J Nat Med DOI 10.1007/s11418-017-1135-0

ORIGINAL PAPER

A novel mode of stimulating platelet formation activity in megakaryocytes with peanut skin extract Takahiro Sato1 · Masako Akiyama1 · Ken‑ichi Nakahama1 · Shujiro Seo2 · Masamichi Watanabe2 · Jin Tatsuzaki2 · Ikuo Morita1 

Received: 11 June 2017 / Accepted: 18 September 2017 © The Japanese Society of Pharmacognosy and Springer Japan KK 2017

Abstract  We report in this study novel biochemical activities of peanut skin extract (PEXT) on thrombocytopoiesis. Peanut skin, derived from Arachis hypogaea L., is a traditional Chinese medicine that is used to treat chronic hemorrhage. We have shown that oral administration of PEXT increases the peripheral platelet levels in mice. Recently, we reported a liquid culture system that is useful for investigating megakaryocytopoiesis and thrombocytopoiesis from human ­CD34+ cells. In this liquid culture system, PEXT was shown to enhance the formation of C ­ D41+/DAPI− cells (platelets), but had no effect on the formation of C ­ D41+/ + DAPI cells (megakaryocytes) or on the DNA content. Furthermore, PEXT selectively stimulated proplatelet formation from cultured mature megakaryocytes and phorbol 12-myristate 13 acetate (PMA)-induced formation of platelet-like particles from Meg01 cells. Despite having no influence on the formation of megakaryocyte colony forming units (CFUs), PEXT increased the size of megakaryocytes during their development from ­CD34+ cells. PEXT showed no effect on the GATA-1 and NF-E2 mRNA levels, which are known to play an important role in thrombocytopoiesis and, based on the results of a pMARE-Luc (pGL3-MAREluciferase) assay, had no influence on NF-E2 activation in Meg01 cells. These results suggest that PEXT accelerates proplatelet formation from megakaryocytes but does not

* Shujiro Seo s‑[email protected] 1

Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan



Functional and Phytochemical Laboratory, Tokiwa Phytochemical Co., Ltd., 158 Kinoko, Sakura‑shi, Chiba 285‑0801, Japan

2



influence the development of hematopoietic stem cells into megakaryocytes. Keywords  Platelet · Megakaryocyte · Peanut skin extract

Introduction Megakaryocytes originate from pluripotent stem cells through a differentiation process that involves stem cell commitment, nuclear polyploidization, and cytoplasmic maturation leading to the production of platelets. The initial stage of megakaryocyte differentiation involves sequential proliferation of ­CD34+ hematopoietic stem cells into proliferating megakaryoblasts and onto erythromegakaryocytic progenitor cells (BFU-E/megakaryocyte) [1]. The second phase involves nuclear polyploidization, increase in cell size, formation of a demarcation membrane system in the cytoplasm, and expression of lineage-specific cell surface markers [2]. The terminal differentiation process involves shedding of proplatelet fragments, which become functional platelets [3]. Proliferation and maturation of megakaryocyte precursors are regulated by several cytokines. Thrombopoietin (TPO) plays a major role in forming megakaryocytes [4]. Recently it has been reported that c-kit ligand (KL), interleukin (IL)3, IL-6, IL-11 and basic fibroblast growth factor indirectly contribute to megakaryocyte formation [5, 6]. Activating signal transducer and activator (STAT)-3 and STAT-5 were proposed to contribute to the activation of the TPO receptor, also known as c-Mpl [7]. The proplatelet formation transcription factor NF-E2 was reported to play an important role in thrombocytopoiesis, but not in megakaryocytopoiesis [8]. NF-E2 binds the Maf recognition element (MARE) via MafG and subsequently activates MARE-dependent target genes. In NF-E2 deficient mice and in cultured NF-E2−/− ES

