Sains Malaysiana 53(4)(2024): 781-794

http://doi.org/10.17576/jsm-2024-5304-05

 

Modulation of Human Bone Marrow Mesenchymal Stem Cells (BMMSC) by Nigella sativa and Trigona Honey: An in vitro Study

(Modulasi Sel Stem Mesenkima Sumsum Tulang Manusia (BMMSC) oleh Nigella sativa dan Madu Trigona: Suatu Kajian in vitro)

 

MASNIZA MUHAMAD LASIM1, NURUL FARIHAH SAMSUDIN1, ZETTY NADIA MOHD ZAIN2, HAYATI ABDUL RAHMAN2, NUR SYAHRINA RAHIM2, ASRAL WIRDA AHMAD ASNAWI2 & NUR FARIHA MOHD MANZOR1,*

 

1Department of Medical Sciences I, Faculty of Medicine and Health Sciences, Universiti Sains Islam Malaysia, 71800 Nilai, Negeri Sembilan, Malaysia

2Department of Medical Sciences II, Faculty of Medicine and Health Sciences, Universiti Sains Islam Malaysia, 71800 Nilai, Negeri Sembilan, Malaysia

 

Received: 27 April 2023/Accepted: 27 February 2024

 

Abstract

Maintenance of bone marrow mesenchymal stem cells (BMMSC) numbers is crucial for tissue repair and regeneration since adult stem cells are often limited in number, expansion capacity and lifespan. It is believed that certain types of foods are beneficial in the preservation and stimulation of stem cells throughout life. Black seeds and stingless bee honey are among the widely investigated functional food with general health promoting benefit. We aim to explore the proliferative, anti-apoptosis and anti-senescence effect of Nigella sativa and Trigona honey on BMMSC. Cell proliferation was evaluated using MTT assay. We performed flow cytometric analysis to verify stem cell surface markers while quantitative PCR was done to measure the relative expression of target genes. Results showed favourable concentration dependent enhancement of cell proliferation by 1 to 10 ug/mL Nigella sativa and 0.097% to 0.195% Trigona honey. Observation on BMMSC’s morphology and surface markers expression revealed no alteration on BMMSC stemness properties. In addition, gene expression analysis supported that there was a significant (P<0.05) increase in proliferation related gene, β-Catenin, and lower expression of apoptotic- and senescence-related gene, BAX and p21, respectively. These preliminary findings suggest a promising effect of Nigella sativaand Trigona honey on stem cell proliferation.

 

Keywords: Apoptosis; black seed; bone-marrow mesenchymal stem cells; proliferation; senescence; stingless bee honey

 

Abstrak

Pengekalan bilangan sel stem mesenkima sumsum tulang (BMMSC) adalah penting untuk pembaikan dan penjanaan semula tisu kerana sel stem dewasa selalunya terhad dalam bilangan, kapasiti pengembangan dan jangka hayat. Jenis makanan tertentu adalah dipercayai bermanfaat dalam pemeliharaan dan ransangan sel stem sepanjang hayat. Habbatus sauda dan madu lebah kelulut adalah antara makanan bermanfaat terhadap kesihatan yang dikaji secara meluas. Penyelidikan ini bertujuan untuk meneroka kesan proliferatif, anti-apoptosis dan anti-penuaan Nigella sativa dan madu Trigona terhadap BMMSC. Proliferasi sel telah dinilai menggunakan ujian MTT. Kami menggunakan analisis aliran sitometriks untuk mengesahkan penanda permukaan sel stem manakala PCR kuantitif dilakukan untuk mengukur pengekspresan relatif gen sasaran. Keputusan menunjukkan proliferasi sel yang menggalakkan dalam kepekatan sebanyak 1 hingga 10 ug/mL Nigella sativa dan 0.096 hingga 0.195% madu Trigona. Pemerhatian pada morfologi dan ekspresi penanda permukaan BMMSC menunjukkan tiada perubahan pada sifat BMMSC. Tambahan pula, analisispengekspresan gen menyokong bahawa terdapat peningkatan yang ketara (P<0.05) dalam gen proliferatif, β-Catenin manakala gen apoptosis dan gen penuaan, BAX dan p21, masing-masing menunjukkan ekspresi yang lebih rendah. Penemuan awal ini mencadangkan kesan memberangsangkan Nigella sativa dan madu Trigona terhadap proliferasi sel stem.

