Radioprotective Effects Of Hydrogen On Cultured Cells And Murine Hematopoietic System

In recent decades, the extensive utilization of atom energy in many fields such as military, civil industry, agriculture, medicine etc brings about great benefits for people, but in the meantime, how to protect human health and safety from severe nuclear radiation become a critical problem in the world. The continuous development and application of nuclear weapons and increase of nuclear weapon-possessing countries have increased the risk of regional nuclear warfare. China is one of the great nuclear countries, possessing strategic nuclear forces such as nuclear weapons, nuclear submarines and a large number of nuclear power stations. Nuclear radiation protection thus is of special significance to protect the health of employees. And strengthening protection of nuclear radiation damage has an important role in the future nuclear war, anti-nuclear terrorist attacks in peacetime, nuclear power plant accident and emergency rescue tasks and there is still a long way to go for our country. Nowadays, with the development of modern biomedicine, the radiation therapy has become an important therapeutic method for malignant diseases, but the damages to some normal tissues induced by radiation limit the application of radiotherapy. It is a novel scientific topic for radiologists to look for high-effective radiation protectants with low toxicity to protect normal tissues from radiation induced damages. In fact, exploitation the ideal radiation protectants has always been emphasis and obstacles in the field of radiation protection research.As we know, the damage of radiation mainly comes from theγray and neutron ray, and the biological effects are divided into two ways: the first is direct effect and the second is indirect effect, which accounts for about 70%-80% of the damages. Since the indirect effect mainly due to the abundant free radicals caused from radiation, so blocking and scavenging of free radicals become our most important protecting strategy.In 2007, Ohsawa et al. found that molecular hydrogen could selectively reduce cytotoxic reactive oxygen species in vitro and exert therapeutic antioxidant activity. From then on, research on hydrogen set off a worldwide upsurge. Hydrogen could selectively reduce hydroxyl radicals excited our interest, because most of irradiation induced injury was caused by hydroxyl radicals. Therefore, we reasoned that hydrogen might have great radioprotective effects.Hydrogen was misunderstood as physiological inert gas because researchers thopught it could not react with any material in vivo. Hydrogen is continuously produced by colonic bacteria in the body and normally circulates in the blood, so it is physiologically safe for humans to inhale hydrogen at a relatively low concentration. Hydrogen gas is dangerous as a kind of flammable gas.We dissolved hysdrogen gas in kinds of solution while using it to solve the problem. Also, it is easy to be used in clinic.There are few reports about the radioprotective effects of hydrogen.Radiation could cause damage on multiple organs, especially on the tissues consisting of rapid proliferation cells. The bone marrow is one of the most susceptible organs to radiation. Ionizing radiation can not only cause damage on hematopoietic stem cells, hematopoietic progenitor cells caused by suppression and destruction, but also on the bone marrow sinusoids. There is a considerable gap in this area in the world. Research on this area will fill the gap, which has great valueContents of study:Hydrogen-rich solution production: Hydrogen was dissolved in PBS/physiological saline for 6 h under high pressure (0.4 MPa) to a supersaturated level using a hydrogen-rich water-producing apparatus produced by our department. We aim at working out a better method of synthesizing and technology.Radioprotective effects of Hydrogen-rich solution on cells: we selected Human Lymphocyte AHH-1 cells to make the dose-survival curve of cells irradiated at different doses of irradiation. We observed the following subjects:â‘ Radioprotective effect of different concentrations of Hydrogen-rich solution on cells irradiated with the same dose ofγ-ray.â‘¡Radioprotective effect of the unique concentration of Hydrogen-rich solution on cells irradiated with different doses ofγ-ray.â‘¢The influence of Hydrogen-rich solution on LDH leakage from irradiated cells.Radioprotective effects of Hydrogen-rich solution on cells murine hematopoietic system:â‘ Radioprotective effect of Hydrogen-rich solution on peripheral white blood cells of irradiated miceâ‘¡Radioprotective effect of the Hydrogen-rich solution on bone marrow nucleated cells of irradiated miceâ‘¢The influence of Hydrogen-rich solution on endogenous spleen colony formation units(CFU-S) of irradiated miceâ‘£The influence of Hydrogen-rich solution on the activities of plasma SOD,GSH of irradiated miceâ‘£The influence of hydrogen-rich solution on plasma MDA of irraditied mice, the oxidative product of lipidPreliminarily exploring the mechanism of radioprotective effects of hydrogen-rich solution: Analyze the effects of the compounds on cell cycle, DNA , 8-OHdG. Methods: 1. Hydrogen-rich solution production: Reference to the literature at home and