| Objective:To investigate the effects of iron overload and related signaling pathways on mesenchymal stem cell(MSC)in patients with myelodysplastic syndrome(MDS),and to reveal the possible mechanism of iron overload injury to the hematopoietic microenvironment of MDS.ContentsThe experiment included 53 newly diagnosed MDS patients and 12 healthy volunteers(NC)in the hematology department of Tianjin medical university general hospital from November 2017 to August 2019,among which were divided into iron overload group(IO)and non-iron overload group(NIO)according to whether the serum ferritin was greater than 1000ng/ml.The isolation of MSC was performed by density centrifugation,and the mononuclear cells(MNCs)were cultured in peri dish.We next observe the basic characteristics of MSC and detect the level of apoptosis.Intracellular iron deposition and iron metabolism-related genes such as DMT1 and ZIP14 were evaluated.Iron overload downstream pathway molecules such as reactive oxygen species(ROS),hypoxia-inducible factor(HIF-1)and its key enzyme proline hydroxylase(PHD2)were investigated.Finally,the levels of MSC-related cytokines such as IL-6,IL-8,VEGF and TGF-βwere detected,as well as the changes of the above indicators after the iron removal and antioxidant treatment were given to the IO group of MSCMethods1.Bone marrow samples of MDS patients and healthy volunteers were collected,and bone marrow mononuclear cells(BMMCs)were isolated by gradient centrifugation method.MSCs were induced and cultured to P3 for subsequent related experiments.2.Inverted microscope was used to observe the morphology of MSC.The purity,apoptosis and ROS levels of MSC were determined by flow cytometry(FCM).3.RT-PCR was used to evaluate the expression levels of DMT1 and ZIP14 iron metabolism-related genes in MSC,the expression levels of downstream signaling molecules of iron overload such as HIF-1αand PHD2,and the levels of IL-6,IL-8,VEGF,TGF-β.4.HIF-1αand PHD2 protein levels in MSC were detected by western blot(WB).5.In IO group,MSC were treated with deferoxamine(DFO)and N-acetylcysteine(NAC)for iron chelation and antioxidant,and the changes in the levels of the iron overload related ROS,iron metabolism related indexes,HIF-1α,PHD2 and related cytokines were detectedResults1.Basic characteristics of MSCFCM was used to detect the purity of MSC,and the results confirmed that the proportion of CD73+CD90+CD105+CD34-CD45-MSCs was above 90%.Observation by inverted microscope showed that the number of MSC,compared with NC-MSC,was significantly reduced,and the morphology was less spindle and irregularly-arranged,FCM showed the apoptosis of MDS-MSC,especially IO-MDS-MSC(42.57±2.78)was significantly higher than NC group(19.92±1.80%,p<0.0001)and NIO group(30.48±2.19,p=0.0011).2.Iron state in MSCIron staining showed a significant decrease in the number of MDS-MSCs,especially IO-MDS-MSC,and significant iron particle deposition in the cytoplasm.PCR was used to evaluate iron metabolize-related genes DMT and ZIP14,the gene expression level of DMT1 in IO group(1.43±0.13)was significantly higher than NC group(0.93±0.08,p=0.0100)and NIO group(1.07±0.11,p=0.0393).ZIP14 gene expression in IO group(1.66±0.15)was significantly higher than that in NC group(0.89±0.10,p=0.0019)and NIO group(1.14±0.13,p=0.0114).These results suggested that the iron metabolism of MSC in MDS patients was changed due to long exposure of iron overload,that lead to increased iron absorption capacity and elevated intracellular iron levels.3.Iron overload causes elevated ROS levels in MSCWe assessed ROS levels through flow cytometry and the results turned out the mean fluorescence intensity(MFI)of ROS in IO group(9.73±0.47*105)was significantly higher than that in NC group(6.74±0.30*10~5,p=0.0035)and NIO group(7.90±0.36*10~5,p<0.0001).The results showed that the increase of iron level in MDS-MSC would lead to the increase of ROS level and further cause cell