 

Kata kunci: Apoptosis; Habbatus sauda; madu lebah kelulut; penuaan; proliferatif; sel stem mesenkima sumsum tulang

 

REFERENCES

Ab Rahman, M.R., Abdul Razak, F. & Mohd Bakri, M. 2014. Evaluation of wound closure activity of Nigella sativa, Melastoma malabathricum, Pluchea indica, and Piper sarmentosum extracts on scratched monolayer of human gingival fibroblasts. Evidence-Based Complementary and Alternative Medicine 2014: 190342. https://doi.org/10.1155/2014/190342

Abd Jalil, M.A., Kasmuri, A.R. & Hadi, H. 2017. Stingless bee honey, the natural wound healer: A review. Skin Pharmacology and Physiology 30(2): 66-75. https://doi.org/10.1159/000458416

Ahmed, A.S.I., Sheng, M.H., Wasnik, S., Baylink, D.J. & Lau, K.H.W. 2017. Effect of aging on stem cells. World Journal of Experimental Medicine 7(1): 1-10. https://doi.org/10.5493/wjem.v7.i1.1

Al-Haj, L., Blackshear, P.J. & Khabar, K.S.A. 2012. Regulation of P21/CIP1/WAF-1 mediated cell-cycle arrest by RNase L and tristetraprolin, and involvement of AU-Rich elements. Nucleic Acids Research 40(16): 7739-7752. https://doi.org/10.1093/nar/gks545

Al-Rahbi, B., Zakaria, R., Othman, Z., Hassan, A., Mohd Ismail, Z.I. & Muthuraju, S. 2014. Tualang honey supplement improves memory performance and hippocampal morphology in stressed ovariectomized rats. Acta Histochemica 116(1): 79-88. https://doi.org/10.1016/j.acthis.2013.05.004

Albajali, A.A., Nagi, A.H., Shahzad, M., Ikram Ullah, M. & Hussain, S. 2011. Effect of Allium sativa L. on pancreatic β. cells in comparison to Nigella sativa L. in streptozotocin induced diabetic rats. Journal of Medicinal Plant Research 5(24): 5779-5784. https://www.researchgate.net/profile/Muhammad-Ikram-Ullah/publication/23521680 7_Effect_of_Allium_sativa_L_ on_ pancreatic_b_cells_incomparison_to_ Nigella_ sativa_L_in_streptozotocininduced _diabetic _rats/links/ 560a7 40e08ae840a08d56442/ Effect-of-Allium-sativa-

Alm, J.J., Koivu, H.M.A., Heino, T.J., Hentunen, T.A., Laitinen, S. & Aro, H.T. 2010. Circulating plastic adherent mesenchymal stem cells in aged hip fracture patients. Journal of Orthopaedic Research 28: 1634-1642. https://doi.org/10.1002/jor.21167

Batista de Sousa, J.M., Leite de Souza, E., Marques, G., de Toledo Benassi, M., Gullón, B., Pintado, M.M. & Magnani, M. 2016. Sugar profile, physicochemical and sensory aspects of monofloral honeys produced by different stingless bee species in Brazilian semi-arid region. LWT 65: 645-651. https://doi.org/10.1016/j.lwt.2015.08.058

Chen, Y., Xiang, L.X., Shao, J.Z., Pan, R.L., Wang, Y.X., Dong, X.J. & Zhang, G.R. 2010. Recruitment of endogenous bone marrow mesenchymal stem cells towards injured liver. Journal of Cellular and Molecular Medicine 14(6 B): 1494-1508. https://doi.org/10.1111/j.1582-4934.2009.00912.x

