cGAS-STING pathway mediates TiO₂ nanoparticle-induced TM4 cell apoptosis via MCUb downregulation and calcium overload

Titanium dioxide nanoparticles (TiO₂ NPs) pose potential reproductive toxicity risks, notably compromising the blood-testis barrier (BTB), for which Sertoli cells are pivotal in maintaining integrity. However, certain research gaps remain regarding the molecular mechanisms by which TiO₂ NPs induce apoptosis in Sertoli cells. This study aims to investigate whether TiO₂ NPs trigger apoptosis through cGAS-STING-mediated down-regulation of MCUb in mouse Sertoli (TM4) cells. TM4 cells were exposed to various concentrations of TiO₂ NPs (0, 25, 50, 75 and 100 μg/mL) for different periods (0, 3, 6, 12 and 24 h). Exposure to TiO₂ NPs generated excessive reactive oxygen species (ROS), caused DNA damage, up-regulated cGAS, STING and TBK1 expression, down-regulated MCUb expression, elevated Ca²⁺ levels in cytoplasm and mitochondria, and enhanced apoptosis in TM4 cells, with statistical significance observed from 50 μg/mL onward. A time-dependent biphasic regulation of STING was observed. The ROS scavenger N-acetyl-L-cysteine (NAC) prevented DNA damage and cGAS-STING activation, while the STING inhibitor C176 restored MCUb expression, normalized Ca²⁺ levels and reduced apoptosis. Exposure to TiO₂ NPs induces an excessive production of ROS, leading to DNA damage, which subsequently influences the expression of the MCUb protein via the cGAS-STING signaling pathway, ultimately causing Ca²⁺ overload and triggering mitochondrial apoptosis in TM4 cells. These findings elucidate a novel molecular mechanism by which TiO₂ NPs induce apoptosis in TM4 cells and provide insights for the prevention of BTB disruption.