| With the rapid development of nanotechnology,nanomaterials have attracted attention in the interdisciplinary application of multiple disciplines.Silica nanoparticles(SiNPs),as one of the main five nanomaterials classified by the Consumer products inventory(CPI).Due to it has the advantage of mature synthesis technology,good stability,good for surface modification,special hollow structure,large specific surface area,strong adsorption capacity and good biocompatibility,and can improve the efficiency of nutrients and drugs through physiological barriers and cell membranes,increase the utilization of effective ingredients and have a slow-release effect,and combine with pathogen antigens to enhance vaccine immunity.Therefore,in animal production,it’s widely used in feed,additives,veterinary medicine,new animal vaccines and any other field.However,the side effects of SiNPs are still unclear.So,the eastablishment of the SiNPs biosafety evaluation system is particularly urgent and important,and it’s also a prerequisite for the actual production of SiNPs.Currently,the studies of SiNPs toxicity are becoming more comprehensive and in-depth.SiNPs can enter blood circulation through a varirty of ways,and pass through the blood-testis barrier,enter the testis and be enriched in the testis,which can cause testicular toxicity.SiNPs induced the decrease the weight of testis,the motility or count of sperm,which also abnormal secretion of testosterone in the blood.Leydig cells are the main place of testosterone secretion,which plays a vital role in spermatogenesis.Although many studies focused on spermatogenesis in male reproductive toxicity,the toxicity and toxicological mechanisms of SiNPs were often overlooked in Leydig cells.Recent years,studies on the toxicological mechanism of SiNPs have found that excessive ROS production plays a vitaly role in the cytotoxicity of SiNPs.The toxicity in vivo or in vitro induced by SiNPs through various pathways such as oxidative stress,endoplasmic reticulum stress,autophagy or apoptosis are closely related to the excessive ROS generation.Studies have confirmed that autophagy participates in SiNPs-induced testicular toxicity and regulates spermatogenesis.Whether SiNPs induce autophagy in leydige cells or the regulation mechanism of autophagy on the secretion of testosterone are still unclear.Therefore,this article takes the primary cultured leydig cells as experimental model,and treated leydig cells with low-concentration of SiNPs.Through immunohistochemical staining,ELISA,Western blot,immunofluorescence technology,lentivirual vector construction technology,flow cytometry,RT-qPCR and other methods to study the effect and mechanism of SiNPs-induced autophagy on testosterone secretion in mouse leydig cells.The main results were as follow:(1)Low concentrations of SiNPs have no significant effect on the cell viability and apoptosis of primary cultured mouse leydig cells,but can regulate the secretion of testosterone.(2)SiNPs can enter the leydig cells and mainly distribute in the cytoplasm,increasing the number of autophagosomes in the cells,and induce autophagy by activating the expression of autophagy-related proteins BECLIN-1 and LC3-II.SiNPs were co-treated leydig cells with autophagy inhibitor 3-MA or CQ,and the autophagy activator RAP,we confirmed that SiNPs can activate autophagy on leydig cells.(3)Low concentrations of SiNPs can regulate testosterone secretion by upregulating the expression of StAR.SiNPs were co-treated leydig cells with autophagy inhibitor 3-MA or CQ,or autophagy activator RAP,which further confirms that SiNPs activated autophagy by upregulating the expression of StAR and promote the testosterone secretion.(4)Successfully constructed Beclin-1 lentiviral interference vector and carried out lentiviral packaging,lentiviral titer determination and interference effect detection.The lentiviral titer determined by the multiple dilution method was 1-10×107 TU/mL,and the interference effect was about 70%,which can provide follow-up research.(5)Leydig cells transfected with Beclin-1 lentivirus can effectively inhibit autophagy induced by SiNPs,the expression of StAR and testosterone secretion.In summary,in this study we confirmed the regulation of SiNPs on the occurrence of autophagy and testosterone secretion,and explored the molecular mechanism of promoting testosterone secretion by SiNPs,providing the theoretical basis and experimental basis for indepth analysis of the reproductive toxicity of SiNPs,and providing new information for further revealing the mechanism of SiNPs induced testicular toxicity and providing theoretical basis for seeking to reduce or inhibit the toxicity of SiNPs. |
