Effect and Involved Mechanism of RSL3-induced Ferroptosis in Acute Leukemia Cells MOLM13 and Drug-resistant Cell Lines / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect and involved mechanism of RSL3 on ferroptosis action in acute leukemia cells MOLM13 and its drug-resistant cells.@*METHODS@#After MOLM13 treated with RSL3, CCK-8 assay was performed to detect cell viability, flow cytometry was used to detect the reactive oxygen species (ROS) level of the cells, RT-qPCR and Western blot were used to detect the expression of glutathione peroxidase 4 (GPX4). After MOLM13/IDA and MOLM13/Ara-C, the drug-resistant cell lines were constructed, the ferroptosis induced by RSL3 was observed. Bone marrow samples were collected from patients with acute monocytic leukemia. RT-qPCR and Western blot were performed to detect the expression of related genes and proteins involved in ferroptosis pathway.@*RESULTS@#RSL3 significantly inhibited the cell viability of MOLM13 and increased the intracellular ROS level, which were partially reversed by ferrostatin-1. The mRNA and protein expression of GPX4 decreased in MOLM13 treated with RSL3. RSL3 inhibited the viability of MOLM13/IDA and MOLM13/Ara-C cells more strongly than that of non-drug resistant cells, also increased the intracellular ROS level . The cytotoxic effects were partially reversed by ferrostatin-1. The mRNA and protein expressions of GPX4 in MOLM13/IDA and MOLM13/Ara-C cells were higher than those in non-drug resistant cells. The mRNA and protein levels of GPX4 in bone marrow of relapsed/refractory acute mononuclear leukemia patients were higher than those of ordinary acute mononuclear leukemia patients.@*CONCLUSION@#RSL3 can induce non-drug resistant cells MOLM13 ferroptosis by inhibiting GPX4 activity. MOLM13/IDA and MOLM13/Ara-C are more sensitive to RSL3 compared with non-drug resistant cells MOLM13, which may be caused by the differences in GPX4 expression. The expressions of GPX4 mRNA and protein in relapsed/refractory acute mononuclear leukemia are higher than those in ordinary acute mononuclear leukemia.

Effects of Combined Infusion of Mesenchymal Stem Cells and Endothelial Progenitor Cells on Lung Injury after Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of combined infusion of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) on lung injury after hematopoietic stem cell transplantation (HSCT).@*METHODS@#The experiment was divided into normal control group, irradiation group, bone marrow cell transplantation group (BMT group), BMT+EPC group, BMT+MSC group and BMT+EPC+MSC group. The model of HSCT was established, on the 30th day after transplantation, the mice were sacrificed. Then lung tissue was taken for testing. The mRNA expression levels of VEGF, IL-18, IL-12b were detected by RT-PCR, and protein expression level of NLRP3 was detected by Western blot. The expression of MPO and CD146 was observed by immunohistochemistry assay.@*RESULTS@#The expression level of VEGF gene in BMT+EPC+MSC group was significantly higher than that in other groups (P＜0.01). The expression level of IL-18 and IL-12b gene was the highest in BMT group and the lowest in BMT+EPC+MSC group, and the difference was statistically significant (P＜0.05). HSCT could increase the expression of NLRP3 protein, and the BMT+EPC+MSC could significantly reduce the level of NLRP3 protein in lung cells, tending to normal. Compared with normal tissues, the BMT+EPC+MSC could improve the lung tissue structure more effectively, the expression of MPO positive cells was lower, and the expression of VEGF positive cells was higher.@*CONCLUSIONS@#The combined infusion of MSC and EPC can promote capillary regeneration, alleviate inflammation and promote lung repair after HSCT, which is superior to single EPC or MSC infusion.

Establishment and Identification of MDS Mouse Model with Irom Overload / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a MDS mouse model with iron overload and to study the effect of iron overload on MDS.</p><p><b>METHODS</b>The exogenous mutant gene RUNX1-S291fs was inserted into the mice bone marrow mononuclear cell's genome in mice by retrovirus and transplanted into C57BL/6 mice irradiated by Co γ-ray. After 8 weeks,intraperitoneal injection of iron was performed to establish an MDS mouse model with iron overload. After 24 weeks of transplantation, the peripheral blood, bone marrow, femur, liver and spleen of mice were taken, then the morphological characteristics of peripheral blood and bone marrow cells were observed by Wright's staining; the liver, spleen and bone marrow were stained with Prussian blue to observe the iron deposition. The surface antigens of bone marrow cells were detected by flow cytometry. Bone marrow mononuclear cells and spleen tissue proteins were detected by Western blot to confirm the transfection of RUNX1-S291fs gene and expression of protein. The blood routine and transplanted cell chimeric rate of mice were monitored periodically.</p><p><b>RESULTS</b>Compared with the empty plasmid control mice, levels of leukocyte and hemoglobin as well as platelet were decreased in RUNX1-S291fs mutant mice; the peripheral blood cells and bone marrow cells showed pathological hematopoiesis; the liver and spleen enlarged significantly; the tissue structure of femur, liver and spleen was abnormal; the expression of bone marrow cell surface antigens was abnormal. Bone marrow cells and spleen tissue expressed the RUNX1-S291fs protein. Compared with the controlled mice injected with normal saline, iron deposition occurred in the bone marrow, liver and spleen stained with Prussian blue in the mice injected with iron agent.</p><p><b>CONCLUSION</b>Mice engineered to carry exogenous mutant gene RUNX1-S291fs and injected with iron showed pathologic features of MDS and iron overload, resulting in establishing MDS iron overloaded mouse model successfully, which lays a foundation for studying the effect of iron overload on MDS.</p>