13

Vol.:(0123456789)



cells, MARE-dependent target genes, such as thromboxane synthase ­A2 and β1 tubulin, have been described [9, 10]. These genes contribute to platelet formation and/or function. Hence, megakaryocytopoiesis from hematopoietic stem cells has been thoroughly investigated; however, the molecular mechanisms and factors that stimulate proplatelet formation, which is the terminal differentiation process of megakaryocytes, remain unclear [11]. Peanuts, Arachis hypogaea L, are an annual legume and an important world-wide crop. The seeds are used to make peanut butter, roasted snack peanuts, peanut confections, and peanut oil. Many studies have reported the many health benefits, such as the prevention of metabolic syndrome, associated with consumption of peanuts. Additional benefits include the control of weight gain and prevention of cardiovascular diseases, and these effects are mainly attributed to the fact that peanuts contain no trans-fatty acids, but are rich in mono- and polyunsaturated fatty acids [12]. The edible parts of peanuts consist of the kernel and protective skin. The skin has a pink-red color and astringent taste and is usually removed before peanut consumption or inclusion in confectionary and snack products. The peanut skin is used to treat chronic hemorrhage and bronchitis in Chinese traditional medicine. Recently, proanthocyanidins and flavonoids have been isolated from the water-soluble phenolic fraction of PEXT [13, 14]. The water-soluble phenolic fraction has been reported to inhibit hyaluronidase and scavenge reactive oxygen species [15]. However, the detailed effect of PEXT on thrombocytopoiesis remains to be clarified. Here, we report the effects of PEXT on megakaryocytopoiesis using a liquid culture system for megakaryocyte terminal differentiation from hematopoietic stem cells into proplatelets. Until now, no reports have described the oral administration of any food or medicine that can potently increase the peripheral amounts of platelets. In this study, we demonstrate that oral administration of PEXT increases the amount of peripheral platelets in mice. We also demonstrate that PEXT accelerates proplatelet formation, but does not influence the development of hematopoietic stem cells into megakaryocytes.

J Nat Med

Dental University (0060309, 070079). Blood (20 µL) was obtained from the retro-orbital plexus of ether-anesthetized mice and diluted in a buffer containing ammonium oxalate (Unopette kits, Becton–Dickinson, Sunnyvale, CA, USA). Both platelet and leukocyte counts were performed using a Z1 Coulter (BECKMAN COULTER, Fullerton, CA, USA) at day 0, 3, 5, 7 and 10. Six-week old mice were divided into the following two groups, control (N = 7) and test (N = 8). Oral delivery of PEXT or control solutions was given to each mouse for 5 days, from day 0 to day 4. PEXT was prepared by TOKIWA Phytochemical Co., Ltd. (Chiba, Japan). In brief, dried peanut skin (250  g) was extracted with aqueous ethanol (3.5 L) at room temperature for 4 h and filtered. The filtrate was concentrated under reduced pressure at below 60 °C to produce an extract (110 mL). Powdered extract (PEXT) (40 g) was obtained after spray drying. PEXT was analyzed by HPLC using Capcell PAK C18 (5 μm, 250 × 4.6-mm, Shiseido, Tokyo, Japan), eluted with 0.1% trifluoroacetic acid (TFA)/H2O— 0.1% TFA/CH3CN (92:8) at a flow rate of 1.0  mL/min. PEXT and each standard compound (Funakoshi, Tokyo, Japan) were dissolved in ­H2O and filtered through a 0.45-μm membrane filter. The detection wavelength was 280 nm. By comparing the retention times between PEXT and standard compounds, procyanidin B1, procyanidin B2, procyanidin B3 and (+) catechin were detected in PEXT (Fig. 1). PEXT was suspended in saline containing 0.5% carboxymethylcellulose (Wako, Osaka, Japan) and administered once daily. Control mice received the same volume of saline containing 0.5% carboxymethylcellulose. Human megakaryocytopoiesis and thrombopoiesis from ­CD34+ cells derived from umbilical cord blood The protocol for human megakaryocytopoiesis and thrombopoiesis from ­CD34+ cells derived from umbilical cord blood (UCB) has been previously described [16]. In brief, UCB samples from normal full-term newborn infants were obtained from Juntendo University (Tokyo, Japan) after receiving informed consent from their mothers. UCB

Materials and methods Animal experimental protocol Male ICR mice (six weeks old), purchased from SLC, Shizuoka, Japan, were maintained under specific pathogen-free conditions until experimentation. All experimental procedures were performed in accordance with guidelines of the Institutional Animal Care and Use Committee, and were approved by the Animal Research Committee, Graduate School of Medical and Dental Science, Tokyo Medical and

