ORIGINAL ARTICLE Adequate stimulation of hematopoietic stem cell proliferation by a polyherbal formulation (EMSA eritin) leading to lymphocyte differentiation in BALB/c mice after radiation Mansur Ibrahim a, *, Edi Widjajanto a , M. Aris Widodo a , Sutiman B. Sumitro b a Department of Biomedicine, Faculty of Medicine, University of Brawijaya, Malang, Jawa Timur, Indonesia b Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, Jawa Timur, Indonesia Received 9 April 2015; received in revised form 23 May 2015; accepted 26 May 2015 Available online 3 August 2015 KEYWORDS EMSA eritin; erythropoiesis; hematopoiesis; lymphocyte; lymphopoiesis Abstract Radiotherapy is still an essential option for treatment of various stages of cancer. However, the irradiation administered would deplete the hematopoietic stem cells in the bone marrow, which would eventually decrease the number of lymphocytes and erythrocytes in the circulatory system. EMSA eritin is a polyherbal formulation that has the ability to stimulate erythropoiesis in mice after radiation therapy. Erythropoiesis is a complex mechanism involving the interaction of dozens of genes that may also be involved in other cellular process, such as lymphopoiesis. In this study, we elucidated the potential of EMSA eritin to stimulate other mechanisms, in addition to being an inducer of erythrocyte production. Bioinformatic analysis results indicated that the EMSA eritin formulation contains multiactive compounds that synergistically drive hematopoiesis and trigger lymphocyte differentiation. We then tested this prediction using an in vivo cell-culture system in a mice model. Experimental re- sults suggested that EMSA eritin is able to induce hematopoietic stem cell proliferation and differentiate these cells into lymphocytes in BALB/c mice after sublethal radiation therapy. Moreover, the polyherbal formulation increased proliferation and survival of B and T lympho- cytes in a dose-dependent manner. The results indicate that the polyherbal formulation is adequate to ameliorate both erythropoiesis and lymphopoiesis. The formulation thus appears * Corresponding author. Department of Biomedicine, Faculty of Medicine, University of Brawijaya, Jalan Veteran, Malang, Jawa Timur 65145, Indonesia. E-mail address: [email protected](M. Ibrahim). http://dx.doi.org/10.1016/j.bgm.2015.05.001 2214-0247/Copyright ª 2015, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved. Available online at www.sciencedirect.com ScienceDirect journal homepage: www.j-bgm.com Biomarkers and Genomic Medicine (2015) 7, 110e115
Transcript
Biomarkers and Genomic Medicine (2015) 7, 110e115
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.j-bgm.com
ORIGINAL ARTICLE
Adequate stimulation of hematopoietic stemcell proliferation by a polyherbalformulation (EMSA eritin) leading tolymphocyte differentiation in BALB/c miceafter radiation
Mansur Ibrahim a,*, Edi Widjajanto a, M. Aris Widodo a,Sutiman B. Sumitro b
a Department of Biomedicine, Faculty of Medicine, University of Brawijaya, Malang, Jawa Timur,Indonesiab Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya,Malang, Jawa Timur, Indonesia
Received 9 April 2015; received in revised form 23 May 2015; accepted 26 May 2015Available online 3 August 2015
Abstract Radiotherapy is still an essential option for treatment of various stages of cancer.However, the irradiation administered would deplete the hematopoietic stem cells in the bonemarrow, which would eventually decrease the number of lymphocytes and erythrocytes in thecirculatory system. EMSA eritin is a polyherbal formulation that has the ability to stimulateerythropoiesis in mice after radiation therapy. Erythropoiesis is a complex mechanisminvolving the interaction of dozens of genes that may also be involved in other cellular process,such as lymphopoiesis. In this study, we elucidated the potential of EMSA eritin to stimulateother mechanisms, in addition to being an inducer of erythrocyte production. Bioinformaticanalysis results indicated that the EMSA eritin formulation contains multiactive compoundsthat synergistically drive hematopoiesis and trigger lymphocyte differentiation. We thentested this prediction using an in vivo cell-culture system in a mice model. Experimental re-sults suggested that EMSA eritin is able to induce hematopoietic stem cell proliferation anddifferentiate these cells into lymphocytes in BALB/c mice after sublethal radiation therapy.Moreover, the polyherbal formulation increased proliferation and survival of B and T lympho-cytes in a dose-dependent manner. The results indicate that the polyherbal formulation isadequate to ameliorate both erythropoiesis and lymphopoiesis. The formulation thus appears
of Biomedicine, Faculty of Medicine, University of Brawijaya, Jalan Veteran, Malang, Jawa Timur
oo.com (M. Ibrahim).
