Silicon Dioxide Nanoparticles Affect Spermatogenesis Via Inducing Cell Cycle Arrest And Apoptosis

Objectives To investigate the toxic effect and mechanism of silicon dioxide nanoparticles（SiO₂NPs）on male reproductive system in mice.Methods Forty 8-week-old C57BL/6 mice were randomly divided into control group（normal saline）and silicon dioxide nanoparticles exposure group（SiO₂NPs）,which were injected intraperitoneally for one week.After the mice were killed,serum and testicular tissue were collected.The content of silicon in serum and testis was detected by inductively coupled plasma mass spectrometry.The pathological changes of testicular tissue were analyzed by hematoxylin and eosin staining.The changes of sex hormone levels in mice were measured by enzyme-linked immunosorbent assay kit.The m RNA expression of androgen receptor and androgen biosynthesis related genes were detected by real-time fluorescence quantitative PCR.The contents of ROS and MDA and the antioxidant enzyme activity of SOD were measured by reactive oxygen species（ROS）,malondialdehyde（MDA）and superoxide dismutase（SOD）kits.The protein expression level of meiotic regulating factors in the testis was detected by Western blot.One step TUNEL apoptosis detection kit was used to detect the apoptosis of various cells in testicular tissue.In vitro cultured mouse spermatocytes（GC-2spd）were divided into control group and SiO₂NPs treatment group（1μg/m L,10μg/m L,100μg/m L）.The activity of GC-2spd cells was detected by cell counting kit-8（CCK-8）.The changes of cytoskeleton and morphology were observed by double staining with phalloidin and 4’,6-diamidino-2-phenylindole（DAPI）.The levels of ROS,MDA and SOD in cells were detected by reactive oxygen species,malondialdehyde and superoxide dismutase kits.The m RNA expression of inflammatory factors（TNF-α,IL-1β,IL-6,IL-10 and IL-17）in cells were detected by real-time fluorescent quantitative PCR.Annexin V-FITC/PI apoptosis detection kit was used to detect the apoptosis rate of GC-2spd cells.The expression levels of cell cycle checkpoint and apoptosis related proteins were detected by Western blot.Results 1 SiO₂NPs accumulate in testis and damage testicular structure.SiO₂NPs reach the testis through blood circulation,resulting in degeneration of interstitial tissue and shrinkage of seminiferous epithelium in the testis.In vitro studies showed that SiO₂NPs caused GC-2spd cytotoxicity.SiO₂NPs significantly inhibited the activity of GC-2spd cells（P<0.05）,resulting in irregular of cell morphology,decrease of intercellular connections and destruction of intracellular mitochondrial structure.2 SiO₂NPs affect spermatogenic cells and inhibit spermatogenesis.The number of spermatogenic cells showed a downward trend and the number of sperm in different stages reduced significantly in exposed mice（P<0.05）.The number of sperm and sperm motility decreased by 56.1%and 56.4%respectively,and the total sperm deformity rate increased significantly（P<0.05）.3 SiO₂NPs caused changes in sex hormones and imbalance of redox system in mice.The results of enzyme-linked immunosorbent assay and real-time fluorescence quantitative PCR showed that SiO₂NPs could significantly reduce the level of sex hormone and the m RNA expression level of androgen biosynthesis related genes in mice（P<0.05）.The levels of ROS in testis of exposed mice increased significantly,resulting in the imbalance of oxidation-reduction system（P<0.05）.In vitro studies showed that SiO₂NPs led to the imbalance of oxidation-reduction system and the significant increase of the m RNA expression levels of inflammatory factors in GC-2spd cells（P<0.05）.4 SiO₂NPs induced cell cycle arrest and apoptosis.Western blot showed that SiO₂NPs significantly inhibited the expression of meiotic regulating factors in testis（P<0.05）.TUNEL staining showed that SiO₂NPs induced spermatogenic cell apoptosis.In vitro studies showed that SiO₂NPs induced GC-2spd cell cycle arrest and apoptosis,which were characterized by down-regulation of cell cycle checkpoint related protein expression and activation of TNF-α/TNFR I apoptosis signaling pathway.Conclusions SiO₂NPs can accumulate in testis and affect spermatogenesis by inducing cell cycle arrest and apoptosis.These findings provide new experimental evidence for the mechanisms of reproductive toxicity caused by NPs and deepened our understanding of the safe application of NPs in medicine and other fields.Figure 16;Table 1;Reference 172...