[The Antiapoptotic Effect of Danggui Buxue Tang and Its Main Components on Hematopoietic Cells in Mice with Bone Marrow Suppression].

OBJECTIVE: To explore the hematopoiesis protection effect of Danggui Buxue Tang (DBT) and its main components Angelica polysaccharide (APS) and Astragalus polysaccharide (ASPS) on myelosuppression mice, and the mechanism of anti-apoptosis of Meg-01 cells. METHODS: Mice were radiated with 4 Gy of 137Csγ ray to establish the model of radiation-induced myelosuppression. DBT, APS or ASPS (10 mg/kg) were injected into irradiated mice. Peripheral blood cell counts were performed on mice before radiation (day 0) and day 7, 14 and 21 after radiation. On the 21st day, poor plasma platelets were collected from mice to detect TPO concentration and then the mice were sacrificed. The femoral bone marrow cells were cultured for colony cell forming units (CFU). Meg-01 cells were cultured without FBS for 24 h to induce apoptosis, and then treated with DBT/APS/ASPS for 72 h. Flow cytometry (FCM) was used to detect early apoptosis (Annexin V), mitochondrial membrane potential (JC-1) and the expression of Caspase-3 to analyze the effect of DBT/APS/ASPS on cell apoptosis. RESULTS: DBT can stimulate the recovery of white blood cells (WBC), red blood cells (RBC) and platelets (PLT) of myelosuppression mice, especially for WBC and PLT (P<0.01, P<0.05). Compared with the control group, the number of BFU-E, CFU-MK and CFU-GM increased after adding DBT (BFU-E & CFU-GM: P<0.05； CFU-MK: P<0.01). The effect of DBT on blood TPO concentration in mice was not obvious (P=0.89). RBC, WBC and PLT were increased in APS group compared with control group (P<0.05). WBC increased after the treatment of ASPS (P<0.05). APS stimulated the formation of CFU-F, CFU-MK and CFU-GM (P<0.05). Only CFU-GM increased in ASPS group(P<0.05). Besides, DBT decreased the apoptosis of Meg-01 cells (P<0.05). The early apoptosis rate and total death rate in APS (100 µg/ml) group were lower than that of control group (P<0.01, P<0.05). The early apoptosis rate of ASPS (100 µg/ml) group was lower than that of control group (P<0.05). JC-1 and Caspase-3 showed that APS (100 µg/ml) significantly reduced apoptosis rate (P<0.01, P<0.05). CONCLUSION: DBT has protective effect on hematopoietic system, especially WBC and PLT, and has anti-apoptotic effect on Meg-01. It was found that the above effects of DBT were mainly caused by APS, and its anti-apoptosis mechanism was carried out mainly through JC-1 and Caspase-3 pathways.

[The Hematopoietic Protective Effect of PDGF-BB on Radiation-Induced Myelosuppression Model Mice].

OBJECTIVE: To investigate the hematopoietic protective effect of platelet-derived growth factor (PDGF)-BB on radiation-induced myelosuppression model mice and effect of anti-apoptosis of megakaryocyte line Meg-01 cells, and its possible mechanism. METHODS: Mice were radiated with 4 Gy of 137Csγ ray to establish the model of myelosuppression. Mice were weighed and peripheral blood cell were counted before radiation (day 0) and day 7, 14 and 21 after radiation. On the 21 st day, the mice were killed. The sternal tissues of the mice were taken for morphological observation, and the femoral bone marrow cells were cultured for the assay of colony cell forming units (CFU). Meg-01 cells were cultured without FBS for 24 h to induce apoptosis, and then treated with PDGF-BB for 48 h. The effects of PDGF-BB on the proliferation were investigated by cell counting. Flow cytometry was used to detect early apoptosis (Annexin V), mitochondrial membrane potential (JC-1) and the expression of caspase-3. RESULTS: Peripheral blood cell counts of mice showed that PDGF-BB stimulated the recovery of white blood cells, red blood cells and platelets after radiation (P<0.05), especially for white blood cells. Morphological examination showed bone marrow hyperplasia in PDGF-BB group, the numbers of megakaryocytes and their progenitor cells were higher than those in the control group. PDGF-BB significantly stimulated the formation of CFU-MK, CFU-GM, BFU-E and CFU-F. PDGF-BB showed a strong proliferation effect in the concentration range of 5-50 ng/ml (P<0.001). PDGF-BB (50 ng/ml) significantly reduced the positive expression of Annexin V (P<0.01). The mitochondrial membrane potential in the control group was decreased when compared with PDGF-BB group, which indicated that the number of apoptotic cells was increased (P<0.01). Besides, the expression of caspase-3 in PDGF-BB group was significantly lower than that in control group (P<0.05). CONCLUSION: PDGF-BB has a protective effect on the hematopoietic system of myelosuppression model mice, especially megakaryocytes and their progenitor cells. PDGF-BB has pro-proliferative and anti-apoptotic effects on Meg-01 cells, and the mechanism may be mediated through JC-1 and caspase-3 pathway.

[Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3].

OBJECTIVE: To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. METHODS: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. RESULTS: The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 µg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 µg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 µg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 µg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 µg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while ß-TG could not. CD36 antibody FA6-152 (25 µg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. CONCLUSION: TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.