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An herbal decoction of Radix astragali and Radix angelicae sinensis promotes hematopoiesis and thrombopoiesis
Journal of Ethnopharmacology 124 (2009) 87–97
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An herbal decoction of Radix astragali and Radix angelicae sinensis promotes hematopoiesis and thrombopoiesis Mo Yang a,∗ , Godfrey C.F. Chan a , Ruixia Deng b , Margaret H. Ng c , Sau Wan Cheng d , Ching Po Lau d , Jie Yu Ye a , Liangjie Wang b , Chang Liu b,∗∗ a
Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China Molecular Chinese Medicine Laboratory, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, PR China d Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, PR China b c
a r t i c l e
i n f o
Article history: Received 30 October 2008 Received in revised form 12 January 2009 Accepted 3 April 2009 Available online 11 April 2009 Keywords: Hematopoiesis Thrombopoiesis Megakaryocytopoiesis Thrombopoietin Apoptosis Radix angelicae sinensis Radix astragali Danggui Buxue Tang (DBT)
a b s t r a c t Ethnopharmacological relevance: A decoction containing Radix angelicae sinensis and Radix astragali (Danggui Buxue Tang, DBT) has been used to raise the “Qi” and nourish the “Blood”. However, its effects on haematopoiesis and particularly thrombopoiesis have not been studied. Aims: This study aims to examine the effects of DBT on hematopoiesis and thrombopoiesis. Materials and methods: A myelosuppression mouse model was treated with DBT (10 mg/kg/day). Peripheral blood cells from DBT and thrombopoietin-treated samples were counted on days 0, 7, 14 and 21. Then CFU assays were used to determine the effects of DBT on the megakaryocytic progenitor cells and other lineages. Last, analyses of annexin V, caspase-3, and mitochondrial membrane potential were conducted in megakaryocytic cell line M-07e. Results: Morphological examination showed that DBT treatment significantly increased the recovery of the megakaryocytic series. DBT significantly enhanced the platelet recovery and CFU-MK formation in vivo. DBT significantly promoted CFU-MK and CFU-F formation. Last, we observed the antiapoptotic effects of DBT on M-07e cells. Conclusion: DBT might promote haematopoiesis and thrombopoiesis in the mouse model through (i) directly promoting the growth of megakaryocytes; (ii) indirectly promoting the growth of bone marrow stromal cells; (iii) inhibiting apoptosis of megakaryocytes. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hematological and cancer patients who experience bone marrow suppression or infiltration resulting from chemotherapy or radiotherapy frequently develop thrombocytopenia, which may lead to hemorrhage and fatality (Ciurea and Hoffman, 2007). In severe cases, platelet transfusion is required to prevent or stop bleeding. However, frequent platelet transfusion can induce the formation of anti-platelet antibodies, and transmit either viral or bacterial infection. Most efforts for the development of thrombopoietic therapy have been focused on thrombopoietin (TPO), the primary growth factor for platelet production (Kaushansky, 2006, 1998). However, the clinical application of TPO produces a neutralizing antibody and induces anti-TPO antibody formation (Li et al., 2001). Therefore, no effective treatment for thrombo-
∗ Corresponding author. Tel.: +852 28199354; fax: +852 28198142. ∗∗ Corresponding author. Tel.: +852 98062110; fax: +852 28175983. E-mail addresses: [email protected] (M. Yang), [email protected] (C. Liu). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.04.007
cytopenia is currently available clinically. Our long-term goal is to identify novel thrombopoietic agents from traditional Chinese medicine (TCM) formulations or products for clinical application. For centuries, dozens of TCM formulations have been used for the promotion of “blood production” and have shown favorable effects on thrombocytopenia (Mei et al., 1991; Wang and Zhu, 1996). One of the most well known formulations is Danggui Buxue Tang (DBT), which consists of two herbs: Radix angelicae sinensis (Danggui, RAS) and Radix astragali (Huangqi, RA). Roots of Angelica sinensis (Oliv.) Diels are the sources of Radix angelicae sinensis and roots of Astragalus membranaceus (Fischer) Bunge and Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K.Hsiao are botanical sources of Radix astragali. We selected DBT for further study for several reasons. First, DBT has been used for “regulating and enriching blood” for almost 800 years (Song et al., 2004), and its clinical efficacies are well established. The standard procedure to prepare DBT includes boiling ∼30 g Radix astragali and 6 g Radix angelicae sinensis in 2 bowls of water by moderate heat until the final volume is reduced to half and daily usage. The decoction
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was thought to be able to raise the “Qi” and nourish the “Blood” of the individual. Second, DBT is a simple formula that consists of two medicinal plants: RAS and RA. Thus, the identification of the active components and their interactions is less complex. Third, the extraction process of DBT (Song et al., 2004) has been standardized, and several chemical compounds such as ferulic acid and ligustilide from RAS, and astragaloside IV, calycosin, and formononetin from RA have been used as marker compounds for quality control (Song et al., 2004). These ensured the chemical consistencies among different preparations. Fourth, more than fifty compounds from RAS and twenty compounds from RA have been identified, which provide a set of candidate compounds for further validation. RAS root is used to invigorate the blood circulation in the treatment of menstrual disorders and to modulate the immune system and RA has been used as an immunostimulating, hepatoprotective, anti-diabetic, analgesic, expectorant and sedative drug (Zheng, 2000). In addition, several previous studies showed DBT can promote hematopoiesis, stimulate cardiovascular circulation, prevent osteoporosis and posses anti-oxidation activity (Mei et al., 1991; Song et al., 2004; Wang and Zhu, 1996). However, no study has been performed to test DBT’s effect on hematopoiesis and particularly, thrombopoiesis. In current study, we examined the effect of DBT on thrombopoiesis as part of the general hematopoietic effects in a mouse model. 2. Materials and methods 2.1. Plant materials and chemical reagents The herbal materials, Astragalus membranaceus var. mongholicus and Astragalus sinensis were provided by Institute of Chinese Medicine, The Chinese University of Hong Kong and were authenticated by us as described previously (Song et al., 2004). Part of the samples are currently deposited in the Molecular Chinese Medicine Laboratory, LKS Faculty of Medicine, University of Hong Kong, China as voucher # mcm-010 for RAS and voucher # mcm-011 for RA. All chemical reagents were purchased from Sigma–Aldrich (St. Louis, MO).
2.3. Quantitative analysis of active constituents Standards including ferulic acid (98% pure) and formononetin (96% pure) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). For calibration, ferulic acid and formononetin were dissolved in 1 ml of MeOH to give serial concentrations from 0.05 to 50 g/ml, and three injections into HPLC were performed for each dilution. HPLC was performed on a 150 mm × 3.9 mm i.d., 4 m Nova-Pak C18 column using an HPLC system consisting of a Waters 626 pump, Waters 600S controller, Waters 717 autosampler, and Waters 486 photodiode array detector. Acetonitrile (Solvent A) and H3 PO4 -acidified water (Solvent B) were used as mobile phases at a flow rate of 1.0 ml/min at room temperature. The gradient profile was 87% B isocratic for 15 min, 87–71% B linear for 5 min, 71% B isocratic for 15 min, 10% B linear for 15 min, and 10% B isocratic for 10 min. Ten microliters of the samples and standards filtered through Millipore filter (0.45 m) were injected, and signals were detected at 254 nm and 313 nm with UV detection. 2.4. Radiation-induced Hematocytopenic Model Seven or eight-week-old male Balb/c mice were obtained from Charles River Japan (Yokohama, Japan) and were given free access to food and water. Ethical permissions for the studies were granted by the Animal Research Welfare Committee, The Chinese University of Hong Kong. Hematocytopenia with thrombocytopenia model was established using 4-Gy-irradiated mice as described previously (Inagaki et al., 2004; Sun et al., 2001). DBT (10 mg/kg/day) and TPO (1 g/kg/day) were given by injection (IP) daily for 21 days starting from the day after radiotherapy in these mice. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) from DBT, TPO, and water control groups were analyzed on days 0, 7, 14, and 21. On day 21, the bone marrow cells were harvested for colonyforming unit (CFU) assays. We measured the plasma TPO levels on day 21. Bone marrow samples were frozen in cryomolds and performed on 5 m sections. The slides were stained with Giemsa staining. Twenty-five random high-power fields from each bone marrow sample were chosen and blindly quantified for histological examination.
2.2. Preparation of DBT extract
2.5. Murine colony-forming unit (CFU) assay
The preparation of DBT extract and the methods of HPLC analyses were based on those reported previously with minor modifications (Dong et al., 2006; Huang, 2005; Ma et al., 2002; Song et al., 2004; Yang and Feng, 1998). Briefly, the plant materials were grounded and powdered by an electric mill (TR-02B, Rong Tsong, Taiwan). Then, 10 g of RA and 2 g of RAS (20 mesh) were accurately weighed and extracted in 8 volume of water by boiling for 2 h. The extraction was filtered and the residue was re-extracted under the same conditions. While nine-tenth of the extract was used in all the following biological experiments, onetenth of the extract was used for HPLC analysis as described below. EtOH was added to this fraction of extract to a final concentration of 70%. The mixture was centrifuged at 4000 × g for 15 min and the precipitate was discarded. The supernatant was dried using a rotavapor (R-210, BUCHI, Switzerland). The residues were dissolved in 5 ml of MeOH and filtered through a Millipore filter (0.45 m). The samples prepared without the EtOH precipitation step could not be passed through the filter. Ten microliters of the sample was injected for HPLC analysis. The detection wave lengths were set at 254 nm and 313 nm for formononetin and ferulic acid, respectively. Peaks were assigned by comparing the retention time and UV spectra of the peaks to that of the standard compounds.