abroad, using the chemical synthesis platform of our laboratory, we optimize the methods of producing hydrogen-rich solution.2. Cells: AHH-1 cells are derived from human lymphoblastoid(Long-term cultured in our laboratory). Cell culture conditions: RMPI-1640 medium (Hyclone Corporation), 10%NCS(GIBCO Corporation, 37℃,5%CO2, Saturated moisture, incubated in cell incubator.3. Irradiation: different doses ofγ-rays by Co-60.4. Determination of cell viability: We assessed the survival rate of different groups of cells by CCK-8. Formula: Viability(%)=OD values of drug group-Background / control OD values-Background)×100%.5. Apoptosis and cell cycle analysis: The cells were stained by using fluorescence staining and Annexinâ…¤/ PI double staining, and analysed by flow cytometry. Cell cycle was assessed by PI single-staining.6. Leukocyte counts and Bone marrow nucleated cells counts: They were counted by using a hemocytometer.7. Analyse concentrations of LDH,SOD,GSH,MDA:They were detected by using commercial kits8. Analyse concentrations of 8-OHdG: Detect the effects of different concentrations of hydrogen rich solution on cells post irradiation by using 8-OHdG elisa kit.9. Cell DNA Detection: To observe the tail under the fluorescence microscope by single-cell gel electrophoresis analysis in each treatment group. The longer the tail is, the more seriously that DNA is damaged.10. Statistical analysis: For single comparisons, we performed a Student's t-test; for multiple comparisons, we used an analysis of variance (ANOVA). We performed experiments for quantification in a blinded fashion. Data were presented as x ? sd, and statistical significance was declared at the P less than 0.05.Results:1. Synthesis and analyze concentration of hydrogen rich solution: Hydrogen was dissolved in PBS/physiological saline 6 hours under high pressure (0.4 MPa) to a supersaturated level using hydrogen-rich water-producing apparatus produced by our department. The concentration was detected by gas chromatography (Biogas Analyzer Systems-1000, Mitleben, Japan). Concentrations of hydrogen rich solution arrived at supersaturation level (hydrogen rich water: 0.823mmol/L; hydrogen rich saline: 0.809mmol/L; hydrogen rich PBS: 0.814 mmol/L)2. Radiobiological effects of of hydrogen rich solution on AHH-1 cells:2.1 Effects of different concentrations of hydrogen rich solution on the survival rate of AHH-1 cell at the same dose of irradiation: We detected the changes of survival rate of AHH-1 cells 48h after 4Gy irradiation. We found among the 0-0.4mmol/L range, with the increase of drug concentration, the cell viability also increased. When the drug concentration was 0.4mmol/L, the cell survival rates were 84.0%. There is a significant difference compared with the irradiation group which were 63.2% (P<0.05).2.2 Effects of the same concentration of hydrogen rich solution on the survival rate of the AHH-1 cells at different doses of irradiation: Pretreated with hydrogen rich solution, the AHH-1 cells viability was detected by CCK-8 48h after different doses of irradiation. The cell survival rates decreased to varying degrees 48h after irradiation of 1,2,4,8 Gy. However, the survival rate of drug-treated cells were higher than group without drugs. The survival rates of administration group and irradiated group were 74.4% and 48.3% under irradiation of 8Gy.2.3 Effects of hydrogen rich solution administered at different times on the survival rate of the AHH-1 cells at the same doses of irradition : the cell survival rate was detected by CCK-8 48h after 6Gy irradiation. The survival rate of irradiated group was 40.1%. When H₂ was administered before irradiaton,the survival rate increased to 89.8%. Compared with the irradiated group, there was significant difference between the groups(P<0.01).The survival rate of the group in which H₂ was administered after irradiaton was 42.3%. Compared with the irradiated group, there was no significant difference (P>0.05).2.4 Effects of hydrogen rich solution on the early apoptosis rate of the AHH-1 cells: Early apoptosis rate of the cells detected by Annexin V / PI double staining at 4Gy irradiation was 21.5%, while early apoptotic rate of treatment group was 10.19%. There was significant difference between the groups (P<0.01). Apoptosis rate of the cells was also detected by fluorescent staining. Apoptotic rate of treatment group was 26.1%, significantly lower than the apoptotic rate of irradiation group,which is 49.3%. There was significant difference between the groups (P<0.01).2.5 Effects of hydrogen rich solution on LDH leakage of the AHH-1 cells: The result indicated that pretreatment with 0.3mmol/L H₂ before irradiation, LDH leakage of AHH-1 cells was detected 4h after exposed under different doses ofγ-radiation by LDH kit,significantly decreased. LDH activity in cell suspension increased to varying degrees 4h after irradiation of 1,2,4,8 Gy. However, the LDH activity of drug-treated cells were lower than group without drugs.There were significant diffrerences between the groups(P<0.01)3. Radioprotective effects of hydrogen rich solution on murine hematopoiesis3.1 Effects of hydrogen-rich solution on WBC of peripheral blood in mice postγ-ray irradiation: After 2,4 Gy irradiation, the number of peripheral white blood