5.05.001Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Hematopoietic stem cell proliferation by EMSA eritin 111
very promising for treatment of patients following radiation therapy. However, this warrantsfurther investigation.Copyright ª 2015, Taiwan Genomic Medicine and Biomarker Society. Published by ElsevierTaiwan LLC. All rights reserved.
Introduction
Anemia can be caused by several factors such as renalchronic diseases1e3 and irradiation in cancer patients.However, radiotherapy is still an essential treatment forpatients with cancer.4,5 Cancer-related anemia is mostlytreated by administering erythropoietin.6 However, eryth-ropoietin administration could potentially accelerate tumorgrowth and jeopardize the survival of these patients.7 TheEMSA eritin is a polyherbal formulation that adequatelystimulates the production of erythrocytes in mice afterradiation therapy. The formulation thus looks promising foruse in safe anemia therapy.8
Erythropoiesis is a complex mechanism that occursthrough the activation of the Janus kinase/signal trans-ducer and activator of transcription (JAK/STAT) signalingpathway9 and Notch signaling.10 The JAK/STAT signalingpathway activation is associated with the interaction ofdozen of genes that might also be associated with othercellular processes, including cell proliferation, migration,immunity, and hematopoiesis.11 The EMSA eritin formula-tion has been proven to stimulate erythropoiesis by tar-geting several genes that are involved in various otherpathways. Therefore, using bioinformatics tools,we explored the potential of EMSA eritin to induceother cellular mechanisms that are beneficial for humanhealth.
Bioinformatics has been used in various applications,including for identifying toxicity of herbs,12 proteinepro-tein interaction,13 and for understanding complex mech-anisms of action of herbal formulations.14 EMSA eritin is amulticomponent formulation and thus has multiple tar-gets. The potency of these polyherbal formulations forvarious purposes can be analyzed using biosystems basedon the interaction of the target gene with the activecompound in these formulations.15 Several studies havebeen successfully using bioinformatic approach to under-stand the mechanism of action of polyherbal products[e.g., Chinese medicines,16 Indian medicine (Ayurveda),Japanese medicine (Kampo), Korean medicine, and Ti-betan medicine14]. In this study, using bioinformatics, wedemonstrate a novel function of EMSA eritin formulation inthe hematopoiesis pathwaydas an “inducer” of erythro-cyte production.
Materials and methods
EMSA eritin formulation
Soybean, coconut and red rice were purchased on October2013 from local market at Makasar, south of Sulawesi,Indonesia.
Soybean and red rice were washed and dried in a vacuumoven at 40�C, and then mashed to obtain particles of 60-mesh size (250 mm). The powder was extracted using waterat a ratio of 1:10 (materials:water) for 2 hours at 50�C. Theextract and coconut water were evaporated by freezedrying for 24 hours at �60�C. Finally, the three materialswere mixed with coconut water powder to produce a pol-yherbal medicine called “EMSA eritin.”
