[Significance of PLSCR1 in Matrine Induced Differentiation of ATRA Resistant APL Cells].

OBJECTIVE: To observe the expression of phospholipid scramblase 1 (PLSCR1) in matrine (MAT) induced differentiation of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) cells, and to explore its correlation to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal pathway. METHODS: NB4 (an APL cell line sensitive to ATRA) and NB4-R1 (a resistant strain of ATRA) were observed as subjects in this study. Effects of combined treatment of 0.1 mmol/L MAT and 1 [mol/L ATRA on the differentiation of two cell lines were detected using nitroblue tetrazolium (NBT) reduction test and flow cytometry (CD11b). Expressions of PML/RARot and PLSCR1 protein/gene were detected using Western blot and Real-time fluorescence quantitative PCR assay. Meanwhile, H89, PKA antagonist, was used to observe cell differentiation antigen and changes of aforesaid proteins and genes. RESULTS: MAT combined ATRA could significantly elevate positive rates of NBT and CD11 b in NB4-R1 cells, and significantly down-regulate the expression of PML/RARapha-fusion protein/gene (P < 0.05, P < 0.01). ATRA used alone could obviously enhance the expression of PLSCRI in NB4 cells at protein and mRNA levels (P < 0.01). But the expression of PLSCR1 was up-regulated in NB4-R1 cells, but with statistical.difference only at the protein level (P <0. 01). In combination of MAT, PLSCR1 protein expression was further elevated in the two cell lines (P < 0.01). Besides, there was statistical difference in mRNA expressions in NB4-R1 cells (P < 0.05). All these actions could be reversed by treatment of 10 micromol/L H89 (P < 0.05, P < 0.01). CONCLUSION: MAT combined ATRA could significantly induce the differentiation of NB4-R1 cells, and inhibit the expression of PML/RARalpha fusion gene/protein, which might be associated with up-regulating PLSCR1 expression.

Combined use of Chinese medicine with allogeneic hematopoietic stem cell transplantation for severe aplastic anemia patients.

OBJECTIVE: To determine the effect of combined treatment with Chinese medicine (CM) and allogeneic hematopoietic stem cell transplantation (allo-HSCT) on patients with severe aplastic anemia (SAA). METHODS: Eleven patients were treated with CM plus allo-HSCT. Nine patients received a conditioning regimen consisting of fludarabine (Flu), anti-thymocyte globulin (pig ALG), or anti-lymphocyte globulin (Rabbit ATG) and cyclophosphamide (CY), and two patients received pig ALG and CY. All patients were treated with Kidney (Shen)-reinforcing, blood-activating, and stasis-removing (KBS) herbal preparation beginning at 1 week before transplantation and ending at 8 weeks after transplantation. Chimerism status was assessed by analyzing short tandem repeat (STR) polymorphisms. RESULTS: All patients recovered hematopoietic function and none had graft failure. The median number of days required for the absolute neutrophil count (ANC) increased to >0.5×10(9)/L was 15 days (12-22 days) and for spontaneous platelet recovery to >20×10(9)/L without post-transplantation transfusion was 17 days (15-27 days). Nine patients were long-term survivors and achieved full donor chimerism. The overall cumulative incidence of acute graft versus host disease (GVHD) grades I-II and III-IV was 18.2% (2/11) and 9.1% (1/11), respectively. The overall accumulated incidence of chronic GVHD was 27.3% and all patients had limited chronic GVHD. At a median follow-up time of 32 months (range: 12-97 months), 9 patients were still alive. The estimated 5-year overall survival (OS) rate was 81.8%. The incidence of treatment-related mortality, 2-year post-transplantation, was 18.2%. Two patients died from GVHD after transplantation. CONCLUSION: Treatment with the KBS formulation may reduce the rate of graft failure and treatment-related mortality and improve the rate of OS in SAA patients with allo-HSCT.

[Research advance on the role of ubiquitin proteasome pathway in acute promyelocytic leukemia].

Acute promyelocytic leukemia (APL) is characterized by PML-RARa expression. Ubiquitin proteasome-pathway (UPP) plays a key role in all-trans retinoid acid (ATRA) and arsenic trioxide (ATO)-induced degradation. In addition, the regulations of cell cycle and transcription are also related to this pathway. Deeply studying the role of ubiquitin-proteasome pathway in APL contributes to elucidate the mechanisms of some drugs and explode the clinical therapeutical insight for APL. In this article, the constitution of UPP, the role of UPP-mediated protein modification in APL, the application of ubiquitination-associated drugs in APL are reviewed.

Bone marrow angiogenesis in patients presenting with differential Chinese medicine syndrome: correlation with the clinico-pathological features of aplastic anemia.