The assay was performed as described previously (Yang et al., 2007). Colony-forming unit-granulocyte macrophage (CFU-GM), burst-forming unit/colony-forming unit-erythroid (BFU/CFU-E), and colony-forming unit-mixed (CFU-GEMM) were cultured in methylcellulose (1%) supplemented with fetal calf serum (FCS, 30%), 1% BSA, 0.1 mM -mercaptoethanol, 3 IU/ml erythropoietin, 10 ng/ml granulocyte macrophage-colony stimulating factor, 10 ng/ml interleukin-3, and 50 ng/ml SCF. Murine bone marrow cells (2 × 105 cells/ml) were seeded in triplicate and incubated for 7 days. Colonies were scored blindly. 2.6. Murine colony-forming unit-megakaryocytes (CFU-MK) assay Murine bone marrow cells (2 × 105 cells) were cultured using the plasma clot culture method (Yang et al., 2007, 2000). The culture medium contained 1% deionized bovine serum albumin (BSA) (Sigma, MO, USA), 0.34 mg CaCl2 , 10% citrated bovine plasma (Sigma), 100 g penicillin, 50 g streptomycin, and IMDM with different concentrations of DBT, TPO, and IL-3 in a total volume of 1 ml. The cells were incubated at 37 ◦ C under 5% CO2 for 7 days, and the number of CFU-MK derived colonies was counted using the acetyl-choline esterase (AchE) staining method
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after 7 days. The colonies were further stained with hematoxylin to count the CFU-GM derived colonies. A CFU-MK colony was defined as a cluster of three or more AchE positive cells, while a CFU-GM colony was considered as a cluster of 40 or more cells. 2.7. Murine bone marrow colony-forming unit-fibroblast (CFU-F) assay The assay was performed as described previously (Yang et al., 2001). Briefly, mouse bone marrow cells (1 × 106 cells) were seeded in 2 ml of IMDM with 10% FCS in triplicates. Cultured cells were incubated at 37 ◦ C and 5% CO2 in a fully humidified atmosphere with or without DBT for 9 days. Fibroblastoid colony-forming cells (CFU-F) assay were used to determine the number of bone marrow-derived fibroblastoid (Yang et al., 2001). Briefly, adherent cells were stained with Giemsa staining. The number of CFU-F colonies was counted under a light microscope. An aggregate containing more than 10 fibroblasts was counted as a CFU-F colony. The effects of DBT and other cytokines such as fibroblast growth factor (FGF, 50 ng/ml), platelet-derived growth factor (PDGF, 50 ng/ml), or vascular endothelial growth factor (VEGF, 50 ng/ml) were also examined using the CFU-F assay. 2.8. Determining TPO levels by ELISA assay TPO level was measured by an ELISA kit (R & D, Minneapolis) as described previously (Sungaran et al., 2000). A monoclonal antibody specific for TPO was pre-coated on to a microplate. Standards and samples were added to the wells. After washing away the unbound substances, enzyme-linked polyclonal antibodies specific for TPO were added to the wells. After removing any unbound antibodies, substrates were added to the wells. The colors developed were in proportion to the amount of TPO bound on the wells. The optical density of each well was measured using a microreader (DNATECH MR 5000) at 450 nm wave length. 2.9. Annexin V, caspase-3, and mitochondrial membrane potential analysis of M-07e cells by flow cytometry The assays were performed as described previously (Yang et al., 2007). Briefly, the megakaryoblastic cell line M-07e (American Type Culture Collection, Manassas, VA, http://www.atcc.org) was maintained in IMDM supplemented with GM-CSF (20 ng/ml) and 10% FCS. Apoptotic cell death was induced by cytokine and serum depletion. We added DBT (200 mg/ml) to the cultures for 72 h. Apoptotic cell death was examined using annexin VFITC/PI, active caspase-3-PE, and JC-1 ApoAlert reagent kits (BD Biosciences, San Diego, http://www.bdbiosciences.com) according to the manufacturer’s instructions. Ten thousand events were acquired for each sample and were analyzed by flow cytometry using Lysis II software (FACScan; BD Pharmingen). 2.10. Statistical analysis Treatment groups were compared using analysis of variance and paired t test or Wilcoxon signed rank test, depending on data distribution, using the JMP software (SAS, Cary, NC). A p value of <0.05 was considered statistically significant. All values were expressed as mean ± SEM. Statistical significances were denoted with three different symbols: “*” (p < 0.05), “#” (p < 0.01) and “+” (p < 0.001).
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3. Results 3.1. Preparation of DBT extract and quantitative analysis of the main constituents The HPLC calibration curves of formononetin and ferulic acid exhibited good linearity in a range from ∼0.05 to ∼50 g/ml. The RSDs were between 1% and 2%. Fig. 1A–D shows the typical HPLC chromatograms of the standard compounds and their relative UV absorption curves, which confirmed the identities of the standard compounds used in the study. The HPLC chromatograms of DBT water extracts detected at 254 nm and 313 nm are shown in Fig. 1E and F, respectively. We identified the peaks for the standard compounds by comparing the retention times as well as the UV absorption curves of the unknown peaks with those of the standards eluted under the same conditions. To quantify the amount of the standard compounds in the DBT extracts, we first constructed the standard curve for each standard. The linear regression equation for formononetin was y = 8E + 06x − 101292, with r2 = 0.9993. For ferulic acid, its equation was y = 514217x − 10751, with r2 = 0.9994. Based on standard curves, the amount of compounds in DBT extracts was found to be 205 mg (for formononetin) and 9 mg (for ferulic acid) per gram of the starting dry DBT materials, respectively, which are similar to those described in previous studies (Dong et al., 2006). It should be pointed out that formononetin is only found in RA, and ferulic acid is only found in RAS. 3.2. Effects of DBT on hematopoiesis and thrombopoiesis in myelosuppressed mice 3.2.1. Effects of DBT on blood cell counts The myelosuppressed mice showed platelet nadir (<300 × 109 /l) on day 7, and then the platelet counts gradually recovered to approximately 80% of that of day 0 by day 21 (Fig. 2A). The platelet counts in the three groups (control, DBT, and TPO, n = 6) on days 0 and 7 did not show significant differences. Mice treated with DBT at 10 mg/kg/day showed a significantly higher platelet number on days 14 (p < 0.01) and 21 (p < 0.01) and accelerated platelet recovery compared with the control mice. Similarly, TPO-treatment at 1 g/kg/day significantly increased the platelet counts as compared to that of the control on days 14 (p < 0.001) and 21 (p < 0.001). This thrombocytopoietic activity of DBT was similar to that of TPO as there were no significant differences in the platelet counts between samples treated with DBT and TPO (Fig. 2A). Although TPO is a primary regulator of megakaryocyte and platelet production in vitro and in vivo, it has also been shown to exert effects on early hematopoietic stem cells (Sitnicka et al., 1996), which can give rises to WBC and RBC. Consequently, we also studied the effects of DBT and TPO on the counts of WBC and RBC. The changes in WBC counts in the experiment are shown in Fig. 2B. No significant differences were seen in the WBC counts on day 0 among the three treatment groups. However, the WBC counts in DBT-treated mice showed a significantly increased recovery on days 7 (p < 0.001), 14 (p < 0.01), and 21 (p < 0.001). Similarly, the WBC counts in TPO-treated mice also showed a significantly increased recovery on days 7 (p < 0.001), 14 (p < 0.01) and 21 (p < 0.001). No significant differences were observed in the WBC counts in the mice treated with DBT and TPO on days 0, 7, 14, and 21, suggesting that DBT and TPO have similar effects. Finally, the changes in RBC counts among the three treatment groups on days 0 and 7 showed no significant difference (Fig. 2C). However, the RBC counts of DBT-treated mice showed a significant increased recovery on days 14 (p < 0.01) and 21 (p < 0.01). Similarly, the RBC counts in the TPO-treated mice also showed a significant increased recovery on days 14 (p < 0.05) and 21 (p < 0.001).
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Fig. 1. Quantification of DBT based on two marker compounds formononetin (a) and ferulic acid (b). HPLC analyses of the standard compound formononetin (A) and ferulic acid (C) are shown. UV spectra (B for formononetin and D for ferulic acid) were used to confirm the identity of the compounds. HPLC analyses of the water extracts of DBT are shown in E and F, respectively. Individual chemical markers are indicated by arrows.
No significant differences were observed in the RBC counts in mice treated with DBT and TPO on days 0, 7, 14, and 21, suggesting that DBT and TPO have similar effects on the recovery of RBCs. 3.2.2. Effects of DBT on the total body weight and organ weight We first tried to determine if DBT and other treatment had any general effects of the animals’ well being. Animal body weights in vehicle-, DBT-, and TPO-treated groups were determined on
days 0, 7, 14, and 21, respectively. The weights showed a gradual increase on days 14 and 21 (Fig. 3A). However, there was a body weight decrease in vehicle- and TPO-treated animals on day 7 after irradiated treatment, but this decrease was not observed in the DBT-treated mice (Fig. 3A). It is possible that DBT protects the loss of body weight resulting from irradiation. The body weights for mice treated with DBT on days 14 (p < 0.05) and 21 (p < 0.05) were significantly smaller than those in the control group. Similarly, TPOtreated mice showed significantly smaller body weights on days 14
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Fig. 2. Effects of DBT on the blood cell counts of irradiated mice model: mice (n = 6) were irradiated on day 0 and then treated with vehicle (control), DBT (10 mg/kg/day, DBT), and TPO (1 g/kg/day, TPO). Blood was taken, and the numbers of platelets, white blood cells (WBC), and red blood cells (RBCs) were counted. As shown, there were significant decreases in all platelet, WBC, and RBC counts on day 7 after the irradiation. (A) DBT-treated mice showed a significantly higher platelet number on days 14 and 21, indicating that they had accelerated platelet recovery as compared to the vehicle treatment. This thrombopoietic activity of DBT was similar to that of TPO examined in the same experiment. (B) WBCs were also decreased by irradiation. Similarly, DBT significantly increased the recovery of WBCs on days 7, 14, and 21 as compared to those treated with vehicle and TPO. In contrast, the TPO treatment also significantly increased the recovery of WBCs on days 7, 14 and 21. (C) The irradiation also induced the decrease in RBC counts. Similarly, both DBT and TPO treatments significantly increased the cell counts on days 14 and 21 compared with those in the control mice. No significant differences were observed in the counts between DBTor TPO-treated samples, suggesting that their effects are similar. * p < 0.05; # p < 0.01 and + p < 0.001.
(p < 0.05) and 21 (p < 0.05). We then investigated if DBT and other treatments had any effects on the ex-bone marrow, hematopoietic organs (Fig. 3B). We found that the spleens in the DBT-treated mice were significantly larger than those in the control and TPOtreated groups (p < 0.05). To determine the net effects of DBT on animal organ size, we normalized the weight of the organ to that of the body (Fig. 3C). As shown, both liver (p < 0.05) and spleen (p < 0.05) from the DBT-treated mice were significantly larger than those from the control group and the TPO-treated group. In contrast, DBT had no effect on the weight of the control organ kidney. This suggested that DBT might promote the hematopoietic functions of liver and spleen. Further study is needed to investigate this possibility.