Czabotar, P.E., Lessene, G., Strasser, A. & Adams, J.M. 2014. Control of apoptosis by the BCL-2 protein family: Implications for physiology and therapy. Nature Reviews Molecular Cell Biology 15(1): 49-63. https://doi.org/10.1038/nrm3722

Dehkordi, F.R. & Kamkhah, A.F. 2008. Antihypertensive effect of Nigella sativa seed extract in patients with mild hypertension. Fundamental and Clinical Pharmacology 22(4): 447-452. https://doi.org/10.1111/j.1472-8206.2008.00607.x

Díez, J.M., Bauman, E., Gajardo, R. & Jorquera, J.I. 2015. Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools. Stem Cell Research and Therapy 6(1): 28. https://doi.org/10.1186/s13287-015-0016-2

Di Renzo, L., Gualtieri, P., Frank, G. & De Lorenzo, A. 2023. Nutrition for prevention and control of chronic degenerative diseases and COVID-19. Nutrients 15(10): 2253. https://doi.org/10.3390/nu15102253

Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F.C., Krause, D.S., Deans, R.J., Keating, A., Prockop, D.J. & Horwitz, E.M. 2006. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4): 315-317. https://doi.org/10.1080/14653240600855905

Eid, A.M., Elmarzugi, N.A., Abu Ayyash, L.M., Sawafta, M.N. & Daana, H.I. 2017. A review on the cosmeceutical and external applications of Nigella sativa. Journal of Tropical Medicine 2017: 7092514. https://doi.org/10.1155/2017/7092514

Franceschi, C., Garagnani, P., Morsiani, C., Conte, M., Santoro, A., Grignolio, A., Monti, D., Capri, M. & Salvioli, S. 2018. The continuum of aging and age-related diseases: Common mechanisms but different rates. Frontiers in Medicine 5: 61. https://doi.org/10.3389/fmed.2018.00061

Ge, Z., Guo, X., Li, J., Hartman, M., Kawasawa, Y.I., Dovat, S. & Song, C. 2015. Clinical significance of high C-MYC and low MYCBP2 expression and their association with ikaros dysfunction in adult acute lymphoblastic leukemia. Oncotarget 6(39): 42300-42311. https://doi.org/10.18632/oncotarget.5982

Gholamnezhad, Z., Keyhanmanesh, R. & Boskabady, M.H. 2015. Anti-inflammatory, antioxidant, and immunomodulatory aspects of Nigella sativa for its preventive and bronchodilatory effects on obstructive respiratory diseases: A review of basic and clinical evidence. Journal of Functional Foods 17: 910-927. https://doi.org/10.1016/j.jff.2015.06.032

Ghonime, M., Eldomany, R., Abdelaziz, A. & Soliman, H. 2011. Evaluation of immunomodulatory effect of three herbal plants growing in Egypt. Immunopharmacology and Immunotoxicology 33(1): 141-145. https://doi.org/10.3109/08923973.2010.487490

Hayati, A.R., Nur Fariha, M.M., Tan, G.C., Tan, A.E. & Chua, K. 2011. Potential of human decidua stem cells for angiogenesis and neurogenesis. Archives of Medical Research 42(4): 291-300. https://doi.org/10.1016/j.arcmed.2011.06.005

Hayflick, L. & Moorhead, P.S. 1961. The serial cultivation of human diploid cell strains. Experimental Cell Research 25(3): 585-621. https://doi.org/10.1016/0014-4827(61)90192-6

He, T.C., Sparks, A.B., Rago, C., Hermeking, H., Zawel, L., da Costa, L.T., Morin, P.J., Vogelstein, B. & Kinzler, K.W. 1998. Identification of C-MYC as a target of the APC pathway. Science 281(5382): 1509-1512. https://doi.org/10.1126/science.281.5382.1509

