Plant Materials and Chemical Reagents
The roots of Angelica sinensis (Oliv.) Diels, (RAS, Chinese name: Danggui) used in our experiments were purchased from Minxian County, Gansu Province, China. These samples are currently deposited in the Molecular Chinese Medicine Laboratory, University of Hong Kong as voucher # mcm-011. The standard RAS materials used for quality control was purchased from National Institute for the Control of Pharmaceutical and Biological Products, China (batch number: 120927-200411). Anthrone and glucose were purchased from Guoyao Enterprise group, Tianjing Damao Chemicals Company (Tianjing, China). Sulfuric acid was purchased from Sigma-Aldrich (St Louis, MO).
Quantitative Analysis of Plant Materials
The experimental and standard RAS materials were dried at 60°C, ground and powdered by an electric mill (TR-02B, Rong Tsong, Taiwan) and sieved through a mesh (size 20). The powders were then ground with KBr (AR, Perkin Elmer, UK) and pressed into pellets using a KBr hand press (Specac, UK). The pellets were analyzed by a Spectrum 100 (PerkinElmer, UK) under the following conditions, wavelength range: 400 to 4000 cm-1; spectral resolution: 4 cm-1. Data analysis was performed using software PerkinElmer Spectrum (Version 6.2.0).
Preparation and Quantitative Analysis of APS Samples
RAS polysaccharides (APS) were prepared according to the method described by Cho et al . Briefly, 100 grams of RAS were extracted three times by boiling in water for four-hours each time. All water extracts were pooled and mixed with ethanol to a final concentration of 75% v/v to precipitate the polysaccharide-enriched fraction (APS). The total amounts of polysaccharides were measured using the anthrone-sulfuric acid assay. Briefly, the standard glucose solution was prepared in MiliQ water at serial concentrations of 0, 0.010, 0.020, 0.040, 0.060 and 0.080 mg/ml. Four ml of anthrone-sulfuric acid solutions (0.2 g/ml) were added to a 1 ml APS solution and each of the six standard solutions respectively. All solutions were kept and mixed on ice. The mixtures were boiled in a water bath for 15 min and left at room temperature for 10 min before they were analyzed using a UV/VIS Spectra Lambda 35 (PerkinElmer). Three replicate analyses were conducted.
Testing Endotoxic Activity by Gel-clot Tachypleus Amebocyte Lysate (TAL) Test
All materials required for TAL test including Tachypleus Amebocyte Lysate, control standard endotoxin (10 EU/ml), TAL reagent water, test tubes and pipette tips free of detectable endotoxin were purchased from Zhanjiang Bokang Marine Biological Co., LTD (Guangdong, P.R.China). The gel-clot TAL tests were preformed according to the method described in Chinese Pharmacopoeia 2010 edition. Briefly, 100 μl series dilutions of control standard endotoxin or APS extracts were mixed with 100 μl TAL reagents. The mixed solutions were incubated at 37°C for 60 minutes. The formation of the gel was scored by turning each test tube upside down. If the gel remained a piece, it is considered a solid gel formation. Otherwise, it is considered a failed gel formation. Endotoxin is measured in Endotoxin Units per milliliter (EU/ml). One EU equals approximately 0.4 ng/ml of endotoxin solution in our experiment.
Radiation-induced Haematocytopenic Model
Seven to eight week-old male Balb/c mice were obtained from Charles River Japan (Yokohama, Japan) and given free access to food and water. The treatment of animals were conducted following protocols provided by Hong Kong government and ethical permissions for the studies were granted by the Animal Research Welfare Committee, The Chinese University of Hong Kong. A haematocytopenia with thrombocytopenia model was established using 4-Gy-irradiated mice as described previously [12, 13]. APS (2.5 mg/day) and TPO (0.25 μg/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 APS, TPO and water control groups were analyzed on days 0, 7, 14, and 21. On day 21, the bone marrow cells were harvested for colony-forming unit (CFU) assays. We measured the plasma TPO levels on day 21. Bone marrow samples were frozen in cryomolds and performed on 5-μm 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.
Murine Colony-Forming Unit (CFU) Assay
The assay was performed as described previously . 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 β-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.
Murine Colony-Forming Unit-Megakaryocytes (CFU-MK) Assay
Murine bone marrow cells (2 × 105 cells) were cultured using the plasma clot culture method [14, 15]. The culture medium contains 1% deionized bovine serum albumin (BSA) (Sigma, Mo USA), 0.34 mg CaCl2, 10% citrated bovine plasma (Sigma), 100 μg penicillin, 50 μg streptomycin and IMDM with different concentrations of APS, 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 acetyl-choline esterase (AchE) staining method after 7 days. The colonies were further stained with haematoxylin to count the CFU-GM derived colonies. A CFU-MK colony was defined as a cluster of 3 or more AchE positive cells and a CFU-GM colony was considered as a cluster of 40 or more cells.
Murine Bone Marrow Colony-Forming Unit-Fibroblast (CFU-F) assay
The assay was performed as described previously [16, 17]. 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 APS for 9 days. Fibroblastoid colony-forming cells (CFU-F) assay were used to determine the number of bone marrow-derived fibroblastoid . 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 were counted as a CFU-F colony. Effects of APS 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.
Annexin V, Caspase 3, and Mitochondrial Membrane Potential Analyses of M-07e Cells by Flow Cytometry
The assays were performed as described previously . 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 APS (200 μg/ml) to the cultures and then the cells were incubated for 72 hours. Apoptotic cell death was examined using the annexin V-FITC/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 analyzed by flow cytometry using the Lysis II software (FACScan; BD Pharmingen).
Treatment groups were compared using analysis of variance, 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).