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Fig. 3. Effects of DBT on animal body size and organ size. The mice (n = 6) were irradiated on day 0 and then treated with vehicle (control), DBT (10 mg/kg/day, DBT), and TPO (1 g/kg/day, TPO) as described in Fig. 2. Their body weight and organ size were then measured. (A) Effects of DBT on animal weights. Significant differences were observed for the animals in the DBT- and TPO-treated groups compared with those in the control group on days 14 and 21. (B) Effects of DBT treatment on the size of the liver, spleen, and kidney. DBT-treated mice had significantly larger spleens compared with those in other groups. (C) We normalized organ size according to the body weight in order to study the change in organ size relative to body weight. The ratios of body weight/organ weight in the DBT-treated mice are significantly larger than those in the control and TPO-treated groups. * p < 0.05; # p < 0.01 and + p < 0.001.
Fig. 4. DBT significantly increased the formation of the CFU of myeloid (CFU-GM), erythroid (BFU-E), mixed (CFU-GEMM), megakaryocytic (CFU-MK), and bone marrow stromal (CFU-F) cells. There was a significant decrease in the colony counts from the control samples (n = 6). DBT and TPO treatments significantly increased the number of colonies compared with those of the control. Furthermore, except for CFU-GM, the effects of DBT and TPO are significantly different. Abbreviations: DBT, Danggui Buxue Tang; BFU/CFU-E, burst-forming unit/colony-forming unit-erythroid; CFU, colony-forming unit; CFU-E, colony-forming unit-erythroid; CFU-F, colony-forming unit-fibroblast; CFU-GM, colony-forming unit-granulocyte macrophage; CFU-GEMM: colony-forming unit-mixed; CFU-MK, colony-forming unit-megakaryocyte. The following symbols * p < 0.05; # p < 0.01; + p < 0.001 are referring to the comparison between the DBT/TPO-treated groups and the control groups. ** Indicates significant difference between the effects of DBT and TPO treatment.
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Fig. 5. Effects of DBT on bone marrow histology. Wright-Giemsa stained bone marrow sections were examined. The number of megakaryocytes was determined and expressed as an average value for every experimental group. In the DBT-treated irradiated mice, the bone marrow was hyperplastic as compared to that of normal mice. The megakaryocyte number was also significantly increased than that of the control, being very close to that of the normal. In both TPO- and DBT-treated groups, there was a significantly increased recovery of the megakaryocytic and granulocytic series by day 21 as compared to the control group.
3.2.3. DBT significantly increased the formation of bone marrow CFU We then tested the in vivo effect of DBT on the hematopoietic CFU of myeloid (CFU-GM), erythroid (BFU/CFU-E), mixed (CFU-GEMM), and megakaryocytic (CFU-MK) lineages and bone marrow stromal cells (CFU-F). The bone marrow cells in the control, DBT-, and TPOtreated animals were collected and cultured for CFU assays. As
Fig. 6. Effects of DBT on in vitro CFU-MK formation: (A) dose response of CFU-MK formation to DBT treatment. DBT significantly increased CFU-MK formation. The results are presented as mean ± SEM (n = 4). (B) Comparison of the effects of DBT and TPO on CFU-MK formation. Addition of DBT (100 g/ml) and TPO (50 ng/ml) significantly enhanced CFU-MK formation (p < 0.001). Abbreviations: DBT, Danggui Buxue Tang; CFU-MK, colony-forming unit-megakaryocyte. * p < 0.05; # p < 0.01 and + p < 0.001.
Fig. 7. Effects of DBT on the formation of CFU-F from murine bone marrow cells. Murine bone marrow cells at 1 × 106 per milliliter were cultured in Iscove’s modified Dulbecco’s medium and 10% fetal calf serum with various doses of DBT and growth factors for 9 days. (A) DBT significantly increased CFU-F formation in a dose-dependent manner. The results are presented as mean ± SEM (n = 5, DBT vs. control). (B) Bone marrow cells were cultured in the presence of VEGF, FGF, or PDGF (50 ng/ml each) with or without DBT (100 g/ml). Individual factors promoted CFU-F formation compared with those of the control (n = 5, treatment vs. control). The addition of DBT significantly enhanced the effect of FGF on CFU-F formation (p < 0.05). Abbreviations: DBT, Danggui Buxue Tang; CFU-F, colony-forming unit-fibroblast; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor. * p < 0.05; # p < 0.01 and + p < 0.001.
shown in Fig. 4, treatment with DBT led to a significant increase in the formation of CFU-GM, BFU-E, CFU-GEMM, CFU-MK, and CFU-F. Similarly, TPO also significantly increased the formation of these cell lineages (+: p < 0.001). The effects of DBT and TPO were also significantly different (as indicated with “**”) in BFU-E, CFU-GEMM, CFU-MK, and CFU-F but not in CFU-GM. More specifically, the effects of TPO were stronger than those of DBT in BFU-E, CFU-GEMM, and CFU-MK cell lineages, but were weaker than DBT for the CFU-F cell lineage. The underlying mechanisms for this discrepancy remain to be elucidated. 3.2.4. Effects of DBT on bone marrow histology We also investigated bone marrow morphology using WrightGiemsa staining (Fig. 5). Twenty-five randomly selected areas of standardized size were examined (4000×) for mean total cell counts (MTC) and mean cell counts in the three cell lineages, erythroid, granulocytic, and megakaryocytic series in each area. In the irradiated mice (control), the hematopoiesis was markedly suppressed as evident from the dramatic drop (∼50%) in the MTC/area concomitant with an increase of necrotic and apoptotic cells as compared to the normal group. The reduction was particularly prominent in the granulocytic (∼60%) and megakaryocytic (∼100%) series, but was much less so in the erythroid series. In the TPOtreated irradiated mice, tri-lineage hematopoiesis was preserved although overall cellularity (MTC/area) was still slightly subnormal (∼20% reduction as compared to normal). The number of granulocytic and megakaryocytic cells was very close to that of the normal group, indicating a better recovery of these cells than the erythroid series, which showed essentially the same number as the control. In the DBT-treated irradiated mice, the bone marrow was hyperplastic (∼70% increase of cellularity) as compared to the normal, which was mainly due to a prominent granulocytic expansion. The
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Fig. 8. Effects of DBT on the expression of annexin V on M-07e cells. Apoptotic cell death was induced by serum depletion (control). DBT (100 g/ml) or TPO (50 ng/ml) was added to the cultures for 72 h. The cells were stained with annexin V-fluorescein isothiocyanate (FL-1) and propidium iodide (PI) (FL-2). R1 denotes late apoptotic and necrotic cells. R2 denotes early apoptotic cells (annexin V positive and PI negative). R1 + R2 represent total dead cells. These data demonstrated that nutrient deprivation significantly increased cell death (n = 6, control vs. normal), and DBT significantly reduced apoptosis and total cell death (n = 6, DBT vs. control). TPO treatment also significantly reduced M-07e cell death (n = 6, TPO vs. control). Abbreviations: DBT, Danggui Buxue Tang; TPO, Thrombopoietin. * p < 0.05; # p < 0.01 and + p < 0.001.
megakaryocyte number was also significantly increased than that of the control, being very close to that of the normal. In conclusion, in both TPO- and DBT-treated groups, there was a significant increased recovery of the megakaryocytic and granulocytic series by day 21 over the control group. 3.3. Effects of DBT on in vitro megakaryocytopoiesis and proliferation of bone marrow stromal cells 3.3.1. DBT enhanced in vitro CFU-MK formation To evaluate the in vitro effect of DBT on CFU-MK formation, mouse bone marrow cells were cultured in a plasma clot system with increasing concentrations of DBT (0, 50, 100, 200 g/ml), then the numbers of CFU-MK colonies were counted (Fig. 6). DBT significantly enhanced the formation of CFU-MK in a dose-dependent manner (n = 4; * p < 0.01; # p < 0.001). TPO also significantly enhanced the formation of CFU-MK (p < 0.001). TPO has an enhancing effect that is significantly stronger than that of DBT (p < 0.01). Furthermore, the effects of DBT and TPO were significantly different, suggesting that they did not significantly enhance the effect of TPO. Lastly, the effect of DBT + TPO treatment was sig-
nificantly different from DBT alone (p < 0.001), suggesting that DBT and TPO might exert their effects through separate pathways. 3.3.2. DBT enhanced murine bone marrow CFU-F formation Fibroblast cells play supporting roles for the differentiation and proliferation of blood cells. Thus we also tested DBT’s effect and found that DBT promoted CFU-F formation from bone marrow cell cultures in a dose-dependent manner. The presence of 100 g/ml DBT increased the number of CFU-F by 0.33-fold (n = 6, p < 0.01, Fig. 7A). In an independent experiment in which single factor DBT, FGF, PDGF, or VEGF was added to the bone marrow cultures, DBT and all cytokines significantly promote CFU-F formation (n = 6, Fig. 7B). Interestingly, DBT + FGF (p < 0.05) showed significantly stronger promoting effects than FGF or DBT alone, suggesting possible synergistic effects between DBT and FGF. 3.4. DBT exerted antiapoptotic effects on M-07e cells As shown above, DBT and TPO both promoted the growth of platelets, WBCs, and RBCs (Fig. 2) and their corresponding progenitor cells CFU-MK, CFU-GM, and BFU-E (Fig. 4). Our previous studies
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Fig. 9. Effects of DBT on the expression of caspase-3 in M-07e cells. M-07e cells were stained with caspase-3-PE (FL-2). These data demonstrated that nutrient deprivation increased caspase-3 expression (M2; n = 6, normal vs. control), and DBT (DBT vs. control) or TPO (TPO vs. control) treatment significantly reduced caspase-3 expression. Abbreviations: DBT, Danggui Buxue Tang; TPO, thrombopoietin. + p < 0.001.
suggest that the increase in cell population might also result from the antiapoptotic effect of the agents in addition to the direct stimulatory effect on cell growth (Yang et al., 2007). Consequently, we tested the antiapoptotic effect of DBT using megakaryocytic cell line M-07e. Early apoptotic cells (annexin V positive, PI negative, R2) and total dead cells (annexin V positive, R1 + R2) were increased in serum- and cytokine-depleted cultures of megakaryoblastic cells M-07e (n = 6, p < 0.001) (Fig. 8). DBT significantly reduced the proportion of these populations (n = 6, p < 0.001) to near-control levels. Similarly, TPO significantly reduced the proportion of these populations (n = 6, p < 0.001). Interestingly, DBT and TPO showed similar effects as there are no significant differences between the R1 and R2 populations in the DBT- and TPO-treated groups. Both DBT and TPO reduced the proportion of R1 + R2 population to a level significantly different from that of the normal (DBT vs. normal, p < 0.05; TPO vs. normal, p < 0.05). Although the anti-apoptotic effects of DBT and TPO based on the proportion of R2 and R1 + R2 populations are not statistically significant, the data shown in Fig. 8 suggest that DBT might have a slightly stronger antiapoptotic effect than TPO.
