The Role Of Autophagy In The Distribution Of SC Cell Cycle Induced By ZEA

Zearalenone(ZEA)is a kind of mycotoxin produced by sickle bacteria,which widely exists in animal feed and human food.It has reproductive toxicity,immune toxicity and great potential risk to the production performance of animal and human health.In recent years,the male reproductive toxicity of ZEA has attracted more and more attention,but so far the ZEA male reproductive toxicity and its toxicity mechanism are poorly understood.For males,the number of Sertoli cells determines the size and potency of adult testicles and play an irreplaceable role in spermatogenesis.In order to reveal the mechanism of ZEA male reproductive toxicity,this experiment used different concentrations of ZEA to treat the SC cells in rats and established an in vitro toxicity test model.With the technique of transmission electron microscopy,flow cytometry,Western Blot and so on,the effect of ZEA on SC cell cycle distribution,the role of autophagy and autophagy related pathways in ZEA induced SC cell cycle distribution have been studied.1.Effect of ZEA on the distribution of SC cell cycle.The primary SC cells of rat were used as materials and the effect of ZEA on the living rate of SC cells was detected by cck-8 method.After selecting the appropriate ZEA concentration,the SC cells were treated with different concentrations(0,0.1,1,10,20,30 ?mol/L).The cell cycle distribution and cell cycle related proteins were determined by flow cytometry,immunofluorescence technique and Western Blot.The results showed that,compared with the control group,the SC cell viability of each infected group was significantly or extreme significantly inhibited with the increase of ZEA concentration.(P<0.05 or P<0.01).When the ZEA concentration was higher than 10?mol/L,the proportion of G0/G1 phase was significantly decreased(P<0.05)while the proportion of G2/M period was extreme significantly increased(P<0.01).For 10 ?mol/L ZEA and the above dose group,p-Cdc2/Cdc2 was significantly up-regulated.In contrast,the protein expression levels of CyclinBl,Cdc25B and p-Histone H3 were down-regulated(P<0.01).The results of the marker protein of mitosis p-Histone H3 expression detected by Immunofluorescence were consistent with Western blot method.The results showed that ZEA could inhibit the proliferation of SC cells in vitro and the cell cycle was arrested in G2 phase,which affected the expression of CyclinB1.The phosphatase kinase activity of p-Cdc2/Cdc2 and Cdc25B indicated that ZEA inhibits the SC cell proliferation and has close relationship with G2/M arrest.2.Effects of ZEA on autophagy of SC cells.After treating the SC cells of rats with different concentrations of ZEA(0,0.1?1?10?20?30 ?mol/L)for 24 hours,the effects of ZEA on the autophagy level of SC cells were detected by immunofluorescence,transmission electron microscopy,Western blot and other techniques.The results showed that 20 ?mol/L ZEA treated SC cells could lead to the formation of autophagy in cytoplasm.With the increasing of ZEA concentration,the number of LC3 aggregation points in SC cells showed an increasing trend,and each group was significantly different from the control group(P<0.01).The late autophagic lysosomes were observed by single Dan sulfonamide(MDC)stain,which found that the number of acid vesicles in each infected group was significantly higher than that in the control group(P<0.01)and was dose-dependent.There were some significant or extremely significant difference between the infected groups and the control group.Compared with the control group,the expression of p62 protein decreased significantly and the expression level of Atg5 was gradually decreased when the concentration of ZEA reached 10 ?mol/L.(P<0.05 or P<0.01).The expression of autophagy associated protein LC3II,beclin-1 and Atg7 was significantly increased in each infected group.In order to further detect the state of autophagy flow,the test was conducted to stimulate the cells by using the late autophagic inhibitor,bafilomycin A1(Baf A1)which showed that the expression quantity of LC3 protein in the group of Bafilomycin A1+20 ?mol/L was significantly increased(P<0.01)compared with that in the treatment group of the individual Bafilomycin A1 treatment group(P<0.01).However,the degradation of ubiquitin protein p62 was significantly lower than that of ZEA alone(P<0.01).In conclusion,ZEA produced cytotoxicity to SC cells and induced autophagy in SC cells.3.The role of PI3K/Akt/mTOR signaling pathway in ZEA induced SC autophagy.After treating the SC cells of rats with different concentrations of ZEA(0,0.1?1?10?20?30 ?mol/L)for 24 hours,the phosphorylation levels of key proteins in PI3K/Akt/mTOR pathway were detected by immunoblotting.To further explore the role of PI3K/Akt/m TOR pathway in the autophagy of ZEA to SC cells,the PI3K pathway inhibitor LY294002(10?mol/L)and 20 pmol/L ZEA were added separately or combined with each other for 24 h,after which,the changes of Akt,m TOR protein phosphorylation level,LC3 content and the clustering of LC3 in cells were detected by western blot and immunofluorescence assay.Results showed that with the rising of the concentrations of ZEA,the phosphorylation level of the PI3K,Akt,mTOR and P70S6K protein gradually declined.Compared with the control group,some significant or extremely significant differences existed in the phosphorylation levels of each protein.Compared with ZEA group,the levels of Akt and m TOR protein phosphorylation of LY294002 and ZEA group were significantly decreased(P<0.01),while the expression level of LC3II was significantly increased(P<0.01).The number of LC3II and the fluorescence intensity increased significantly.In conclusion,ZEA could activate the PI3K/AKT/mTOR signaling pathway,which negatively regulated the autophagy of SC cells in ZEA.4.The role of the activation of ZEA induced autophagy and PI3K/AKT/mTOR signaling pathway in SC cell cycle distribution.To investigate the role of autophagy in ZEA on the cycle of SC cells and the specific mechanism of ZEA on the cycle distribution of SC cells after the treatment of SC cells by ZEA(20 ?mol/L),autophagy inhibitor-chloroquine(CQ),promoter-Rapamycin(RAPA)and the autophagy correlation pathway PI3K pathway inhibitor(LY294002),the cell cycle distribution and periodic correlation protein expression were detected by flow cytometry,immunofluorescence technique and Western blot.The results showed that compared with ZEA group the proportion of G2/M in CQ+ ZEA cell cycle was decreased(P<0.05)while the proportion of G2/M in the cell cycle of RAPA+ZEA group was increased(P<0.05).The expression of CyclinBl,Cdc2/p-Cdc2 and other proteins in CQ+ ZEA group decreased(P<0.05).However,that in the RAPA+ ZEA group significantly increased(P<0.05).Compared with ZEA group,the proportion of cell G2/M arrested in LY294002+ZEA group decreased(P<0.05).The activity of Cdc2/p-Cdc2 in cell cycle was enhanced(P<0.05).In conclusion,after activating and down-regulating the PI3K/Akt/mTOR signaling pathway of autophagy,the arresting of ZEA induced SC G2/M phase could be partially reversed.In conclusion,ZEA can inhibit cell proliferation,affect the distribution of SC cell cycle,and induce the G2 arrest.In a certain concentration range,ZEA can induce the autophagy by activating and down-regulating the PI3K/AKT/mTOR signaling pathway,thereby partially reverse the arresting of ZEA induced G2/M phase in SC cells.