The Effect Of Iron Overload On The Hematopoiesis Of Hematopoietic Stem And Progenitor Cells By Inducing The Generation Of Reactive Oxygen Species

Objective:To establish a model of hematopoietic stem and progenitor cells with iron overload derived from bone marrow (BM) and umbilical cord blood (UCB) cells and explore the effects of oxidative stress (reactive oxygen species,ROS) on the hematopoiesis of hematopoietic stem and progenitor cells with iron overload.Methods:1.The iron overload model was set by adding different concentration of ferric citrate (FAC) into the mononuclear cells derived from BM and UCB. Then cultured for different times, and confirmed the model by detecting labile iron pool (LIP).2.Then detect the changes of ROS, apoptosis and the percentage and the numbers of CD34+,CD33+,GlyA+cells of hematopoietic stem and progenitor cells with iron overload by flow cytometry. The ability of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM and CFU-mix) of these cells were detected by colony culture in vitro.The expression of the ROS-associated proteins,p38MAPK/p-p38MAPK and AKT was detected by western blot.3.The differences of these index were tested after the hematopoietic stem and progenitor cells with iron overload were treated with deferasirox (DFO) or antioxidants (N-acetyl-L-cysteine,NAC and L-Glutathione, GSH).Results:1.BM cells were cultured with the adding of FAC at different concentrations for different times and then we found the LIP increased with time and concentration dependent manners. The intracellular LIP reaches maximum when cultured at400μmol/L of FAC for24hours. The ROS of total cells, leukocytes and erythrocytes increased markedly when cells were cultured at400μmol/L of FAC for24hours, which was up to1.77,1.75and2.12fold separately compared with normal control. DFO and NAC could reduce the ROS efficiently (p<0.05).2. UCB cells were cultured with the addition of FAC at different concentrations for different times. The level of total ROS increased with time and concentration-dependent manners. The intracellular level of ROS peaked when cultured at200μmol/L of FAC for24hours. Cells were treated with antioxidants NAC or GSH after cultured with200μmol/L FAC for24hours. Then the ROS levels of total cells, myeloid cells and erythroid cells decreased markedly versus normal controls. The LIP of total cells, myeloid cells and erythroid cells increased markedly when cells were cultured at200μmol/L of FAC for24hours versus normal controls (p<0.05). NAC and GSH had no effect on the level of LIP.3.The cells treated with FAC promoted strong expression of the p38MAPK, p-p38MAPK and AKT compared with control group.The levels of the expression were suppressed when treated with DFO and NAC.4.The apoptotic rates of the FAC treated cells derived from BM[(24.80±2.99)%] increased significantly compared with normal control [(8.90±0.96)%](p<0.05). The ability of hematopoietic colony forming in FAC treated cells decreased markedly compared with normal control (p<0.05).The percentage of CD34+cells of FAC treated cells [(0.39±0.07)%] also decreased significantly compared with normal control [(0.91±0.12)%](p<0.05). The apoptotic rates of FAC-treated cells derived from UCB[(20.90±3.45)%] increased significantly versus normal controls [(9.20±1.29)%](p<0.05). The capacity of hematopoietic colony forming in FAC treated cells decreased markedly versus normal controls. The percentage and numbers of CD34+, CD33+, GlyA+cells of FAC-treated cells also decreased significantly versus normal controls (p<0.05).And these changes could be recovered by adding NAC,GSH or DFO.Conclusion:The model of hematopoietic stem and progenitor cells with iron overload could be established when excess extracellular iron come into cells.Iron overload could affect the hematopoiesis of hematopoietic stem and progenitor cells by inducing the generation of ROS, which is associated with p38MAPK/p-p38MAPK and AKT.This damage could be decreased by removing excess iron and ROS of these cells.