The expression of active caspase-3 (M2, Fig. 9), a downstream effector protein of apoptosis, was significantly increased in nutrient-depleted cells (n = 6; p < 0.0001). The treatment with DBT (p < 0.001) or TPO (p < 0.001) significantly reduced caspase-3 expression. There is no significant difference between the effects of DBT and TPO (p = 0.558). Furthermore, both DBT and TPO reduced caspase-3 expression to that of the normal. Mitochondria with normal mitochondrial membrane potential ( m ) concentrate JC-1 into aggregates (R1: red/orange fluorescence in FL2, Fig. 10), whereas in depolarized mitochondria, JC-1 forms monomers (R2: green fluorescence in FL1). Compared with normal M-07e cells, serum-depleted control cultures had an increased proportion of cells containing JC-1 monomers (R2, green fluorescence), indicating a drop in m (n = 7, p < 0.001). This population of apoptotic cells was significantly decreased in cultures containing DBT (p < 0.001) or TPO (p < 0.01). The total population of apoptotic cells (R1 + R2) showed similar patterns in samples from the normal, DBT-treated, and TPO-treated groups (p < 0.001) as compared to the control group. However, R1, a transitional cell subset containing both monomers and aggregates, was not significantly
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Fig. 10. Effects of DBT on the mitochondria membrane potential of M-07e cells. M-07e cells were stained with JC-1 reagent. Control cultures had increased proportion of cells containing JC-1 monomers (R2, green fluorescence FL1). As shown, the percentage of R2 populations is significantly higher in the normal, DBT-, and TPO-treated groups as compared to that in the control group (n = 7). R1, a transitional cell subset containing both monomers and aggregates, was not significantly affected by the reagents. R2: a cell subset showing mitochondria membrane damage. Abbreviations: DBT, Danggui Buxue Tang; TPO, thrombopoietin. * p < 0.05; # p < 0.01 and + p < 0.001.
affected by these agents. DBT and TPO had similar antiapoptotic effects as both reduced the proportion of R2 populations to levels that are not significant to that of the normal group. 3.5. The effects of DBT might be exerted through a pathway independent of TPO To determine if DBT changes the expression level of TPO, we used ELISA to measure the TPO concentration in the plasma of different groups of mice. As shown in Fig. 11, no significant differences in TPO concentration were observed in the plasma from the normal, control mice, and DBT-treated mice, suggesting that the effects observed for DBT were not mediated via up-regulating the protein production of TPO. 4. Discussion Fig. 11. No change in TPO levels after DBT treatment. The levels of TPO in the control, normal, and DBT-treated samples were measured using ELISA. The differences among TPO levels are not statistically significant.
Thrombocytopenia is a common clinical problem, and no effective and safe treatment is clinically available (reviewed in (Ciurea
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and Hoffman, 2007)). First-generation thrombopoietic agents, recombinant TPO, were found to develop neutralizing antibodies and cause thrombocytopenia in patients (Andemariam et al., 2007; Li et al., 2001). Second-generation thrombopoietic agents include AMG531, which is a peptide, and Eltrombopag and AKR501, which are small-molecule, nonpeptide TPO-R agonists (Andemariam et al., 2007). These thrombopoietic agents are still undergoing clinical development (Andemariam et al., 2007; Bussel et al., 2006, 2007; Kuter, 2007). The present study has for the first time demonstrated the specific thrombopoietic effect in addition to the general hematopoietic effects of DBT, justifying future endeavors aiming to identify, isolate, and develop novel thrombopoietic compounds from DBT. One of the unresolved questions is how DBT promotes thrombopoiesis. Megakaryocytopoiesis is a multi-stage biological process which involves the differentiation of hematopoietic stem cells into immature megakaryocytes and of immature megakaryocytes to mature megakaryocytes, which finally produce platelets. The events are regulated by TPO and cytokines such as IL-1 and IL3 (Kaushansky, 2006; Yang et al., 2000), and probably IL-6 and IL-11 (Kaushansky, 1998, 2006). It is conceivable that the chemical components from DBT promoted thrombopoiesis by activating the above cytokines and/or their downstream signaling pathways alone or synergistically. Another question is whether or not DBT promotes thrombopoiesis specifically, as shown in Fig. 2B and C, DBT also promoted the production of WBC and RBC. This is of no surprise considering that TPO can also enhance the differentiation and production of hematopoietic stem cells, which can differentiate into all blood cell lineages (Sitnicka et al., 1996). By analogy, DBT might also exert its effects on earlier hematopoietic stem cells and further research is needed to test this hypothesis. Dozens of chemical compounds, fifty from RAS and twenty from RA, have been isolated and partially characterized (estimated from a database at http://www.cintcm.com). Based on the chemical structures and previous biological studies on these compounds, several groups of the compounds are likely to be responsible for the thrombopoietic activity. First, the polysaccharide components of DBT might be responsible for the effects. The results obtained from our laboratory have shown that (i) Angelica Polysaccharide (APS) significantly enhanced TPO-induced megakaryocytopoiesis in vitro; and (ii) APS enhanced megakaryocytopoiesis and platelet production in vivo in mice (unpublished results). The structures of APS share similarity with those of heparin and several other glycosaminoglycans (GAGs), which have been found to enhance the growth of hematopoietic progenitors and megakaryocytopoiesis (Chen et al., 1999; Gordon et al., 1987; Han et al., 1996; Or et al., 1996; Shen et al., 1994). Second, RAS is rich in vitamin B12 and Folic acid. Both are well known to be required in maintaining DNA stability (Duthie et al., 2002). Since blood cells are under rapid production, any deficiency in vitamin B12 and Folic acid will lead to anemia. This can at least partially explain the “blood production” function of DBT. In the future, specific bioassays will be developed to screen for the active components that contribute to specific biological activities and to elucidate the molecular mechanisms underlining DBT’s functions. 5. Conclusions In this study, we have shown that DBT, a traditional Chinese medicine formula, has thrombopoietic activities in vivo in a mouse model. These effects are likely attributed to three different mechanisms. First, DBT promotes the proliferation of megakaryocytes directly. Second, DBT promotes the proliferation of megakaryocytes indirectly by promoting the proliferation of bone marrow stromal cells. Finally, DBT promotes thrombopoiesis by inhibiting apoptosis of megakaryocytes.
Conflict of interest The authors declare that there are no commercial interests related to this study. Acknowledgements We are also grateful to the following colleagues: Prof. Karen Li from The Chinese University of Hong Kong for help with the flow cytometry experiments; Mr. N.H. Pong, from The Chinese University of Hong Kong for technical assitance in the animal study, Prof. Allan S.Y. Lau from The University of Hong Kong for helpful advice and provisions of academic support in the Molecular Chinese Medicine Program, Ms. Cindy Cheung, Mr. Stanley Chi-Chung Chik and Mr. Keith Hei Kiu Wong from The University of Hong Kong for technical assitance with HPLC experiments. The authors acknowledge the grant of the Seed Funding Programme of HKU (M Yang), Research Grant Council of Hong Kong (Area of Excellence to K.P. Fung and CERG HKU7526/06 M to C. Liu). References Andemariam, B., Psaila, B., Bussel, J.B., 2007. Novel thrombopoietic agents. Hematology/the Education Program of the American Society of Hematology. American Society of Hematology Education Program, 106–113. Bussel, J.B., Cheng, G., Saleh, M.N., Psaila, B., Kovaleva, L., Meddeb, B., Kloczko, J., Hassani, H., Mayer, B., Stone, N.L., Arning, M., Provan, D., Jenkins, J.M., 2007. Eltrombopag for the treatment of chronic idiopathic thrombocytopenic purpura. The New England Journal of Medicine 357, 2237–2247. Bussel, J.B., Kuter, D.J., George, J.N., McMillan, R., Aledort, L.M., Conklin, G.T., Lichtin, A.E., Lyons, R.M., Nieva, J., Wasser, J.S., Wiznitzer, I., Kelly, R., Chen, C.F., Nichol, J.L., 2006. AMG 531, a thrombopoiesis-stimulating protein, for chronic ITP. The New England Journal of Medicine 19, 1672–1681. Chen, Q.S., Wang, Z.Y., Han, Z.C., 1999. Enhanced growth of megakaryocyte colonies in culture in the presence of heparin and fibroblast growth factor. International Journal of Haematology 70, 155–162. Ciurea, S.O., Hoffman, R., 2007. Cytokines for the treatment of thrombocytopenia. Seminars in Hematology 44, 166–182. Dong, T.T., Zhao, K.J., Gao, Q.T., Ji, Z.N., Zhu, T.T., Li, J., Duan, R., Cheung, A.W., Tsim, K.W., 2006. Chemical and biological assessment of a Chinese herbal decoction containing Radix astragali and Radix angelicae sinensis: determination of drug ratio in having optimized properties. Journal of Agricultural and Food Chemistry 54, 2767–2774. Duthie, S.J., Narayanan, S., Brand, G.M., Pirie, L., Grant, G., 2002. Impact of folate deficiency on DNA stability. The Journal of Nutrition 132, 2444S–2449. Gordon, M.Y., Riley, G.P., Watt, S.M., Greaves, M.F., 1987. Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment. Nature 326, 403–405. Han, Z.C., Bellucci, S., Shen, Z.X., Maffrand, J.P., Pascal, M., Petitou, M., Lormeau, J., Caen, J.P., 1996. Glycosaminoglycans enhance megakaryocytopoiesis by modifying the activities of hematopoietic growth regulators. Journal of Cellular Physiology 168, 97–104. Huang, L.F., 2005. The applications of HPLC fingerprints. In: Xie, P.S. (Ed.), Chromatographic Fingerprints of Chinese Medicine. People’s Medical Publishing House, Beijing, pp. 284–288. Inagaki, K., Oda, T., Naka, Y., Shinkai, H., Komatsu, N., Iwamura, H., 2004. Induction of megakaryocytopoiesis and thrombocytopoiesis by JTZ-132, a novel small molecule with thrombopoietin mimetic activities. Blood 104, 58–64. Kaushansky, K., 2006. Lineage-specific hematopoietic growth factors. The New England Journal of Medicine 354, 2034–2045. Kaushansky, K., 1998. Thrombopoietin. The New England Journal of Medicine 339, 746–754. Kuter, D.J., 2007. New thrombopoietic growth factors. Blood 109, 4607–4616. Li, J., Yang, C., Xia, Y., Bertino, A., Glaspy, J., Roberts, M., Kuter, D.J., 2001. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood 98, 3241–3248. Ma, X.Q., Shi, Q., Duan, J.A., Dong, T.T., Tsim, K.W., 2002. Chemical analysis of Radix astragali (Huangqi) in China: a comparison with its adulterants and seasonal variations. Journal of Agricultural and Food Chemistry 50, 4861–4866. Mei, Q.B., Tao, J.Y., Cui, B., 1991. Advances in the pharmacological studies of Radix Angelica sinensis (Oliv) Diels (Chinese Danggui). Chinese Medical Journal 104, 776–781. Or, R., Elad, S., Shpilberg, O., Eldor, A., 1996. Low molecular weight heparin stimulates megakaryocytopoiesis in bone-marrow transplantation patients. American Journal of Hematology 53, 46–48. Shen, Z.X., Basara, N., Xi, X.D., Caen, J., Maffrand, J.P., Pascal, M., Petitou, M., Lormeau, J.C., Han, Z.C., 1994. Fraxiparin, a low-molecular-weight heparin, stimulates megakaryocytopoiesis in vitro and in vivo in mice. British Journal of Haematology 88, 608–612.
