Radioprotective Effect And Mechanism Of Nitroxides R-1

BackgroundIonizing radiation consists of particles or electromagnetic waves that are energetic enough to detach electrons from atomsor molecules, and it is omnipresent in nature and our living environment. With the rapid development and widespread application of nuclear technology, people will be more likely to be exposed to ionizing radiation under such a situation. After being exposed to ionizing radiation, series of biological effects will occur in the body. Low doses of ionizing radiation will induce hormesis effect and adaptive response, while excessive dose of ionizing radiation will result in serious injuries and even death. The negative effects of radiation bring people into shadow of unpleasant and cause a certain degree of panic, in addition, the lack of the effective radioprotective drug. In order to reduce or relieve the damage, people look forward to a simple and effective method to prevent ionizing radiation. Therefore, the scientists have been looking for radioprotective drugs with efficiency and low toxicity and achieved good results. Currently, the Radioprotective agents that were clinically applied with the satisfactory effect is scarce in the world. Thus, the development of radioprotective agents has been an urgent mission. Based on the previously study, we selected nitroxide derivative as a candidate radioprotective agent to explore the effect of radiation protection.ObjectiveThe new synthetic compounds nitroxides R-1 was selected to investigate the protective effect and preliminary mechanism on human gastric epithelial cell(GES) and human liver cell(L-02) exposed to ionizing radiation. Some basic experimental data will be provided for the development of new radioprotective agent.MethodsToxicity of nitroxides R-1 at 24, 48 and 72h for L-02 cell and GES cell was determined using MTT assay and the appropriate concentrations were selected for following experiments. The cell viability of L-02 cell and GES cell with different concentrations of nitroxides R-1 were measured at 72h by MTT methods after 0,1, 2, 4and 8Gy (60)~Co-γray irradiation. WR-2721 at the terminal concentration of 4mmol/L was used as positive control. Accordingly, the cell viability of L-02 cell and GES cell were detected at 10d and 12d respectively by colony formation assay after 0, 1, 2, 4 and 8Gy (60)~Co-γray irradiation. The concentrations of 0.25μM and 0.125μM for L-02 cell and GES cell respectively was chosen to pretreat cells according to the result of MTT and colony formation. The L-02 cells and GES cells were divided into four groups:①the control group,②the drug group,③the irradiation group and④the combination group. The drug were given 0.25μM and 0.125μM for L-02 cell and GES cell respectively, the irradiation groups were 4Gy of (60)~Coγ-ray irradiation, the combination group received both treatment, whereas the control group received sham exposure. 24, 48 and 72h later inverted microscopy, Hoechst 33258 staining and flow cytometry were used observe the morphology or apoptosiss of each group in L-02 cell and GES cell. Meantime, the cell cycle was detected by flow cytometry, the enzyme activity of SOD and GSH were detected by chemiluminescence and spectrophotometric method respectively, the concentration of MDA and ROS were measured by TBA and DCFH-DA method, the expression of P53, Bcl-2, Bax and Caspase-3 protein were observed by western blot in each group of L-02 cell and GES cell.Results1. Cell toxicity test: Compared with the 0μM group, Nitroxides R-1 did not inhibit the viability of L-02 cells and GES cells when the drug concentration was less than 1μM, OD value were not significantly changed (P>0.05). whereas it could inhibit L-02 cells and GES cell growth when the concentration was higher than 2μM, OD value decreased significantly with increasing concentration (P<0.05). Therefore, 0.1, 0.125, 0.25, 0.5, 1μM concentrations were selected for the experiment of radioprotection.2. Radioprotective experiment: Compared with the 0μM group, the OD value and colony formation rate of L-02 cell and GES cell at different concentration of nitroxides R-1 were increased markedly (P<0.05). The 0.25μM and 0.125μM concentration groups were selected for the follow experiment because the effects of the two were more significant.3. The cell morphology experiment: Compared with the control group, both morphology and structure of L-02 cell and GES cell in the drug group did not change significantly. After 4Gy of (60)~Co-γray irradiation L-02 cell and the GES cell had clear outline, increased refraction, decreased apoptotic cells, the number of micronucleus and nuclear fragmentation were reduced in combination group. 4. The results of the cell cycle: compare with the control group in the same phase, the drug group of L-02 cells and GES were not significantly changed (P>0.05). Compared with the irradiation group, the G2 cell cycle arrest was relieved of L-02 cells and GES cells in the combination group. The differences were significant (P<0.05).5. The variation of SOD, GSH, MDA and ROS in each group: the results showed that compared with the control group, the value of SOD, GSH, MDA and ROS in the drug group of L-02 cells and GES cells were change little. The differences were not significant (P>0.05). Compared with the irradiation group, the enzyme activity of SOD and GSH were increased markedly, the concentration of MDA and ROS were descended in the combination group of the two cells. The differences were significant (P <0.05).6. The result of western blot: compare with the irradiation group, the expression of P53, Bax and Caspase-3 were decreased significantly, while the expression of Bcl-2 was increased in the combination group of L-02 cells and GES cellsConclusionNitroxides R-1 can protect L-02 cells and GES cells from (60)~Co-γrays induced injury effectively. Scavenging free radicals in cells, alleviating the G2 arrest, changing the protein expression relevant to apoptosis are involved in the underlying radioprotective mechanism of nitroxides R-1.