Toxicity Of Silica Nanoparticles To Mouse Follicular Granulosa Cells

In recent years,with the promotion of nanotechnology,the development and application of nanomaterials represented by silica nanoparticles（SNPs）in the animal husbandry and veterinary industry have developed rapidly.A series of nano-formulations with SNPs as carriers are widely used in feed additives,veterinary drugs,and new animal vaccines.With the wide application of SNPs,their toxic and side effects have received extensive attention,due to its small particle size,it can penetrate the physiological barrier and enter the blood circulation to reach and enrich various organs and tissues.During animal production,it was found that SNPs can cause follicular atresia,induce reproductive toxicity,and reduce animal reproductive capacity.Studies have found that SNPs can accumulate in the ovary,induce apoptosis of follicular granulosa cells and induce follicular atresia,induce reproductive toxicity,and reduce animal reproductive capacity.However,how SNPs induce apoptosis in follicles granulosa cells remains unclear.Therefore,this paper takes mouse ovarian granulosa cells as the research object to explore the toxic effects of SNPs on them.The test results are as follows:（1）In order to detect the ovary toxicity of SNPs in mice,21 days old ICR female mice were randomly divided into control group and SNPs group（25 mg/kg）,and ovaries and uterus were taken for follow-up experiments after 7 days of acute experiment.The organ coefficient was determined;H＆E and TUNEL were used to detect the pathological changes of the ovary.The mouse follicular granulosa cells were cultured in vitro,treated with different concentrations of SNPs,the cell viability was detected by CCK-8,and the apoptosis rate was detected by flow cytometry.The in vivo test results showed that compared with the control group,the follicular granulosa cells in the SNPs group had obvious pyknosis and deep staining,and obvious green fluorescence indicated that the granulosa cells had undergone apoptosis.The results of in vitro cultured follicular granulosa cells treated with SNPs showed that compared with the control group,the cell viability of SNPs was significantly decreased,and the apoptosis rate was significantly increased in a dose-dependent manner.The above results indicated that SNPs could induce apoptosis of granulosa cells in follicles and induce follicular atresia;SNPs could significantly reduce the activity of follicular granulosa cells and promote apoptosis.（2）In order to detect the regulatory effect of SNPs on ER stress,follicular granulosa cells were treated with different concentrations of SNPs（Control,200,400,and 800 μg/mL）,and Western Bolt was used to detect the expressions of ER stress-related proteins and IP3R;Realtimequantitative PCR The expression of IP3R was detected;the calcium ion concentration in cells was detected by flow cytometry.The results showed that SNPs significantly increased the expressions of ATF6,ATF4,CHOP,and EROla in ER stress compared with the control group in a dose-and time-dependent manner;the expression of IP3R1 increased significantly in a dose-dependent manner,and calcium ions Concentrations increased significantly in a time-and dose-dependent manner in the 6-12 h range.These results suggest that SNPs can activate endoplasmic reticulum stress and cause calcium disturbance in granulosa cells.（3）In order to examine the regulatory effect of PERK signaling pathway on SNPs induced calcium release and apoptosis,inhibtion of IP3R1,ATF4,CHOP,and ERO1α contreated follicular granulosa cells with SNPs.Western Bolt was used to detect the expression of endoplasmic reticulum stress and apoptosis-related proteins and IP3R1;flow cytometry was used to detect the calcium ion concentration and apoptosis rate in cells.The results showed that compared with the shNC plus SNPs group,inhibition of ATF4,CHOP,and ERO1α after contreated with SNPs could significantly reduce the expression of ATF4,CHOP,and ERO1αin endoplasmic reticulum stress;inhibition of IP3R1,ATF4,CHOP,and ERO1α after contreated with SNPs significantly reduced the expression of IP3R1 and cleaved Caspase-3 and the ratio of BAX/BCL-2,and significantly reduced the calcium ion concentration and apoptosis rate caused by SNPs.These results suggest that SNPs can regulate IP3R1-mediated calcium disturbance by regulating the PERK/eIF2α/ATF4/CHOP/ERO1α pathway during endoplasmic reticulum stress,thereby promoting granulosa cell apoptosis.Taken together,SNPs can activate endoplasmic reticulum stress and promote the apoptosis of ovarian follicular granulosa cells through IP3R1-dependent calcium release from the endoplasmic reticulum mediated by the PERK/eIF2α/ATF4/CHOP/ERO1α pathway.In this paper,the toxicity of mouse ovarian granulosa cells by silica nanoparticles was studied,provides new ideas for the study of reproductive toxicity and independent mechanism of SNPs,and provides data supports for a comprehensive understanding of the toxic and side effects of SNPs.The healthy development of animal husbandry and the veterinary industry is of great significance.