[The antioxidant system mediated by Nrf2 in C2C12 cells responding to H₂O₂ stimulus under different oxygen...

To apply hypoxia of different oxygen concentration on C2C12 cells to study the changes of Nrf2 antioxidant system under H₂O₂.

The perfect simulative effect time and concentration of H₂O₂ were chosen. Cell vitality was tested after C2C12 cells cultured in 0.1 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 0.75 mmol/L, 1 mmol/L and 2 mmol/L H₂O₂ for 1 or 2 h respectively. The C2C12 cells were divided into different oxygen concentration group: 21%O₂, 12%O₂, 8%O₂, 5%O₂ respectively. And then cells were treated with H₂O₂ for 1 h, and collected for determination. Immunofluorescence of Nrf2 and the protein expression of Nrf2 were detected. The expressions of antioxidant enzymes superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase(CAT), NADPH quinine oxidoreductase-1 (NQO-1), glutathione peroxidase-1 (GPX-1), Heme oxygenase-1 (HO-1) mRNA and cellular ROS levels were tested by high quality fluorescence assay.

0.5 mmol/L H₂O₂ for 1 h was selected as the conditions of H₂O₂stimulation. Compared with 21% O₂ group, the expressions of Nrf2 mRNA and protein, antioxidant enzymes SOD1, SOD2, CAT, HO-1, NQO-1, GPX-1 mRNA were increased significantly (P＜0.05 or P＜0.01), and ROS level was lower (P＜0.01) in 12%O₂ group cells; only the expression of GPX-1 mRNA was increased (P＜0.05) in 8%O₂ group; the expressions of Nrf2 mRNA and protein expression, antioxidant enzymes SOD1, SOD2, NQO-1, GPX-1 mRNA were decreased significantly(P＜0.05 or P＜0.01), and ROS level was higher (P＜0.01) in 5%O₂ group.

Hypoxia can affect the Nrf2 antioxidant system, and the different oxygen concentrations have different impact. In addition, 12% O₂ for 12 h could promote the Nrf2 antioxidant system, and 5% extremely low oxygen may inhibit it.