Abstract: Objective To study the protective effects of nitroxides against human keratinocytes oxidative damage induced by H₂O₂ and its possible mechanisms. Methods Normal human keratinocyte cultures obtained from foreskin were served as test-system. Human keratinocytes were cultured in human keratinocytes growth media (KGM) without serum and supplemented with 0.1 mmol/L Ca²⁺. Experiments were performed in culture when the cells grew to fuse. Oxidative damage was induced by adding H₂O₂ directly to the culture media at different concentrations in the present and absence of nitroxides [(2, 2, 6, 6-tetramethylpiperidine (TEMPO) a nd 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine (TEMPOL)]. The cell viability was monitored and the intracellular level of reduced glutathione (GSH), the activities of glutathione-peroxidase(GSH-Px), superoxide dismutase (SOD), and catal ase were evaluated. Results ①H₂O₂ could cause damage to human keratinocytes directly. There was a significantly negative correlation between H₂O₂ concentration and cell viability. ②The level of GSH in keratinocytes lowered with treatment of H₂O₂. The activities of GSH-Px, SOD and catalase decreased. ③Pretreatment o f the cells with TEMPO and TEMPOL inhibited the damaging effects of H₂O₂ on cell viability and on cell antioxidant enzymatic systems. Conclusion The results of the study suggest that nitroxides may provide protection for cultured human kera tinocytes against H₂O₂-induced oxidative injury. It is proposed that the effects of nitroxides against cell oxidative damage be related to their protection of cellular enzymatic activities and maintaining cellular antioxidant systems.

Key words: Nitrogen oxides, Keratinocytes, Antioxidants