The ROS mediates MCUb in mitochondria-regulated apoptosis of TM4 cells induced by titanium dioxide nanoparticles

Abstract Titanium dioxide nanoparticles (TiO₂ NPs) can cause mitochondrial apoptosis of TM4 cells, but the mechanisms behind this process are not yet clear. The aim of this study was to evaluate if the accumulation of ROS caused by TiO₂ NPs inhibits MCUb expression, causing mitochondrial calcium overload and ultimately leading to 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 hours. We measured the cell viability, ROS level, MCUb and VDAC1 expression, mitochondrial and cytoplasmic Ca²⁺ level, MMP, apoptosis rate, and the key proteins related to apoptosis via the mitochondria pathway (Bcl-2, Bax, Caspase 3, Caspase 9, p53 and Cyt c). The effect of NAC on MCUb expression, calcium homeostasis, and cell apoptosis were also measured in this study. The results showed that compared to TM4 cells in control group, TiO₂ NPs significantly increased ROS level, downregulated MCUb expression, prompted the Ca²⁺ level in mitochondria and cytoplasm, and enhanced the mitochondria-regulated apoptosis, starting from the 50 µg/mL TiO₂ NPs group. However, NAC significantly increased the expression of MCUb, attenuated Ca²⁺ level in mitochondria and cytoplasm, and reduced the mitochondria-related apoptosis of TM4 cells compared with those in TiO₂ NPs group cells. In conclusion, TiO₂ NPs induced ROS accumulation which inhibits the expression of MCUb. The deceased MCUb level leads to Ca²⁺ overload in mitochondria, which causes TM4 cells apoptosis through the mitochondrial pathway. The results of this research elucidate the role of ROS in regulating mitochondrial calcium overload through MCUb for the first time when TM4 cells were exposed to TiO₂ NPs, and the results also supplement the molecular mechanism of cell apoptosis induced by TiO₂ NPs.