Study On The Role Of PI3K/AKT Signaling Pathway In TiO₂ NPs-induced Cell Cycle Arrest And Apoptosis Of Mouse Spermatogonial Cells

Objective:Titanium dioxide nanoparticles（TiO₂NPs）could induce male reproductive toxicity and its mechanisms have gained much attention in academia.However,there is a lack of research on whether TiO₂NPs induce germ cell cycle arrest and its related mechanisms.In this study,taking cell cycle arrest as the entry point,we analyzed the effects of TiO₂NPs on the cell cycle and apoptosis of mouse spermatogonia（GC-1）,and investigated the role of PI3K/AKT signaling pathway in GC-1 cell cycle arrest and apoptosis induced by TiO₂NPs.This research is designed to provide novel insights to explore the molecular mechanisms of male reproductive toxicity induced by TiO₂NPs,and also has great theoretical value for the safety evaluation criteria for TiO₂NPs.Methods:Firstly,after GC-1 cells were treated with TiO₂NPs（0,25,50,75 and 100μg/m L）for 12,24 and48 h,the cell activity was analyzed by CCK-8 experiment to select the optimal intervention time.Then,to investigate the effects of TiO₂NPs on cell growth,cell cycle,apoptosis and PI3K/AKT signaling pathway,we treated GC-1 cells with TiO₂NPs for 24 h.Here,we observed cell morphology by inverted phase contrast microscope.Flow cytometry was used to analyzed the uptake of TiO₂NPs by cells,cell cycle and apoptosis.Western Blot was utilized to detect the expression of cell cycle-related proteins and PI3K/AKT signaling pathway proteins.Finally,we respectively treated GC-1 cells with PI3K agonist IGF-1 and PI3K inhibitor LY294002 to further verify the regulation of PI3K/AKT signaling pathway in cell cycle arrest and apoptosis induced by TiO₂NPs.Results:1.Cell activity and morphology The results of CCK-8 assay found that,compared with that of the control group,the cell viability of GC-1 cells treated with TiO₂NPs（25,50,75 and 100μg/m L）for 12 h showed a slight downward trend,but there was no significant difference（P>0.05）.After treatment for 24 h and 48 h,the cell viability of TiO₂NPs groups was significantly decreased compared with that of the control group（P<0.05）,showing a dose-dependent manner.In addition,the cell viability of GC-1 cells treated with 25,50,75 and 100μg/m L TiO₂NPs for 24 h and 48 h was lower than that treated for 12 h,and the cell viability of GC-1 cells treated with TiO₂NPs for 48 h was lower than that treated for 24 h.These results suggested that there was a dose-dependent manner and a time-dependent manner.After GC-1 cells were treated with TiO₂NPs for 24 h,the results of cell morphology showed that the high dose of TiO₂NPs could severely damage the morphology of GC-1 cells and decrease the cell number.In addition,flow cytometry found there was a dose-dependent manner for the uptake of nanoparticles.2.Cell cycle related indicators After GC-1 cells were treated with TiO₂NPs for 24 h,the results of flow cytometry showed that compared with that of the control group,TiO₂NPs could induce GC-1 cell cycle arrest in G0/G1 phase in a dose-dependent manner.Western Blot results indicated that TiO₂NPs significantly decreased the expression of cycle-related proteins CDK2,CDK4,Cyclin E1 and Cyclin D1,and increased the expression of p21 and p53 proteins,showing a dose-dependent manner（P<0.05）.3.Apoptosis related indicators After cells were treated with TiO₂NPs for 24 h,the apoptosis rates of GC-1 cells were detected by flow cytometry.We found that,compared with that of the control group,the apoptosis rates were significantly increased in 50,75 and 100μg/m L TiO₂NPs treatment groups（P<0.01）.4.The role of PI3K/AKT signaling pathway After GC-1 cells were treated with TiO₂NPs at different doses for 24 h,Western Blot results showed that TiO₂NPs significantly decreased the expression levels of p-PI3K,p-AKT,p-m TOR and p-P70S6K proteins（P<0.05）,but had no significant influence on the expression of PI3K,AKT and m TOR proteins.The inhibitory effect of TiO₂NPs on PI3K/AKT signaling pathway was further assessed by the combination of PI3K agonist IGF-1 and PI3K inhibitor LY294002.The CCK-8 assay results showed that,compared with that of the TiO₂NPs group,the cell viability was significantly increased in the IGF-1+TiO₂NPs group（P<0.05）,but showed a decreasing trend in cell viability（P<0.05）in the LY294002+TiO₂NPs group.Western Blot results showed that,the protein expression levels of p-AKT and p-m TOR in the TiO₂NPs+IGF-1 group were significantly increased compared with that of the TiO₂NPs group,while the protein expression levels in the TiO₂NPs+LY294002group were significantly decreased than those in the TiO₂NPs group.The results of flow cytometry showed that,compared with those of the TiO₂NPs group,the cell cycle arrest and apoptosis were significantly alleviated in the TiO₂NPs+IGF-1 group,while they were further aggravated in the TiO₂NPs+LY294002group.Moreover,Western Blot results found that compared with those of the TiO₂NPs group,the expression of CDK2,CDK4,Cyclin E1 and Cyclin D1 were up-regulated and the expression of P21 and p53proteins were down-regulated in the TiO₂NPs+IGF-1 group.On the contrary,the expressions of CDK2,CDK4,Cyclin E1 and Cyclin D1 were significantly decreased in TiO₂NPs+LY294002 group,while the expressions of P21 and p53 proteins were significantly increased.Conclusion:TiO₂NPs could decrease the activity of GC-1 cells and induce cell cycle arrest in G0/G1phase by inhibiting the PI3K/AKT signaling pathway,thereby resulting in cell apoptosis,and cell cycle arrest mediated by PI3K/AKT signaling pathway might be one of the important mechanisms of TiO₂NPs-induced apoptosis of GC-1 cells.