Radioprotective Effects And Mechanism Of Hydrogen On Human Cells And Mice

With the development of science and technology and the progress of society, ionizingradiation is widely used in medical devices, research institutions, nuclear reactors and theirsupport facilities. Radiation therapy is the mainly treatment of malignant tumors. Whilekilling of tumor cells, normal tissue also has been damaged. While nuclear power is using,improper operation will produce severe nuclear contamination, such as the leakage of ThreeMile Island Nuclear Power Plant in the United States, the Chernobyl nuclear accident and theJapan’s Fukushima nuclear accident. At the same time, the using of nuclear weapons inmodern warfare has been a great threat to people’s life. In Kosovo War, the U.S. military useso many depleted uranium, and the number of patients with acute radiation disease risesharply. The threat of radiation has become increasingly widely on human health,and ionizingradiation is recognized as the fourth largest polluter, following the water, air and noisepollution. Ionizing radiation protection, prevention and treatment of various types of radiationsickness have become important issues for today’s need.Intestinal epithelial cells is one of the fastest proliferative organization in vivo, andgastrointestinal tract is more susceptible to radiation damage. In the gastrointestinal tract,intestinal crypt epithelial cells are most vulnerable to radiation-induced apoptosis. Thecondition of the intestinal type of radiation sickness developed rapidly. The patient oftenoccur nausea, diarrhea and other symptoms. Bone marrow hematopoietic system has notchanged in the incidence of intestinal type of radiation sickness, and the necrosis of theintestinal mucosa are the major damage.The heart irradiation can cause chronic damage of the heart pump function and heartdisease. And the fibrosis can be defined as a continuous signal transmitting wound repair inthe organization.Currently, the thiol compound is one of the most effective radiation protectants.However, they have significant side effects, including the route of administration of relativelyhigh toxicity and adverse. Accordingly, we need a safer and more effective anti-radiationtherapy.The ionizing radiation effects on tissue are mainly by increasing the production ofradical-mediated hydroxyl. It was found that hydrogen could reduce cytotoxic reactiveoxygen species selectively, such as OH and ONOO–. We have shown that hydrogen has thepotential anti-radiation effect before. Now, we have studied whether the hydrogen-rich solution has the anti-radiation effect on cells and mice.Contents of research:1. Hydrogen-rich culture medium radioprotective effects of human intestinal epithelialcells: we selected human intestinal crypt epithelial cell line. We observed the followingindicators: HIEC cell survival after gamma-ray radiation. HIEC cell apoptosis rate andmorphological changes after the gamma-ray radiation. DNA strand breaks of HIEC cells aftergamma-ray radiation.2. Hydrogen-rich solution effects on the irradiated intestine of mice:â‘ Changes ofendogenous antioxidants in the small intestine after the gamma-ray radiationâ‘¡Changes ofoxidative damage after the gamma-ray radiation.â‘¢Pathological changes of the smallintestine after gamma-ray radiationâ‘£Changes of apoptosis of intestinal cells aftergamma-ray radiation.⑤Changes of the hydrogen concentration in the small intestine.3. Hydrogen-rich culture medium radioprotective effects on normal human umbilicalvein endothelial cells. We selected Human Umbilical Vein Endothelial Cells. We observed thefollowing indicators: HUVEC cell survival after gamma-ray radiation. HUVEC cell apoptosisrate and morphological changes after the gamma-ray radiation.4. Hydrogen-rich solution effects on the irradiated heart of mice.â‘ The survival rate ofgamma-ray radiation Balb/c mice after30days.â‘¡Changes of endogenous antioxidants inheart after the gamma-ray radiation.â‘¢Changes of oxidative damage after the gamma-rayradiation.â‘£Pathological changes of heart after gamma-ray radiation.⑤Changes ofmyocardial fibrosis after the gamma-ray radiation.â‘¥Changes of DNA strand breaks of heartafter gamma-ray radiation5. The radioprotective mechanism of hydrogen-rich solution:â‘ The use of fluorescencedetection to detect the effect of reducing hydroxy radical by Hydrogen-rich solution.