Analysis of lymphocyte proliferation in vitro
Lymphocytes were isolated from the spleen of mice thatwere maintained in a pathogen-free chamber. The micewere killed by cervical dislocation, and the spleen wasremoved and washed two times with phosphate-bufferedsaline (PBS) in a sterile Petri dish. The organs were pressedclockwise using a sterile syringe, and the homogenateswere mixed with 10 mL PBS to isolate lymphocytes. Thecell suspense was centrifuged at 2500 rpm, at 10�C for 5minutes. The supernatant was discarded, but the pelletsformed were taken and resuspended in Roswell Park Me-morial Institute-1640 medium supplemented with 2-mercaptoetanol (final concentration, 50 mL), 10% fetalbovine serum, 10 mL a-CD3 (supernatant), and lipopoly-saccharide (10 ng/mL). The cells (1.5e2 � 106 cells/mL)were cultured in 48-well plates and classified into fourgroups based on formulation treatment: three groups weretreated with different concentrations of EMSA eritin(0.025 mg/mL, 0.25 mg/mL, and 2.5 mg/mL), and onegroup did not receive this formulation and served as thecontrol group. The cells were cultured in an incubator(culture conditions, 5% CO2 atmosphere at 37�C) for 3days. The cells were then harvested and stained with50 mL of fluorescein isothiocyanate (FITC)-conjugatedantimouse CD4 (clone GK1.5), phycoerythrin (PE)-conju-gated antimouse CD25 (clone PC61.5), PE-conjugatedantimouse CD8 (clone 53-6.7), and PE-conjugated anti-mouse B220 (clone RA3-6B2) antibody. The cell numberswere counted using a flow cytometer (FACSCalibur; BDBiosciences, New Jersey, USA), and calculated using BDCellQuest PRO software.
In vivo analysis of hematopoietic stem celldifferentiation
Regular BALB/c mice (8e9-week old) were divided into sixgroups: five groups received sublethal total body irradiation(600 rad) from an open-beam cobalt 60 gamma source andone group served as the control. Ten hours later, the irra-diated mice were force fed with EMSA eritin at doses of0 mg/g body weight (BW), 1 mg/g BW, 3 mg/g BW, and9 mg/g BW, respectively. The EMSA eritin was given once a
112 M. Ibrahim et al.
day for 2 weeks. Peripheral blood was monitored everyother day by counting blood cells using a hemocytometer.The mice were maintained in the pathogen-free facility ofthe Department of Biology, Faculty of Mathematic andNatural Sciences, Brawijaya University, Malang, Indonesia.Two weeks after the treatment, the bone marrow cellswere isolated. The cell-surface molecules were stainedusing FITC-conjugated antimouse CD34 (clone RAM34), PE/Cy5-conjugated antimouse CD135 (clone A2F10), PerCP-conjugated antimouse CD117 (clone 2B8). Data obtainedwere analyzed by CellQuest and tested by statistical anal-ysis (analysis of variance) with p < 0.05 consideredsignificant.
Bioinformatic analysis for identification of EMSAeritin-targeted protein and its network
Bioinformatic analysis was carried out by mapping theinteraction of the target genes with the active compound inthe formulation using HitPick and the Search Tool for theRetrieval of Interacting Genes/Proteins (STRING) data-base.17 First, the active compound in coconut water, redrice, and soybean were curated through references. TheSimplified Molecular-Input Line-Entry system structure ofthe active compounds identified was drawn using PubChemSketcher V2.4 (https://pubchem.ncbi.nlm.nih.gov). Thestructure was used to predict the molecular or protein
Figure 1 Interaction of EMSA eritin targeted genes. Active commatopoiesis and erythropoiesis. The figure indicates that EMSA ertopoiesis and erythropoiesis. The network analysis was carried outProteins database according to experimental data, data retrieved
target using HITPICK, which was developed by MonicaCampillos, PhD (Helmholtz Center Munich, Germany).
The protein targets were analyzed using the STRINGdatabase (http://string-db.org/) based on their interactionwith another protein that plays a role in erythropoiesis andhematopoiesis. Molecular interactions were predicted usingthe STRING database, which contains experimental dataand predictions of more than 5 million proteins and 200million protein interactions. Proteineprotein interactionswere identified using a filter-based pathway (Kyoto Ency-clopedia Gene and Genome pathway) that has been vali-dated previously.
