribution and associated proteins in TM4 cellsThe cell proliferation was analyzed by the xCELLigence system in real time and cell viability was analyzed using cell counting kit-8(CCK-8)assay,the cell cycle distribution was analyzed using flow cytometry,the expressions of cell cycle regulatory proteins were detected by Western Blot.The data has shown that after using different concentrations of ZEA(1,10,20,40,60,80 and 100 μM ZEA)for 24 h,compared with control group,the cell viability was decreased;the cell numbers in the cycle was inhibited in the G0/G1 phase were decreased(P<0.05)and the cell numbers in G2/M phase were increased(P<0.05 or P<0.01)in a dose-dependent manner;the expressions of cell cycle associated proteins including CyclinB1,CyclinD1,CDK2 and CDK4 were decreased(P<0.05 or P<0.01)in a dose-dependent manner.Taken together,ZEA could inhibit the growth of TM4 cells through influencing the cell cycle distribution and the expressions of cell cycle regulatory proteins.2.ZEA influenced cell cycle distribution and induced apoptosis though ROS generation in TM4 cellsIn order to detect the the molecular mechanisms of ZEA-induced cell cycle arrest and apoptosis in TM4 cells,the apoptotic parameters were assessed by flow cytometry,Western Blot and transmission electron microscopy(TEM),the effects of ZEA on levels of Superoxide dismutase(SOD),Malondialdehyde(MDA)and reactive oxygen species(ROS)were analyzed by using related assay kit.The data has shown that after using different concentrations of ZEA(1,10,20,40,60,80 and 100 μM ZEA)for 24 h,compared with control group,the release of LDH was increased;the rate of apoptosis was also increased(P<0.05 or P<0.01);the ration of active Capase-3 was increased(P<0.05 or P<0.01);the rate of Bax/Bcl-2,and the expressions of Cleaved-caspase-3 and Cleaved-caspase-9 were increased(P<0.05 or P<0.01)the intracellular levels of SOD,MDA and ROS were increased(P<0.05 or P<0.01)after treatment with ZEA.After co-treaed with NAC the accumulation of cells in the G2/M phase was decreased,and the rate of apoptosis was decreased significantly compared with cells treated with ZEA alone.These data suggested that ZEA induced cell cycle arrest and cell apoptosis through oxidative stress signal pathway.3.ER stress and ATP/AMPK pathways were involved in ROS-mediated cell cycle arrest and apoptosis induced by ZEA in TM4 cellsIn order to further explore the mechanisms of ZEA induced cell cycle arrest and apoptosis,the effects of ZEA on the ER stress,distribution of cell cycle and apoptosis were analyzed.ER stress associated proteins and AMPK pathway family proteins were analyzed by using Western Blot;the level of intracellular ATP was analyzed by using ATP assay.The data has indicated that after exposure to ZEA,the level of intracelluar Ca2+was increased(P<0.05);ER stress associated proteins,such as,BiP,PERK,ATF6 and CHOP were increased(P<0.05 or P<0.01);the rate of p-AMPKα/AMPKα was increased(P<0.05 or P<0.01);the level of ATP was gradually decreased(P<0.05 or P<0.01)in a dose manner;using NAC could alleviate the level of ER stress;the accumulation of cells in the G2/M phase and the rate of apoptosis were decreased after co-treated with 4-PBA and ZEA;co-treatmnet with AMPK inhibitor dorsomorphin could alleviate the accumulation of cells in the G2/M phase and decrease the rate of apoptosis.Taken together,this study revealed that ZEA induced TM4 cell cycle G2/M arrest and cell apoptosis through ROS-and ER-stress and the ATP/AMPK pathway.4.The role of autophagy and oxidative stress in ZEA destroyed the cytoskeletal structure in TM4 cellsThe aim of this study is to detect the role of autophagy and oxidative stress in ZEA destroyed the cytoskeletal structure in TM4 cells.The changes by ZEA on the cytoskeletal structure,EGFP-LC3 and mRFP-LC3 colocalization,and formation of LC3 puncta were detected by confocal immunohistochemistry.Using Western Blot analyzed the cytoskeleton and autophagy regulatory proteins.The autophagic vacuoles were observed by TEM.The detection of autophagic vacuoles by using MDC.The data indicated that ZEA can damage the cytoskeletal structure and reduce the expressions of vimentin and N-cadherin(P<0.05 or P<0.01);compared to the control group LC3-positive puncta was remarkably increased after treament with ZEA;ZEA stimulated the formation of lysosomes as well as autophagosomes in TM4 cells;LC3II were increased(P<0.05 or P<0.01);promoting the level of autophagy could aggravate the damage the cytoskeletal structure,and inhibit the level of autophagy could alleviate the damage the cytoskeletal structure;NAC co-treatment could alleviate the disruption of the cytoskeletal structure and increase the expressions of Vimentin and N-cadherin.These results suggested that ZEA can destroy the cytoskeletal structure,and autophagy and oxidative stress were involved in the process of ZEA-destroyed cytoskeletal structure in TM4 cells.5-The role of PI3K-AKT-mTOR and MAPK pathways in ZEA induced autophagy in TM4 cellsIn order to investigate the role of PI3K-AKT-mTOR and MAPK pathways in ZEA-induced autophagy in TM4 cells.Western Blot was used to analyze the expressions of PI3K-AKT-mTOR,MAPK and autophagy associated proteins.The results suggested that compared with the control group,the ratios of p-PI3K/PI3K,p-AKT/AKT,P-mTOR/mTOR and MAPK family proteins were decreased(P<0.05 or P<0.01)after the treatment with ZEA;after co-treated with LY294002 and ZEA,compared with the cells exposed to ZEA alone group,the proteine expressions of LC3.II and Beclin-1 were significantly decreased(P<0.05 or P<0.01),and the expression of the P62 protein was significantly increased(P<0.05);the level of autophagy was increased and the p-mTOR/mTOR was decreased(P<0.05)after treatment with U0126 and siRNA of ERK2;the ration of p-ERX1/2/ERK1/2 was significantly increased(P<0.05)in cells co-treated with 4-PBA and ZEA,compared with cells treated with ZEA alone.These data suggested that the ER stress stimulated autophagy via inhibiting the ERK signal way to suppress the phosphorylation of mTOR.