Hou, Y., Dan, X., Babbar, M., Wei, Y., Hasselbalch, S.G., Croteau, D.L. & Bohr, V.A. 2019. Ageing as a risk factor for neurodegenerative disease. Nature Reviews Neurology 15(10): 565-581. https://doi.org/10.1038/s41582-019-0244-7

Junaid, R., Wahid, M., Waseem, F.S., Habib, R. & Hasan, A. 2021. Effect of glucose mediated oxidative stress on apoptotic gene expression in gingival mesenchymal stem cells. BMC Oral Health 21(1): 1-13. https://doi.org/10.1186/s12903-021-02007-y

Kaatabi, H., Bamosa, A.O., Badar, A., Al-Elq, A., Abou-Hozaifa, B., Lebda, F., Al-Khadra, A. & Al-Almaie, S. 2015. Nigella sativa improves glycemic control and ameliorates oxidative stress in patients with type 2 diabetes mellitus: Placebo controlled participant blinded clinical trial. PLoS ONE 10(2): e0113486. https://doi.org/10.1371/journal.pone.0113486

Kang, I.N., Lai, S.I., Masniza, M.L., Fong, S.W., Abdul Rahman, I.G. & Yvone-Tee, G.B. 2015. Identification of valid reference genes for reliable RT-QPCR in human normal and cancer brain cell lines. Health and the Environment Journal 6(1): 31-44. https://www.researchgate.net/publication/292971297_Identification_of_Valid_Reference_Genes_for_Reliable_RT-qPCR_in_Human_Normal_and_Cancer_Brain_Cell_Lines

Karimian, A., Ahmadi, Y. & Yousefi, B. 2016. Multiple functions of P21 in cell cycle, apoptosis and transcriptional regulation after DNA damage. DNA Repair 42: 63-71. https://doi.org/10.1016/j.dnarep.2016.04.008.

Kek, S.P., Chin, N.L., Tan, S.W., Yusof, Y.A. & Chua, L.S. 2017. Classification of honey from its bee origin via chemical profiles and mineral content. Food Analytical Methods 10(1): 19-30. https://doi.org/10.1007/s12161-016-0544-0

Kim, K., Vance, T.M., Chun, O.K. & Kim, D.O. 2017. Effect of green tea extract on circulating endothelial progenitor cells: A pilot study. Cell Proliferation 50(2): e12323.

Knösel, T., Altendorf-Hofmann, A., Lindner, L., Issels, R., Hermeking, H., Schuebbe, G., Gibis, S., Siemens, H., Kampmann, E. & Kirchner, T. 2014. Loss of P16(INK4a) is associated with reduced patient survival in soft tissue tumours, and indicates a senescence barrier. Journal of Clinical Pathology 67(7): 592-598. https://doi.org/10.1136/jclinpath-2013-202106

Li, C.Y., Wu, X.Y., Tong, J.B., Yang, X.X., Zhao, J.L., Zheng, Q.F., Zhao, G.B. & Ma, Z.J. 2015. Comparative analysis of human mesenchymal stem cells from bone marrow and adipose tissue under xeno-free conditions for cell therapy. Stem Cell Research and Therapy 6(1): 55. https://doi.org/10.1186/s13287-015-0066-5

Li, Y., Gao, Q., Yin, G., Ding, X. & Hao, J. 2012. WNT/β-catenin-signaling pathway stimulates the proliferation of cultured adult human sertoli cells via upregulation of C-myc expression. Reproductive Sciences 19(11): 1232-1240. https://doi.org/10.1177/1933719112447126

Li, Y-M., Schilling, T., Benisch, P., Zeck, S., Meissner-Weigl, J., Schneider, D., Limbert, C., Seufert, J., Kassem, M., Schütze, N., Jakob, F. & Ebert, R. 2007. Effects of high glucose on mesenchymal stem cell proliferation and differentiation. Biochemical and Biophysical Research Communications 363(1): 209-215. https://doi.org/10.1016/j.bbrc.2007.08.161

