Effect And Mechanism On Bone Marrow Microenvironment Affected By Iron Overload

Objective: To explore the effect and mechanism of iron overload on hematopoiesis of bone marrow microenvironment.Methods: 1. Iron overload mouse model was established by giving 4Gy γ-ray total body irration and/or injecting iron dextran intraperitoneally. To evaluate the efficacy of the model, hepatic, splenic and bone marrow(BM) iron deposits were assessed and the level of labile iron pool(LIP) of BM-MSCs were detected. 2. The BM-MSCs vialibity was assessed by doubling-time(DT) and cell counting kit 8(CCK8). For osteogenic differentiation, cells were plated in the osteogenic induction medium, for adipogenic differentiation, cells were plated in the adipogenic induction medium. The osteogenic ability was evaluated by ALP activity and Alizarin red. The adipogenic ability was evaluated by oil red staining(ORO). The co-culture system was established to assess the hematopoiesis supporting function of BM-MSCs. Immunohistochemical staining was performed to evaluated the expression of hematopoiesis supporting factor. 3. Cellular ROS level was tested with fluorescent probe 2’, 7’-dichlorofluoresin diacetate(DCFH-DA) and the expression of FOXO3 and PI3 K were determined through Real-time PCR to explore the mechanism.The expression of osteogenic gene(RUNX2, OCN and COL II) and adipogenic gene(PPARg, ap2 and Adipsin were also detected by Real-time PCR. 4. Whether iron overload could be partially reversed by DFX/ NAC treatment.Results: 1. Iron deposits in liver, spleen and BM cells were easy to see and LIP level in BM-MSCs was increased, which showed iron overload model was established successfully. 2. Under IO condition, the cell viability was diminished, and the DT of BM-MSCswas(2.07±0.14) d, which was significantly longer than that of control(1.03±0.07) d(P<0.05). Iron overload could be partially reversed by deferasirox(DFX)/N-acetyl-L-cysteine(NAC) treatment. IO inhibited osteogenic differentiation and increased adipogenic differentiation of BM-MSCs. Compared with the control group, the expression of ALP in the IO group was decreased. After 4 weeks of induction, the mineralized nodules formed in the IO group were less than that of the control group. Besides,lipid accumulation significantly increased in the IO group compared with that of control. After co-administration with DFX and NAC, this effect could be partly alleviated. 3. IO impaired the supporting function of BM-MSCs.Cells obtained at 1week after co-culture with iron-overloaded BM-MSCs showed an expected decrease in CFU-GM, CFU-E, BFU-E, and CFU-mix(P<0.05). 4. Immunohistochemical analysis and Real-time PCR demonstrated that the levels of SCF, CXCL12, and VEGF were lower in bone marrow sections from IO mice compared to those from control mice. Moreover, IO injury effects could be alleviated by iron–chelation and anti-oxidative therapy.Conclusions: 1. The iron-overloaded model could be set successfully by intraperitoneal administration of iron dextran, which could damage hepatic, splenic and bone marrow hematopoietic function. 2. The dose of 4Gy total body irradiation can cause bone marrow damage and iron overload occurred based on this injury model, which could damage bone marrow microenvironment hematopoietic function aggravatingly. 3. Iron overload is closely related to increased ROS level in BM-MSCs. This damage could be decreased by removing excess iron and ROS.