| Objective In this study, we aimed to explore the effect and mechanism of iron over load on macrophage and erythropoiesis in myelodysplastic syndrome(MDS).Method(1) The cell was treated by different concentrations(0, 5, 10, 20, 40, 80 ?mol/Lrespectively) of ferric ammonium citrate(FAC).The control group was the group of without FAC. We detected the number of cells and the cellular state,metabolic activity, the change of phagocytosis, the level of ROS and reactive nitrogen and changes of related oxidative stress signaling pathways in different groups. We also treated cell with deferasirox(DFX) and the antioxidant N-acetylcysteine(NAC) to clear excess oxidative stress, and detected changes in the above index.(2)In this study, we first established an IO mice model by administering iron dextran(25 mg/ml) in wild and MDS mouse. The mouse were devided into four grups: the wild mouse without IO(Ctrl group), the wild mouse with IO(IO group), MDS mouse without IO(MDS group), the MDS mouse with IO(MDS+IO group). The establishment of IO mice models were confirmed by both the iron deposits staining of bone marrow(BM), liver, spleen and the labile iron pool level of bone marrow mononuclear cells( BMMNCs). We dete cted the counts of BMMNCs, the hematopoietic colony-forming unit and the proportion of different stages BM erythroblasts by flow cytometry plots.The seindexes confirm that IO damage the erythropoiesis in MDS mice. We detected the the ROS level, the mRNA expression levels of NOX4 and GPX1 and TGF-? family. The concentration of GDF11 in peripheral serum was detected by ELISA method.Results(1) We found the level of labile iron pool(LIP) in macrophage co-cultivated with iron was increasing. The LIP level treated with 80?mol/L FAC showed the highest in these groups.(2) We also found that with the increase of FAC concentration, the metabolic activity of macrophages in 5 groups decreased to 51.58%, 40.98%, 16.23%,3.46% and 0.05% compared to the control group(P < 0.05) in all the experimental groups). And the group with the metabolic activity decreased to 16.23% was selected as the iron overload group for the following experiments.(3) Compared with the control group, the number and phagocysis of macrophages in iron overload group reduced to the 32.80% of the control group(P<0.05), and the state of cells changed from adherence to partial suspension. The phagocysis of macrophages in iron overload group reduced to the 20.40%of the control group(P<0.05).(4) Our further study showed that the levels of ROS and the activity nitrogen in iron overload groups increased 7.71 and 1.45 fold compared with control group(P <0.05). Here wedetected that the genes NOX4 and iNOS related to ROS and reactive nitrogen production respectively showed high expression,The GPX1 gene which participated with ROS clearance had a low express level. Our datas also presented PI3K gene involved in the oxidative stress signaling pathway in iron overload group was upregulated, while FOXO3 showe dadownregulation level compared with control group.(5) After iron chelation and antioxidant treatment in iron overload group, the damage were partially reversed compared with iron overload group.(6) The establishment of IO mice models were confirmed by both the iron de posits staining of bone marrow(BM), liver, spleen and the labile iron poollev el of bone marrow mononuclear cells(BMMNCs).(7) The counts of BMMNCs in IO group((29.40±1.74)*106)and MDS group((2.95±2.25)*106)were less than that in Ctrl group in Right tibia(P>0.05).There were no significant differences. The counts of BMMNCs in MDS+IO group((19.12±5.18)*106)was less than that in Ctrl group(P<0.05) and less than that in IO group and MDS group((P>0.05).(8) It was found that the hematopoietic colony-forming unit(CFU-E, BFU-E) in IO group and MDS group MDS+IO group were less than that in Ctrl group(P <0.05). The CFU-E, BFU-E in MDS+IO group were less than that in Ctrl group(P <0.05).(9) The proportion of late red cell in IO group(4.04±0.49),MDS group(3.46±0.31) and MDS+IO group(1.80±0.59) were less than that in Ctrl group(5.64±0.83)(P<0.05). The proportion of late red cell in MDS+IO group was less than that in IO group and MDS group(P>0.05).(10) The ROS levels in IO group(54757±5982),MDS group(65778±8186)and MDS+IO group(107757±6690) in late red cell were higher than that in Ctrl group(42994±3292)(P<0.05). The ROS level in MDS+IO group was higher than that in IO group and MDS group(P<0.05).(11) RT-PCR was used to detect the mRNA expression levels of the gene NOX4 related to ROS generation and GPX1 related with ROS clearance. It was found that in red late cell the NOX4 expression levels were the highest in MDS+IO group compared with other groups,while the GPX1 mRNA level was the least in MDS+IO group.(12) We found that the mRNA levels of GDF11, GDF15, Activin B, Acvr2b and ALK5 in IO group, MDS group and MDS+IO group were higher than that in Ctrl group in late red cell(P<0.05).These levels in MDS+IO group were higher than that in Ctrl group in late red cell(P<0.05).While the mRNA levels of Activin A, ALK4 and BMP8 had no significant change in these groups. We found that the change of GDF11 level was the most significant in MDS+IO group compared with other groups.The GDF11 mRNA level in IO group, MDS group and MDS+IO group were 3.56,3.89,5,24 fold compared with that in Ctrl group.(13) The concentration of GDF11 in peripheral serum was detected by ELISA method. It was found that the concentration of GDF11 in IO group((796.6±103.4)pg/ml), MDS group((678.0±78.14)pg/ml) and MDS+IO group((1525.0±78.37)pg/ml) were higher than that in Ctrl group((342.0±26.09)pg/ml)(P<0.05). The concentration of GDF11 in MDS+IO group were higher than that in Ctrl group(P<0.05).Conclusions The damages of iron overload on macrophages was mediated by inducing ROS production and activating oxidative stress signaling pathways. And chronic IO damage the erythropoiesis in MDS mouse by increasing ROS level and the GDF11 level which inducedby ROS. Our established model provides method to explore the mechanism of iron overload on macrophage and erythropoiesis, and new sight in iron overload therapy. |
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