Chronic Iron Overload Damage the Erythropoiesis in MDS Mouse By Increasing GDF11 and ROS Levels

Iron overload (IO) is a kind of disease with an excess of iron depositing in the body's organs and tissues, resulting in the dysfunction of these organs and tissues, which contains hereditary hemochromatosis and secondary IO such as ineffective hematopoiesis in aplastic anemia and myelodysplastic syndrome (MDS) and blood transfusion. Numerous researches and our previous study found that IO can damage the hematopoietic function in bone marrow. MDS is one of the most common IO diseases and IO can further injure the hematopoietic function especially on erythropoiesis in MDS.However, it is indeterminacy on the mechanism behind this stiuation. This study is to explore the possible mechanism of IO on the injury of MDS erythropoiesis though establishing an IO MDS mice model.

In this study, we first established an IO mice model by administering iron dextran(25 mg/ml) by intraperitoneal injection every 3 days for 2 months in wild and MDS mouse(NUP98-HOXD13 transgenic 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). After 6 months, 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 next found that IO damage the erythropoiesis in MDS mice. We detected the proportion of different stages BM erythroblasts by flow cytometry plots of Ter119⁺ CD71⁺ cells (R1, basophilic erythroblasts, poly red cells, immature red blood cells) and Ter119⁺ CD71^- cells (R2, late erythroblast and reticulocyte ). It was found that the hematopoietic colony-forming unit (CFU-E, BFU-E) in MDS+IO group were less than that in other groups. The proportion of R1(10.39±1.43) and R2(1.58±0.54) in MDS+IO group were the least compared with Ctrl group(R1 17.36±3.45,R2 4.59±1.57),IO group(R1 14.53±2.65,R2 2.56±0.76) and MDS group(R1 13.64±3.27,R2 2.35±0.78). And the reticulocytes proportion in MDS+IO group(11.36±3.65) was the highest compared with Ctrl group(5.159±1.537), IO group(7.61±1.36)) and MDS group(7.35±1.39).

Then we further explored the mechanism of IO damage on MDS erythropoiesis. It was reported that TGF-β family and oxidative stress (ROS) played an important role on erythropoiesis. Our previous studies also had shown that the ROS and GDF11(a member of TGF-β family) levels were raised in BMMNCs on C57BL/6 mice with IO. Similarly, we detected them on the BM erythroblast in this study. Firstly, we sorted the R1 and R2 erythroblasts,then we performed RT-PCR to analyze mRNA levels of the TGF-β family. We found that the mRNA levels of GDF11, GDF15, Activin B, Acvr2b and ALK5 in MDS+IO group were the highest compared with that of other groups, while the mRNA levels of Activin A, ALK4 and BMP8 had no significant change in these groups. As we found that the change of GDF11 level was the most significant in MDS+IO group compared with other groups, we focused on the study of GDF11 in the following experiment. The concentration of GDF11 in peripheral serum was detected by ELISA method. It was found that the concentration of GDF11 was the highest in MDS+IO group compared with other groups. And also, the GDF11 related signal pathway proteins (Smad 2, Smad 3, p-Smad 2 and p-Smad 3) were significantly elevated in R1 and R2 erythroblast in MDS+IO group compared with other groups,which indicated that GDF11 related signal pathway was involved in the deficient of BM erythropoiesis in MDS+IO mice. Furthermore, We found that the ROS levels in Ter119⁺ cells in MDS+IO group, MDS group and IO group were increased 4.15 folds, 2.16 folds , 2.63 folds compared with Ctrl group. 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 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.

In conclusion, our study indicated that chronic IO damage the erythropoiesis in MDS mouse by increasing GDF11 and ROS levels which would provide an theoretical basis to find new targets for the treatment of IO in MDS patients with erythropoiesis dysfunction.