Toxicity Of Silica Nanoparticles To Mouse Primary Leydig 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 has developed rapidly.SNPs as carriers a range of nanomaterials are widely used in feed additives,veterinary drugs and novel animal vaccines.At the same time,the contact routes of SNPs with animals are gradually increasing.The study found that SNPs can penetrate the blood-testis barrier(BTB)and accumulate in the testes due to their special physical and chemical properties such as small particle size and large specific surface area,causing apoptosis of Ley dig cells,resulting in male reproductive toxicity and affecting reproductive ability.Previous research in our group found that SNPs can lead to a decrease in testosterone secretion,however,the toxic side effects of SNPs on the male reproductive system need to be further confirmed.Therefore,this paper takes mouse testes as the main research object to explore the safe dose of SNPs on the male reproductive system in production practice,and the test results are as follows:(1)In vivo,male ICR mice were randomly divided into control groups,12.5,25 and 50 mg/mL SNP groups for 15 d,30 d and 60 d(Only processed once on the first day),respectively.The weight of mice was regularly measured and the change in mouse behavior was observed.Detection of toxic damage of SNPs to mouse testicles by organ coefficient detection,H&E and TUNEL staining.The results of experiments showed that compared with the control group,after 15 days of treatment,apoptosis of Ley dig cells in the 12.5 mg/mL SNPs treatment group was not obvious,and apoptosis phenomena such as cytoplasmic solidification and cell volume reduction were more and more obvious with the increase of dose,and the mesenchymal cells gradually decreased,and obvious green fluorescence appeared and showed dose dependence.After 30 days of SNP treament,compared with the control group,there were no obvious lesions in the 12.5 mg/mL SNP group and the 25 mg/mL SNP group,and there was still apoptosis and reduction of mesenchymal cells in the 50 mg/mL SNP group,green fluorescence was not obvious in the 12.5 mg/mL SNP group and 25 mg/mL SNP group,and green fluorescence was prominent in the 50 mg/mL SNP group.After 60 days of SNP treament,compared with the control group,the SNP group had no obvious lesion features and no obvious green fluorescence.The results showed that the SNPs were injected through the tail vein at low doses(12.5 mg/kg)and had no toxic side effects on the testes of mice and were safe doses.High doses(25 and 50 mg/kg)had a toxic effect on testicular development during short treatment(15 and 30 days),but the toxic side effects became smaller with increasing treatment time(60 days).(2)In vitro,different concentrations of SNPs(100,200,400,600,800,1000 and 1200 μg/mL)were treated with Leydig cells.The toxic effects of SNPs on Ley dig cells were detected by CCK-8,flow cytometry and Western blotting.The results of CCK-8 showed that SNPs could reduce the viability of Leydig cells and were dose-dependent.The flow cytometry results showed that the degree of apoptosis of Leydig cells increased after treatment with 200,400 and 800 μg/mL SNPs,and the Western blot results showed no significant difference in apoptosis levels in the 200μg/mL SNPs group,but apoptosis levels increased significantly in a dose-dependent manner between the 400 and 800 μg/mL SNPs groups.The results indicate that the SNPs have no toxic side effects on in vitro cultured Leydig cells at low concentrations(less than 200 μg/mL),but show cytotoxicity as concentrations increase.In vitro,200,400 and 800 μg/mL SNPs were treated with Leydig cells,the changes of autophagosomes in mesenchymal cells were detected by MDC method,and the expression of autophagy-related proteins were detected by Western blotting.The MDC results showed that with the gradual increase of the dose of SNPs,the aggregation degree of autophagic vacuoles in PLCs gradually increased.The combination of early autophagy inhibitor 3-MA,late inhibitor CQ and activator RAP were used to detect autophagy and apoptosis-related proteins by flow cytometry and Western blotting,and the expression of autophagy and apoptosis-related proteins were detected by flow cytometry and Western blotting after knockout of BECLIN-1 gene.The Western blot results showed that the 3-MA treatment group could inhibit autophagy and the apoptosis level was significantly increased.The CQ treatment group was able to inhibit autophagy,and the apoptosis level was significantly increased.The RAP-treated group was able to activate autophagy with a significant reduction in apoptosis levels.Knockout of the BECLIN-1 gene significantly reduced autophagy and increased apoptosis.The results show that the activation of autophagy(using autophagy activators)can effectively inhibit SNP-induced Leydig cytotoxicity.In summary,SNPs are injected through the tail vein and have no toxic side effects on the testes of mice at low doses,which is a safe dose;High doses have a toxic effect on testicular development after a short period of treatment,but the toxic side effects disappear with the increase of treatment time.SNPs have no toxic side effects on in vitro cultured Leydig cells at low concentrations,and the addition of autophagy activators can effectively inhibit SNPs-induced Leydig cytotoxicity.By studying the male reproductive toxicity of SNPs,this paper explores the safe dose and action time of SNPs in vivo and ex vivo,which has important guiding significance for ensuring the healthy development of animal husbandry.