The Role Of FOXO3A In Ionizing Irradiation-Induced Hematopoietic Cell Injury

Objective The possibility of nuclear accidents are increasing today,which can induce damage to human health.Both short-term high dose or long-term low dose radiation exposure can induce hematopoietic system damage.The damage of hematopoietic system induced by ionizing irradiation is mainly manifested as follows:acute bone marrow suppression and long-term bone marrow suppression.Acute bone marrow suppression is mainly due to apoptosis of hematopoietic progenitor cells(HPCs)and a small number of hematopoietic stem cells(HSCs).Its clinical manifestations recently have been more successfully managed by the use of hematopoietic growth factors(HGFs).Long-term bone marrow suppression is mainly due to the senescence of hematopoietic stem cells.At present,there is no effective treatment and the mortality is high.Therefore,the research on the protection and treatment of hematopoietic cell injury induced by irradiation attract the attention of researchers.Irradiation-induced reactive oxyen species(ROS)can promote apoptosis,senescence and abnormal differentiation of hematopoietic stem cells,lead to a decrease in the number and self-renewal ability of hematopoietic stem cells.Under physiological conditions,there is an an effective ROS scavenging mechanism in cells.Forkhead box transcription factor O 3(FOXO3/FOXO3A)plays an important role in the regulation of oxidative stress.The transcriptional activation of FOXO3A mediated by ROS can promote the expression of antioxidant enzymes,scavenge ROS and alleviate oxidative stress injury.Previous studies have found that FOXO3A plays an important role in homeostasis regulation of hematopoietic stem cells.The role and the mechanism of FOXO3A in hematopoietic cells injury induced by irradiation need to be further study.In this study,we used FOXO3A knockout mouse to explore the role of FOXO3A in hematopoietic cells injury induced by irradiation.Methods FOXO3A knockout mouse(FVB/N and C57BL/6J background)were provided and bred in SPF grade laboratory animal center,the homozygous mouse(FOXO3A-/-)and wild mouse(FOXO3A+/+,WT)were identified by polymerase chain reaction(PCR)and bred for subsequent experiments.Mouse of FVB/N background were used in subsequent experiments.In vitro,undifferentiated bone marrow cells(Lin-BMCs)were separated from FOXO3A-/-mouse and WT mouse(FVB/N).Lin-BMCs were respectively exposed to different dose of X-ray(1 Gy,4Gy),the dose rate was 0.9Gy/min,and then cell viability,colony forming unit-granulocyte and macrophage(CFU-GM)and the expression of?HaAX in HPCs/HSCs were detected,to observe the effect of FOXO3A gene knockout on the radiosensitivity of Lin-BMCs and the injury of HPCs/HSCs induced by irradiation.In vivo,FOXO3A-/-mouse and WT mouse(FVB/N)were divided into four groups:wild-type mouse control group(WT),FOXO3A-/-mouse control group(FOXO3A-/-),wild-type mouse irradiation group(WT+IR),and FOXO3A-/-mouse irradiation group(FOXO3A-/-+IR).Mouse received 4Gy X-ray total body irradiation(TBI),the dose rate was 0.9Gy/min,and then the number of peripheral blood,bone marrow cell counts,organ index,bone marrow cell phenotype and CFU-GM of hematopoietic progenitor cells were quantified 2 weeks after TBI,to observe the effect of FOXO3A gene knockout on the sensitivity and recovery of hematopoietic radiation damage.Results FOXO3A gene knockout mouse(FVB/N and C57BL/6J background)were successfully bred.The genotype of FOXO3A knockout mouse were identified by PCR.There are no significant differences in the growth and development between the offspring of FOXO3A gene knockout mouse and the wild-type mouse.Under physiological conditions,Lin-BMCs of FOXO3A-/-mouse showed decreased cell viability,CFU-GM and the expression of ?H2AX compared to those in WT mouse(P<0.05).Also,bone marrow nucleated cell count decreased and the proportion of HPCs increased in FOXO3A-/-mouse(P<0.05),but there was no significant difference in peripheral blood count and organ index(P>0.05).It is showed that cell viability decreased in Lin-BMCs of FOXO3A-/-mouse and WT mouse after 1Gy,4Gy radiation exposure,in a time and dose dependent manner.Further analysis found that,the level of cell viability reduction decreased 11.16%in Lin-BMCs of FOXO3A-/-mouse 6h after 1Gy X-ray exposure when compared to WT mouse(P<0.001),and decreased 16.13%in Lin-BMCs 12h after 4Gy X-ray exposure(P<0.05).And CFU-GM significantly reduced in Lin-BMCs of FOXO3A-/-mouse and WT mouse afterlGy,4Gy ionizing irradiation(P<0.05),in a dose dependent manner However,there was no significant difference in the decrease percentage of CFU-GM between FOXO3A-/-mouse and WT mouse after 1Gy,4Gy X-ray irradiation(P>0.05).DNA damage experimental results showed the expression of ?H2AX increased in HPCs/HSCs of FOXO3A-/-mouse and WT mouse after X-ray exposure,in a dose dependent manner.Further analysis found that the level of the increase in ?H2AX expession significant elevated in HPCs after 1Gy X-ray radiation exposure when compared to WT mouse(P<0.001),and significant elevated in HPCs/HSCs after 4Gy X-ray radiation exposure(P<0.01).In vivo,it is showed that peripheral blood cell counts,bone marrow cell count and spleen index as well as the proportion and number of HPCs increased in FOXO3A-/-mouse and WT mouse 2 weeks after 4Gy X-ray TBI(P<0.05).Further analysis found that the level of the reduction in the proportion of HPCs/HSCs significant decreased in FOXO3A-mouse(P<0.05),but an inhibition in the decrease of bone marrow nucleated cells and CFU-GM(P<0.001)after 4Gy X-ray TBI when comparied to those in WT mouse.However,there was no significant difference in white blood cell count,red blood cell number,hemoglobin content,platelet count,lymphocyte and neutrophil proportion between FOXO3A-/-mouse and WT mouse after 4Gy TBI(P>0.05).Also,There was no significant difference in the decrease of spleen index between FOXO3A-/-mouse and WT mouse after 4Gy TBI(P>0.05).Conclusions Our experimental results showed that:1)Under physiological conditions,FOXO3A gene knockout damaged the homeostasis maintainance of hematopoietic system,caused hematopoietic cells injury.2)Lin-BMCs of FVB/N strain mouse received 1Gy and 4Gy X-ray exposure,FOXO3A gene knockout increased DNA damage of hematopoietic cells.3)When mouse received 4Gy X-ray TBI,FOXO3A gene knockout aggravated the decline in the proportion of HPCs\HSCs,but inhibited bone marrow nucleated cells decrease and the ability of colony formation recession in HPCs,which suggested that FOXO3A gene knockout can destroy the homeostasis of hematopoietic cells and show differences in stress response after ionizing radiation exposure.To sum up,FOXO3A gene knockout aggravated HPCs and HSCs injury induced by irradiation,and has a certain impact on the homeostasis maintenance and radiatosensitivity of hematopoietic cells.The role of FOXO3A in the regulation of hematopoietic system damage induced by radiation exposure and whether it can be used as a target for the prevention and treatment of hematopoietic system damage induced by ionizing irradiation remain to be further studied.