M. Yang et al. / Journal of Ethnopharmacology 124 (2009) 87–97 Sitnicka, E., Lin, N., Priestley, G.V., Fox, N., Broudy, V.C., Wolf, N.S., Kaushansky, K., 1996. The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells. Blood 87, 4998–5005. Song, Z.H., Ji, Z.N., Lo, C.K., Dong, T.T., Zhao, K.J., Li, O.T., Haines, C.J., Kung, S.D., Tsim, K.W., 2004. Chemical and biological assessment of a traditional Chinese herbal decoction prepared from Radix astragali and Radix angelicae sinensis: orthogonal array design to optimize the extraction of chemical constituents. Planta Medica 70, 1222–1227. Sun, L., Liu, X., Qiu, L., Wang, J., Liu, M., Fu, D., Luo, Q., 2001. Administration of plasmid DNA expressing human interleukin-6 significantly improves thrombocytopoiesis in irradiated mice. Annals of Hematology 80, 567–572. Sungaran, R., Chisholm, O.T., Markovic, B., Khachigian, L.M., Tanaka, Y., Chong, B.H., 2000. The role of platelet alpha-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells. Blood 95, 3094–3101. Wang, Y., Zhu, B., 1996. The effect of angelica polysaccharide on proliferation and differentiation of hematopoietic progenitor cell. Zhonghua yi xue za zhi 76, 363–366.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
An herbal decoction of Radix astragali and Radix angelicae sinensis promotes hematopoiesis and thrombopoiesis Mo Yang a,∗ , Godfrey C.F. Chan a , Ruixia Deng b , Margaret H. Ng c , Sau Wan Cheng d , Ching Po Lau d , Jie Yu Ye a , Liangjie Wang b , Chang Liu b,∗∗ a
Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China Molecular Chinese Medicine Laboratory, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, PR China d Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, PR China b c
a r t i c l e
i n f o
Article history: Received 30 October 2008 Received in revised form 12 January 2009 Accepted 3 April 2009 Available online 11 April 2009 Keywords: Hematopoiesis Thrombopoiesis Megakaryocytopoiesis Thrombopoietin Apoptosis Radix angelicae sinensis Radix astragali Danggui Buxue Tang (DBT)
a b s t r a c t Ethnopharmacological relevance: A decoction containing Radix angelicae sinensis and Radix astragali (Danggui Buxue Tang, DBT) has been used to raise the “Qi” and nourish the “Blood”. However, its effects on haematopoiesis and particularly thrombopoiesis have not been studied. Aims: This study aims to examine the effects of DBT on hematopoiesis and thrombopoiesis. Materials and methods: A myelosuppression mouse model was treated with DBT (10 mg/kg/day). Peripheral blood cells from DBT and thrombopoietin-treated samples were counted on days 0, 7, 14 and 21. Then CFU assays were used to determine the effects of DBT on the megakaryocytic progenitor cells and other lineages. Last, analyses of annexin V, caspase-3, and mitochondrial membrane potential were conducted in megakaryocytic cell line M-07e. Results: Morphological examination showed that DBT treatment significantly increased the recovery of the megakaryocytic series. DBT significantly enhanced the platelet recovery and CFU-MK formation in vivo. DBT significantly promoted CFU-MK and CFU-F formation. Last, we observed the antiapoptotic effects of DBT on M-07e cells. Conclusion: DBT might promote haematopoiesis and thrombopoiesis in the mouse model through (i) directly promoting the growth of megakaryocytes; (ii) indirectly promoting the growth of bone marrow stromal cells; (iii) inhibiting apoptosis of megakaryocytes. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hematological and cancer patients who experience bone marrow suppression or infiltration resulting from chemotherapy or radiotherapy frequently develop thrombocytopenia, which may lead to hemorrhage and fatality (Ciurea and Hoffman, 2007). In severe cases, platelet transfusion is required to prevent or stop bleeding. However, frequent platelet transfusion can induce the formation of anti-platelet antibodies, and transmit either viral or bacterial infection. Most efforts for the development of thrombopoietic therapy have been focused on thrombopoietin (TPO), the primary growth factor for platelet production (Kaushansky, 2006, 1998). However, the clinical application of TPO produces a neutralizing antibody and induces anti-TPO antibody formation (Li et al., 2001). Therefore, no effective treatment for thrombo-
∗ Corresponding author. Tel.: +852 28199354; fax: +852 28198142. ∗∗ Corresponding author. Tel.: +852 98062110; fax: +852 28175983. E-mail addresses: [email protected] (M. Yang), [email protected] (C. Liu). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.04.007
cytopenia is currently available clinically. Our long-term goal is to identify novel thrombopoietic agents from traditional Chinese medicine (TCM) formulations or products for clinical application. For centuries, dozens of TCM formulations have been used for the promotion of “blood production” and have shown favorable effects on thrombocytopenia (Mei et al., 1991; Wang and Zhu, 1996). One of the most well known formulations is Danggui Buxue Tang (DBT), which consists of two herbs: Radix angelicae sinensis (Danggui, RAS) and Radix astragali (Huangqi, RA). Roots of Angelica sinensis (Oliv.) Diels are the sources of Radix angelicae sinensis and roots of Astragalus membranaceus (Fischer) Bunge and Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K.Hsiao are botanical sources of Radix astragali. We selected DBT for further study for several reasons. First, DBT has been used for “regulating and enriching blood” for almost 800 years (Song et al., 2004), and its clinical efficacies are well established. The standard procedure to prepare DBT includes boiling ∼30 g Radix astragali and 6 g Radix angelicae sinensis in 2 bowls of water by moderate heat until the final volume is reduced to half and daily usage. The decoction
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was thought to be able to raise the “Qi” and nourish the “Blood” of the individual. Second, DBT is a simple formula that consists of two medicinal plants: RAS and RA. Thus, the identification of the active components and their interactions is less complex. Third, the extraction process of DBT (Song et al., 2004) has been standardized, and several chemical compounds such as ferulic acid and ligustilide from RAS, and astragaloside IV, calycosin, and formononetin from RA have been used as marker compounds for quality control (Song et al., 2004). These ensured the chemical consistencies among different preparations. Fourth, more than fifty compounds from RAS and twenty compounds from RA have been identified, which provide a set of candidate compounds for further validation. RAS root is used to invigorate the blood circulation in the treatment of menstrual disorders and to modulate the immune system and RA has been used as an immunostimulating, hepatoprotective, anti-diabetic, analgesic, expectorant and sedative drug (Zheng, 2000). In addition, several previous studies showed DBT can promote hematopoiesis, stimulate cardiovascular circulation, prevent osteoporosis and posses anti-oxidation activity (Mei et al., 1991; Song et al., 2004; Wang and Zhu, 1996). However, no study has been performed to test DBT’s effect on hematopoiesis and particularly, thrombopoiesis. In current study, we examined the effect of DBT on thrombopoiesis as part of the general hematopoietic effects in a mouse model. 2. Materials and methods 2.1. Plant materials and chemical reagents The herbal materials, Astragalus membranaceus var. mongholicus and Astragalus sinensis were provided by Institute of Chinese Medicine, The Chinese University of Hong Kong and were authenticated by us as described previously (Song et al., 2004). Part of the samples are currently deposited in the Molecular Chinese Medicine Laboratory, LKS Faculty of Medicine, University of Hong Kong, China as voucher # mcm-010 for RAS and voucher # mcm-011 for RA. All chemical reagents were purchased from Sigma–Aldrich (St. Louis, MO).
