The Bone Marrow-Derived Mesenchymal Cells Were Injured By Iron Overload through Catalyzing the Production of Reactive Oxidative Stress in Mice

Iron overload (IO) is a pathological phenomenon characterized by excessive iron depositing in tissue and caused by hereditary hemochromatosis or repeated blood transfusions in some diseases such as beta thalassemia, MDS. IO can induce organ dysfunctions, such as hepatic fibrosis, diabetes and cardiac diseases and even hematopoiesis suppression. Bone marrow-derived mesenchymal cells (BM-MCs), which located in the hematopoietic niche, functions as maintaining the stability of the hematopoietic microenvironment and supporting hematopoiesis via secreting many cellular factors and differentiating into other stromal cells. Our preliminary researches suggest that IO may induce murine hematopoietic cells injury and suppress hematopoiesis of IO patients. (Lu et al., Eur J Haematol, 2013; Chai et al., blood, 2013 abstract). Here we observed the inhibitory effects of IO onthe BM-MCs and preliminarily discussed the related mechanism.

Mice were divided into four equal groups and designed as non-treated control mice group (control): mice were treated with isovolumic saline by intraperitoneal injection everyday. IO mice group (IO): mice were treated with 25mg/ml iron dextran by intraperitoneal injection every 3 days. The iron-chelation mice group (Deferasirox, DFX): IO mice were treated with 125mg/kg DFX by gavage everyday. Anti-oxidative group: IO mice were treated with 40 mM N-acetyl-cysteine (NAC, anti-oxidative regent) in drinking water. All experiments were carried at the same time for 4 weeks. Then BM-MCs were isolated from compact bone at the end of forth week according to the reference (Zhu H et al, Nature Protocol, 2010). Iron deposits in BM-MCs were observed by morphological study and labile iron pool (LIP) level of BM-MCs was detected using the calcein-AM method. The results suggest that the iron deposits and LIP level of BM-MCs in IO mice was significantly increased, which could be partly alleviated by DFX (p<0.05).

Then, the biological characteristic and supporting function on normal bone marrow mononuclear cells (BMMNCs) of BM-MCs were evaluated. Firstly, the proliferation ability of BM-MCs was assessed by double time (DT) and cell counting kit-8(CCK8) assay. BM-MCs of IO mice showed a longer DT (2.07±0.14)d compared to that of control group (1.03±0.07)d, which could be partly reversed by DFX (1.52±0.07)d and NAC (1.68±0.03)d (P<0.05),respectively. The similar results were obtained by CCK8 assay,too. Secondly, the osteoblastic differentiation ability of BM-MCs was evaluated via ALP staining and Alizarin Red S staining, as well as osteogenic gene expression. IO inhibited the ALP expression and mineralized nodules formation of osteoblast. Meanwhile, we observed a significant decrease in ALP and RUNX2 mRNA expression by RT-PCR, which also can be reversed by iron-chelation and anti- oxidative therapy. However, Oil red O (ORO) staining indicated that lipid accumulation notablely increased in IO group lipoblast compared with control group, which meaned that IO increased adipogenic differentiation of BM-MCs. Thirdly, the supporting function of IO BM-MCs on normal BMMNCs is assessed by the colony-forming unit (CFU) assay. Normal control or IO BM-MCs were co-cultured with the same pool of normal BMMNCs, After 7 days, co-cultures containing normal BM-MCs formed more CFU than that of control group. These results suggest that the proliferation potential and the differentiation potential of BM-MCs were decreased in the IO environment.

Further more, we also explored the possible mechanism of this phenomenon. Our results showed a marked increase in reactive oxidative species (ROS) level of IO group than that of control group, which could be reversed by NAC and DFX treatments, respectively (P<0.05). Quantitative RT-PCR analysis of genes associated with ROS were performed, the results showed that FOXO3 decreased in IO group compared with control group (p<0.01). However there was no significant difference in PI3K expression between IO group and control group. All above implied that FOXO3 may play an important role in IO catalyzed oxidative stress.

In conclution, dysfunction of BM-MCs, an important component of hematopoietic microenvironment, may play a crucial role in the suppression of hematopoiesis during IO. Improving the function of BM-MCs may serve as a new strategy to enhance normal hematopoiesis in IO bone marrow .