13

Procyanidin B3 Procyanidin B1

Fig. 1  HPLC profile of PEXT

(+)-catechin Procyanidin B2

J Nat Med

samples were diluted twofold with phosphate-buffered saline (PBS) and separated by centrifugation (800 × g for 30 min) on Ficoll–Paque (d = 1.077 g/mL; Pharmacia Biotech AB, Uppsala, Sweden) to obtain mononuclear cell preparations. ­CD34+ cells were purified from these preparations using a human CD34 MicroBead kit (Miltenyi Biotec, CA, USA) and autoMACS separation system (Miltenyi Biotec, Auburn,CA, USA). Flow cytometric analysis of purified cell preparations using a phycoerythrin-conjugated anti-CD34 monoclonal antibody (clone BIRMA-K3; DAKO, Glostrup, Denmark) showed that more than 95% of the selected cells were positive for CD34. These cells were cultured in Xvivo-20 (BioWhittaker, Walkersville, MD, USA) containing 50  ng/mL TPO (PeproTech EC, London, UK) and 40 ng/mL KL (Biosource, Camarillo, CA, USA) at a density from 2 to 5 × 104 cells/well. Cultures were maintained at 37 °C in a humidified 5% ­CO2 atmosphere. The cultured cells were treated with 50 ng/ mL TPO and 40 ng/mL KL for 8 days with or without 1 μg/mL PEXT. After cultivation, cells of each size were counted using a Z1 Coulter. For analysis of proplatelet formation from matured megakaryocytes, cultured cells treated with 50 ng/mL TPO and 40 ng/mL KL for 8 days were labeled with 10 nM CellTracker Orange (Molecular Probes, Eugene, OR, USA) for 1 h at 37 °C in a humidified 5% ­CO2 atmosphere. After incubation, the cells were washed two times with PBS. Labeled cells were seeded into wells of a 96-well plate (1–2 × 103 cells/well), with each well containing 100 μL of Xvivo-20, 50 ng/mL TPO, and 40 ng/mL KL with or without PEXT (0.01–1 μg/mL), and were then incubated for 4 days. After cultivation, cells were fixed with 2% formaldehyde, and the fluorescent particles were analyzed using Image SXM software. Flow cytometry A total of ­105–106 cells were suspended in 2% formaldehyde in PBS for 1 h and were washed with PBS three times. Cells were incubated in 0.1% bovine serum albumin (BSA) containing PBS for 30 min with fluorescein isothiocyanate (FITC)-conjugated mAb CD41-FITC (clone 5B12; DAKO, Glostrup, Denmark) or isotype-matched antibodies ­(IgG1 FITC conjugated; YLEM, Roma, Italy) to serve as a control. After incubation, cells were washed with PBS containing 0.1% BSA three times and were then suspended in 1 mL of PBS containing 0.1% BSA, 200 µg/mL RNase A (Sigma, St. Louis, MO, USA) and 5 µg/mL 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR, USA). Cell-associated immunofluorescence was analyzed by LSR II using Cell Quest software (Becton–Dickinson, San Jose, CA, USA).