2.3. Quantitative analysis of active constituents Standards including ferulic acid (98% pure) and formononetin (96% pure) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). For calibration, ferulic acid and formononetin were dissolved in 1 ml of MeOH to give serial concentrations from 0.05 to 50 g/ml, and three injections into HPLC were performed for each dilution. HPLC was performed on a 150 mm × 3.9 mm i.d., 4 m Nova-Pak C18 column using an HPLC system consisting of a Waters 626 pump, Waters 600S controller, Waters 717 autosampler, and Waters 486 photodiode array detector. Acetonitrile (Solvent A) and H3 PO4 -acidified water (Solvent B) were used as mobile phases at a flow rate of 1.0 ml/min at room temperature. The gradient profile was 87% B isocratic for 15 min, 87–71% B linear for 5 min, 71% B isocratic for 15 min, 10% B linear for 15 min, and 10% B isocratic for 10 min. Ten microliters of the samples and standards filtered through Millipore filter (0.45 m) were injected, and signals were detected at 254 nm and 313 nm with UV detection. 2.4. Radiation-induced Hematocytopenic Model Seven or eight-week-old male Balb/c mice were obtained from Charles River Japan (Yokohama, Japan) and were given free access to food and water. Ethical permissions for the studies were granted by the Animal Research Welfare Committee, The Chinese University of Hong Kong. Hematocytopenia with thrombocytopenia model was established using 4-Gy-irradiated mice as described previously (Inagaki et al., 2004; Sun et al., 2001). DBT (10 mg/kg/day) and TPO (1 g/kg/day) were given by injection (IP) daily for 21 days starting from the day after radiotherapy in these mice. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) from DBT, TPO, and water control groups were analyzed on days 0, 7, 14, and 21. On day 21, the bone marrow cells were harvested for colonyforming unit (CFU) assays. We measured the plasma TPO levels on day 21. Bone marrow samples were frozen in cryomolds and performed on 5 m sections. The slides were stained with Giemsa staining. Twenty-five random high-power fields from each bone marrow sample were chosen and blindly quantified for histological examination.
2.2. Preparation of DBT extract
2.5. Murine colony-forming unit (CFU) assay
The preparation of DBT extract and the methods of HPLC analyses were based on those reported previously with minor modifications (Dong et al., 2006; Huang, 2005; Ma et al., 2002; Song et al., 2004; Yang and Feng, 1998). Briefly, the plant materials were grounded and powdered by an electric mill (TR-02B, Rong Tsong, Taiwan). Then, 10 g of RA and 2 g of RAS (20 mesh) were accurately weighed and extracted in 8 volume of water by boiling for 2 h. The extraction was filtered and the residue was re-extracted under the same conditions. While nine-tenth of the extract was used in all the following biological experiments, onetenth of the extract was used for HPLC analysis as described below. EtOH was added to this fraction of extract to a final concentration of 70%. The mixture was centrifuged at 4000 × g for 15 min and the precipitate was discarded. The supernatant was dried using a rotavapor (R-210, BUCHI, Switzerland). The residues were dissolved in 5 ml of MeOH and filtered through a Millipore filter (0.45 m). The samples prepared without the EtOH precipitation step could not be passed through the filter. Ten microliters of the sample was injected for HPLC analysis. The detection wave lengths were set at 254 nm and 313 nm for formononetin and ferulic acid, respectively. Peaks were assigned by comparing the retention time and UV spectra of the peaks to that of the standard compounds.
The assay was performed as described previously (Yang et al., 2007). Colony-forming unit-granulocyte macrophage (CFU-GM), burst-forming unit/colony-forming unit-erythroid (BFU/CFU-E), and colony-forming unit-mixed (CFU-GEMM) were cultured in methylcellulose (1%) supplemented with fetal calf serum (FCS, 30%), 1% BSA, 0.1 mM -mercaptoethanol, 3 IU/ml erythropoietin, 10 ng/ml granulocyte macrophage-colony stimulating factor, 10 ng/ml interleukin-3, and 50 ng/ml SCF. Murine bone marrow cells (2 × 105 cells/ml) were seeded in triplicate and incubated for 7 days. Colonies were scored blindly. 2.6. Murine colony-forming unit-megakaryocytes (CFU-MK) assay Murine bone marrow cells (2 × 105 cells) were cultured using the plasma clot culture method (Yang et al., 2007, 2000). The culture medium contained 1% deionized bovine serum albumin (BSA) (Sigma, MO, USA), 0.34 mg CaCl2 , 10% citrated bovine plasma (Sigma), 100 g penicillin, 50 g streptomycin, and IMDM with different concentrations of DBT, TPO, and IL-3 in a total volume of 1 ml. The cells were incubated at 37 ◦ C under 5% CO2 for 7 days, and the number of CFU-MK derived colonies was counted using the acetyl-choline esterase (AchE) staining method
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after 7 days. The colonies were further stained with hematoxylin to count the CFU-GM derived colonies. A CFU-MK colony was defined as a cluster of three or more AchE positive cells, while a CFU-GM colony was considered as a cluster of 40 or more cells. 2.7. Murine bone marrow colony-forming unit-fibroblast (CFU-F) assay The assay was performed as described previously (Yang et al., 2001). Briefly, mouse bone marrow cells (1 × 106 cells) were seeded in 2 ml of IMDM with 10% FCS in triplicates. Cultured cells were incubated at 37 ◦ C and 5% CO2 in a fully humidified atmosphere with or without DBT for 9 days. Fibroblastoid colony-forming cells (CFU-F) assay were used to determine the number of bone marrow-derived fibroblastoid (Yang et al., 2001). Briefly, adherent cells were stained with Giemsa staining. The number of CFU-F colonies was counted under a light microscope. An aggregate containing more than 10 fibroblasts was counted as a CFU-F colony. The effects of DBT and other cytokines such as fibroblast growth factor (FGF, 50 ng/ml), platelet-derived growth factor (PDGF, 50 ng/ml), or vascular endothelial growth factor (VEGF, 50 ng/ml) were also examined using the CFU-F assay. 2.8. Determining TPO levels by ELISA assay TPO level was measured by an ELISA kit (R & D, Minneapolis) as described previously (Sungaran et al., 2000). A monoclonal antibody specific for TPO was pre-coated on to a microplate. Standards and samples were added to the wells. After washing away the unbound substances, enzyme-linked polyclonal antibodies specific for TPO were added to the wells. After removing any unbound antibodies, substrates were added to the wells. The colors developed were in proportion to the amount of TPO bound on the wells. The optical density of each well was measured using a microreader (DNATECH MR 5000) at 450 nm wave length. 2.9. Annexin V, caspase-3, and mitochondrial membrane potential analysis of M-07e cells by flow cytometry The assays were performed as described previously (Yang et al., 2007). Briefly, the megakaryoblastic cell line M-07e (American Type Culture Collection, Manassas, VA, http://www.atcc.org) was maintained in IMDM supplemented with GM-CSF (20 ng/ml) and 10% FCS. Apoptotic cell death was induced by cytokine and serum depletion. We added DBT (200 mg/ml) to the cultures for 72 h. Apoptotic cell death was examined using annexin VFITC/PI, active caspase-3-PE, and JC-1 ApoAlert reagent kits (BD Biosciences, San Diego, http://www.bdbiosciences.com) according to the manufacturer’s instructions. Ten thousand events were acquired for each sample and were analyzed by flow cytometry using Lysis II software (FACScan; BD Pharmingen). 2.10. Statistical analysis Treatment groups were compared using analysis of variance and paired t test or Wilcoxon signed rank test, depending on data distribution, using the JMP software (SAS, Cary, NC). A p value of <0.05 was considered statistically significant. All values were expressed as mean ± SEM. Statistical significances were denoted with three different symbols: “*” (p < 0.05), “#” (p < 0.01) and “+” (p < 0.001).
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3. Results 3.1. Preparation of DBT extract and quantitative analysis of the main constituents The HPLC calibration curves of formononetin and ferulic acid exhibited good linearity in a range from ∼0.05 to ∼50 g/ml. The RSDs were between 1% and 2%. Fig. 1A–D shows the typical HPLC chromatograms of the standard compounds and their relative UV absorption curves, which confirmed the identities of the standard compounds used in the study. The HPLC chromatograms of DBT water extracts detected at 254 nm and 313 nm are shown in Fig. 1E and F, respectively. We identified the peaks for the standard compounds by comparing the retention times as well as the UV absorption curves of the unknown peaks with those of the standards eluted under the same conditions. To quantify the amount of the standard compounds in the DBT extracts, we first constructed the standard curve for each standard. The linear regression equation for formononetin was y = 8E + 06x − 101292, with r2 = 0.9993. For ferulic acid, its equation was y = 514217x − 10751, with r2 = 0.9994. Based on standard curves, the amount of compounds in DBT extracts was found to be 205 mg (for formononetin) and 9 mg (for ferulic acid) per gram of the starting dry DBT materials, respectively, which are similar to those described in previous studies (Dong et al., 2006). It should be pointed out that formononetin is only found in RA, and ferulic acid is only found in RAS. 3.2. Effects of DBT on hematopoiesis and thrombopoiesis in myelosuppressed mice 3.2.1. Effects of DBT on blood cell counts The myelosuppressed mice showed platelet nadir (<300 × 109 /l) on day 7, and then the platelet counts gradually recovered to approximately 80% of that of day 0 by day 21 (Fig. 2A). The platelet counts in the three groups (control, DBT, and TPO, n = 6) on days 0 and 7 did not show significant differences. Mice treated with DBT at 10 mg/kg/day showed a significantly higher platelet number on days 14 (p < 0.01) and 21 (p < 0.01) and accelerated platelet recovery compared with the control mice. Similarly, TPO-treatment at 1 g/kg/day significantly increased the platelet counts as compared to that of the control on days 14 (p < 0.001) and 21 (p < 0.001). This thrombocytopoietic activity of DBT was similar to that of TPO as there were no significant differences in the platelet counts between samples treated with DBT and TPO (Fig. 2A). Although TPO is a primary regulator of megakaryocyte and platelet production in vitro and in vivo, it has also been shown to exert effects on early hematopoietic stem cells (Sitnicka et al., 1996), which can give rises to WBC and RBC. Consequently, we also studied the effects of DBT and TPO on the counts of WBC and RBC. The changes in WBC counts in the experiment are shown in Fig. 2B. No significant differences were seen in the WBC counts on day 0 among the three treatment groups. However, the WBC counts in DBT-treated mice showed a significantly increased recovery on days 7 (p < 0.001), 14 (p < 0.01), and 21 (p < 0.001). Similarly, the WBC counts in TPO-treated mice also showed a significantly increased recovery on days 7 (p < 0.001), 14 (p < 0.01) and 21 (p < 0.001). No significant differences were observed in the WBC counts in the mice treated with DBT and TPO on days 0, 7, 14, and 21, suggesting that DBT and TPO have similar effects. Finally, the changes in RBC counts among the three treatment groups on days 0 and 7 showed no significant difference (Fig. 2C). However, the RBC counts of DBT-treated mice showed a significant increased recovery on days 14 (p < 0.01) and 21 (p < 0.01). Similarly, the RBC counts in the TPO-treated mice also showed a significant increased recovery on days 14 (p < 0.05) and 21 (p < 0.001).
