Role Of Autophagy In Zinc Oxide Nanoparticles (ZnO NPs)-induced Apoptosis Of Mice GC-1 Spg Cells

Objective:Zinc oxide nanoparticles(ZnO NPs)have been extensively used in various industries and reported to inhibit spermatogenesis and induce damage of liver,heart and spleen.During the process of spermatogenesis,spermatogonium can continuously undergo mitosis to form primary spermatocyte,then through meiosis to form spermatid,and finally to form spermatozoon,suggesting that spermatogonium play an essential role in spermatogenesis.The aim of this study is to observe the effect of ZnO NPs on GC-1 spg cells and to investigate the potential role of autophagy in ZnO NPs-induced apoptosis of GC-1 spg cells.Methods:MTT was used to observe the effect of ZnO NPs on viability of mouse GC-1 spg cells.Western blot and Annexin V-FITC/PI double staining were respectively utilized to survey the impact of ZnO NPs on apoptosis of mouse GC-1 spg cells.Western blot and Transmission electron microscopy(TEM)were exploited to investigate the impact of ZnO NPs on autophagy of mouse GC-1 spg cells.Oxidative stress of mouse GC-1 spg cells was detected by taking advantage of the the active oxygen reagents test kits.After the inhibiton of oxidative stress with NAC,western blot was used to test the effect of ZnO NPs on apoptosis and autophagy of mouse GC-1 spg cells.In addition,Annexin V-FITC/PI double staining and TEM were separately utilized for detection of impact on apoptosis and autophagy of mouse GC-1 spg cells treated with ZnO NPs.After inhibiting autophagy,the effect of ZnO NPs on apoptosis of mouse GC-1 spg cells were tested by western blot and Annexin V-FITC/PI double staining.Results:The cells were respectively treated with 0,1,2,4 ?g/ml ZnO NPs for 24 h.The results showed that ZnO NPs inhibited viability of GC-1 spg cells in a dose-dependent manner(P<0.05).The expression of cleaved Caspase-8,cleavedCaspase-3 and Bax proteins distinctly increased and the expression of Bcl-2 protein decreased in GC-1 spg cells,suggesting that ZnO NPs might induce apoptosis of mouse GC-1 spg cells.Annexin V and PI double staining further identified the induction of apoptosis by ZnO NPs.The results of reactive oxygen species assay proved that ZnO NPs induced oxidative stress in GC-1 spg cells.Western blot's results revealed that ZnO NPs significantly increased the content of LC3 and the ratio of LC3-II to LC3-I,as well as the protein levels of Atg 5 and Beclin 1.The effect of ZnO NPs on autophagy was further investigated by TEM,which indicated that ZnO NPs might induce autophagy of mouse GC-1 spg cells.However,the induction of apoptosis and the inhibition of cell viability by ZnO NPs could be significantly rescued by the inhibition of oxidative stress by N-Acetyl-L-cysteine(NAC).These results indicated that oxidative stress might be involved in ZnO NPs-induced apoptosis of mouse GC-1 spg cells.Similarly,the inhibition of oxidative stress by NAC was shown to rescue the induction of autophagy,suggesting that oxidative stress might also be involved in ZnO NPs-induced autophagy of mouse GC-1 spg cells.With treatment of autophagy inhibitor 3-Methyladenine(3-MA),the inhibition of cell viability and induction of apoptosis on GC-1 spg cells were visibly reduced.Conclusions:These results showed that oxidative stress was involved in apoptosis and autophagy of mouse GC-1 spg cells induced by ZnO NPs.Inhibition of autophagy could significantly reduce ZnO NPs-induced apoptpsis of the cells.