3.1 Characterization of TiO2 NPs
The TiO2 NPs exhibit spherical, nearly spherical, or polygonal shapes with an average particle size of 20.71 ± 2.23 nm, as displayed through TEM (Figure S2A, B). Additionally, XRD analysis revealed the dominant peak at two-theta peak of 25.856, 37.819, and 48.313, which is consitent with the anatase crystal structure of the TiO2 NPs (Figure S2C).
3.2 Effects of TiO2 NPs on the morphology and viability of TM4 cells
TM4 cells in the control group showed good growth status, exhibiting high density and spindle-shaped morphology. However, in the experimental group, the cell morphology deteriorated with increasing concentration of TiO2 NPs increased. The abnormol morphology, including unclear cell boundaries, reduced cell density, and increased intercellular space was observed (Figure S3A).
The cell viability of TM4 cells decreased in a concentration-dependent manner with the increase of TiO2 NPs concentration, compared to the control group cells. The decrease in cell viability of TM4 cells was statistically significant from the 50 µg/mL TiO2 NPs group (P < 0.01) (Figure S3B).
3.3 TiO2 NPs induced excessive ROS accumulation in TM4 cells
The intracellular ROS level in TM4 cells induced by TiO2 NPs was concentration-dependent. The difference was statistically significant from 50 µg/mL compared with the TM4 cells in the control group (P < 0.05) (Fig. 1A, B).
3.4 Effect of TiO 2 NPs on the protein expression of MCUb and calcium levels in TM4 cells
The expression level of MCUb protein in TM4 cells was significantly reduced in a concentration-dependent manner after 24 hours of treatment with varying concentrations of TiO2 NPs,, compared to that of the control group. The difference was statistically significant from the 50 µg/mL TiO2 NPs group (P < 0.01) (Fig. 2A, B). However, the expression level of VDAC1 protein in TM4 cells exposed to TiO2 NPs did not show significant changes compared to cells in the control group (Fig. 2C, D).
TiO2 NPs significantly increased the mitochondrial Ca2+ level at concentrations of 50 µg/mL (P < 0.01) compared to the control group cells. TiO2 NPs induced increase of mitochondrial Ca2+ level in TM4 cells in concentration-dependent manner (Fig. 2E, F).
After being treated with TiO2 NPs, the cytoplasmic Ca2+ level of TM4 cells increased significantly, showing a TiO2 NPs concentration dependency. The increase of Ca2+ level in cytoplasm was statistically significant from 50 µg/mL TiO2 NPs group, compared to the control group cells (P < 0.01) (Fig. 2G, H).
3.5 TiO2 NPs damaged mitochondrial membrane potential (MMP) in TM4 cells
As the concentration of TiO2 NPs increased, the green fluorescence became lighter and exhibited concentration dependence (Fig. 3A - C). The results of the quantitative analysis suggested that compared with the cells in control group, the decrease of MMP in TM4 cells was statistically significant, starting from 25 µg/mL TiO2 NPs (P < 0.01) (Figure S4A).
3.6 TiO2 NPs induced TM4 cell apoptosis through mitochondrial pathway
The cell apoptosis rate significantly increased in a concentration-dependent manner after 2the TiO2 NPs treatment for 24 hours, compared to that of the control group. This increase was statistically significant from the 50 µg/mL TiO2 NPs group (P < 0.01) (Fig. 4A, B).
Compared with that of the control group cells, TiO2 NPs inhibited the expression of the anti-apoptotic protein Bcl-2 in a concentration-dependent manner, and the difference started to be statistically significant in 75 µg/mL TiO2 NPs group (P < 0.05) (Fig. 4C, D). Exposure to TiO2 NPs led to a concentration-dependent upregulation of pro-apoptotic proteins Bax, Caspase 3, Caspase 9, p53 and Cyt c in TM4 cell. The increase of Bax and Caspase 3 expression levels began to be statistically significant in 50 µg/mL TiO2 NPs group, compared with those of the control group (P < 0.05) (Fig. 4E-H). The increase of Caspase 9 and p53 expression levels began to be statistically significant in 25 µg/mL and 100 µg/mL TiO2 NPs group, respectively, compared with the control group cells (P < 0.01) (Fig. 4I-L). Compared with cells in control group, the increase of Cyt c was statistically significant, starting from 75 µg/mL TiO2 NPs group (P < 0.01) (Fig. 4M, N).
