Fig. 6
ROS overproduction induced by montmorillonites (Mts) in vitro and in vivo, and the effects of NAC pretreatment on Mt-induced cytotoxicity in vitro. A, B Under CLSM, ROS levels of HCEC-B4G12 in vitro were investigated by H2DCF-DA staining (green) at 2, 6, 12 and 24 h after exposure to Na-Mt (A) and C-H-Na-Mt (B) applied in various concentrations (0, 3.125, 12.5, and 50 μg/mL). D, E Quantification of the ROS fluorescence intensity in (A, B). C Representative DHE staining (green) of the corneal section of rats after their exposure to different concentrations (2 and 10 mg/mL) of Na-Mt or C-H-Na-Mt for 7 days in vivo. Nuclei were stained by DAPI (blue). F Quantification of ROS fluorescence intensity of different groups in (C). Data are presented as the mean ± SD. * p < 0.05 compared with the control. G, J Representative images of ROS levels of HCEC-B4G12 monitored by CLSM. HCEC-B4G12 cells were pretreated with 10 mM NAC for 1 h before exposing them to different concentrations (12.5 and 50 μg/mL) of Na-Mt or C-H-Na-Mt for additional 6 h. H, K Quantification of ROS fluorescence intensity in (G, J). I, L ATP content was measured at 12 h post-exposure to Na-Mt and C-H-Na-Mt, with and without pretreatment with NAC. Data points are the mean ± SD from three independent experiments, with three parallel samples per concentration in each experiment. *p < 0.05 compared with the control lacking the NAC pretreatment. #p < 0.05 compared with the control receiving the NAC pretreatment. &p < 0.05 between treatments with and without NAC pretreatment at the same concentration of different Mt. Scale bars: 50 μm