Toxicity Study Of Silica Nanoparticles On Mouse Ovary

As one of the most widely used nanomaterials,silica nanoparticles(SNPs)play an important role in material science,biomarkers,medicine,and other fields.In the field of veterinary medicine,the number of probes,drugs,vaccines,and other products prepared with SNPs as carriers are increasing.With the deepending of the study on SNPs,their toxicity problems have also been exposed and gradually paid attention.Due to its small partical size,SNPs penetrate the vascular barrier and accumulate in tissues and organs to cause damage.Previous studies have demonstrated that SNPs cause damage and apoptosis in animal germ cells,resulting in the reduction of animal productivity and threatening the development of animal husbandry.The safe dose and duration of action of SNPs on the ovaries in animal applications are currently unknown.Therefore,this paper conducts experiments on mouse ovaries as the research objectFirstly,the in vivo study was conducted.The 5-week-old ICR female rats were randomly divided into the normal saline group(control group)and the SNP groups(12.5,25,and 50 mg/kg).After a single tail vein injection,their ovaries were collected for subsequent study at 15,30,and 90 d respectively.Their weight and behavior changes regularly were recorded;organ coefficient was calculated;the minimum SNPs toxic dose and toxicity elimination time were detected via H&E and TUNEL staining.The ovarian granulosa cells of the 3-week-old mice were cultured in vitro and treated with 75,150,300,450,600,750,and 900 μg/mL SNPs.CCK-8 was used to detect the lowest dose that interferes with cell activity;flow cytometry was used to detect the concentration and toxic duration of SNPs that affect cell apoptosis rate after different concentrations of SNPs(150,300 and 600 μg/mL)treatment;Western Blot was used to detect the levels of the apoptosis-related protein cleaved Caspase-3 and the BAX/BCL2 ratio,as well as autophagy-related proteins BELLIN-1,LC3-II,P62,and mature lysosomal hydrolase pro-CTSD at different concentrations and treatment times,in order to determine the safe dose and duration of action of SNPs;LysoTracker Red and AO(Acridine Orange)staining were used to detect lysosomal damage;immunofluorescence staining of CTSD was used to detect lysosomal hydrolase activity.SNPs,combined with autophagy activator RAP,and autophagy inhibitor 3-MA,CQ were exposed to the ovarian granulosa cells,to detect the rate of apoptosis and the levels of autophagy and apoptosis-related proteins,verifying effective ways to reduce the toxicity of SNPs.The results are as follows:(1)The safe dose and time of SNPs acting on the ovaries of mice in vivo:The results of the in vivo study showed that there was no statistical difference in the weight,as well as the shape and size of the ovaries of the mice between the SNP groups and the normal saline group.Compared to the saline treated group,under 12.5 mg/kg SNPs treatment,there was almost no damage to the ovaries.However,after 25 and 50 mg/kg SNPs treatment,the granulosa cells of the mouse ovaries showed significant nuclear deep staining and pyknosis,and follicular atresia.The ovaries of SNP treated mice for 15 and 30 days showed more severe cell apoptosis and follicular atresia compared to the saline group.There was no significant difference in damage between the groups after 60 days,indicating that the SNPs used in this experiment were safe at a dose of 12.5 mg/kg,and the toxicity to the ovaries of mice disappeared after 60 days of in vivo treatment.The TUNEL staining in the SNPs group showed an increase in green fluorescence,indicating cell apoptosis.The fluorescence results of each group confirmed the above conclusion.(2)The safe concentration and time of SNPs acting on mouse ovarian granulosa cells:CCK-8 and flow cytometry results showed that below 150 μg/mL SNPs,the cell viability was not affected,but significantly decreased with increasing treatment concentration,and the apoptosis rate significantly increased.Western Blot results showed that after treatment with 150 μg/mL SNPs,there was no significant change in the expression of apoptosis-related protein cleaved Caspase-3 and the BAX/BCL-2 ratio,but both significantly increased under 300 and 600μg/mL SNPs treatments.After more than 6 hours,with the prolongation of treatment time,the expression levels of cleared Caspase-3 and the ratio of BAX/BCL-2 both increased.The above results indicate that the SNPs are at low concentrations(below the dose of 150 μg/mL,it has no toxic or side effects on ovarian granulosa cells cultured in vitro,but shows cytotoxicity as the concentration increases.(3)The minimum concentration and time for activation of autophagy by SNPs:Western blot results showed that compared to the control group,after 150 μg/mL SNP treatment and above,the expression of BECLIN-1 and LC3-Ⅱ significantly increased in a dose-dependent manner in ovarian granulosa cells.The level of P62 was increased after 300 and 600 μg/mL SNP treatments,while decreased after 150 μg/mL SNP treatment.The level of P62 was significantly increased after 300 μg/mL SNP treatment for 12 and 24 h,while decreased for 6 h.Indicating only when a level below 150 μg/mL can induced autophagy be avoided as much as possible,and within 6 hours is the effective time to avoid autophagy activation.The levels of P62 and LC3-II were significantly increased after SNP treatment,combined with BAFA 1.Immunofluorescence staining results showed that green fluorescence of CTSD was weakened and dispersed,indicating that lysosome membrane was damaged,hydrolase was escaped,and activity was decreased.Lyso Tracker Red and AO staining enhanced red fluorescence,indicating lysosomal acidification.The above results indicate that SNPs can damage the lysosomes of ovarian granulosa cells and produce cytotoxicity.(4)Interference with autophagy reduces SNPs induced apoptosis of ovarian granulosa cells:Flow cytometry results showed that the apoptotic rate was significantly increased after SNPs combined with RAP or CQ,while decreased after SNPs combined with 3-MA.Western Blot results showed that the level of LC3-II was significantly increased after SNPs combined with RAP and CQ,while the level of BECLIN-1 was significantly increased after SNPs combined with RAP,the level of BECLIN-1 was significantly decreased after SNPs combined with CQ,and the levels of LC3-II and P62 was significantly decreased after SNPs combined with 3-MA.The BAX/BCL-2 ratio was significantly increased after SNPs combined with RAP or CQ,and the level of cleaved Caspase-3 and the BAX/BCL-2 ratio were significantly decreased after SNPs combined with 3-MA.BECLIN-1 depletion or the inhibition of ROS,the apoptotic rate induced by SNPs was significantly decreased in ovarian granulosa cells.The above results indicate that inhibiting autophagy(adding autophagy inhibitors or interfering with autophagy activation)or clearing ROS(adding NAC)can effectively inhibit SNPs induced ovarian granulosa cell toxicity.In conclusion,SNPs injected through the tail vein have no toxic or side effects on the ovaries of mice at low doses(12.5 mg/kg),making them a safe dose;High doses have toxic effects on ovarian development after short-term treatment(within 60 d),but the toxic side effects disappear as the treatment time increases.SNPs have no toxic side effects on ovarian granulosa cells cultured in vitro at low concentrations(150 μg/mL)and short time(within 6 h),but as the concentration increases,SNPs can produce cytotoxicity by damaging lysosomes.Adding autophagy inhibitors or clearing ROS can effectively inhibit SNPs induced ovarian granulosa cell toxicity.This paper studies the female reproductive toxicity produced by SNPs,and explores the safe dose and action time of SNPs in vivo and in vitro,which has important guiding significance for ensuring the healthy development of animal husbandry.