The Protective Effect And Mechanism Of Antioxidants On Radiation-induced Damage Of Hematopoietic System In Mice

Objective The body damage caused by ionizing radiation is through either direct or indirect effects.The direct effect is that the great energy produced by ionizing radiation acts directly on the biological macromolecules and results in damage.The indirect effect is that ionizing radiation first ionized the water in human body and generated a large number of reactive oxygen species which cause damage to biological macromolecules and biofilms.The hematopoietic system is especially sensitive to total body irradiation(TBI),and myelosuppression is one of the major effects of TBI.Astaxanthin(ATX)is a powerful natural antioxidant with low toxicity which can effectively scavenge reactive oxygen species.The effect of astaxanthin on hematopoietic system injury after TBI was investigated in our study.Methods C57BL/6 mice were divided into five groups:control,4Gy alone and 4Gy+25mg/kg,50mg/kg,100mg/kg ATX.The numbers of peripheral blood cells and bone marrow cells were counted by hemocytometer to explore the optimal concentration of astaxanthin as C57BL/6 mice were randomly divided into four groups:control,ATX,TBI and TBI+ATX,which were administered with ATX 3 days before 4Gy or 6Gy irradiation and up to 7 days after irradiation.The effect of ATX on acute and persistent hematopoietic systems injury caused by radiation was examined 12 or 60 days after TBI.The numbers of peripheral blood cells and bone marrow cells were counted by hemocytometer.Flow cytometry was used to detect the proportion of hematopoietic progenitor cells and hematopoietic stem cells,the level of intracellular ROS,DNA damage and apoptosis,the expression of NRF2-related proteins and cytochrome C.Immunofluorescence staining was used to detect NRF2 translocation.Western blot analysis was used to evaluate the expression of NRF2 and apoptotic-related proteins.Enzymatic activity assay kits were used to analyze SOD,CAT,and GPX1 activities.Colony formation ability of HPC was measured by CFU-GM,and the self-renewal capacity of HSC was analyzed by bone marrow competitive regeneration assay.Results Compared with TBI+25mg/kg and 100mg/kg astaxanthin,50mg/kg astaxanthin could significantly increase the numbers of peripheral blood cells and bone marrow cells after irradiation which demonstrated the best radio-protective effect in our experimental condition.In addition,compared with TBI,50mg/kg astaxanthin provided a radio-protective role by increasing the numbers of peripheral blood cells and bone marrow cells and accelerating the self-renewal and proliferative abilities of hematopoietic stem and progenitor cells in mice with acute and persistent hematopoietic injury.The radio-protective effect of ATX was probably attributable to the scavenging of ROS and the reduction of DNA damage and cell apoptosis.These changes were associated with increased activation of Nrf2 pathway and regulation of apoptotic related proteins.Conclusion Astaxanthin protects bone marrow cells of mice against radiation induced acute and persistent hematopoietic injury by reducing the level of intracellular ROS and cell apoptosis,which provides experimental basis for exploring a new radio-protective candidate drugs with low toxicity and high efficiency.Objective The body damage caused by indirect effect of ionizing radiation is mainly attributed to the ionization of water and the generation of large amounts of reactive oxygen species(ROS),which result in the DNA double strand breaks and oxidative damage.The hematopoietic stem cell is especially sensitive to ROS produced by total body irradiation(TBI)and myelosuppression is one of the major effects of TBI.Unfortunately,there is no safe and effective radio-protective agent so far.Hydrogen-rich water(HW)contains high concentrations of H2,which plays an antioxidative effect by specifically react with hydroxyl radical(·OH)-one of strong oxygen free radicals.The aim of this study was to explore the protective effect of hydrogen-rich water on radiation-induced hematopoietic stem cells injury and its underlying mechanisms.Methods C57BL/6 mice were randomly divided into four groups:control,HW,4Gy total body irradiation and TBI+HW.All mice were administrated HW by gavage 10 minutes before and up to 7 days after TBI and sacrificed 15 days after TBI.Hemocytometer was used to count the number of peripheral blood cells and bone marrow cells.Flow cytometry was used to detect the proportion of hematopoietic progenitor cells(HPCs)and hematopoietic stem cells(HSCs),the level of intracellular ROS,cell cycle,cell apoptosis and NRF2 related proteins.Immunofluorescence staining was used to detect y-H2AX and 8-oxoG.Western blot analysis was used to evaluate the expression of P21.Enzymatic activities assay kits were used to analyze SOD,GPX and ·OH activities.Colony formation ability of HPC was measured by CFU-GM,and the self-renewal capacity of HSC was analyzed by competitive repopulation assay.Results HW may increase the 30-day survival rate of mice exposed to lethal dose TBI.Compared with TBI,the number of peripheral blood cells wsa increased and the skewed differentiation of leukocyte was improved.The number of bone marrow cells increased by 35.4%.CFU-GM of HW increased 47.8%.Bone marrow competitive repopulation assay showed that HW improved the self-renewal ability of hematopoietic stem cells.The radio-protective effect of HW was probably attributable to the scavenging of ROS,the improvement of cell cycle and the reduction of cell apoptosis.These changes were associated with increased activation of Nrf2 and its downstream anti-oxidative proteins and regulation of apoptotic related proteins.Conclusions Hydrogen-rich water may be used as a potent therapeutic agent to protect the hematopoietic stem cells aginst TBI-induced myelosuppression according to reduce ROS,cell cycle arrest and apoptosis in mice.