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Fig. 1. Quantification of DBT based on two marker compounds formononetin (a) and ferulic acid (b). HPLC analyses of the standard compound formononetin (A) and ferulic acid (C) are shown. UV spectra (B for formononetin and D for ferulic acid) were used to confirm the identity of the compounds. HPLC analyses of the water extracts of DBT are shown in E and F, respectively. Individual chemical markers are indicated by arrows.
No significant differences were observed in the RBC counts in mice treated with DBT and TPO on days 0, 7, 14, and 21, suggesting that DBT and TPO have similar effects on the recovery of RBCs. 3.2.2. Effects of DBT on the total body weight and organ weight We first tried to determine if DBT and other treatment had any general effects of the animals’ well being. Animal body weights in vehicle-, DBT-, and TPO-treated groups were determined on
days 0, 7, 14, and 21, respectively. The weights showed a gradual increase on days 14 and 21 (Fig. 3A). However, there was a body weight decrease in vehicle- and TPO-treated animals on day 7 after irradiated treatment, but this decrease was not observed in the DBT-treated mice (Fig. 3A). It is possible that DBT protects the loss of body weight resulting from irradiation. The body weights for mice treated with DBT on days 14 (p < 0.05) and 21 (p < 0.05) were significantly smaller than those in the control group. Similarly, TPOtreated mice showed significantly smaller body weights on days 14
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Fig. 2. Effects of DBT on the blood cell counts of irradiated mice model: mice (n = 6) were irradiated on day 0 and then treated with vehicle (control), DBT (10 mg/kg/day, DBT), and TPO (1 g/kg/day, TPO). Blood was taken, and the numbers of platelets, white blood cells (WBC), and red blood cells (RBCs) were counted. As shown, there were significant decreases in all platelet, WBC, and RBC counts on day 7 after the irradiation. (A) DBT-treated mice showed a significantly higher platelet number on days 14 and 21, indicating that they had accelerated platelet recovery as compared to the vehicle treatment. This thrombopoietic activity of DBT was similar to that of TPO examined in the same experiment. (B) WBCs were also decreased by irradiation. Similarly, DBT significantly increased the recovery of WBCs on days 7, 14, and 21 as compared to those treated with vehicle and TPO. In contrast, the TPO treatment also significantly increased the recovery of WBCs on days 7, 14 and 21. (C) The irradiation also induced the decrease in RBC counts. Similarly, both DBT and TPO treatments significantly increased the cell counts on days 14 and 21 compared with those in the control mice. No significant differences were observed in the counts between DBTor TPO-treated samples, suggesting that their effects are similar. * p < 0.05; # p < 0.01 and + p < 0.001.
(p < 0.05) and 21 (p < 0.05). We then investigated if DBT and other treatments had any effects on the ex-bone marrow, hematopoietic organs (Fig. 3B). We found that the spleens in the DBT-treated mice were significantly larger than those in the control and TPOtreated groups (p < 0.05). To determine the net effects of DBT on animal organ size, we normalized the weight of the organ to that of the body (Fig. 3C). As shown, both liver (p < 0.05) and spleen (p < 0.05) from the DBT-treated mice were significantly larger than those from the control group and the TPO-treated group. In contrast, DBT had no effect on the weight of the control organ kidney. This suggested that DBT might promote the hematopoietic functions of liver and spleen. Further study is needed to investigate this possibility.
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Fig. 3. Effects of DBT on animal body size and organ size. The mice (n = 6) were irradiated on day 0 and then treated with vehicle (control), DBT (10 mg/kg/day, DBT), and TPO (1 g/kg/day, TPO) as described in Fig. 2. Their body weight and organ size were then measured. (A) Effects of DBT on animal weights. Significant differences were observed for the animals in the DBT- and TPO-treated groups compared with those in the control group on days 14 and 21. (B) Effects of DBT treatment on the size of the liver, spleen, and kidney. DBT-treated mice had significantly larger spleens compared with those in other groups. (C) We normalized organ size according to the body weight in order to study the change in organ size relative to body weight. The ratios of body weight/organ weight in the DBT-treated mice are significantly larger than those in the control and TPO-treated groups. * p < 0.05; # p < 0.01 and + p < 0.001.
Fig. 4. DBT significantly increased the formation of the CFU of myeloid (CFU-GM), erythroid (BFU-E), mixed (CFU-GEMM), megakaryocytic (CFU-MK), and bone marrow stromal (CFU-F) cells. There was a significant decrease in the colony counts from the control samples (n = 6). DBT and TPO treatments significantly increased the number of colonies compared with those of the control. Furthermore, except for CFU-GM, the effects of DBT and TPO are significantly different. Abbreviations: DBT, Danggui Buxue Tang; BFU/CFU-E, burst-forming unit/colony-forming unit-erythroid; CFU, colony-forming unit; CFU-E, colony-forming unit-erythroid; CFU-F, colony-forming unit-fibroblast; CFU-GM, colony-forming unit-granulocyte macrophage; CFU-GEMM: colony-forming unit-mixed; CFU-MK, colony-forming unit-megakaryocyte. The following symbols * p < 0.05; # p < 0.01; + p < 0.001 are referring to the comparison between the DBT/TPO-treated groups and the control groups. ** Indicates significant difference between the effects of DBT and TPO treatment.
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Fig. 5. Effects of DBT on bone marrow histology. Wright-Giemsa stained bone marrow sections were examined. The number of megakaryocytes was determined and expressed as an average value for every experimental group. In the DBT-treated irradiated mice, the bone marrow was hyperplastic as compared to that of normal mice. The megakaryocyte number was also significantly increased than that of the control, being very close to that of the normal. In both TPO- and DBT-treated groups, there was a significantly increased recovery of the megakaryocytic and granulocytic series by day 21 as compared to the control group.
3.2.3. DBT significantly increased the formation of bone marrow CFU We then tested the in vivo effect of DBT on the hematopoietic CFU of myeloid (CFU-GM), erythroid (BFU/CFU-E), mixed (CFU-GEMM), and megakaryocytic (CFU-MK) lineages and bone marrow stromal cells (CFU-F). The bone marrow cells in the control, DBT-, and TPOtreated animals were collected and cultured for CFU assays. As
Fig. 6. Effects of DBT on in vitro CFU-MK formation: (A) dose response of CFU-MK formation to DBT treatment. DBT significantly increased CFU-MK formation. The results are presented as mean ± SEM (n = 4). (B) Comparison of the effects of DBT and TPO on CFU-MK formation. Addition of DBT (100 g/ml) and TPO (50 ng/ml) significantly enhanced CFU-MK formation (p < 0.001). Abbreviations: DBT, Danggui Buxue Tang; CFU-MK, colony-forming unit-megakaryocyte. * p < 0.05; # p < 0.01 and + p < 0.001.
Fig. 7. Effects of DBT on the formation of CFU-F from murine bone marrow cells. Murine bone marrow cells at 1 × 106 per milliliter were cultured in Iscove’s modified Dulbecco’s medium and 10% fetal calf serum with various doses of DBT and growth factors for 9 days. (A) DBT significantly increased CFU-F formation in a dose-dependent manner. The results are presented as mean ± SEM (n = 5, DBT vs. control). (B) Bone marrow cells were cultured in the presence of VEGF, FGF, or PDGF (50 ng/ml each) with or without DBT (100 g/ml). Individual factors promoted CFU-F formation compared with those of the control (n = 5, treatment vs. control). The addition of DBT significantly enhanced the effect of FGF on CFU-F formation (p < 0.05). Abbreviations: DBT, Danggui Buxue Tang; CFU-F, colony-forming unit-fibroblast; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor. * p < 0.05; # p < 0.01 and + p < 0.001.
shown in Fig. 4, treatment with DBT led to a significant increase in the formation of CFU-GM, BFU-E, CFU-GEMM, CFU-MK, and CFU-F. Similarly, TPO also significantly increased the formation of these cell lineages (+: p < 0.001). The effects of DBT and TPO were also significantly different (as indicated with “**”) in BFU-E, CFU-GEMM, CFU-MK, and CFU-F but not in CFU-GM. More specifically, the effects of TPO were stronger than those of DBT in BFU-E, CFU-GEMM, and CFU-MK cell lineages, but were weaker than DBT for the CFU-F cell lineage. The underlying mechanisms for this discrepancy remain to be elucidated. 3.2.4. Effects of DBT on bone marrow histology We also investigated bone marrow morphology using WrightGiemsa staining (Fig. 5). Twenty-five randomly selected areas of standardized size were examined (4000×) for mean total cell counts (MTC) and mean cell counts in the three cell lineages, erythroid, granulocytic, and megakaryocytic series in each area. In the irradiated mice (control), the hematopoiesis was markedly suppressed as evident from the dramatic drop (∼50%) in the MTC/area concomitant with an increase of necrotic and apoptotic cells as compared to the normal group. The reduction was particularly prominent in the granulocytic (∼60%) and megakaryocytic (∼100%) series, but was much less so in the erythroid series. In the TPOtreated irradiated mice, tri-lineage hematopoiesis was preserved although overall cellularity (MTC/area) was still slightly subnormal (∼20% reduction as compared to normal). The number of granulocytic and megakaryocytic cells was very close to that of the normal group, indicating a better recovery of these cells than the erythroid series, which showed essentially the same number as the control. In the DBT-treated irradiated mice, the bone marrow was hyperplastic (∼70% increase of cellularity) as compared to the normal, which was mainly due to a prominent granulocytic expansion. The
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Fig. 8. Effects of DBT on the expression of annexin V on M-07e cells. Apoptotic cell death was induced by serum depletion (control). DBT (100 g/ml) or TPO (50 ng/ml) was added to the cultures for 72 h. The cells were stained with annexin V-fluorescein isothiocyanate (FL-1) and propidium iodide (PI) (FL-2). R1 denotes late apoptotic and necrotic cells. R2 denotes early apoptotic cells (annexin V positive and PI negative). R1 + R2 represent total dead cells. These data demonstrated that nutrient deprivation significantly increased cell death (n = 6, control vs. normal), and DBT significantly reduced apoptosis and total cell death (n = 6, DBT vs. control). TPO treatment also significantly reduced M-07e cell death (n = 6, TPO vs. control). Abbreviations: DBT, Danggui Buxue Tang; TPO, Thrombopoietin. * p < 0.05; # p < 0.01 and + p < 0.001.
