Toxic Effects Of Human Chorionic Trophoblast Cells Induced By AgNPs And Its Potential Mechanism

[Objective]With the widespread application of silver nanoparticles（Ag NPs）in manufaturing industry,medicine,health care industry,and daily necessities,the health risks of humans constantly exposed to various commercial products containing Ag NPs residues are increasing.Currently,an increased number of studies have focused on the toxic effects induced by Ag NPs and their potential mechanisms,but studies on the em-bryonic developmental toxicity induced by Ag NPs are very limited.The placenta plays a key role in fetal growth,and placental dysfunction and damage are associated with embryo/fetal toxicity.Therefore,the purpose of this study was to evaluate the placental toxicity induced by Ag NPs and reveal its potential molecular mechanism,so as to pro-vide a theoretical basis for evaluating the potential embryonic developmental toxicity of nano-materials.[Methods]In vitro experiments,human chorionic trophoblast cells（HTR-8/SVneo）were used as the in vitro model and exposed to 20 nm Ag NPs at dose levels（0.5,1,2,4μg/m L）.The activity of HTR-8/SVneo cells was estimated by MTT,RTCA,and lactate dehydrogenase（LDH）methods,and then intracellular reactive oxygen spe-cies（ROS）production was also determined by Reactive Oxygen Species Assay Kit.The supernatant was used in the enzymatic assays to determine the activities of super-oxide dismutase（SOD）,malondialdehyde（MDA）andglutathione（GSH）.Cells were exposed to Ag NPs was investigated by focusing on cell apoptosis,autophagy and ne-crosis.Furthermore,the m RNA expression levels of the key factors of placental growth（PLGF and ASCL2）and inflammatory factors（IL-6,IL-8 and TNF-α）were determined by fluorescence quantitative PCR.In order to further verify the inflammatory effect of Ag NPs induced placenta,pregnant mice were used as an in vivo model,which was administered by gavage at low,medium and high doses of Ag NPs（25,50 and 100mg/kg）.Placentas were collected at 18.5 days of gestation for immunohistochemical staining to analyze the positive expression of inflammatory factors（IL-6 and TNF-α）.[Results]In vitro cytotoxicity data showed that HTR-8/SVneo cell exposed to Ag NPs would induce significant cytotoxicity.In particular,the cell viability induced by Ag NPs（≥1μg/m L）did change significantly as compared with the control group,and the media inhibiting concentration（IC50）was 1.81×10^-6 g/m L.The degree of cell mor-phological damage was consistent with cell viability and LDH release,showing an ob-vious dose-dependent effect.Compared with the control group,the levels of ROS,SOD and MDA were significantly increased when cells exposed to Ag NPs,these data show that Ag NPs can induce the increase of intracellular oxidative stress level,resulting in the unbalance of intracellular redox balance.And the early apoptosis and necrosis of HTR-8/SVneo cells also increased significantly with the increase of Ag NPs exposure dose,especially in the high dose group（4μg/m L）,the cells exhibited nuclear contrac-tion,cytoplasmic condensation,and cell rupture.In addition,cells exposed to 2μg/m L Ag NPs could cause a significant increase in the expression level of a key protein in autophagy（LC3）compared with other treatment groups,suggesting that the cells pro-duced severe autophagy.Notably,cells exposed to 2μg/m L Ag NPs could cause a sig-nificant increase in the expression levels of placental growth factor（Pl GF）and cyto-trophoblast specific factor（ASCL2）.Similarly,cells exposed to 2μg/m L Ag NPs also significantly increased the expression levels of interleukin-6（IL-6）,interleukin-8（IL-8）and tumor necrosis factor-α（TNF-α）compared to the control group.Thus,the ex-pression levels of PLGF and ASCL2 factors in trophoblast cells induced by Ag NPs are consistent with the expression levels of cellular inflammatory factors（IL-6,IL-8,and TNF-α）,suggesting that Ag NPs induce human chorionic trophoblast cells to produce an immune-inflammatory response.In addition,the immunohistochemical analysis of placental tissue showed that the positive expressions of IL-6 and TNF-αinflammatory factors also increased significantly after Ag NPs exposure to ICR pregnant mice,which further verified that Ag NPs could induce placental inflammatory response.[Conclusion]（1）Exposure to 20 nm Ag NPs can induce a decline in human chori-onic trophoblast cell viability even at a lower exposure level,and cell membrane dam-age and cell structure were damaged in a dose-dependent manner.（2）Ag NPs can trigger cellular oxidative stress,decreased mitochondrial mem-brane potential and dysfunction,leading to adverse outcomes such as apoptosis,ne-crosis,and autophagy.（3）Ag NPs exposure induces significant increases in transcript levels of key fac-tors（PLGF and ASCL2）and inflammatory factors（IL-6,IL-8 and TNF-α）in placental trophoblastic cells;The positive inflammatory factors（IL-6,TNF-α）expression in pla-cental tissues showed a dose-dependent effect with the nanosilver exposure concen-tration,indicating that placental damage caused by Ag NPs exposure was related to the immune-inflammatory response of human chorionic trophoblast cells.