Injurious Effect Of Iron Overload Mediated Ferroptosis Pathway On Erythropoiesis Function In Mice With Non-Transfusion Dependent Thalassemia

Objective: 1.To evaluate the injurious effect of iron overload on the erythropoiesis function of bone marrow in mice with non-transfusion dependent thalassemia;2.To investigate whether iron overload mediated Ferroptosis pathway to damage the erythropoiesis function in mice with non-transfusion dependent thalassemia.Methods: 1.The 12 months old non-transfusions dependent thalassemia mice were selected as thalassemia group and the normal mice in the same litters as control group.The whole blood of the mice was taken for complete blood count to evaluate the anemia status of the mice.2.The peripheral blood serum of mice was isolated.Serum iron was detected by colorimetry and serum ferritin was detected by ELISA to evaluate the circulating iron load status of mice.The heart,liver,spleen,duodenum and distal femur of mice were taken for histopathological sections and Prussian blue staining.The iron deposits in organs and bone marrow were observed under light microscope,to assess the iron load status of the organs and bone marrow of mice.3.RT-PCR was used to detect the m RNA expression of Hepcidin gene in mouse liver cells to evaluate the regulation of iron metabolism.4.The whole blood of mice was taken for reticulocyte count,and serum was separated to detect the level of erythropoietin(EPO).Antibodies CD71 and TER119 were labeled on isolated mouse bone marrow mononuclear cells,and the cell population was analyzed by flow cytometry.The erythrocyte groups were delineated in the FSC vs SSC dot plot,and then the erythrocyte groups were divided into three groups according to the labeling of antibodies: normoblast(CD71+,Ter119-),basophilic normoblast and polychromatic normoblast(CD71+,Ter119 +),and orthochromatic normoblast and reticulocyte(CD71-,Ter119 +),to understand the proportion of each group of erythrocyte groups,so as to evaluate the erythropoiesis activity of bone marrow.5.Bone marrow mononuclear cells separated and the semi-solid medium for methyl cellulose containing EPO and IL-3 cytokines such as were mixed and vaccination it in 6 orifice plate culture.The number of colony forming unit-erythroid(CFU-E)and burst forming unit-erythroid(BFU-E)of the two groups of mice were observed and counted under light microscope after48 hours and 9 days of culturing.And flow cytometry was used to detect the apoptosis rate of mice bone marrow mononuclear cells to evaluate the erythropoiesis function damage of mice bone marrow.After counting the number of colonies,the BFU-E and CFU-E colony cells were eluted from the culture medium,and the mitochondrial morphology of the colony cells was observed under transmission electron microscopy.6.Index of Ferroptosis level was detected: the three groups of erythroid Cells after sorting were marked with Cell Rox (?) Deep Red Reagent and Bodipy (?) 661/676 molecular probe.The ROS level and lipid peroxidation level were detected by flow cytometry.The glutathione level of bone marrow mononuclear cells of mice was detected by the glutathione detection kit.RT-PCR was used to detect the m RNA expression level of GPX4 gene of bone marrow mononuclear cells of mice.The cultured BFU-E and CFU-E colony cells were eluted from the medium,and the mitochondrial morphology of the colony cells was observed under transmission electron microscopy in order to evaluate the level of Ferroptosis of bone marrow cells.Results: 1.Compared with control group,hemoglobin(Hb),and the mean corpuscular volume(MCV)and the mean corpuscular hemoglobin(MCH)of peripheral blood of the mice in thalassemia group were significantly decreased(P<0.05),serum iron and serum ferritin were significantly higher than that of control group(P<0.05),and the iron deposits in the heart,liver,spleen,duodenum and bone marrow of mice in the thalassemia group were heavy,while the blue iron particles were not seen in other organs except the spleen in the control group.In addition,the expression level of iron metabolism regulator hepcidin gene in mice of thalassemia group was decreased compared with control group(P<0.05).2.Serum proportions of EPO and reticulocyte(RET)in thalassemia group were significantly higher than those in control group,with statistical significance(P<0.05).The proportion of myeloid proerythrocytes and early,middle and juvenile erythrocytes in thalassemia group was significantly higher than that in control group(P<0.05),but there was no difference in late juvenile erythrocytes and reticulocytes(P>0.05).The number of BFU-E colonies in the thalassemia group was lower than that in the control group(P<0.05),but there was no significant difference in the number of CFU-E colonies between the two groups(P>0.05).The early,late and total apoptosis rates of bone marrow erythrocytes in the thalassemia group were higher than those in the control group,and the differences were statistically significant(P<0.05).3.The proportion of ROS positive cells and the mean fluorescence intensity of Bodipy (?) 665/676 molecular probe labeled positive cells in the normoblast,basophilic normoblast and polychromatic normoblast,and orthochromatic normoblast and reticulocyte three group of mice in thalassemia group were significantly higher than those in control group(P<0.05).There was no significant difference in the level of lipid peroxidation among the three groups of the normoblast,basophilic normoblast and polychromatic normoblast,and orthochromatic normoblast and reticulocyte in the control group,but the lipid peroxidation level of the normoblast in thalassemia group was significantly higher than that of basophilic normoblast and polychromatic normoblast,and orthochromatic normoblast and reticulocyte in thalassemia group(P=0.004,P=0.003).But there was no significant difference in the level of lipid peroxidation between basophilic normoblast and polychromatic normoblast,and orthochromatic normoblast and reticulocyte in thalassemia group mice(P=0.878).The level of glutathione(GSH)in bone marrow mononuclear cells of mice in thalassemia group was significantly lower than that in control group(P<0.05).Cell morphology under transmission electron microscopy showed that the mitochondrial membrane of bone marrow BFU-E and CFU-E colony cells in the thalassemia group was defective,the continuity of mitochondrial cristal membrane was broken,and the density of cristal membrane was increased,and the number of mitochondrial cristae was reduced compared with that in the control group,while the morphology of mitochondria in the control group was normal.Conclusion: 1.There is a significant secondary iron overload state in bone marrow of non-transfusions dependent thalassemia mice model,and iron overload damages the erythropoiesis function of bone marrow cells and increases the apoptosis rate of bone marrow cells.2.It was confirmed for the first time that iron overload can damage the erythropoiesis function in mice model of non-transfusions dependent thalassemia by mediated Ferroptosis pathway.