Protective Mechanism Of Cimetidine Against Oxidative Damage Induced By Irradiation

The rapid development of nuclear energy utilization puts forward higher requirements for radiation safety.The use of nuclear energy in people's lives and production is becoming more extensive,and it alsobrings a lot of radiation pollution to the environment.People are often exposed to low-dose ionizing radiation,including medical radiological diagnosis and treatment,background radiation in the natural environment,and a wide range of radioactive materials widely used in industry.With the widespread use of ionizing radiation for therapeutic applications,the risk of radiation damage has increased in varying degrees.Thus,the health effect of low-dose radiation(LDR)has received a great deal of attention from radiobiologists.There is also evidence that radiation can stimulate various repair mechanisms to reverse radiation damage at some dose.Some low-dose radiation exposure is also likely to stimulate the occurrence of cancer[3~5].Because of the low dose of ionizing radiation induced by the mechanism of injury is controversial,low-dose ionizing radiation protection drug research is rarely reported.Therefore,it is necessary to carry out study for protective drugs aganist low-dose ionizing radiation.The pharmacodynamic study of cimetidine,the novel low-dose ionizing radiation protective drug,was carried out by our group,which results showed that cimetidine had a good protective effect on oxidative damage induced by low dose ionizing radiation in mice.In order to study the antioxidative mechanism of cimetidine more deeply,this paper has conducted the following research:(1)the protective effect of cimetidine on the low-dose long-term irradiation on beagle dogs;(2)the protective effects of cimetidine on ionizing radiation in human normal umbilical vein endothelial cell(HUVEC)(3)the protective effects of cimetidine on oxidative damage induced by ionizing radiation in HUVEC cells;(4)the protective mechanism of cimetidine against oxidative damage caused by ionizing radiation.The main research contents and results are as follows: 1.The protective effect of cimetidine on low-dose long-term irradiation induced damage on beagle dogsBeagle dogs were selected as the model of CMTD pharmacodynamics study.After exposed to 0.2-1.0 Gy cumulative irradiation,the pheripharal blood of beagle dogs was counted and the results showed that it was fluctuation in the normal range except several WBC values were higher than normal,which might be stimulated by stress.Compared with the normal control group(CG),the number of bone marrow hematopoietic stem cells(HSCs)was significantly decreased after 24 h of cumulative irradiation at 1.0 Gy(p <0.01).Compared with the irradiation model group,HSCs of the positive and CMTD groups were increased,especially in the middle dose group(p <0.05).It was indicated that CMTD had a good protective effect on bone marrow hematopoietic stem cells.In the micronucleus test,the micronucleus rate increased slightly after irradiation.Although the micronucleus rate of the positive group and the cimetidine(53.33mg/10kg·d)group is lower than the normal control group and the irradiation model group,it was not significant different between the treatment groups and the normal control group..The slight damage was observed in the liver and spleen of the irradiated beagle dogs by pathological assay,showing a slight increase in cell arrangement and a slight increase in the number of red blood cells,.CMTD showed a better protective effect on the spleen than on the liver.The middle dose of CMTD has the best protective effect among three CMTD treatment groups.The results of serum antioxidant enzymes assay showed that the activities of SOD,CAT,GPx and GST in the serum were significantly decreased(p <0.01,p <0.05)after radiation exposure.Compared with the irradiation model group,the activity of SOD and CAT in CMTD groups were significantly increased(p <0.01).The GPx and GST activities of high dose of CMTD groups were also significantly increased(p < 0.01,p <0.05).These results showed that CMTD had protective effect on activities of antioxidant enzymes,while the effectson SOD and CAT activities were better than that on GPx and GST.(p <0.01).Compared with the irradiation model group,the serum MDA content in the positive group and middle/high dose groups were significantly decreased(p <0.01),indicating that CMTD could help decrease lipid oxidation induced by irradiation The above results demonstrated that CMTD had a good protective effect on the antioxidant system in irradiated beagle dogs.2.The protective effect of cimetidine on ionizing radiation induced damage in HUVECcellsCytotoxicity of CMTD and the protective effect of CMTD on radiation induced damage in HUVEC cells).The results showed CMTD had no cytoxicity and could increase cell survival rate and clonal formation rate.The cell survival rate of CMTD group(200 umol/L)is 61.3%,which was 12% higher than that of irradiated group.Hochest / PI double staining and Annexin V / PI method were used to observe the apoptosis of HUVEC cells together with fluorescence microscopy and flow cytometry.The results showed that cimetidine could significantly reduce cell apoptosis induced by irradiation and has a protective effect on both early and late apoptosis of cells.Gamma-H2 AX,a marker of DNA damage repair,was detected by flow cytometry.It was found that CMTD could significantly decreased ?-H2 AX level induced by irradiation..The results showed that cimetidine had a good protective effect on intracellular DNA damage caused by irradiation.3.The protective effects of cimetidine on oxidative damage induced by ionizing radiation in HUVEC cellsThe ROS level,antioxidant enzyme activity,lipid peroxidation product and DNA oxidative damage product content(8-OHdG)in irradiated cells(8.0 Gy acute irradiation)were detected.The results showed that CMTD significantly reduced ROS,MDA and 8-OHdG induced by ionizing radiation,while increased the activity of SOD,CAT,GPx and GST in the irradiated cells.The results suggested that cimetidine protects against oxidative damage induced by removing free radicals in the irradiated cells,protecting the activity of intracellular antioxidant enzymes,reducing radiation-induced lipid peroxidation and DNA oxidative damage.4.Preliminary study on mechanism of cimetidine against oxidative damage caused by ionizing radiationBased on the previous study,in which it was found that CMTD could reduce the level of ROS in irradiated cells,protect the antioxidant enzyme activity of irradiated animals and cells,reduce the production of oxidized products,alleviate the damage of irradiated blood hematopoietic stem cells,reduce irradiated cell apoptosis and DNA damage,improve its survival rate of radiation protection,the mechanism of CMTD was explored in this study.The results showed that CMTD had scavenging effect on superoxide anion radical,hydroxyl radical,alkyl radical and DPPH free radicals,especially on the scavenging hydroxyl radicals.Furthermore,we tested the Nrf2 nuclear transcription factor and its downstream antioxidant genes in the endogenous anti-oxidative stress pathway.Nrf2 was detected by immunofluorescence assay.It was found that CMTD could promote the translocation of Nrf2 from the cytoplasm to the nucleus.Real-time quantitative PCR showed the expression of SOD2 and GPx1 in the downstream of Nrf2 was up-regulated by CMTD,which was 3.55 and 6.45 times higher than that of the normal group,while the expression of Nrf2 gene was not changed significantly.The above results suggest that cimetidine may promote the activation of Nrf2 into the nucleus,regulate the downstream antioxidant enzyme-related genes expression,improve the body's antioxidant capacity and reduce oxidative damage induced by irradiation.