The Experimental Study Of Oxidized Coenzyme NAD+ On Anti-Radiation Damage In Mice

BackgroundRadiation therapy is an important means of cancer treatment,It is not only useful to malignant tumors, but also to benign tumors. The best efficacy of radiation therapy is to maximize anti-tumor cell by minimizing the radiation damage to normal tissue cells. That means to kill tumor cells mostly under the tolerance of normal tissue. But it is hard to avoid damage to normal tissue while killing tumor. With the development of science technology and radiation, more and more research were engaged in radiation protection. Radiation workers and patients exposed to radiation both were injured while curing tumor. Radiation scholars pay more attention to decreasing radiation damage to normal tissue, increasingly.Recent studies on the anti-radiation agents include more substances, which mainly in decreasing radiation injury to normal tissue, improving the sensitivity of tumor cells to radiation, repairing damaged cells to eliminate free radicals. The reaserch on anti-radiation agents focused on the following aspects (1) sulfur compounds, cysteamine, aminoethyl-isothiourea, cysteine, S-2-(3-amino-propylamine-based)-ethyl phosphorothioates (WR2721) and so on. (2),hormones, estradiol valerate, estradiol cyclopentane propionic acid, palmitic acid diester of estradiol, estradiol benzoate, ethinyl estradiol,500,522,523, and male hormones-hung ene-diol, etc.. (3) plant, ginseng, polysaccharides, alkaloids, phenols saponins, coumarins. (4) cytokines, such as GM-CSF, G-CSF, IL-3, EPO, TPO, IL-11, SCF and so on. (5) new drugs of the current research, CBLB502, meloxicam, ramipril, captopril, specific immune globulin. However, there is not an ideal anti-radiation agent which can be used in clinical,because of toxic side effects of-oxidants, the network characteristics of cytokines, and difficulty of herbal ingredients to purify. It is a key problem to find a new effective radiation protection drug with low toxic side effects on radiation protection science.Oxidized coenzyme NAD+, also called nicotinamide adenine dinucleotide, in mammals plays an important role in metabolic processes, by regulating the activity of SIRT1 to control chromosomes transcription, is an important coenzyme of cellular energy metabolism, involved in cell oxidation-reduction reactions and electron transfer of respiratory chain. In the mitochondrial, NAD+accepts electronic delivery and is reduced to NADH, can inhibit the production of free radical by electronic transfer, increase glutathione content, inhibit the release of cytochrome C from mitochondria, while generating ATP required in cell metabolism.1 molecule NADH is oxidized to generate 3 molecule ATP. NADH can inhibit free radical generation in the mitochondria and decrease mitochondrial membrane potential and so on. Nerve cells lack of NADH can lead to neuronal degeneration and necrosis, and there is a certain association with genetic susceptibility of Eercihai. The changes of regulation of expression of NADH in Neuroblastoma cells may be related to the occurrence of neuroblastoma. Abnormal of NADH alternative splicing may be closely related to the occurrence and development of cervical squamous cell carcinoma. The NADH content in epidermal cells after ultraviolet irradiation is related to the development of precancerous lesions of epidermal cells. Decreased cellular repair capacity after irradiation were caused by reduced intracellular NADH levels, inactivation of PARP involved in cell repairation.Exogenous NADH, can increase the release of the neurotransmitter dopamine nerve cells, improve Parkinson's disease; resis to cell injury induced by rotenone and regulate gene expression related to cell apoptosis. In summary, NADH can regulate radiation injury on cell. But NADH is expensive and difficult to produce,to protect.NAD+has't above shortcomings, the mitochondrial NAD+accept electronic transfer to NADH, therefore,The purpose of this experiment of NAD+anti-radiation damage in mice is to develop a new radiation protective agent.PurposeThe purpose of the study is to provide a new idea and method for a new type of anti-radiation protective agent by studying the influence from NAD+to hematopoietic function, immune function and the organ function of mice injured by radiation, exploring its recovery capacity against radiation damage in mice of kunmingMethods1. Kunming mice were randomly divided into three groups, normal control groupⅠirradiation control groupⅡ, NAD+irradiation groupⅢ, with a one-time total body irradiation using linear accelerator, and a irradiation dose of 6 Gy. In the same time, intraperitoneal administration per second per day 3 days before irradiation was also needed.2.1,7,15 days after irradiation, changes in peripheral white blood cells of mice was observed by automatic blood cell counter.3.24 hours after irradiation, counted the number of nucleated cells of bone marrow cells. 