Figure 4.

Protective effect of CoCl₂ on H₂O₂-induced cytotoxicity, repression of HIF-1α, Nrf-2, Keap1, and Prdx6 expression, and ROS accumulation. HEI-OC1 cells were preincubated with 200 μM CoCl₂ for 6 h before exposure to 25 μM H₂O₂ for 9 h. (A) The CCK-8 assay was used to determine cell viability. Data are presented as means ± SDs for three independent experiments expressed as a percentage of the untreated control (* p < 0.05, compared with the untreated control, ^# p < 0.05, CoCl₂ only versus H₂O₂ only or CoCl₂ plus H₂O₂). (B) Representative immunoblot showing HIF-1α, Nrf-2, Keap1, and Prdx6 expression. Individual bands were quantified densitometrically and normalized to Lamin B (HIF-1α and Nrf-2) or GAPDH (Prdx6 and Keap1). Values in the graphs are presented as fold changes relative to the untreated control, expressed as means ± SDs of three independent experiments (* p < 0.05, compared with the untreated control, ^# p < 0.05, CoCl₂ only versus H₂O₂ only or CoCl₂ plus H₂O₂). (C) After the treatment as described above, the levels of ROS were determined by measuring CM-H₂DCFDA. Data are presented as fold changes relative to the untreated control, expressed as ± SDs of three independent experiments (* p < 0.05, compared with the untreated control, ^# p < 0.05, CoCl₂ only versus H₂O₂ only or CoCl₂ plus H₂O₂).