cells in treatment group and irradiation group decreased to different levels within 30 days, but the number of peripheral white blood cells in treatment group which was intraperitoneal injected 0.6mmol / L hydrogen-rich solution, were higher than the irradiation group. (P <0.05)3.2 Effects of hydrogen-rich solution on bone marrow nucleated cells in mice postγ-ray irradiation: After 4 Gy irradiation, the number of bone marrow nucleated cells in treatment group and irradiation group decreased to different levels, but the number of bone marrow nucleated cells in treatment group which was intraperitoneal injected 0.6mmol / L hydrogen-rich solution, were higher than the irradiation group. (P <0.05)3.3 Effects of hydrogen-rich solution on endogenous spleen colony formation units in mice postγ-ray irradiation:After 7 Gy irradiation, On the 11st day, treatment group and irradiation group colonies increased to different levels, but the number of colonies in treatment group which was intraperitoneal injected 0.6mmol / L hydrogen-rich solution, were higher than the irradiation group. (P <0.05)3.4 Effects of hydrogen-rich solution on CFU-GM in mice postγ-ray irradiation:After 7Gy irradiation, bone marrow nucleated cells were isolated and counted to culture. On the 7th day, treatment group and irradiation group colonies decreased to different levels, but the number of colonies in treatment group which was intraperitoneal injected 0.6mmol / L hydrogen-rich solution, were higher than the irradiation group. (P <0.05)3.5 Effects of hydrogen-rich solution on survival rate of mice postγ-ray irradiation: Mice were given hydrogen rich water. 30 days survival was checked and scored daily for 30 days. Survival rate of the mice in hydrogen group was significantly higher than the group without hydrogen. (P<0.05)4. The Mechanism of Radiobiological Effects of Hydrogen-rich Solution on AHH-1 Cells 4.1 The effects of hydrogen-rich solution on the concentration of 8-OHdG in AHH-1 cells after irradiation: The levels of 8-OhdG in the groups irradiated with and without drugs are detected by the 8-OhdG detection kit which reflects the degrees of cell damage. The results showed that 8-OhdG concentrations decreased significantly in the group treated with drugs compared with group treated without drugs in AHH-1 cells. (P<0.05).4.2 The effects of different concentrations of Hydrogen-rich Solution on DNA in AHH-1 cells post irradiation: we detected DNA double-strand break damage of AHH-1 cells through the neutral single cell gel electrophoresis. We analysis the results by CAPS-1 software. The results showed that normal cells in neutral electrophoresis revealing almost no tailing. Irradiation groups with and without drugs have some degrees of tailing. However, cell tailing in the treatment group was significantly decreased.(P<0.01)4.3 Effects of hydrogen-rich solution on SOD and GSH of blood in mice postγ-ray irradiation: 12h after 4,8 Gy irradiation, SOD and GSH in treatment group and irradiation group decreased to different levels, but SOD and GSH in treatment group which was intraperitoneal injected 0.6mmol / L hydrogen-rich solution, were higher than the irradiation group. (P <0.05)4.4 Effects of hydrogen-rich solution on MDA of blood in mice postγ-ray irradiation: 12h after 4,8 Gy irradiation, MDA in treatment group and irradiation group decreased to different levels, but MDA in treatment group which was intraperitoneal injected 0.6mmol / L hydrogen-rich solution, were higher than the irradiation group. (P <0.05)Discussion and Preliminary Conclusions:Radiation damage to the body can be divided into direct damage and indirect damage,The main point of radiation protection is against indirect damage by clearing the radiation-induced free radicals. Recently, it was found that Hydrogen gas has the ability to selectively scavenge hydroxyl radicals. But because hydrogen is inflammable, explosive, which greatly affected the application of its biological effects.To resolve the problem of its flammable, explosive characters, We dissolved hydrogen gas in solution at high pressure (normal saline, PBS, water, etc.) to made the supersaturated solution.We found that hydrogen rich PBS could improve the cell survival rate and reduce early apoptosis of AHH-1 cells after irradiation in cell biology experiments. We also found that hydrogen rich solution also has strong radioprotective effects on murine hematopoietic system. Administrated before irradiation, it could improve peripheral blood WBCs and bone marrow nucleated cells, and also could increase mouse CFU-S and CFU-GM's formation. We furtherly studied the mechanism of the radioprotective effects of hydrogen-rich solution. We found that administrating hydrogen-rich solution before radiation could be effective on reducing the 8-OhdG levels and DNA damage of the AHH-1 cells. It could protect antioxidant enzymes (SOD, GSH) levels in mice, and reduce the formation of lipid oxidation products MDA.Conclusion: hydrogen rich solution has good radioprotection effects on AHH-1 cells and hematopoiesis system.Its mechanism related to its ability of scavenging free radicals and reducing the oxidative stress caused by free radicals. Because hydrogen has no side effects, it has good prospect in the field of radiation protection.