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Accumulation of silica nanoparticles (SNPs) in the testes leads to male reproductive toxicity. However, little is known about the effect and mechanistic insights of SNP-induced autophagy on apoptosis in Leydig cells. In this study, we aimed to verify the role of SNP-induced autophagy in apoptosis and explore the possible underlying mechanism in mouse primary Leydig cells (PLCs). H&E staining showed that SNPs changed the histological structures of the testes, including a reduction in the Leydig cell populations in vivo. CCK-8 assay showed that SNPs decreased cell viability, and flow cytometry showed that SNPs increased cell apoptosis, both in a dose-dependent manner in vitro. Additionally, Western blotting further found that SNPs activated autophagy by an increase in BECLIN-1, ATG16L, and LC3-II levels and promoted the intrinsic pathway of apoptosis by an increase in the BAX/BCL-2 ratio, cleaved the caspase 8 and caspase 3 levels. Furthermore, autophagy decreased SNP-induced apoptosis via regulation of the caspase 8 level combined with rapamycin, 3-methyladenine, and chloroquine. BECLIN-1 depletion increased the caspase 8 level, leading to an increase in SNP-induced cell apoptosis. Collectively, this evidence demonstrates that SNPs activated BECLIN-1-mediated autophagy, which prevented SNP-induced testicular toxicity via the inhibition of caspase 8-mediated cell apoptosis in Leydig cells.

Silica nanoparticles (SNPs) can cause abnormal spermatogenesis in male reproductive toxicity. However, the toxicity and toxicological mechanisms of SNPs in testosterone synthesis and secretion in Leydig cells are not well known. Therefore, this study aimed to determine the effect and molecular mechanism of low doses of SNPs in testosterone production in Leydig cells. For this, mouse primary Leydig cells (PLCs) were exposed to 100 nm Stöber nonporous spherical SNPs. We observed significant accumulation of SNPs in the cytoplasm of PLCs via transmission electron microscopy (TEM). CCK-8 and flow cytometry assays confirmed that low doses (50 and 100 μg/mL) of SNPs had no significant effect on cell viability and apoptosis, whereas high doses (more than 200 μg/mL) decreased cell viability and increased cell apoptosis in PLCs. Monodansylcadaverine (MDC) staining showed that SNPs caused the significant accumulation of autophagosomes in the cytoplasm of PLCs. SNPs activated autophagy by upregulating microtubule-associated protein light chain 3 (LC3-II) and BCL-2-interacting protein (BECLIN-1) levels, in addition to downregulating sequestosome 1 (SQSTM1/P62) level at low doses. In addition, low doses of SNPs enhanced testosterone secretion and increased steroidogenic acute regulatory protein (StAR) expression. SNPs combined with rapamycin (RAP), an autophagy activator, enhanced testosterone production and increased StAR expression, whereas SNPs combined with 3-methyladenine (3-MA) and chloroquine (CQ), autophagy inhibitors, had an opposite effect. Furthermore, BECLIN-1 depletion inhibited testosterone production and StAR expression. Altogether, our results demonstrate that low doses of SNPs enhanced testosterone secretion via the activation of autophagy in PLCs.