The Mechanisms Of Damage To Bone Marrow By Iron Overload In Pancytopenic Patients With Positive BMMNC-Coombs Test

Objective:To investigate the mechanisms of damage to bone marrow by iron overload in pancytopenic patients with positive BMMNC-Coombs test (IRP).Methods:1. Twenty-one iron overloading IRP patients,26non iron overloading IRP patients and10normal controls were enrolled in this study. The expressions of ROS, Bcl-2, Caspase-3and apoptosis of BMMNC were analyzed by flow cytometry (FCM).2. To the iron overloading IRP BMMNC,5mmol/L NAC was added into the RPMI1640culture media for24h, and then the changes of the expression of ROS, Bcl-2, Caspase-3and apoptosis of BMMNC were detected by FCM.3. The number and ratio of the CD4+T lymphocytes and CD8+T lymphocytes in peripheral blood of the47IRP patients were detected by FCM. The apoptosis of the CD4+T lymphocytes and CD8+T lymphocytes in bone marrow of the47IRP patients were detected by FCM too.4. The apoptosis of the erythrocytes and the stem cells coated with auto-antibodies in bone marrow of the iron overloading IRP patients were detected by FCM.Results:1.①The expressions of ROS in BMMNC, granulocytes, erythrocytes and stem cells of the iron overloading IRP patients(109.11±34.81,120.49±36.32,51.77±19.62and64.64±25.87)were significantly higher than those of non iron overloading IRP patients (57.42±27.33,70.28±31.77,31.87±13.67and39.46±17.96) and normal controls (45.60±23.78,64.13±29.61,23.21±11.72and41.76±20.39)(P＜0.05). There were no difference between non iron overloading IRP patients and normal controls (P＞0.05).②The expressions of Bcl-2in the BMMNC, erythrocytes and stem cells of the iron overloading IRP patients (32.33±16.49,81.17±25.39and73.19±15.88) were significantly lower than those of non iron overloading IRP patients (65.53±20.62,130.43±33.66and95.99±23.02) and those of normal controls (56.05±25.35,133.14±38.87and106.19±26.33)(P＜0.05). The expression of Bcl-2in granulocytes of the non iron overloading IRP patients(23.75±8.65) was significantly lower than that of normal controls (38.53±13.75)(P＜0.05). The expressions of Bcl-2in the BMMNC, erythrocytes and stem cells of the non iron overloading IRP patients were lower than those of normal controls (P＞0.05).③The expressions of Caspase-3in granulocytes, erythrocytes and stem cells in BM of the iron overloading IRP patients(27.97±9.70,8.00±2.92and9.61±2.49) were significantly higher than those of non iron overloading IRP patients (19.71±5.49,5.17±1.83and6.56±1.65) or normal controls (15.85±6.32,4.00±1.39and5.48±1.31)(P<0.05). There were no significantly difference between non iron overloading IRP patients and normal controls (P＞0.05)④The percentage of apoptotic cells of BMMNC, granulocytes, erythrocytes and stem cells in BM of the iron overloading IRP patients (49.52±14.40)%,(49.88±16.86)%,(84.55±7.92)%and (55.37±13.49)%were significantly higher than those of non iron overloading IRP patients[(29.78±12.96)%,(23.79±10.42)%,(75.01±10.69)%and(43.49±10.79)%]and normal controls[(24.09±8.05)%,(22.29±7.37)%,(53.29±10.27)%and (38.94±13.62)%](P＜0.05). The apoptosis in the erythrocytes of the non iron overloading IRP patients (75.01±10.69)%was significantly higher than that of normal controls [(53.29±10.27)%](P＜0.05), There were no significantly difference between non iron overloading IRP patients and normal controls (P＞0.05)2.