Liu, X. & Zhou, X. 2013. Effect of Wnt/β-catenin and NF-ΚB signaling pathways on mucus secretion with hypertonicity in 16HBE cells. Brazilian Archives of Biology and Technology 56(4): 567-574. https://doi.org/10.1590/S1516-89132013000400006

Majdalawieh, A.F., Hmaidan, R. & Carr, R.I. 2010. Nigella sativa modulates splenocyte proliferation, Th1/Th2 cytokine profile, macrophage function and NK anti-tumor activity. Journal of Ethnopharmacology 131(2): 268-275. https://doi.org/10.1016/j.jep.2010.06.030

Masniza, M.L., Zetty Nadia, M.Z., Nur Syahrina, R., Hayati, A.R., Asral Wirda, A.A., Fadlul Azim Fauzi, M. & Nur Fariha, M.M. 2020. Date palm fruit (var. Ajwa) promotes proliferation of human bone marrow mesenchymal stem cells: Potential natural booster for endogenous stem cells growth. Fruits 75(4): 161-169. https://doi.org/10.17660/th2020/75.4.3

Minutolo, A., Grelli, S., Marino-Merlo, F., Cordero, F.M., Brandi, A., MacChi, B. & Mastino, A. 2012. D(-)lentiginosine-induced apoptosis involves the intrinsic pathway and is p53-independent. Cell Death and Disease 3(7): e358. https://doi.org/10.1038/cddis.2012.97

Nakamura, T., Sakai, K., Nakamura, T. & Matsumoto, K. 2011. Hepatocyte growth factor twenty years on: Much more than a growth factor. Journal of Gastroenterology and Hepatology (Australia)26(Suppl 1): 188-202. https://doi.org/10.1111/j.1440-1746.2010.06549.x

Pfaffl, M.W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids. Res. 29(9): e45.

Prasetyaningtyas, W.E., Romadhon, D.P., Susana, F., Djuwita, I. & Mohamad, K. 2016. Black seed (Nigella sativa) extract induce in vitro proliferation and differentiation of rat pancreatic and bone cells. Jurnal Veteriner 17(3): 337-346. https://www.cabdirect.org/cabdirect/abstract/20173048253

Puca, F., Fedele, M., Rasio, D. & Battista, S. 2022. Role of diet in stem and cancer stem cells. International Journal of Molecular Sciences 23(15): 8108. https://doi.org/10.3390/ijms23158108

Randhawa, M.A. & Alghamdi, M.S. 2011. Anticancer activity of Nigella sativa (black seed) - A review. American Journal of Chinese Medicine 39(6): 1075-1091. https://doi.org/10.1142/S0192415X1100941X

Rayess, H., Wang, M.B. & Srivatsan, E.S. 2012. Cellular senescence and tumor suppressor gene p16. International Journal of Cancer 130(8): 1715-1725. https://doi.org/10.1002/ijc.27316

Reya, T. & Clevers, H. 2005. Wnt signalling in stem cells and cancer. Nature 434: 843-850. https://doi.org/10.1038/nature03319

Rodrigues, M., Griffith, L.G. & Wells, A. 2010. Growth factor regulation of proliferation and survival of multipotential stromal cells. Stem Cell Research and Therapy 1(4): 32. https://doi.org/10.1186/scrt32.

Rutledge, G.A., Fisher, D.R., Miller, M.G., Kelly, M.E., Bielinski, D.F. & Shukitt-Hale, B. 2019. The effects of blueberry and strawberry serum metabolites on age-related oxidative and inflammatory signaling in vitro. Food and Function 10(12): 7707-7713. https://doi.org/10.1039/c9fo01913h

Sasaki, M., Abe, R., Fujita, Y., Ando, S., Inokuma, D. & Shimizu, H. 2008. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. The Journal of Immunology 180(4): 2581-2587. https://doi.org/10.4049/jimmunol.180.4.2581

Scheller, M., Huelsken, J., Rosenbauer, F., Taketo, M.M., Birchmeier, W., Tenen, D.G. & Leutz, A. 2006. Hematopoietic stem cell and multilineage defects generated by constitutive β-catenin activation. Nature Immunology 7(10): 1037-1047. https://doi.org/10.1038/ni1387