Megakaryocyte colony forming unit assay The quantification of megakaryocyte colony forming units (CFU-megakaryocyte) was carried out using the Megacult-C Kit (Stem Cell Technologies, Vancouver, Canada). Briefly, ­CD34+ cells (5000 cells/slide) were incubated with serum-free medium containing 50 ng/mL TPO, 10 ng/mL IL-6, 10 ng/mL IL-3 and 1.1 mg/mL collagen for 14 days at 37 °C in a humidified 5% C ­ O2 atmosphere. Colonies containing CD41 positive megakaryocytes were identified by immunostaining with an anti-CD41 antibody using the alkaline-phosphatase anti-alkaline phosphatase technique as previously described [17, 18]. Megakaryocyte colonies consisting of clusters containing more than three cells were analyzed to determine the number of megakaryocyte CFUs, subdivided small colonies (3–20 cells per colony) or large colonies (more than 20 cells per colony). Treatments were continued throughout the 14 day culture period. Platelet‑like particle formation assay Meg01 cells (ATCC, Rockville, MD, USA) are a human megakaryoblastic cell line, which was established from the bone marrow of a patient with chronic myelogenous leukemia. Meg01 cells express the megakaryocyte marker platelet glycoprotein IIB/IIIa on their cell surface and possess no markers for B or T lymphocytes or for myeloid cells. Furthermore, Meg01 cells produce platelet-like particles when stimulated with PMA (BIOMOL, Plymouth Meeting, PA, USA) [19, 20]. Meg01 cells were grown in RPMI 1640 supplemented with 10% FCS. Cultures were maintained at 37 °C in a humidified 5% C ­ O2 atmosphere. Meg01 cells were collected from maintenance cultures, washed, and used in the indicated experiments. Platelet-like particle (PLP) formation was analyzed as follows. Maintenance cultured Meg01 cells were seeded into the wells of a 48-well plate (5 × 104 cells/well), with each well containing 500 μL of RPMI1640 containing 10% FCS, 10 nM PMA and with or without PEXT (0.1–10 μg/mL), and were cultured for 2 days. After cultivation both platelet-sized particles and Meg01 cell-sized particles were counted using a Z1 Coulter. Total RNA extraction and real‑time PCR Cultured cells treated with or without several doses of PEXT (1–10 μg/mL) were washed two times with PBS prior to RNA isolation. RNA was isolated from the cells using the TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Two micrograms of total RNA were subjected to reverse-transcription (ReverTra Ace, TOYOBO, Japan). The cDNA samples were amplified with Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA, USA). All data were analyzed

13



J Nat Med

by the comparative Ct Method (7500 Fast Real-Time PCR System, Applied Biosystems, Foster City, CA, USA). The following sequences were used. HPRT forward primer: 5′-TGG​ T GG​ AGA​T GA​T CT ​ C TC​ A A-3′, HPRT reverse primer: 5′-GGC​TTA​TAT​CCA​ACA​CTT​GG-3′, CD41 forward primer: 5′-AGC​CCC​TCC​CCC​ATT​CAC​C-3′, CD41 reverse primer: 5′-CCA​CCA​GCA​CCC​ACC​AGA​TTG-3′. GATA-1 forward primer: 5′-GGC​AGG​TAC​TCA​GTG​CAC​ CA-3′, GATA-1 reverse primer: 5′-CAC​TGG​CAT​TTC​TCC​ GCC-​ 3′, NF-E2 forward primer: 5′-CGAC ​ TCA ​ GGA ​ TTA ​ TC​ CCT​CAAC-3′ and NF-E2 reverse primer: 5′-CTG​GTC​TAG​ AGA​ACT​CAG​CTC​CTT​-3′.

Perkin-Elmer, Branchburg, NJ, USA). All other reagents were of analytical grade.

Plasmid construction and luciferase assay

Results

The construction of pMARE-Luc was modified as described previously [21]. Briefly, an oligonucleotide containing three copies of the β-globin enhancer NF-E2 site (5′-TCG ACC​ CGA​AAG​GAG​CTG​ACT​CAT​GCT​AGCCC-3′) and SpeI and BglII restriction enzyme sites was amplified. The PCR products were digested with SpeI and BglII and ligated into a pGL3-basic promoter (Promega, Madison, WI, USA) using Ligation High (Toyobo, Osaka, Japan). Meg01 cells (1 × 106 cells) were co-transfected with 1 μg of the MAREluciferase construct or the pGL3-basic promoter (pTAL) and 0.1 μg of phRL-TK (internal control plasmid, Promega, USA) using the human C ­ D34+ cell Nucleofector kit (amaxa, Gaithersburg, MD, USA). After transfection, cells were cultured in 48-well plates with wells containing RPMI1640, 10% FCS and 10 nM PMA with or without 3 μg/mL PEXT for 24 h. The luciferase activities in the cell lysates were analyzed according to the manufacturer’s instructions (Dual Luciferase Kit, Promega, 1420 ARVOmx/Light,

To confirm the effect of PEXT on platelet production, 150 mg/kg PEXT was orally administered to mice once daily for 5 days from day 0 to day 4, and blood samples were taken over the next 6 days. The number of peripheral platelets was significantly increased from day 5 to day 10 (p