â‘¡Changes of caspase-3activity of the small intestine of mice after radiation.â‘¢Hydrogen-richsolution changes the expression of apoptosis-related proteins.â‘£Hydrogen-rich solutionchanges the level of genes by real-time PCR.6.The effect of hydrogen-rich solution of HUVEC gene expression.Methods:1. The H₂-rich saline or H₂-rich culture solution preparation: physiological saline or aculture medium placed in a sealed bag filled with a sufficient amount of hydrogen pressure.H₂-rich culture fluid stored in the refrigerator at4degrees, changed once a week to ensure thatthe hydrogen concentration of0.6mmol/L. 2.Cells: HIEC and HUVEC cells were maintained in DMEM with10%FBS. Andincubated at37°C,5%CO2incubator.3. Cells and Mice radiation models: gamma-ray,1.0Gy/min.4. Radiation-induced hydroxyl radical elimination: Using the the HPF probe,fluorescence intensity was observed under a fluorescence microscope analysis.5. Detection of cell survival after radiation: CCK-8assay kit and crystal violet stainingcell clones were used after radiation. Hoechst/PI staining and DAPI/PI staining were used forthe detection of nuclear morphology and cell viability.6. Apoptosis rate detection: After Annexin V/PI staining, the cells were analysed by flowcytometry.7. DNA damage detection:Comet assay for the detection of DNA fragmentation.8. Detection of the hydrogen concentration of the small intestine: The hydrogenconcentration of different time points were detected by the hydrogen concentration detector.9. Determination of oxidation-related indicators: Using SOD, GSH, MDA, PC and8-OHdG kit to detect.10. Changes in the structure of the small intestine of mice heart tissue: Using HE stainingto observe pathological changes.11. Intestinal cell apoptosis detection: TUNEL staining detection kit.12. The detection of apoptosis-related proteins: The apoptosis-related protein expressionsof intestinal epithelial cell after radiation were detected by western blot analysis.13. Gene expression after gamma-ray irradiation: The gene expressions of umbilical veinendothelial cells after gamma-ray irradiation were detected by real-time PCR.14. The effect of hydrogen-rich solution of HUVEC gene expression: Affymetrix genechip.15. Statistical analysis: For comparison of the difference between the two groups, weused the Student’s t-test. For comparison of the difference between more than two groups, ananalysis of variance（ANOVA） was used. Data were described as x±sem, and statisticalsignificance P was less than0.05.Results:1. Hydrogen-rich culture medium pretreatment reduce radiation damages of humanintestinal epithelial cell.HIEC cells were compared with radiation alone group after each dose gamma-rayradiation.The Hydrogen-rich culture medium can significantly improve the HIEC cell survival and cloning efficiency after irradiation. Hydrogen-rich culture medium can also reduce therate of apoptotic cells after irradiation. Compared with radiation alone group, the comet taillength of hydrogen-rich culture medium pretreatment HIEC cells was significantly reduced.These results suggest that hydrogen-rich culture medium has good radiation protection onHIEC cells.2. Hydrogen-rich saline pretreatment reduce radiation damages of the smallintestine of mice.The peripheral blood SOD, GSH activities of hydrogen-rich saline group mice wassignificantly higher than the irradiation-alone group. And hydrogen-rich saline can significantlyreduce the concentration of MDA,8-OHdG and PC in the intestinal tissue of mice after radiation.The intestinal tissues were stained by HE staining, and Chiu score statistics show thathydrogen-rich saline treatment can significantly reduce the radiation-induced small intestinalmucosal injury. TUNEL staining showed that hydrogen-rich saline can reduce the apoptosis ofintestinal epithelial cell.3. Hydrogen-rich culture medium pretreatment reduce radiation damages ofHUVEC cell.The hydrogen-rich culture medium can significantly improve the HUVEC cell survivaland cloning efficiency after irradiation. HUVEC cells were stained by Hochest33342/PIstaining kit. The blue fluorescence was enhanced and the number of bright red cells wereincreased in the irradiation alone group, whereas hydrogen-rich culture medium pre-treatmentcan significantly reduce the the HUVEC cell apoptosis and death.4. Hydrogen-rich saline pretreatment reduce radiation damages of the heart ofmice.The heart homogenates SOD, GSH activities of hydrogen-rich