Effects of Cyclin A1 on the Proliferation of SKM-1 Cells and Its Potential Role in Myelodysplastic Syndrome / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Cyclin A1 on the proliferation of SKM-1 cells and its underlying role in myelodysplastic syndrome (MDS).</p><p><b>METHODS</b>Cyclin A1 was knocked down with its small interfering RNA (siRNA). The efficiency of siRNA transfection was measured by Western blot and RT-PCR. Then the proliferation of SKM-1 cells and the expression of CDK2,RUNX1 and SRSF2 with and without knockdown of Cyclin A1 recorded and analysed respectively.</p><p><b>RESULTS</b>Cyclin A1 was knocked down by siRNA after transfected for 48 h. The kncokdown of Cyclin A1 inhibited the proliferation of SKM-1 cells and down-regulated the expression of CDK2, RUNX1 and SRSF2, and these effects were at least partially mediated through RUNX1 and SRSF2 signaling pathway.</p><p><b>CONCLUSION</b>Cyclin A1 plays an important role in the proliferation of SKM-1 cells. These findings provide new insights into the pathogenesis of MDS, and it may be a potential target in the treatment of MDS.</p>

Establishment of an mouse model of iron-overload and its impact on bone marrow hematopoiesis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a mouse model of iron overload by intraperitoneal injection of iron dextran and investigate the impact of iron overload on bone marrow hematopoiesis.</p><p><b>METHODS</b>A total of 40 C57BL/6 mice were divided into control group, low-dose iron group (12.5 mg/ml), middle-dose iron group (25 mg/ml), and high-dose iron group (50 mg/ml). The control group received normal saline (0.2 ml), and the rest were injected with intraperitoneal iron dextran every three days for six weeks. Iron overload was confirmed by observing the bone marrow, hepatic, and splenic iron deposits and the bone marrow labile iron pool. In addition, peripheral blood and bone marrow mononuclear cells were counted and the hematopoietic function was assessed.</p><p><b>RESULTS</b>Iron deposits in bone marrow, liver, and spleen were markedly increased in the mouse models. Bone marrow iron was deposited mostly within the matrix with no significant difference in expression of labile iron pool.Compared with control group, the ability of hematopoietic colony-forming in three interventional groups were decreased significantly (P<0.05). Bone marrow mononuclear cells counts showed no significant difference. The amounts of peripheral blood cells (white blood cells, red blood cells, platelets, and hemoglobin) in different iron groups showed no significant difference among these groups;although the platelets were decreased slightly in low-dose iron group [(780.7±39.60)×10(9)/L], middle dose iron group [(676.2±21.43)×10(9)/L], and high-dose iron group [(587.3±19.67)×10(9)/L] when compared with the control group [(926.0±28.23)×10(9)/L], there was no significant difference(P>0.05).</p><p><b>CONCLUSIONS</b>The iron-overloaded mouse model was successfully established by intraperitoneal administration of iron dextran. Iron overload can damage the hepatic, splenic, and bone marrow hematopoietic function, although no significant difference was observed in peripheral blood count.</p>

Effect and mechanism of iron-catalyzed oxidative stress on mesenchymal stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore effect of iron overload on the proliferation and apoptosis of mesenchymal stem cell(MSCs) and the possible mechanism.</p><p><b>METHODS</b>Iron overload model of MSCs was established by adding ferric ammonium citrae (FAC) into the culture medium at different concentrations (100, 200, 400 Μmol/L) and incubated for different lengths of time (12, 24, 48 h). The levels of labile iron pool (LIP) and reactive oxygen species (ROS) were measured to confirm oxidative stress state in the model. Changes in cell proliferation and apoptosis after iron overload were measured through population double time(DT)and annexin V-PI assay. Finally, the expressions of phosphorylated p38 mitogen activated protein kinase (P-p38MAPK), p38MAPK, protein kinase B (AKT), and p53 were determined through Western blot analysis to investigate which ROS-mediated signaling pathway was involved in this process.</p><p><b>RESULTS</b>The LIP level of MSCs was significantly increased by FAC treatment at 400 Μmol/L (mean fluorescence intensity 482.49±20.96 vs. 303.88±23.37, P<0.05). The level of intracellular ROS was positively correlated with the concentration of FAC and reached a peak level when cultured with 400 Μmol/L FAC (P<0.05).After treatment with 400 Μmol/L FAC at different time points (12 h, 24 h, and 48 h), the DT of MSCs was (1.47± 0.11) d, (1.80±0.13) d, and (2.04±0.14) d, respectively, which was signifcantly longer than that of the control, which was(1.20±0.05)d (P<0.05).The apoptosis rate was also significantly higher in iron overload group[(3.51±1.17)% vs.(0.66±0.62)%, P<0.05]with consequent increase in the expressions of P-p38MAPK, p38MAPK, and p53 proteins in iron overload group, while no significant difference was found in the expression of AKT.</p><p><b>CONCLUSION</b>Iron overload can inhibit the proliferation of MSCs and induce their apoptosis through the generation of ROS, which is probably due to the stimulation of p38MAPK- p53 signaling pathway.</p>