3.7 NAC treatment restored cell ability and normal morphology in TM4 cells
The cells exposed to TiO2 NPs showed a decrease in cell viability, compared to the cells in the control group (P < 0.01) (Figure S5A). However, the cell viability of the NAC + TiO2 NPs group cells significantly improved in comparison to the cells in TiO2 NPs group (P < 0.01) (Figure S5A). Furthermore, as shown with red circles, the cells with abnormal morphology could be seen in the TiO2 NPs group. However, the cells with abnormal morphology of TM4 cells in the NAC + TiO2 NPs group was improved (Figure S5B).
3.8 Effect of NAC treatment on the protein level of MCUb and calcium levels in TM4 cells
The expression level of MCUb protein in TM4 cells was significantly inhibited in TiO2 NPs group compared to the control group cells (P < 0.01). However, MCUb protein expression level of the TM4 cells in NAC + TiO2 NPs group was significantly promoted, compared to that of the TiO2 NPs group (P < 0.01) (Fig. 5A, B). The expression level of VDAC1 protein in TM4 cells remained unchanged in each group (Fig. 5C, D).
Compared with the control group cells, the mitochondrion Ca2+ level of TiO2 NPs treatment group cells was significantly increased (P < 0.01). NAC + TiO2 NPs treatment significantly alleviated the mitochondrion Ca2+ level in TM4 cells, compared to TiO2 NPs group cells (P < 0.01) (Fig. 5E, F).
The cytosolic Ca2+ level of the TiO2 NPs group showed a significant increase as compared to that of the control group (P < 0.01).The cytosolic Ca2+ level was statistically reduced (P < 0.01) in the NAC + TiO2 NPs group compared to that of the TiO2 NPs group (P < 0.01) (Fig. 5G, H).
3.9 NAC treatment increased MMP inhibited by TiO2 NPs in TM4 cells
The fluorescence images of the MMP in TM4 cells were shown in Fig. 6. The MMP in TM4 cells was significantly reduced in the TiO2 NPs group, when compared to that of the control group (P < 0.01) (Figure S5B). However, after joint treatment with TiO2 NPs and NAC, the MMP level increased significantly as compared to the TiO2 NPs group cells (P < 0.05) (Figure S4B).
3.10 NAC inhibited apoptosis promoted by TiO2 NPs in TM4 cells
Compared with that of the control group, the apoptosis rate of TiO2 NPs treated group significantly increased (P < 0.001). The apoptosis rate of the NAC and TiO2 NPs combined treatment group was significantly lower than that of the TiO2 NPs treatment group cells (P < 0.001) (Fig. 7A, B).
TiO2 NPs significantly inhibited the expression level of the anti-apoptotic protein Bcl-2, compared to that of the control group cells (P < 0.05). In contrast, the expression of Bcl-2 was significantly increased in the NAC + TiO2 NPs group compared to the TiO2 NPs group cells (P < 0.05) (Fig. 7C, D). Furthermore, the expression levels of pro-apoptotic proteins including Bax, Caspase 3, Caspase 9, and p53 were significantly upregulated in the TM4 cells of the TiO2 NPs group, compared to those of the control group cells. However, the expression levels of these apoptotic proteins (Bax, Caspase 3, Caspase 9, and p53) were significantly downregulated in the cells treated with NAC and TiO2 NPs, compared to the TiO2 NPs group cells (Fig. 7E-L). Additionally, the expression of Cyt c protein in the TiO2 NPs group was significantly higher than that of the control group cells (P < 0.001). However, the Cyt c protein level in the NAC + TiO2 NPs group was significantly reduced compared to the TiO2 NPs group cells (P < 0.01) (Fig. 7G).