dysfunction.4.ROS can increase HIF-1 level by inhibiting PHD2 in MSCRT-PCR was used to evaluate the gene expression of HIF-1αand PHD2.The data indicate that the expression of HIF-1αin IO-MSC group was significantly increased(1.97±0.18)than controls(0.88±0.11,p=0.0003)and NIO-group(1.44±012,p=0.0165).The PHD2 was known to hydroxylate in order to catalytic degradation,its expression was reduced in both IO(0.91±0.08,p=0.0279)and NIO-MSC(1.05±0.11,p=0.0050)group than controls(0.68±0.07),and IO-group showed lowest levels.The Western Blot also proved HIF1αprotein was increased in MDS-MSC,especially in IO-MDS-MSC.The protein level of PHD2 was lowest in IO-group than NC and NIO group.The Correlation analysis showed there were positive correlation between ROS and HIF-1αin IO-MDS-MSC(r=0.563,p=0.002).These results support that high level of ROS could induce HIF-1αexpression through inhibiting the PHD2 in MDS-MSC.5.Increased HIF-1 can lead to changes in cytokine levels in MSCWe used KEGG Pathway Database to screen out some specific cytokines,include IL-6,IL-8,VEGF,TGFβthat usually act as the downstream molecular of HIF-1α,and also are related to bone marrow hematopoiesis and microenvironment.We performed RT-PCR to analysis these specific cytokines of MDS-MSCs.Proinflammatory cytokine IL-6 was higher in IO-MDS-MSCs groups(2.03±0.18)than NIO(1.54±0.14,p=0.0343)-and NC(0.97±0.10,p=0.0003)-MSCs.The gene expression of negative-regulated hematopoietic factors such as TGF-βwas increased in MDS-MSCs,and highest in IO-group(1.81±0.16)compared to controls(0.96±0.07,p=0.0007)and NIO-MDS-MSC(1.32±0.10,p=0.0098).VEGF which was associated with angiogenesis in the pathogenesis of MDS was significantly increased in IO-MDS-MSCs(1.80±0.17)than NC(0.98±0.08,p=0.0022)and NIO group(1.35±0.12,p=0.0360).The expression of IL--8,a cytokine associated with the progression of MDS disease,in IO=MDS-MSC(1.64±0.14)was higher than that in NC(1.04±0.08,p=0.0058)and NIO(1.40±0.10,NS).For these results,we conclude that ROS/HIF-1 pathway was involved in regulating the cytokine level of MDS-MSC.6..Iron chelation and antioxidant treatment can reduce the level of cytokines in MSCIO-MDS-MSC(n=10)were treated with DFO and NAC for iron chelation and antioxidant.The results showed that after treatment,intracellular ROS level significantly decreased(p=0.0356,p=0.0430),PHD2 level recovered than before(p=0.0457,p=0.0462),and HIF-1αlevel decreased(p=0.0333,p=0.0415).The expression levels of IL-6(p=0.0163,p=0.0498),IL-8(p=0.0031,p=0.0008),TGF-β(p=0.0403,p=0.0355)and VEGF(p=0.0222,p=0.0458)were all decreased compared with those before treatment.Conclusion1.There was no significant difference as to the surface markers of BM-MSC between MDS patients and normal MSCs,but the number of cells was reduced in MDS-MSC,and the morphology of MDS-MSC was less spindle and irregular-arranged.2.Iron overload in MDS patients can affect the bone marrow microenvironment,leading to enhanced iron absorption of BM-MSC and increased intracellular iron level,resulting in cell dysfunction.3.Iron overload in MDS leads to an increase intracellular ROS level,which has a strong redox activity.ROS can down-regulate PHD2 level.Decrease of PHD2 level can further inhibit HIF-1 degradation,ultimately leading to an increase in HIF-1 level.4.The downstream pathway of HIF-1 was various,among which IL-6,IL-8,TGF-βand VEGF are one of its typical downstream molecules,which have the functions of promoting inflammation and angiogenesis,inhibiting immunity and hematopoiesis,etc.The increase of HIF-1 level will cause the corresponding increase of the levels of these four cytokines in MSC,leading to the dysfunction of MSC,and abnormal of bone marrow microenvironment.5.Iron chelation and antioxidant treatment could reduce the apoptosis of IO-MDS-MSC,and reduce the levels of ROS,further decrease the level of HIF-1 and IL-6,IL-8,TGF-βand VEGF. |
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