Effect And Mechanism On The Hematopoiesis Of Bone Marrow Affected By Iron Overload

ObjectivesTo explore the effect and mechanism on the hematopoiesis of bone marrow affected by iron overload.Methods1. Iron overload mouse model was established by giving4Gy γ-ray total body irration and/or injecting iron dextran intraperitoneally.To confirm the efficacy of the model, hepatic, splenic and bone marrow (BM) iron deposits were assessed and the level of Labile iron pool (LIP) of BMMNCs were detected.2. Peripheral blood cells and the number of BMMNCs and frequencies of different linage cells in BMMNCs were monitored.Hematopoietic colony-forming counts (CFU-E, BFU-E, CFU-GM and CFU-mix) were used to measure the clonogenic function of HPCs. Single-cell colony assay and competitive transplantation experiment were used to observe hematopoietic stem cell function.3. Cellular ROS level was tested using fluorescent probe2’,7’-dichlorofluoresin diacetate (DCFH-DA) and the expression of NOX4and GPX were determined through Real-time PCR to explore the mechanism.4. Whether iron overload could be partially reversed by DFX/NAC treatment.Results1. Iron deposits in liver, spleen and BM cells were easy to see and LIP level in BM was increased, which showed iron overload model was established successfully.2. Compared with control group, the number of BMMNCs and the frequencies of different linage cells in iron overload group had no significant difference. The frequencies of HSCs and LT-HSCs, Hematopoietic colony-forming counts and Single-cell colony assay (32.00±5.25vs.51.00±4.24) in iron overload group caused a sustained reduction (P<0.05). Competitive repopulation assay revealed that the donor cell engraftment was decreased by1.46fold of total cells,2.41fold of myeloid cells,1.68fold of T cells, respectively(P<0.05). Iron overload elevated reactive oxygen species (ROS) levels. ROS level in iron overload BM was increased by1.42fold in erythroid cells and1.43fold in BMMNCs,1.94fold in HPCs and3.48fold in HSCs, respectively (P<0.05). Iron overload could be partially reversed by deferasirox (DFO)/N-acetyl-L-cysteine (NAC) treatment.3. Compared with control group, the number of platelets and BMMNCs and the frequency of erythroid cells and myeloid cells were decreased.Moreover, hematopoietic colony forming units, Single-cell cloning counts (12.50±1.00vs.16.00±0.80) and hematopoietic reconstitution capacity were decreased significantly in irradiation group. Compared with irradiation group, the number of platelets and the frequency of erythroid cells and myeloid cells were decreased.Moreover, hematopoietic colony forming units,Single-cell cloning counts (4.75±1.10vs.12.50±1.00) and hematopoietic reconstitution capacity were decreased significantly in irradiation+iron group (P<0.05). Compared with irradiation group, ROS level was increased by1.94fold in BMMNCs,1.93fold in erythroid cells and2.70fold in myeloid cells, respectively (P<0.05).Conclusions1. The iron-overloaded model could be set successfully by intraperitoneal administration of iron dextran. It could damage hepatic, splenic and bone marrow hematopoietic function,while no significant difference was observed in peripheral blood count.2. The dose of4Gy total body irradiation can cause bone marrow damage and iron overload occurred based on this injury model, which could damage bone marrow hematopoietic function aggravatingly. But in the early time iron overload can be compensatory by extramedullary hematopoiesis.3. Iron overload is closely related to increased ROS level in BMMNCs. This damage could be decreased by removing excess iron and ROS. The findings would be helpful to further study the injury mechanism of iron overload on the hematopoiesis of bone marrow.