Result
Results of bioinformatic analysis indicated that EMSA eritinhas 15 protein targets from 14 active compounds. Furtheranalysis using the STRING database showed that theseproteins interacted with each other to form a complexnetwork. Interestingly, the protein complex also interactedwith genes that play a crucial role in erythropoiesis andhematopoiesis (Figure 1). The data obtained suggest thatthe mixture of active compounds in EMSA eritin was able tostimulate hematopoiesis, despite erythropoiesis activation.Several target genes (NOS and ESR) are strongly responsiblefor hematopoiesis and innate immune systemhomeostasis.18,19
pounds in EMSA eritin targeted genes that are involved in he-itin is able to simultaneously and synergistically induce hema-using the Search Tool for the Retrieval of Interacting Genes/
from databases, and text-mining prediction methods.26e30
Figure 2 EMSA eritin promotes T- and B-cell proliferation and survival. Spleen cells (2� 106) were obtained from 7-week-oldwild-type BALB/c mice and cultured in 48-well plates for 96 hours in a medium containing anti-CD3 supernatant (1.5%). EMSA eritinat different concentrations (dose 1 = 0.025 mg/mL; dose 2 = 0.25 mg/mL; dose 3 = 2.5 mg/mL) was added as indicated in panels.After culture, cells were subjected to cell-surface molecule staining with anti-B220, anti-CD4, anti-CD8, and anti-CD25 antibodies.Control is a simple culture without the addition of EMSA eritin. The panels show the percentage of cells positively stained with anti-B220, anti-CD4, anti-CD8, anti-CD25 antibodies. Data are presented as mean � standard deviation (nZ 5 mice in each group).
Hematopoietic stem cell proliferation by EMSA eritin 113
The capacity of EMSA eritin to regulate the differentia-tion, proliferation, and survival of lymphocytes was eluci-dated in the cell-culture system. The polyherbalformulation increased proliferation and survival of lym-phocytes B220þ, CD4þ, CD8þ, and CD25þ (Figure 2). Pro-liferation of lymphocytes was found to increase along withthe augmentation of the EMSA eritin doses in a dose-dependent manner. The results indicate that EMSA eritinis able to stimulate proliferation, differentiation, and sur-vival of both B and T lymphocytes. Therefore, we specu-lated that the EMSA eritin formulation has the potential toinduce lymphopoiesis. We next examined its potential toinduce hematopoietic stem cell differentiation in a micemodel.
Regular BALB/c mice were irradiated (600 rad) to depletehematopoietic stem cells in the bone marrow. The irradi-ated mice were treated with EMSA eritin each day for 2weeks. The polyherbal formulation increased the prolifera-tion of hematopoietic progenitor cells (CD34þCD117þ) inBALB/c mice after radiation. The cell-surface moleculeCD117 plays a role in triggering cell proliferation, differen-tiation, and survival.20 Moreover, the formulation triggeredthe differentiation of the stem cells into lymphopoiesis lin-eages (CD34þCD135þ; Figure 3). CD135 is necessary fordevelopment of lymphocytes (B and T cells), but not for the
development of other blood cells (myeloid development).These results prove that the EMSA eritin is able to stimulatelymphopoiesis in BALB/c mice after radiation.
Discussion
The results of this study indicate that the active compoundsin the EMSA eritin polyherbal formulation are able tostimulate hematopoiesis, leading to differentiation oflymphocyte. This is consistent with the results of in silicoanalysis that the active compounds interact with genesinvolved in hematopoiesis. The study result also corre-sponds with a previous report indicating that coconut waterimproves homeostasis.21 Moreover, the other componentsof red rice presented higher phenolic contents that exhibitantioxidant activities.22 Soybean is an excellent source ofdietary peptides that have beneficial effects on immunefunction, brain function, and neurochemicals in humans.23
Experimental results also illustrate that the EMSA eritinformulation can regulate homeostasis.
Indeed, the polyherbal formulation is able to betterrecover depletion of bone marrow stem cells after radi-ation therapy than erythropoietin. This can be attributedto the multiactive compounds in the formulation that
Figure 3 EMSA eritin increases bone marrow cell survival, proliferation, and differentiation. Sublethally irradiated (600 rad) micereceived an oral administration of different concentrations of EMSA eritin (dose 1 = 0.025 mg/mL; dose 2 = 0.25 mg/mL; dose 3 = 2.5mg/mL) for 15 days as indicated or erythropoietin (EPO) injection (according to company protocol). Bone marrow cells (2� 106)were obtained from the treated mice, and then subjected to cell-surface staining with anti-CD34, anti-CD135, and anti-CD117antibodies. Controls are regular-type mice without manipulation. (A) Panels showing the percentage of cells positively stainedwith CD34 and CD117 (CD34þCD177þ). (B) Panels showing the percentage of cells positively stained with CD34 and CD135(CD34þCD135þ) in the panels. Data are presented as mean � standard deviation (nZ 4 mice in each group).
