The ROS Mediates MCUb in Mitochondria-Regulated Apoptosis of TM4 Cells Induced by Titanium Dioxide Nanoparticles.

Titanium dioxide nanoparticles (TiO₂ NPs) can cause mitochondrial apoptosis of TM4 cells associated with reactive oxygen species (ROS) accumulation and Ca²⁺ overload, but the relations among these processes remain unclear. This study aimed to evaluate whether the accumulation of ROS caused by TiO₂ NPs inhibits MCUb expression, leading to mitochondrial calcium overload and subsequent cell apoptosis through the mitochondrial pathway. TM4 cells were exposed to different concentrations of TiO₂ NPs (0, 25, 50, 75, 100 μg/mL) for 24 h. We assessed cell viability, ROS level, MCUb and VDAC1 expression, mitochondrial and cytoplasmic Ca²⁺ levels, mitochondrial membrane potential (MMP), apoptosis rate, and key proteins related to mitochondrial apoptosis (Bcl-2, Bax, Caspase 3, Caspase 9, p53 and Cyt c). Additionally, the effect of N-acetylcysteine (NAC) on MCUb expression, calcium homeostasis, and cell apoptosis was evaluated. Compared to control group, TiO₂ NPs significantly increased ROS level, downregulated MCUb expression, elevated Ca²⁺ levels in mitochondria and cytoplasm, and enhanced mitochondria-regulated apoptosis, starting from the 50 μg/mL TiO₂ NPs group. However, NAC significantly increased MCUb expression, attenuated Ca²⁺ levels in mitochondria and cytoplasm, and reduced mitochondria-related apoptosis. In conclusion, TiO₂ NPs induced ROS accumulation, which inhibited the expression of MCUb. The decreased MCUb level led to Ca²⁺ overload in mitochondria, causing TM4 cell apoptosis via the mitochondrial pathway. This research elucidates, for the first time, the role of MCUb and its relation with ROS in apoptosis of TM4 cells induced by TiO₂ NPs, which supplementing the molecular mechanism of cell apoptosis caused by TiO₂ NPs.