ABSTRACT
Objcetive To investigate the protective effects of the nitroxides R-1 on human liver cells exposed to ionizing radiation.Methods Human liver cells L-02 were cultured and irradiated with 60Co γ-rays at the doses of 0,1,2,4,and 8 Gy,in order to screen the proper irradiation dose.WR2721 at the terminal concentration of 4 mmol/L was used as positive control.L-02 cells irradiated with 4 Gy were added with R-1 at the terminal concentration of 0.25 μmol/L at 30 min before irradiation or immediately after irradiation.MIT method was used to screen the proper conditions for follow-up experiment 72 h later.L-02 cell culture fluid was added with R-1 at the concentrations of 0,0.125,0.25,0.5,and 1 μmol/L,respectively for 30 min before irradiation at the doses of 0,1,2,4,and 8 Gy to ealculate clone formation rate at 10 d post-irradiation.L-02 cells were cultured and divided into 4 groups:control group without any treatment.drug group pretreated by 0.25 μmol/L R-1 only,irradiation group,irradiated at 4 Gy only,and drug + irradiation group with combination of 0.25 μmol/L R-01 and 4 Gy irradiation.The inverted microscopy and Hoechst 33258 staining and flow eytometry were used to observe the apoptosis of the cells at 24,48,and 72 h later.Results Nitroxides R-1 did not inhibit the viability of L-02 cell when its concentration was less than 1 μmol/L and it inhibited the L-02 cell growth when the concentration wu higher than 2 μmoL/L.The A value and colony formation rate of different concentration of R-1 groups were all higher than those of the irradiation group,and the effect of the 0.25 μmol/L drug concentration group was the most significant.Consequently,the concentration 0.25 μmoL/L was selected for follow-up experiment.Compared with the irradiation group,the L-02 cells of the pretreatment group showed solid adherence, increased refraction,clear outline,less apoptotic and dead cells at 4 Gy post-irradiation.Conclusions Nitroxides R-1 can protect the human liver cells from 60Coγ-ray induced injury effectively.The mechanism of its protective effect may be the reduction of apoptosis.