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work in synergy to improve hematopoiesis in the bonemarrow. Moreover, the EMSA eritin formulation also trig-gers lymphocyte differentiation, in addition to its po-tential to act as an inducer of hematopoietic stem cellproliferation and erythropoiesis. The polyherbal can beattributed to the activation of multiple targets (genes) bythe polyherbal formulation, which in-turn interact with agroup of genes that are responsible for hematopoiesis anderythropoiesis. We then assumed that this polyherbalformulation is adequate to ameliorate both erythropoiesisand lymphopoiesis. The formulation also works as animmunomodulator, which is very beneficial to cure can-cer. Thus, the EMSA eritin formulation has dual benefitsfor cancer patients: upregulating erythrocyte productionand stimulation of the immune system to attack cancercells.
Previous studies have demonstrated that herbs such asashwagandha24 and xiao-ban-xia25 contain complex mole-cules that have better efficacy compared with a single iso-lated active compound. The multitarget capability of herbsmay simultaneously and synergistically induce molecularmechanisms in cells. Based on these reports, we believe thatEMSA eritin improves homeostasis systemically. However,the effects of EMSA eritin demonstrated in this study war-rant further investigation for its possible use as a stimulatorof hematopoiesis in humans in future clinical trials.
Conclusion
EMSA eritin is adequate to stimulate hematopoiesis, whicheventually leads to lymphopoiesis. This ability is confirmed by
Hematopoietic stem cell proliferation by EMSA eritin 115
the increasing numbers of CD34þCD135þ cells in BALB/c micefollowing irradiation. Besides, EMSA eritin increased prolif-eration and differentiation of lymphocytes. Our study resultsindicate that EMSA eritin is a promising option for stimulatinghematopoiesis in cancer patients after radiotherapy.
Conflicts of interest
The authors declare that they have no conflicts of interest.
References
1. Fernandez H, Singh AK. Management of anemia in chronickidney disease. In: Kimmel PL, Rosenberg ME, eds. ChronicRenal Disease. San Diego, CA: Academic Press; 2015:624e633.
2. Barbieri C, Mari F, Stopper A, et al. A new machine learningapproach for predicting the response to anemia treatment in alarge cohort of end stage renal disease patients undergoingdialysis. Comput Biol Med. 2015;61:56e61.
3. Cyganek A, Pietrzak B, Kociszewska-Najman B, et al. Anemiatreatment with erythropoietin in pregnant renal recipients.Transplant Proc. 2011;43:2970e2972.
4. Morere J-F. Role of epoetin in the management of anaemia inpatients with lung cancer. Lung Cancer. 2004;46:149e156.
5. Prosnitz RG, Yao B, Farrell CL, et al. Pretreatment anemia iscorrelated with the reduced effectiveness of radiation andconcurrent chemotherapy in advanced head and neck cancer.Int J Radiat Oncol Biol Phys. 2005;61:1087e1095.
6. Ferrario E, Ferrari L, Bidoli P, et al. Treatment of cancer-related anemia with epoetin alfa: a review. Cancer TreatRev. 2004;30:563e575.
7. Cao Y. Erythropoietin in cancer: a dilemma in risk therapy.Trends Endocrinol Metab. 2013;24:190e199.
8. Rifa’i M, Khasanah Q. Activity of EMSA-ERITIN therapy onerythropoiesis of Balb/C mice post total body irradiation (TBI).In: Proceedings of the 5th Annual Basic Science InternationalConference 2015 (BaSIC 2015). Faculty of Mathematics andNatural Sciences, Brawijaya University, Indonesia; February2015.
9. Okonko DO, Marley SB, Anker SD, et al. Erythropoietin resis-tance contributes to anaemia in chronic heart failure and re-lates to aberrant JAKeSTAT signal transduction. Int J Cardiol.2013;164:359e364.