megakaryocyte number was also significantly increased than that of the control, being very close to that of the normal. In conclusion, in both TPO- and DBT-treated groups, there was a significant increased recovery of the megakaryocytic and granulocytic series by day 21 over the control group. 3.3. Effects of DBT on in vitro megakaryocytopoiesis and proliferation of bone marrow stromal cells 3.3.1. DBT enhanced in vitro CFU-MK formation To evaluate the in vitro effect of DBT on CFU-MK formation, mouse bone marrow cells were cultured in a plasma clot system with increasing concentrations of DBT (0, 50, 100, 200 g/ml), then the numbers of CFU-MK colonies were counted (Fig. 6). DBT significantly enhanced the formation of CFU-MK in a dose-dependent manner (n = 4; * p < 0.01; # p < 0.001). TPO also significantly enhanced the formation of CFU-MK (p < 0.001). TPO has an enhancing effect that is significantly stronger than that of DBT (p < 0.01). Furthermore, the effects of DBT and TPO were significantly different, suggesting that they did not significantly enhance the effect of TPO. Lastly, the effect of DBT + TPO treatment was sig-
nificantly different from DBT alone (p < 0.001), suggesting that DBT and TPO might exert their effects through separate pathways. 3.3.2. DBT enhanced murine bone marrow CFU-F formation Fibroblast cells play supporting roles for the differentiation and proliferation of blood cells. Thus we also tested DBT’s effect and found that DBT promoted CFU-F formation from bone marrow cell cultures in a dose-dependent manner. The presence of 100 g/ml DBT increased the number of CFU-F by 0.33-fold (n = 6, p < 0.01, Fig. 7A). In an independent experiment in which single factor DBT, FGF, PDGF, or VEGF was added to the bone marrow cultures, DBT and all cytokines significantly promote CFU-F formation (n = 6, Fig. 7B). Interestingly, DBT + FGF (p < 0.05) showed significantly stronger promoting effects than FGF or DBT alone, suggesting possible synergistic effects between DBT and FGF. 3.4. DBT exerted antiapoptotic effects on M-07e cells As shown above, DBT and TPO both promoted the growth of platelets, WBCs, and RBCs (Fig. 2) and their corresponding progenitor cells CFU-MK, CFU-GM, and BFU-E (Fig. 4). Our previous studies
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Fig. 9. Effects of DBT on the expression of caspase-3 in M-07e cells. M-07e cells were stained with caspase-3-PE (FL-2). These data demonstrated that nutrient deprivation increased caspase-3 expression (M2; n = 6, normal vs. control), and DBT (DBT vs. control) or TPO (TPO vs. control) treatment significantly reduced caspase-3 expression. Abbreviations: DBT, Danggui Buxue Tang; TPO, thrombopoietin. + p < 0.001.
suggest that the increase in cell population might also result from the antiapoptotic effect of the agents in addition to the direct stimulatory effect on cell growth (Yang et al., 2007). Consequently, we tested the antiapoptotic effect of DBT using megakaryocytic cell line M-07e. Early apoptotic cells (annexin V positive, PI negative, R2) and total dead cells (annexin V positive, R1 + R2) were increased in serum- and cytokine-depleted cultures of megakaryoblastic cells M-07e (n = 6, p < 0.001) (Fig. 8). DBT significantly reduced the proportion of these populations (n = 6, p < 0.001) to near-control levels. Similarly, TPO significantly reduced the proportion of these populations (n = 6, p < 0.001). Interestingly, DBT and TPO showed similar effects as there are no significant differences between the R1 and R2 populations in the DBT- and TPO-treated groups. Both DBT and TPO reduced the proportion of R1 + R2 population to a level significantly different from that of the normal (DBT vs. normal, p < 0.05; TPO vs. normal, p < 0.05). Although the anti-apoptotic effects of DBT and TPO based on the proportion of R2 and R1 + R2 populations are not statistically significant, the data shown in Fig. 8 suggest that DBT might have a slightly stronger antiapoptotic effect than TPO.
The expression of active caspase-3 (M2, Fig. 9), a downstream effector protein of apoptosis, was significantly increased in nutrient-depleted cells (n = 6; p < 0.0001). The treatment with DBT (p < 0.001) or TPO (p < 0.001) significantly reduced caspase-3 expression. There is no significant difference between the effects of DBT and TPO (p = 0.558). Furthermore, both DBT and TPO reduced caspase-3 expression to that of the normal. Mitochondria with normal mitochondrial membrane potential ( m ) concentrate JC-1 into aggregates (R1: red/orange fluorescence in FL2, Fig. 10), whereas in depolarized mitochondria, JC-1 forms monomers (R2: green fluorescence in FL1). Compared with normal M-07e cells, serum-depleted control cultures had an increased proportion of cells containing JC-1 monomers (R2, green fluorescence), indicating a drop in m (n = 7, p < 0.001). This population of apoptotic cells was significantly decreased in cultures containing DBT (p < 0.001) or TPO (p < 0.01). The total population of apoptotic cells (R1 + R2) showed similar patterns in samples from the normal, DBT-treated, and TPO-treated groups (p < 0.001) as compared to the control group. However, R1, a transitional cell subset containing both monomers and aggregates, was not significantly
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Fig. 10. Effects of DBT on the mitochondria membrane potential of M-07e cells. M-07e cells were stained with JC-1 reagent. Control cultures had increased proportion of cells containing JC-1 monomers (R2, green fluorescence FL1). As shown, the percentage of R2 populations is significantly higher in the normal, DBT-, and TPO-treated groups as compared to that in the control group (n = 7). R1, a transitional cell subset containing both monomers and aggregates, was not significantly affected by the reagents. R2: a cell subset showing mitochondria membrane damage. Abbreviations: DBT, Danggui Buxue Tang; TPO, thrombopoietin. * p < 0.05; # p < 0.01 and + p < 0.001.
affected by these agents. DBT and TPO had similar antiapoptotic effects as both reduced the proportion of R2 populations to levels that are not significant to that of the normal group. 3.5. The effects of DBT might be exerted through a pathway independent of TPO To determine if DBT changes the expression level of TPO, we used ELISA to measure the TPO concentration in the plasma of different groups of mice. As shown in Fig. 11, no significant differences in TPO concentration were observed in the plasma from the normal, control mice, and DBT-treated mice, suggesting that the effects observed for DBT were not mediated via up-regulating the protein production of TPO. 4. Discussion Fig. 11. No change in TPO levels after DBT treatment. The levels of TPO in the control, normal, and DBT-treated samples were measured using ELISA. The differences among TPO levels are not statistically significant.
Thrombocytopenia is a common clinical problem, and no effective and safe treatment is clinically available (reviewed in (Ciurea
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and Hoffman, 2007)). First-generation thrombopoietic agents, recombinant TPO, were found to develop neutralizing antibodies and cause thrombocytopenia in patients (Andemariam et al., 2007; Li et al., 2001). Second-generation thrombopoietic agents include AMG531, which is a peptide, and Eltrombopag and AKR501, which are small-molecule, nonpeptide TPO-R agonists (Andemariam et al., 2007). These thrombopoietic agents are still undergoing clinical development (Andemariam et al., 2007; Bussel et al., 2006, 2007; Kuter, 2007). The present study has for the first time demonstrated the specific thrombopoietic effect in addition to the general hematopoietic effects of DBT, justifying future endeavors aiming to identify, isolate, and develop novel thrombopoietic compounds from DBT. One of the unresolved questions is how DBT promotes thrombopoiesis. Megakaryocytopoiesis is a multi-stage biological process which involves the differentiation of hematopoietic stem cells into immature megakaryocytes and of immature megakaryocytes to mature megakaryocytes, which finally produce platelets. The events are regulated by TPO and cytokines such as IL-1 and IL3 (Kaushansky, 2006; Yang et al., 2000), and probably IL-6 and IL-11 (Kaushansky, 1998, 2006). It is conceivable that the chemical components from DBT promoted thrombopoiesis by activating the above cytokines and/or their downstream signaling pathways alone or synergistically. Another question is whether or not DBT promotes thrombopoiesis specifically, as shown in Fig. 2B and C, DBT also promoted the production of WBC and RBC. This is of no surprise considering that TPO can also enhance the differentiation and production of hematopoietic stem cells, which can differentiate into all blood cell lineages (Sitnicka et al., 1996). By analogy, DBT might also exert its effects on earlier hematopoietic stem cells and further research is needed to test this hypothesis. Dozens of chemical compounds, fifty from RAS and twenty from RA, have been isolated and partially characterized (estimated from a database at http://www.cintcm.com). Based on the chemical structures and previous biological studies on these compounds, several groups of the compounds are likely to be responsible for the thrombopoietic activity. First, the polysaccharide components of DBT might be responsible for the effects. The results obtained from our laboratory have shown that (i) Angelica Polysaccharide (APS) significantly enhanced TPO-induced megakaryocytopoiesis in vitro; and (ii) APS enhanced megakaryocytopoiesis and platelet production in vivo in mice (unpublished results). The structures of APS share similarity with those of heparin and several other glycosaminoglycans (GAGs), which have been found to enhance the growth of hematopoietic progenitors and megakaryocytopoiesis (Chen et al., 1999; Gordon et al., 1987; Han et al., 1996; Or et al., 1996; Shen et al., 1994). Second, RAS is rich in vitamin B12 and Folic acid. Both are well known to be required in maintaining DNA stability (Duthie et al., 2002). Since blood cells are under rapid production, any deficiency in vitamin B12 and Folic acid will lead to anemia. This can at least partially explain the “blood production” function of DBT. In the future, specific bioassays will be developed to screen for the active components that contribute to specific biological activities and to elucidate the molecular mechanisms underlining DBT’s functions. 5. Conclusions In this study, we have shown that DBT, a traditional Chinese medicine formula, has thrombopoietic activities in vivo in a mouse model. These effects are likely attributed to three different mechanisms. First, DBT promotes the proliferation of megakaryocytes directly. Second, DBT promotes the proliferation of megakaryocytes indirectly by promoting the proliferation of bone marrow stromal cells. Finally, DBT promotes thrombopoiesis by inhibiting apoptosis of megakaryocytes.
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