OBJECTIVE: To explore differences in bone marrow angiogenesis seen in aplastic anemia (AA) patients presenting with differential Chinese medicine (CM) syndrome, and to correlate these differences with clinical pathology. METHODS: Thirty-five patients were enrolled, including 18 with "yang deficiency syndrome" and 17 with "yin deficiency syndrome." Bone marrow biopsies and serum were collected. Microvessel density (MVD) and positive expression of vascular endothelial-derived growth factor (VEGF) were detected by immunohistochemisty. Hypoxia inducible factor -1α (HIF-1α), and VEGF expression were assayed by enzyme-linked immunoabsorbent assay (ELISA), serum lactate dehydrogenase (LDH) was tested by enzyme method and liquid chip technology was used to detected the expression of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. RESULTS: Counts for leukocytes, absolute neutrophils and platelets in "yin deficiency syndrome" were lower than those found in "yang deficiency syndrome" (P<0.05). MVD and VEGF expression, and the positive rate of CD34 and VEGF in bone marrow were lower in AA, especially in "yin deficiency syndrome" (P<0.01 or P<0.05). "Yin deficiency syndrome" displayed decreased VEGF and LDH expression, and enhanced expression of HIF-1α as compared to "yang deficiency syndrome" (P<0.05). Levels of IL-4 and IL-6 were higher in AA (P<0.01), but IL-10 was decreased (P<0.05). High TNF-α expression was seen in "yang deficiency syndrome" and IFN-γ expression was decreased in "yin deficiency syndrome" as compared with normals (P <0.01 and P<0.05, respectively). CONCLUSION: AA patients have lower MVD than normals, especially in "yin deficiency syndrome." MVD might differentially correlate to disease severity, and could be dependent on bone marrow or serum VEGF expression and LDH. Additionally, IL-2, IL-10, IL-4 and IFN-γ were negatively associated while IL-6 and TNF-α were positively associated with MVD.

[Study on matrine alleviating retinoic acid resistance in acute promyelocytic leukemia].

OBJECTIVE: To explore the role of matrine (MAT) alleviating all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukemia (APL) and its mechanism. METHODS: ATRA sensitive strain of APL (NB4) and resistant strain (NB4-R1, NB4-R2) were used in this study. The low toxic dosage of MAT was established by MTT test, and ATRA IC(50) of the cell strains (cultured with or without 0.1 mmol/L MAT) were obtained to confirm the reversal index (RI); the influence of MAT (10, 8, 6, 4, 2, 1, 0.1, 0.01, 0.001 mmol/L) combine with 1 µmol/L ATRA on the differentiation of the three cell strains were observed by nitro blue tetrazolium chloride (NBT) test and morphologic changes. The apoptosis rate of cells treated with different concentration of MAT combined with 1 µmol/L ATRA was tested by flow cytometry with Annexin V/PI staining. RESULTS: (1) The toxicity of MAT to NB4, NB4-R1, and NB4-R2 cells was increased with the concentration, IC(50) value was (0.661 ± 0.035) mmol/L, (0.673 ± 0.132) mmol/L and (0.329 ± 0.020) mmol/L, respectively; (2) After treated with 0.1 mmol/L MAT, the ATRA resistance factor of NB4-R1 decreased markedly (RI = 4.96 ± 1.15), but did not of NB4-R2(RI = 0.66 ± 0.17); (3) The differentiation capacity of NB4 and NB4-R1 was enhanced with increase of MAT, and peaked at 0.1 mmol/L (P < 0.05), but did not of NB4-R2; (4) After treated with MAT, the ATRA (1 µmol/L) induced apoptosis rate in NB4 and NB4-R1 increased significantly (P < 0.05 and P < 0.01, respectively). CONCLUSION: MAT can reverse the ATRA resistance of NB4-R1, which may relate to the effect of MAT on differentiation and apoptosis. Treatment with MAT plus ATRA may exaggerate the cells resistance potency.

Protective effect of Radix Astragali injection on immune organs of rats with obstructive jaundice and its mechanism.

AIM: To observe the protective effect of Radix Astragali injection on immune organs (lymph nodes, spleen and thymus) of rats with obstructive jaundice (OJ) and its mechanism. METHODS: SD rats were randomly divided into sham-operation group, model control group and Radix Astragali treatment group. On days 7, 14, 21 and 28 after operation, mortality rate of rats, pathological changes in immune organs, expression levels of Bax and nuclear factor (NF)-kappaB p65 proteins, apoptosis indexes and serum tumor necrosis factor (TNF)-alpha level in spleen and thymus were observed, respectively. RESULTS: Compared to model control group, the number of dead OJ rats in Radix Astragali treatment group decreased (P > 0.05). The TNF-alpha level (27.62 +/- 12.61 vs 29.55 +/- 18.02, 24.61 +/- 9.09 vs 31.52 +/- 10.95) on days 7 and 21, the pathological severity score for spleen [0.0 (0.0) vs 0.0 (2.0) on days 7 and 14 and for lymph nodes [0.0 (1.0) vs 1.0 (2.0), 1.0 (0.0) vs 2.0 (1.0)] on days 21 and 28, the product staining intensity and positive rate of Bax protein in spleen [0.0 (0.0) vs 1.0 (2.0), 0.0 (1.0) vs 2.0 (1.5) and thymus [0.0 (0.0) vs 1.0 (2.0), 0.0 (1.0) vs 2.0 (1.5)] on days 14 and 28, the apoptotic indexes [0.0 (0.0) vs 0.0 (0.01)] in spleen and thymus [0.0 (0.0) vs 0.0 (0.01) on days 14 and 21 were significantly lower in Radix Astragali treatment group than in model control group (P < 0.05). CONCLUSION: Radix Astragali has protective effects on immune organs of OJ rats by relieving the pathological changes in immune organs, reducing TNF-alpha level and inhibiting Bax expression and apoptosis in spleen and thymus.