4.24 hours after irradiation measuring the rate of apoptosis of bone marrow cells, spleen lymphocyte apoptosis by flow cytometry.5.24 hours after irradiation, detecting the Caspase-3 activity of bone marrow cells, spleen lymphocytes by microplate reader.6.10 days after irradiation, measuring spleen index, thymus index and after HE staining, observing the changes in organizational structure of spleen, thymus under light microscope.7.10 days after irradiation, after HE staining, observing the changes in organizational structure heart, liver and kidney under light microscope.8.30 days after irradiation, survival rate of mice were counted.9 Statistical analysis Experimental data obtained were processed by SPSS 15.0 statistical software. The white blood cell count of each group and the time groups were underwent analysis of difference by factorial design analysis of variance, the rest of the data using single-factor analysis of variance. Measurement data by x±s, P<0.05 for the difference was statistically significant.Results:1.NAD+on the hematopoietic function in irradiated mice.1.1 The effects of NAD+on peripheral blood leukocytes in rats exposed to radiation.Seven days after irradiation,, the count of WBC of mice in each group dropped to the lowest, followed by a gradual recovery. Fifteen days after irradiation, WBC has been restored to 92% of pre-exposure in NAD+irradiation group, while WBC recovered to73%pre-exposure in irradiation group. 1.2 The effects of NAD+on the number of nucleated cells in the bone marrow of irradiated mice.NAD+irradiated femur bone marrow nucleated cell count lower than the normal control group, but higher than irradiated control group.1.3 The changes of apoptosis rate of bone marrow cells in mice.24 hours after exposure, the apoptosis rate of bone marrow cells in NAD+ irradiation group is lower than that in irradiation control group.1.4 The effects of NAD+on the activity of Caspase-3 in bone marrow cells of irradiated mice.24 hours after exposure, Caspase-3 activityof bone marrow cells in NAD+ irradiated group significantly decreased comparing with the irradiation control group.1.5 The effects of NAD+on the survival rate of irradiated miceMice in normal control group all survived, while in NAD+-irradiation group survival was significantly higher than that in irradiation alone group.2 NAD+on the immune function in irradiated mice2.1. The effects of NAD+on the spleen, thymus Index in Kunming mice after exposion.Spleen index and thymus index in NAD+irradiation treatment group were both higher than that in irradiated control group,2.2.The results of apoptosis rate of splenic lymphocyte in miceTen days after irradiation, the count of spleen lymphocytes was measured by flow cytometry, apoptosis rate of splenic lymphocyte in the radiation control group was higher than the normal control group, apoptosis rate of splenic lymphocyte in the NAD+radiation treatment group were lower than the radiation control group.we can see that NAD+can inhibit the radiation damage to spleen lymphocytes apoptosis.2.3 NAD+on Caspase-3 activity of spleen lymphocytes in irradiated mice.Ten days after irradiation, in irradiated control group, Caspase-3 activity of spleen lymphocytes is higher than the normal control group, while Caspase-3 activity in spleen lymphocytes of mice in NAD+irradiated group significantly reduced comparing with the irradiated control group.2.4 Histological changes of spleenAfter HE staining, spleen body in irradiation control group compared with normal control group and NAD+irradiation group was significantly reduced, germinal center is not obvious, spleen central artery stenosis, and bleeding associated with same stuff. The splenic corpuscle in NAD+irradiation group is more than that in the irradiated control group while less than that in the normal control group,.3 The effects of NAD+on the radiation damage of heart, liver, kidney in mice3.1. Cardiac histological changesTen days after irradiation, mouse heart HE staining showed that, there were no significant difference in the organizational structure and cell morphology of heart among each group.3.2. The liver histological changesTen days after irradiation, in irradiated control group, mouse liver HE staining showed that the majority of liver tissue appeared degeneration, necrosis, and balloon-like change are the most. In the NAD+-irradiation group, necrosis of liver tissue is significantly better than that in irradiated control group. 3.3. Renal histological changesTen days after irradiation, mouse kidney HE staining showed that volume of irradiated kidney reduced, the ratio of cortical decreased, the gap between epithelial cells of renal tubular increased, vacuole-like structure in intracytoplasmic increased, and part of the epithelial cell showed swelling and necrosis. Glomeruli did not change significantly in the optical microscope observation, NAD+-irradiation changes in renal tubular cells in the control group significantly reduced compared with irradiation control group.ConclusionIn this study, irradiated mice by hematopoietic function, immune function results show that:NAD+can be against radiation-induced decline in white blood cells in mice,can increase the number of bone marrow nucleated cells and the survival rate of irradiated mice, can reduce the small mouse bone marrow cells, spleen lymphocyte apoptosis and bone marrow cells, spleen lymphocytes Caspase-3 activity, can increase the spleen, thymus index, inhibit radiation on the spleen, liver and kidney damage,and have no significant effect on the heart.