The Protective Effect Of Theaflavin On Ionizing Radiation-Induced Hematopoietic System Injury

Objective Nuclear technology has been widely applied in many fields,such as industry,energy,environment,food,agriculture,medicine,military and so on.However,radiation exposure caused by nuclear accidents threatens social stability and seriously damages human life and health.Acute radiation injury of hematopoietic system is the main reason of death caused by nuclear accidents,is also a major complication after radiotherapy for tumor patients.Ionizing radiation-induced hematopoietic stem cell(HSC)injury is the key cause of the injury of hematopoietic system.Theaflavin(TF)is a polyphenol compound extracted from black tea.It can scavenge reactive oxygen species(ROS)and regulate the endogenous antioxidant system in cells.The purpose of this study was to investigate the effects of TF on ionizing radiation-induced hematopoietic system injury.Methods1.Experiments about screening administration does:C57BL/6 mice were randomly divided into control group,total body irradiation(TBI)group,TBI+25mg/kg TF group,TBI+50mg/kg TF group,TBI+100mg/kg TF group.All the irradiated mice were exposed to 4Gy TBI once.1 days before irradiation and up to 7 days after irradiation,mice were daily treated with different doses of TF solution or the equal volume vehicle.On the 15th day after irradiation,the body weight and organ weight of mice were measured,the organ index was calculated,the total number of thymocytes was counted,and the peripheral blood cell counts were detected by hematology analyzer.2.Experiments about thymus,peripheral blood and bone marrow:according to the optimum dosage,C57BL/6 mice were randomly divided into control group,TF group,TBI group and TBI+TF group.All the irradiated mice were exposed to 4Gy or 6.5Gy TBI once.On the 15th day after 4Gy TBI thymus was separated,then hematoxylin and eosin were stained to observe the morphological changes of thymus,and the CD4CD8 T cell subsets in the thymus were detected by flow cytometry.The percentages of B cells,T cells and myeloid cells in peripheral blood were detected by flow cytometry.The the total number of bone marrow cells in unilateral femoral was counted,and the frequencies of hematopoietic stem and progenitor cells in bone marrow were detected by flow cytometry.Hematoxylin and eosin were stained to observe the morphological changes of bone marrow.3.Endogenous spleen nodules:C57BL/6 mice were randomly divided into control group,TF group,7Gy group and 7Gy+TF group.The spleen of mice was separated on the 7th day after irradiation,and the number of endogenous spleen nodules was observed and counted.4.Competitive bone marrow transplantation:C57BL/6 mice(CD45.1),as donor mice,were randomly divided into control group,TF group,4Gy group and 4Gy+TF group.On the 15th day after irradiation,bone marrow cells from donor mice were mixed with bone marrow cells from competitors C57BL/6 mice(CD45.1+ CD45.2+),and then the mixed cells were transplanted into the corresponding recipicents C57BL/6 mice(CD45.2)which had received 8Gy TBI.The donor chimerism in the peripheral blood of recipicents and the percentage of B cells,T cells and myeloid cells in donor-derived cells were detected by 2 months after transplantation.5.Experiments about apoptosis and oxidative stress:C57BL/6 mice were randomly divided into control group,TF group,4Gy group and 4Gy+TF group.On the 15th day after irradiation,the apoptosis rate and ROS level of hematopoietic stem and progenitor cells were detected by flow cytometry.In addition,Lineage-c-kit+ cells were sperated from bone marrow;the expression of yH2AX and 8-oxoguanine(8-oxoG)in Lineage-c-kit+ cells was examined by immunofluorescence;the expression of nuclear factor E2 related factor 2(NRF2)signaling pathway and superoxide dismutase 2(SOD2),glutathione peroxidase(GPX1)and NADPH oxidase 4(NOX4)in Lineage-c-kit+ cells was examined by Western Blot.6.Experiments about senescence and peripheral blood:C57BL/6 mice were randomly divided into group control,6.5Gy group,and 6.5Gy+TF group.2 months after irradiation,the percentages of B cells,T cells and myeloid cells in peripheral blood and the fruquency and ROS level of LSKs in bone marrow were detected by flow cytometry.Lineage-c-kit+cells were sperated from bone marrow,and the percentage of P-Galactosidase((3-Gal)+ cells and the mMRA level of p16Ink4a in Lineage-c-kit+ cells were detected.7.Experiments about survival rate:wild type(WT)C57BL/6 mice and Nrf2-/-C57BL/6 mice received 7.2Gy TBI,and were daily administered with 50mg/kg TF or equal volume of vehicle 1 days before irradiation and up to 7 days after irradiation.The survival rate of mice in 2 months was observed and recorded.8.Experiments about Nrf2-/-mice experiment:Nrf2-/-mice were randomly divided into control group,TF group,4Gy group and 4Gy+TF group.On the 15th day after irradiation,the ROS level and frequency of hematopoietic stem and progenitor cells of Arf2-/-mice were detected by flow cytometry.9.Statistical analysis:the measurement data are expressed as Mean±SEM.For more than 2 sets of data,the differences between groups were analyzed by one-way ANOVA,followed by a post-hoc student Newman-keuls test for multiple comparisons.The idenpendent-sample t test was uesd to compare two sets of data.Kaplan-Meier meathod was used for survival analysis and Log-rank test was used to compare the survival curves.Differences were considered significant at P<0.05.Results1.50mg/kg TF provided the best protection for body weight,organ index,total number of thymus cells and peripheral blood cell counts in irradiated mice.50mg/kg was used as the dosage of TF in the follow-up experiments.2.TF attenuated the morphological changes of the irradiated thymus,promoted the recovery of CD4CD8 T cell subsets in irradiated thymus,and increased the number of endogenous splenen nodules in irradiated mice.3.TF attenuated the myeloid skewing in irradiated mice,increased the number of bone marrow cells,LSKs and HPCs in irradiated bone marrow,and mitigated the morphological changes of bone marrow in irradiated mice.4.TF enhanced the engraftment ability of irradiated bone marrow cells.5.TF could not change the apoptosis rate of hematopoietic stem and progenitor cells in irradiated bone marrow.But it reduced the oxidative stress and the accumulation of DNA damage in irradiated hematopoietic stem and progenitor cells.6.TF activated the NRF2 signaling pathway,and up-regulated the expression of SOD2,GPX1 and down-regulated the expression of NOX4 in irradiated hematopoietic stem and progenitor cells.7.TF inhibited ionizing radiation-induced HSC senescence.8.TF improved the survival of WT irradiated mice,but it did not improve the survival of Nrf2-/-irradiated mice.9.TF could not increase the number and reduce the oxidative stress of hematopoietic stem and progenitor cells in the bone marrow of Nrf2-/-irradiated mice.Conclusion TF can ameliorate ionizing radiation-induced hematopoietic system injury,and it has the potential to be a radioprotective agent.