Effects of humanized interleukin 21 on anti-leukemic activity of cytokine induced killer cells and the mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of humanized interleukin 21 (IL-21) on anti-leukemic activity of cytokine induced killer(CIK) cells derived from peripheral blood(PB) and the mechanism.</p><p><b>METHODS</b>Mononuclear cells were separated from peripheral blood and cultured with cytokines to induce CIK cells. Proliferation of CIK cells with or without IL-21 stimulation and their cytotoxic activity against K562 cells was measured by MTT method. IL-21 receptor (IL-21R) and immunophenotypes of CIK cells were measured by flow cytometry. The expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), perforin, granzyme A, granzyme B, FasL and NKG2D mRNA were measured by semi-quantitative RT-PCR. FasL on the surface of CIK cells and intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry. The concentration of IFN-γ and TNF-α in the cultured supernatant were measured by enzyme immunoassay. JAK-STAT signalling pathway of CIK cells were measured by Western-blot.</p><p><b>RESULTS</b>After IL-21 stimulation, the proportion of CIK cells increased from (17.5 ± 4.7)% to (26.5 ± 2.1)%. Cytotoxic activity against K562 cells by CIK cells increased from (22.8 ± 2.8)% to(44.6 ± 8.3)%. The expression of IL-21R increased about 2 folds. The mRNA expression of IFN-γ increased almost 2 folds from (0.3760 ± 0.2358) to (0.7786 ± 0.2493), TNF-α increased almost 2 folds from (0.6557 ± 0.1598) to (1.3145 ± 0.2136), perforin increased almost 1.5 folds from (0.6361 ± 0.1457) to (0.9831 ± 0.1265), granzyme B increased almost 2 folds from (0.4084 ± 0.1589) to (0.7319 ± 0.1639), FasL increased almost 2 folds from (0.4015 ± 0.2842) to (0.7381 ± 0.2568), the expression of granzyme A, TNF-β and NKG2D were similar with control. Flow cytometry analysis showed that the expression of FasL of CIK cells was higher than that of control (0.19% vs 0.04%), the expression of perforin increased from 35.28% to 53.16%, and the expression of granzyme B increased from 43.16% to 78.82%. The concentration of IFN-γ in the culture supernatant increased almost 2 folds from (25.8 ± 6.1) ng/L to (56.0 ± 2.3) ng/L, and TNF-α increased almost 3 folds from (5.64 ± 0.61) µg/L to (15.14 ± 0.93) µg/L. Western blot showed that the expression of STAT1 and STAT5a had no significant differences, but the expression of STAT3 and STAT5b were higher than that of control.</p><p><b>CONCLUSION</b>Humanized IL-21 could enhance the anti-leukemic activity of CIK cells via increasing IL-21R, perforin, granzyme B, FasL, IFN-γ and TNF-α, as well as activating JAK-STAT signaling pathway. These data indicate that IL-21 has a potential clinical value in the enhancement of anti-leukemic immunotherapy.</p>

Effect of oxidative stress on bone marrow mesenchymal stem cells / 中国医学科学院学报

Bone marrow mesenchymal stem cells (MSCs) are somatic stem cells that can differentiate into progenies of multiple lineages. They play an important role in hematopoiesis and stem cell therapy due to their multi-lineage potentials and immunomodulatory properties. Oxidative stress is a disturbed redox state caused by accumulation of reactive oxygen species. It can induce the senescence and apoptosis of MSCs via phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and p53 pathways, and inhibit the proliferation and differentiation of MSCs through apurinic/apyrimidinic endonuclease/redox factor 1 (APE/REF-1) and extracellular signal-regulated kinase (ERK) pathways. Furthermore, using anti-stress medication and hypoxic preconditioning, the functions of MSCs can be further enhanced. Accordingly, further studies on the effect of oxidative stress on MSCs and its signaling pathways may be meaningful for the treatment of hematologic diseases and for improving stem cell therapy.