10. Oh P, Lobry C, Gao J, et al. In vivo mapping of Notch pathwayactivity in normal and stress hematopoiesis. Cell Stem Cell.2013;13:190e204.
11. Hou SX, Zheng Z, Chen X, et al. The JAK/STAT pathway inmodel organisms: emerging roles in cell movement. Dev Cell.2002;3:765e778.
12. Huang S-H, Tung C-W, Fulop F, et al. Developing a QSAR modelfor hepatotoxicity screening of the active compounds intraditional Chinese medicines. Food Chem Toxicol. 2015;78:71e77.
13. Tao W, Xu X, Wang X, et al. Network pharmacology-basedprediction of the active ingredients and potential targets of
Chinese herbal radix curcumae formula for application tocardiovascular disease. J Ethnopharmacol. 2013;145:1e10.
14. Pelkonen O, Pasanen M, Lindon JC, et al. Omics and its po-tential impact on R&D and regulation of complex herbalproducts. J Ethnopharmacol. 2012;140:587e593.
15. Ouedraogo M, Baudoux T, Stevigny C, et al. Review of currentand “omics” methods for assessing the toxicity (genotoxicity,teratogenicity and nephrotoxicity) of herbal medicines andmushrooms. J Ethnopharmacol. 2012;140:492e512.
16. Li S, Zhang B. Traditional Chinese medicine network pharma-cology: theory, methodology and application. Chin J Nat Med.2013;11:110e120.
17. Widodo N. Bioinformatics: cheap and robust method to explorebiomaterial from Indonesia biodiversity. AIP Conf Proc. 2015;1649:148e151.
18. Sanchez-Aguilera A, Arranz L, Martın-Perez D, et al. Estrogensignaling selectively induces apoptosis of hematopoietic pro-genitors and myeloid neoplasms without harming steady-statehematopoiesis. Cell Stem Cell. 2014;15:791e804.
20. Edling CE, Hallberg B. c-Kitda hematopoietic cell essentialreceptor tyrosine kinase. Int J Biochem Cell Biol. 2007;39:1995e1998.
21. DebMandal M, Mandal S. Coconut (Cocos nucifera L.: Areca-ceae): in health promotion and disease prevention. Asian Pac JTrop Med. 2011;4:241e247.
22. Paiva FF, Vanier NL, Berrios JDJ, et al. Physicochemical andnutritional properties of pigmented rice subjected to differentdegrees of milling. J Food Compost Anal. 2014;35:10e17.
23. Yimit D, Hoxur P, Amat N, et al. Effects of soybean peptide onimmune function, brain function, and neurochemistry inhealthy volunteers. Nutrition. 2012;28:154e159.
24. Widodo N, Takagi Y, Shrestha BG, et al. Selective killing ofcancer cells by leaf extract of ashwagandha: components,activity and pathway analyses. Cancer Lett. 2008;262:37e47.
25. Li Z, Xiao S, Ai N, et al. Derivative multiple reaction monitoringand single herb calibration approach for multiple componentsquantification of traditional Chinese medicine analogousformulae. J Chromatogr A. 2015;1376:126e142.
26. Effiong GS, Ebong PE, Eyong EU, et al. Amelioration of chlor-amphenicol induced toxicity in rats by coconut water. J App SciRes. 2010;6:331e335.
27. Tian S, Nakamura K, Kayahara H. Analysis of phenolic com-pounds in white rice, brown rice, and germinated brown rice. JAgric Food Chem. 2004;52:4808e4813.
28. Yodmanee S, Karrila TT, Pakdeechanuan P. Physical, chemicaland antioxidant properties of pigmented rice grown in South-ern Thailand. Int Food Res J. 2011;18:901e906.
29. Paganga G, Miller N, Rice-Evans CA. The polyphenolic contentof fruit and vegetables and their antioxidant activities. Whatdoes a serving constitute? Free Radic Res. 1999;30:153e162.
30. Plaza L, de Ancos B, Cano PM. Nutritional and health-relatedcompounds in sprouts and seeds of soybean (Glycine max),wheat (Triticum aestivum. L) and alfalfa (Medicago sativa)treated by a new drying method. Eur Food Res Technol. 2003;216:138e144.
