Role And Mechanism Of Autophagy In Zinc Oxide Nanoparticles(Zno Nps)-Induced Apoptosis Of Mice Leydig Cells

Objectives:Zinc oxide nanoparticles(ZnO NPs)has been widely used in industry and reported to have a toxic effect on the male reproductive system besides hepatotoxicity and neurotoxicity,but the precise mechanism is yet to be elucidated.The aim of this study is to observe the effect of ZnO NPs on the mouse testis tissue,and try to investigate the role and mechanism of autophagy in ZnO NPs-induced apoptosis of Leydig cells.Methods:Hematoxylin and eosin(HE)staining was used to observe testicular and epididymal toxicology.Western blot utilized to investigate the effect of ZNO NPs on apoptosis and autophagy in mouse testis tissue.The active oxygen reagents test kits was taken advantage of detecting whether ZNO NPs inducing oxidative stress of mouse testis tissue and Leydig cells.ELISA assay was used to test the effect of ZNO NPs on the testosterone content of mouse serum.MTT was utilized to observe the effect of ZNO NPs and H2O2 on viability of mouse Leydig cells.Western blot and annexin V-FITC/PI double staining were exploited to survey the impact of ZNO NPs on the apoptosis of mouse Leydig cells.Western blot and Transmission electron microscopy(TEM)were used to detect the impact of ZNO NPs and H2O2 on autophagy of mouse Leydig cells.Results:Male mice were received the gavages at the dosages of 0,100,200,400 mg ZnO NPs /kg/day for 28 days.Compared with the control group,ZnO NPs had no effect on the testis and epididymal coefficient in mice(P>0.05).The testes of vehicle-treated mice showed normal seminiferous tubules lined by both spermatogenic cells and sertoli cells and an absence of germ cells detached in the tubular lumen.In the 100 mg ZnO NPs /kg/day exposure group,there was no significant morphologic changeswere observed of the seminiferous epithelium.However,the seminiferous tubule illustrated mild disorganized histo-architecture in the 200 mg ZnO NPs/kg/day group.In the 400 mg ZnO NPs/kg/day exposure group,seminiferous tubules exhibited disintegration of seminiferous epithelium,germ cell depletion and reduction in the round sperms.There was a significant decrease in sperm density following exposure to either 100,200 or 400 mg ZnO NPs/kg/day in mice compared with the vehicle control exposure group,which indicated that ZnO NPs could inhibit spermatogenesis.The contents of apoptosis-related proteins cleaved Caspase-8,cleaved Caspase-3 and Bax and the protein level of Bcl-2 were increased and decreased in the ZnO NPstreated testis tissue,respectively,which indicated that ZnO NPs could induce apoptosis of mouse testis tissue.Meanwhile,ZnO NPs significantly increased both LC3-II and the ratio of LC3-II/LC3-I,and the contents of autophagy proteins Atg5 and Beclin1.These results showed that ZnO NPs could induce autophagy of the testis tissue.ZnO NPs significantly increased MDA level in the testis in a dose-dependent manner;whereas the content of GSH,activities of antioxidant enzymes SOD and GSH-PX were dramatically decreased in the ZnO NPs-treated cells,respectively.These results indicated that ZnO NPs could induce oxidative stress in mouse testis tissue.ZnO NPs inhibited testosterone output in a dose-dependent manner,which indicated that ZnO NPs might affect the function of Leydig cells.In vitro studies showed that ZnO NPs markedly inhibited viability in a dosedependent manner(P<0.05)and increased the contents of apoptosis-related proteins cleaved Caspase-8,cleaved Caspase-3 and Bax and decreased the protein level of Bcl-2,respectively,which indicated that ZnO NPs might induce apoptosis of mouse Leydig TM3 cells.AnnexinV-FITC and PI double staining further identified ZnO NPs could induce apoptosis of the cells.A significant increase in the oxidative stress responses was induced in the ZnO NPs-treated cells.H2O2 also inhibited viability and induced apoptosis of the cells;while inhibition of oxidative stress by N-Acetyl-L-cysteine(NAC)could rescue the inhibition of cell viability and induction of apoptosis by ZnO NPs.In summary,oxidative stress might be involved in ZnO NPs-induced apoptosis of mouse Leydig TM3 cells.Meanwhile,ZnO NPs and H2O2 were shown to induce autophagy of mouse Leydig TM3 cells;while inhibition of oxidative stress could also inhibit autophagy,which indicated that oxidative stress was also involved in ZnO NPs-induced autophagy of the cells.However,inhibition of autophagy further aggravated the inhibition of cell viability and increased apoptosis induced by ZnO NPs.Conclusion:These results showed that oxidative stress was involved in ZnO NPs-induced apoptosis and autophagy of mouse testis tissue and mouse Leydig TM3 cells;and inhibition of autophagy increased ZnO NPs-induced apoptosis of the cells.