Radioprotective Effects Of Hydrogen On Human Liver Cells And Liver Of Mice

In recent decades, the extensive utilization of atom energy in many fields such asmilitary, civil industry, agriculture, medicine etc brings about great benefits for people, but inthe meantime, how to protect human health and safety from severe nuclear radiation become acritical problem in the world. The former memory of accident in Soviet Chernobyl nuclearpower plant with disastrous consequences to the human has not completely eliminated, theFukushima nuclear power plant radioactive nuclear leak caused by a strong earthquake inJapan in March2001caused a worldwide panic of radiation again.So human have to thinkabout how to effective use of the atom energy while ensuring that the health of human is notcompromised.The liver is an important organ with numerous functions including the production of bile,metabolism of ingested nutrients, elimination of waste products, glycogen storage, and proteinsynthesis. However, on one hand the normal liver cell damage in the radiation therapy (RT) ofliver cancer cannot be ignored and is also the major limitation to the use of radiation in thetreatment of intrahepatic cancers is radiation-induced liver toxicity. And on the other hand theliver is often incidentally irradiated during RT for tumors in the upper abdomen,right lowerlung, distal esophagus, or whole abdomen or whole-body RT. It is a novel scientific topic forradiologists to look for high-effective radiation protectants with low toxicity to protect normaltissues from radiation induced damages.In2007, Ohsawa et al. found that molecular hydrogen could selectively reduce cytotoxicreactive oxygen species in vitro and exert therapeutic antioxidant activity. From then on,research on hydrogen set off a worldwide upsurge. Hydrogen could selectively reducehydroxyl radicals excited our interest, because most of irradiation induced injury was causedby hydroxyl radicals. As we know, the damage of radiation mainly comes from the γ ray andneutron ray, and the biological effects are divided into two ways: the first is direct effect andthe second is indirect effect, which accounts for about70%-80%of the damages. Since theindirect effect mainly due to the abundant free radicals caused from radiation, so blocking andscavenging of free radicals become our most important protecting strategy. In2009, theradioprotective effects of hydrogen had been demonstrated by our department in recentstudies. We dissolved H2into saline, PBS or medium then under0.4MPa pressure more than6hours to make it reach beyond saturation levels. This H2-rich solution is safe and effectiveas a radioprotectant. Nowadays, with the development of modern biomedicine, the radiation therapy hasbecome an important therapeutic method for malignant diseases, but the damages to somenormal tissues induced by radiation limit the application of radiotherapy. In fact, exploitationthe ideal radiation protectants has always been emphasis and obstacles in the field of radiationprotection research.Recent research shows that H2suppresses hepatic injury caused byischemia-reperfusion through reducing oxidative stress. And H2was reported as a novelantioxidant which is safe, effective, and with no known side effects, being widely used inmedical applications. In the present study, we investigated whether H2could protect liverfrom radiation-induce injury in vitro and in vivo.Contents of study:1. Hydrogen-rich solution production: Hydrogen was dissolved in physiologicalsaline/medium for6h under high pressure (0.4MPa) to a supersaturated level using ahydrogen-rich water-producing apparatus produced by our department.2. Radioprotective effects of Hydrogen-rich solution on cells: we selected human normalliver cell line HL-7702(L-02) cells cells to make the dose-survival curve of cells irradiated atdifferent doses of irradiation. We observed the following subjects: Radioprotective effect ofHydrogen-rich solution on cells irradiated with γ-ray reflected by cell viability, apoptotic.3.Radioprotective effects of Hydrogen-rich solution on the liver of mice:①Radioprotective effect of the Hydrogen-rich solution on liver function.②Radioprotectiveeffect of the Hydrogen-rich solution on architecture of liver.③The effect of theHydrogen-rich solution on the apoptosis of hepatocyte.4. Preliminarily exploring the mechanism of radioprotective effects of hydrogen-richsolution:①Using fluorescein to detect the effect of reducing hydroxy radical by H2-richsolution after radiation②The effect of the Hydrogen-rich solution on the level of PC and8-OHdG.③Analyze the effect of H2on the Bcl-2family of proteins by western blotting.④Thehydrogen concentration in the liver of mice after intraperitoneal injection of H2-rich solution.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 ofproducing hydrogen-rich solution.2.Cells: Human normal liver cell line HL-7702(L-02) cells were obtained from the CellResource Center (Shanghai Institutes for Biological Sciences, China) and were maintained inRPMI1640with10%FBS and at37°C in a5%CO2humidified chamber. 3. Irradiation: different doses of γ-rays by Co-60.4. Detect the effect of reducing hydroxy radical by H2-rich solution: Detection ofirradiation-induced OH by fluorescein.5. Determination of cell viability: We assessed the survival cells by using fluorescencedouble staining Hoechst33342and PI.6. Apoptosis analysis: The cells were stained by using fluorescence staining and AnnexinⅤ/PI double staining, and analysed by flow cytometry.7. Detect the level of H2: The concentration was detected by gas chromatography in liverof mice8. Evaluation of hepatic function: ALT and AST activities were assessed by anautomated procedure in Department of inspection.9. Analyze concentrations of8-OHdG and PC: Detect the effects of hydrogen richsolution on liver of mice post irradiation by using8-OHdG and PC elisa kit.10. Histopathological analysis: Stained with hematoxylin–eosin (HE) used forhistopathological examination and used electron microscope to observe the microscopicchanges in the structure.11. Apoptosis of hepatocyte: Detected by TUNEL stain.12. The expression of apoptosis associated protein: Detected by western blotting13. Statistical analysis: For single comparisons, we performed a Student’s t-test; formultiple comparisons, we used an analysis of variance (ANOVA). We performed experimentsfor quantification in a blinded fashion. Data were presented as x±sd, and statisticalsignificance was declared at the P less than0.05.Results:The hydrogen-rich solution used in this date, using hydrogen-rich water-producingapparatus produced by our department. The concentration was detected by gaschromatography (Biogas Analyzer Systems-1000, Mitleben, Japan). Concentrations ofhydrogen rich solution arrived at supersaturation level (hydrogen rich water:0.823mmol/L;hydrogen rich saline:0.809mmol/L; hydrogen rich PBS:0.814mmol/L).1. Radioprotective effects of Hydrogen-rich solution on L-02cells:We confirmed that H2can preserve cell viability by manually counting thedouble-stained cells with a fluorescence microscope at24h after irradiation. And H2also canreduce the apoptotic in irradiated cells which detected by flow cytometry analysis. These datasuggested that H2can protect cultured cells from IR damage by increasing cell viability and reducing the apoptotic.2. Radioprotective effects of Hydrogen-rich solution on the liver of mice2.2Effects of hydrogen rich solution on irradiation liver of mice: To determine the changesin hepatic function after irradiation, we analyzed the levels of serum ALT and AST. Afterexpose to IR, serum ALT and serum AST both significantly increased, whereas pretreatedwith H2-rich saline decreased the elevations levels of them. Hepatic architectural changesinduced by IR were studied by HE-stained and electron microscope to observe themicroscopic changes in the structure. In the radiation control group damages were foundamong the architecture of liver together with pyknotic cells. However pretreated with H2-richsaline not only improved hepatic architectural but also decreased the number of pyknotic cells.The TUNEL result showed that H2can reduces apoptosis of hepatocyte.3. The mechanism of radioprotective effects of hydrogen-rich solution3.1H2reduces OH induced by irradiation: We assessed the green fluorescence producedby oxidized HPF which is a marker of OH.After exposed to irradiation, the fluorescencesignals increased in cells of normal medium. While the signals significantly decreased amongcells in H2-rich medium. Thus, we confirmed that H2can reduce OH induced by irradiation inliving liver cells.3.2H2reduced oxidative stress injury induced by radiation: We analyzed the levels of8-OHdG and PC to determine the oxidative stress injury induced by IR. The levels of8-OHdG and PC both significantly increased after exposed to radiation. However pretreatedwith H2-rich saline could decrease the elevations levels of them.3.3Regulative effects of H2against apoptosis induced by IR in the cells: In the cells wedetermined the expression of Bcl-2family proteins. In our study we found that irradiationupregulated the expression of proapoptotic protein Bax and downregulated the expression ofantiapoptotic protein Bcl-2. H2treatment significantly reduced the apoptotic cell death whichdepends on the intrinsic mitochondrial apoptotic pathway by reduce the ROS to regulate theactivation of Bcl-2family proteins.3.4Regulative effects of H2against apoptosis induced by IR in the liver of mice: In theliver of mice we determined the expression of Bcl-2family proteins. In our study we foundthat irradiation upregulated the expression of proapoptotic protein Bax and downregulated theexpression of antiapoptotic protein Bcl-2. H2treatment significantly reduced the apoptotic celldeath which depends on the intrinsic mitochondrial apoptotic pathway by reduce the ROS toregulate the activation of Bcl-2family proteins. 3.5Detect the level changes of H2in the liver of mice: After intraperitoneal injection ofH2-rich solution, the hydrogen concentration in the liver of mice came to the top at5min andat15min it came back to the normal.Discussion and Conclusions:The major limitation to the use of radiation in the treatment of intrahepatic cancers isradiation-induced liver toxicity. Radiation damage to the body can be divided into directdamage and indirect damage，The main point of radiation protection is against indirectdamage by clearing the radiation-induced free radicals. Recently, it was found that Hydrogengas has the ability to selectively scavenge hydroxyl radicals. And our department had provedthat the hydrogen-rich solution has a good radioprotective effect in previous studies, played asignificant role in the protection of the mouse hematopoietic system and intestinal tissue andno side effect. So in the present study, we hope to get the same result in protect effect on liverfrom radiation-induce injury in vitro and in vivo. In cells, H2can reduce OH induced byirradiation in living liver cells and protected cultured cells from IR damage by increasing cellviability and reducing the apoptotic. We also found that hydrogen rich solution also has strongradioprotective effects on the liver of mice. H2can protect hepatic function changes andattenuate the damage of architecture in liver induced by IR. And also H2reduced apoptosis ofhepatocyte. We furtherly studied the mechanism of the radioprotective effects ofhydrogen-rich solution. We found that administrating hydrogen-rich solution before radiationcould be effective on reducing the8-OhdG and PC levels in the liver of mice. The other handH2treatment significantly reduced the apoptotic cell death which depends on the intrinsicmitochondrial apoptotic pathway by reduce the ROS to regulate the activation of Bcl-2familyproteins.Conclusion: we showed that H2, safety and with no known toxic side effects, effectivelyhepatic-protective against oxidative stress and damage caused by IR in cells and liver of mice.H2dissolved in saline could easily be delivered intravascularly and rapidly across cellmembranes reacted with cytotoxic ROS. Thus, with development of H2treatment will relievethe radiation-induced liver disease in future.