①The expressions of ROS in BMMNC, granulocytes, erythrocytes and stem cells after being treated with antioxidants (52.96±28.29),(66.73±30.52),(30.29±14.89) and (37.21±20.39) were significantly lower than those of being untreated with antioxidants (103.91±33.98),(118.17±41.85),(55.42±16.92) and (72.01±30.97)(P＜0.05)②The expressions of Bcl-2in BMMNC, granulocytes and erythrocytes after being treated with antioxidants (64.57±16.18),(42.87±13.10) and (106.89±24.36) were higher than those of being untreated with antioxidants (38.06±17.58),(20.35±9.67) and(78.47±25.92)(P＜0.05). There was no significant differences between stem cells (P＞0.05) ③The expressions of Caspase-3in granulocytes, erythrocytes and stem cells after being treated with antioxidants (16.81±4.25),(5.54±1.72) and (7.75±2.74) were lower than those of being untreated with antioxidants (29.69±6.31),(7.33±1.76) and (8.66±2.38), particularly in granulocytes and erythrocytes (P＜0.05)④The apoptosis of BMMNC, erythrocytes and stem cells after being treated with antioxidants (37.94±14.22)%,(47.58±10.91)%and (31.99±14.19)%were lower than those of being untreated with antioxidants (49.87±13.50)%,(84.59±9.12)%and (52.81±14.49)%(P＜0.05). There was no significant differences between granulocytes (P＞0.05)3.①The percentage of the CD4+T lymphocytes in PB of the iron overloading IRP patients (40.86±8.74)%was significantly higher than that of non iron overloading IRP patients[(35.96±7.03)%] and normal controls[(28.00±6.73)%]; The percentage of the CD4+T lymphocytes in PB of the non iron overloading IRP patients was significantly higher than that of normal controls (P＜0.05)②The percentage of the CD8+T lymphocytes in PB of the iron overloading IRP patients (29.14±6.77)%were significantly lower than that of non iron overloading IRP patients[(33.42±7.49)%]and normal controls[(35.40±5.27)%](P＜0.05); The percentage of the CD8+T lymphocytes in PB of the non iron overloading IRP patients was lower than that of normal controls (P＞0.05)③The ratio of CD4+T lymphocytes/CD8+T lymphocytes in PB of the iron overloading IRP patients (144.00±36.23)%were significantly higher than that of non iron overloading IRP patients[(113.85±37.31)%]and normal controls[(79.50±21.27)%](P＜0.05); The ratio of CD4+T lymphocytes/CD8+T lymphocytes in PB of the non iron overloading IRP patients was significantly higher than that of normal controls (P＜0.05)④The apoptosis of the CD8+T lymphocytes in bone marrow (27.35±10.76)%was significantly higher than that of the non iron overloading IRP patients [(15.47±8.99)%](P<0.05)⑤The apoptosis of the CD4+T lymphocytes in bone marrow (12.10±5.57)%was higher than that of the non iron overloading IRP patients [(10.49±4.14)%](P>0.05).⑥The apoptosis of the CD8+T lymphocytes/CD4+T lymphocytes in bone marrow(251.50±80.60)%was significantly higher than that of the non iron overloading IRP patients [(139.30±47.10)%](P＜0.05)4.①Among47IRP patients.24patients’ erythrocytes coated with auto-antibodies, the apoptosis of the erythrocytes coated with auto-antibodies in BM of the iron overloading IRP. patients (87.76±6.61)%was significantly higher than that of the non iron overloading IRP patients[(66.11±18.78)%](P＜0.05)②Among47IRP patients,27patients’ stem cells coated with auto-antibodies, the apoptosis of the stem cells coated with auto-antibodies in BM of the iron overloading IRP patients (56.03±14.20)%was significantly higher than that of the non iron overloading IRP patients[(43.29±10.56)%](P＜0.05)Conclusions:Iron overload damaging the bone marrow hematopoiesis might be through the following mechanisms.1. The increased ROS induced by excessive iron deposition affected the expression of Caspase-3and Bcl-2, which caused more BMMNC apoptosis;2. The abnormal number and ratio of T lymphocytes caused by iron overload aggravated the abnormality of immunity of IRP;3. Iron overload may increase the damage to erythrocytes and stem cells coated with auto-antibodies.