saline group mice wassignificantly higher than the irradiation-alone group. And hydrogen-rich saline can significantlyreduce the concentration of MDA,8-OHdG in the heart tissue of mice after radiation. Thepathological changes of heart were observed by the HE staining method1day and100days afterradiation. In1day, the control group and the hydrogen-rich saline group did not changesignificantly. And in100days, the control group had myocardial degradation, whereas nosignificant pathological changes in the hydrogen-rich saline group. The MASSON result showedthat hydrogen-rich saline can reduce the myocardial fibrosis in mice in100days afterradiation.5. Hydrogen-rich solution radioprotective effects mechanism. 5.1Scavenging effect of hydrogen-rich culture medium on the radiation generatedhydroxyl radical. We used fluorescence microscope to observe the change in fluorescenceintensity after HPF staining. It was found that the fluorescence intensity in the hydrogen-richculture medium group was significantly lower than the irradiation group. It shows thathydrogen-rich culture medium has a strong effect of scavenging the radiation generatedhydroxyl radical.5.2Hydrogen-rich saline changes the expression of caspase3activity in the smallintestine of irradiated mice. The test results showed that caspase3activity hydrogen-richsaline group in the small intestine of mice within each time point decreased significantly thanthe control group. The result shows that hydrogen-rich saline can reduce apoptosis caused byradiation, and reduce radiation-induced intestinal injury.5.3Hydrogen-rich medium pretreatment of human intestinal epithelial cells changes theexpression of apoptosis related proteins after radiation. The protein of HIEC cells wasextracted24h after12Gy radiation for Western blot. The results showed that hydrogen-richmedium group significantly up-regulated the expression of Bcl-2, and Bax expression wassignificantly down-regulated.5.4Detect the gene expression after gamma-ray irradiation in HUVEC cells: HUVECcells RNA was extracted4h after4Gy γ ray irradiation. After reverse transcription, Real-timePCR was detected. The results showed that Bcl-2in the hydrogen-rich group was significantlyincreased, while the generation of Bax was significantly reduced.6. Gene expression profiling: Hydrogen-rich medium could regulate cell cycle, reduceapoptosis and death, reduce inflammation, increase cellular resistance, improve the ability ofcell proliferation.Discussion and Conclusions:Ionizing radiation can cause serious injury on the body. The role of it divided into twokinds. One is the direct effect, which causes DNA strand breaks.And the other is indirecteffect, which can can produce large amounts of hydroxyl radicals, causing lipid peroxidation,oxidative DNA damage and protein carbonylation, ultimately leading to apoptosis and death.It was found by Ohsawa et al. that hydrogen could reduce cytotoxic reactive oxygen speciesselectively, such as hydroxyl radical. Our previous studies also proved hydrogen-rich solutionhas a good role in radiation protection. In the experiment on the HIEC and HUVEC cells, wefound that hydrogen-rich culture medium pre-treatment can increase the survival rate of thecells, enhance cell proliferation and reduce the level of apoptosis after radiation. Hydrogen-rich saline can significantly improve the survival rate of mice. It can improve thebody’s antioxidant capacity, reduce mouse intestinal epithelial injury and apoptosis ofintestinal epithelial cells. It can also reduce the radiation-induced heart damage, and inhibitethe myocardial cells fibrosis after radiation. According to the study, we found that thehydrogen-rich solution can effectively remove the radiation generated hydroxyl radicals. Italso can prevent lipid peroxidation, protein oxidation and DNA peroxide, and can regulate theexpression of apoptosis-related proteins. Finally, hydrogen-rich solution can inhibit cell deathand apoptosis.Conclusion: Hydrogen-rich solution has a good radiation protective effect on humanintestinal epithelial cells, human umbilical vein endothelial cells and mice small intestine andheart. Its mechanism of action may be associate with effectively removing radiation generatedhydroxyl radicals and the regulation of apoptosis-related protein expression. Thus, because ofits safety and with no side effects, the rich H₂solution will become a new radioprotectors.