Activation of Autophagy by Low-Dose Silica Nanoparticles Enhances Testosterone Secretion in Leydig Cells

Abstract

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.

BECLIN-1-Mediated Autophagy Suppresses Silica Nanoparticle-Induced Testicular Toxicity via the Inhibition of Caspase 8-Mediated Cell Apoptosis in Leydig Cells

“…The average diameter of SNPs was 109.0 ± 14.5 nm which was determined by randomly counting and measuring more than 500 particles ( Figure 1 B). The hydrodynamic diameter of SNPs and the zeta potential of SNPs can be obtained from our previous study [ 26 ].…”

“…Two BECLIN-1 shRNA vectors (shBecl1-1 and shBecl1-2) and a negative control shRNA vector (shNC) were preserved in our laboratory [ 26 ]. The recombinant BECLIN-1 lentiviruses were packaged using HEK 293T cells as described previously [ 27 ].…”

“…MDC staining was performed to monitor the activation of autophagy in the PLCs after SNP treatment as described previously [ 26 ]. Briefly, the PLCs were cultured on sterile cover slips and then treated with various doses of SNPs for 12 h. Next, the PLCs were incubated with MDC solution in the dark.…”

Hydroxysafflor Yellow A (HSYA) Protects Endplate Chondrocytes Against IL-1β-Induced Injury Through Promoting Autophagy

“…It has been reported that LC-3 could promote the formation of autophagosomes, and MDC level reflected the formation of autophagosomes [ 20 , 21 ]. Thus, LC-3 and MDC staining were performed to investigate the effect of HSYA on cell autophagy.…”

Silica Nanoparticles Promote Apoptosis in Ovarian Granulosa Cells via Autophagy Dysfunction

“…Oxidative stress induces autophagy, resulting in apoptosis [ 10 ]. Our recent studies show that SNPs (110 nm, spherical) reduce the population of Leydig cells and disorganization of spermatogenic cell layers under 25 mg/kg dose in vivo, enhance testosterone secretion under 100 μg/mL [ 11 ], and suppress Leydig cell apoptosis via the activation of caspase-8 under 400 μg/mL in vitro [ 12 ]. Autophagy dysfunction results in the accumulation of impaired organelles and unfolded/misfolded proteins, ultimately leading to cell death [ 13 , 14 ].…”