The Role Of Ros In Apoptosis Of Erythrocytes And MSCS From MDS Patients With Iron Overload And Related-mechanism Research

Objective:The aims of this study are to evaluate the role of ROS in the damage of MDS/AML/MSCs,to investigate the role of HIF-1a/ROS pathway in the erythroid apoptosis of MDS patients,and the role of AMPK/MFF/Drp1 pathway in the apoptosis of MDS-MSCs.Methods:(1)FAC was used to set up IO models in MDS/AML/MSCs in vitro.LIP and ROS levels were detected by flow cytometry in MDS/AML/MSCs after incubation with FAC.Next,the cell apoptosis,viability and cycle of MDS/AML/MSCs were tested under the condition of IO.Then,MDS/AML/MSCs with IO were treated with DFO,NAC or Catalase to determine the role of ROS in the damage of MDS/AML/MSCs caused by IO.(2)The protein levels of HIF-1a were tested by Western blotting in MDS/AML cells after incubation with FAC.MDS/AML cells were transfected with HIF-1a shRNA or HIF-1a overexpression virus,then ROS levels were detected by flow cytometry.ROS levels,apoptosis,viability and cell cycle were determined in MDS/AML cells after transfecting with HIF-1a overexpression virus under the condition of IO.To evaluate the role of HIF-1a in the change of cellular function caused by IO from MDS/AML cells.BMMNCs were isolated from bone marrow of MDS patients and cultured in vitro.ROS levels,apoptosis and the protein levels of HIF-1a were detected from CD235a+ erythrocytes of BMMNCs to determine the role of HIF-1a/ROS pathway in the erythroid apoptosis caused by IO from MDS patients.(3)MSCs were isolated from bone marrow of MDS patients and cultured in vitro.Mitochondrial morphology,the levels of autophagy,ATP concentrations,the activity of ETC complex I/II/III and apoptosis,viability were detected in health MSCs and MDS-MSCs under the environment of IO.Then,to evaluate the role of ROS in above effects caused by IO in MSCs after treatment with DFO,NAC or Catalase.Western blotting was used to analyze the proteins levels of AMPK/MFF/Drp1 pathway in health MSCs after incubation with FAC.Next,MSCs were transfected with AMPK CRISP/Cas9 or MFF shRNA virus to determine the role of AMPK/MFF/Drp1 pathway in the change of cellular function caused by IO from MSCs.Last,Western blotting was used to analyze the proteins levels of AMPK-related pathway to evaluate the role of AMPK/MFF/Drp1 pathway in the apoptosis caused by IO from MDS-MSCs after treatment with DFO,NAC or Catalase.Results:(1)FAC increased LIP and ROS levels in MDS/AML/MSCs,accompanied by apoptosis increased,viability decreased,and cell cycle blocked.However,ROS levels and apoptosis decreased,viability enhanced,and cell cycle promoted in MDS/AML/MSCs after treatment with DFO,NAC or Catalase.These results suggest that IO could cause the damage of MDS/AML/MSCs through up-regulation of ROS levels.(2)The ROS levels were increased or decreased in MDS/AML cells after transfecting with HIF-1a shRNA or HIF-1a overexpression virus.The protein levels of HIF-1a decreased in MDS/AML cells after incubation with FAC.Especially,not only the ROS levels decreased,but also the apoptosis decreased,viability enhanced,and cell cycle promoted in MDS/AML cells after transfecting with HIF-1a overexpression virus under the condition of IO.More importantly,HIF-1a/ROS pathway plays a key role in the erythroid apoptosis caused by IO in MDS patients.(3)In health MSCs,IO decreased ATP concentrations through up-regulation of ROS levels and down-regulation of ETC complex II/III activity,in turn stimulating the AMPK/MFF/Drp1 pathway and lead to mitochondrial fission,autophagy enhanced,apoptosis increased,and viability decreased in MSCs.What’s more,we found that IO caused down-regulation of ATP concentrations through up-regulation of ROS levels,accompanied by apoptosis increased,viability decreased,mitochondrial fission,and autophagy enhanced in MDS-MSCs.More interestingly,IO activated the AMPK-related pathway in MDS-MSCs.Thus,our data determine that AMPK/MFF/Drp1 pathway plays an important role in the damage of MDS-MSCs caused by IO.Conclusions:IO caused apoptosis increased,viability decreased,and cell cycle blocked in MDS/AML/MSCs through up-regulation of ROS levels;IO decreased the expression levels of HIF-1a proteins,and lead to up-regulation of ROS levels in erythrocytes of MDS,then caused high erythroid apoptosis;High ROS levels,caused by IO,triggered the AMPK/MFF/Drp1 pathway,and lead to mitochondrial damage and cell apoptosis in MDS-MSCs.