Shalini, S., Dorstyn, L., Dawar, S. & Kumar, S. 2015. Old, new and emerging functions of caspases. Cell Death and Differentiation 22: 526-539.  https://doi.org/10.1038/cdd.2014.216

Shen, H., Wang, Y., Zhang, Z., Yang, J., Hu, S. & Shen, Z. 2015. Mesenchymal stem cells for cardiac regenerative therapy: Optimization of cell differentiation strategy. Stem Cells International 2015: 524756 https://doi.org/10.1155/2015/524756

Shibuya, M. 2013. Vascular endothelial growth factor and its receptor system: Physiological functions in angiogenesis and pathological roles in various diseases. Journal of Biochemistry 153(1): 13-19. https://doi.org/10.1093/jb/mvs136

Shin, J.S., S.W., Hong, Lee, S.L., Kim, T.H., Park, I.C., An, S.K., Lee, W.K., Lim, J.S., Kim, K.I., Yang, Y., Lee, S.S., Jin, D.H. & Lee, M.S. 2008. Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells. International Journal of Oncology 32(2): 435-439. https://doi.org/10.3892/ijo.32.2.435

Stolzing, A., Coleman, N. & Scutt, A. 2006. Glucose-induced replicative senescence in mesenchymal stem cells. Rejuvenation Research 9: 31-35. https://doi.org/10.1089/rej.2006.9.31

Tan, A.W., Liau, L.L., Chua, K.H., Ahmad, R., Akbar, S.A. & Pingguan-Murphy, B. 2016. Enhanced in vitro angiogenic behaviour of human umbilical vein endothelial cells on thermally oxidized TiO2 nanofibrous surfaces. Scientific Reports 6: 21828. https://doi.org/10.1038/srep21828

Xu, S.Z., Zhong, W., Watson, N.M., Dickerson, E., Wake, J.D., Lindow, S.W., Newton, C.J. & Atkin, S.L. 2008. Fluvastatin reduces oxidative damage in human vascular endothelial cells by upregulating Bcl-2. Journal of Thrombosis and Haemostasis 6(4): 692-700. https://doi.org/10.1111/j.1538-7836.2008.02913.x

Yi, T. & Song, S.U. 2012. Immunomodulatory properties of mesenchymal stem cells and their therapeutic applications. Archives of Pharmacal Research 35: 213-221. https://doi.org/10.1007/s12272-012-0202-z

Zaid, S.S.M., Sulaiman, S.A. Sirajudeen, K.N.M. & Othman, N.H. 2010. The effects of Tualang honey on female reproductive organs, tibia bone and hormonal profile in ovariectomised rats - Animal model for menopause. BMC Complementary and Alternative Medicine 10: 82. https://doi.org/10.1186/1472-6882-10-82

Zainol, M.I., Mohd Yusoff, K. & Mohd Yusof, M.Y. 2013. Antibacterial activity of selected Malaysian honey. BMC Complementary and Alternative Medicine 13: 129. https://doi.org/10.1186/1472-6882-13-129

Zhang, D.Y., Wang, H.J. & Tan, Y.Z. 2011. Wnt/β-catenin signaling induces the aging of mesenchymal stem cells through the DNA damage response and the p53/p21 pathway. PLoS ONE 6(6): e21397. https://doi.org/10.1371/journal.pone.0021397

Zhang, R., Liu, Y., Yan, K., Chen, L., Chen, X.R., Li, P., Chen, F.F. & Jiang, X.D. 2013. Anti-inflammatory and immunomodulatory mechanisms of mesenchymal stem cell transplantation in experimental traumatic brain injury. Journal of Neuroinflammation 10: 871. https://doi.org/10.1186/1742-2094-10-106

Zhao, S. & Fernald, R.D. 2005. Comprehensive algorithm for quantitative real-time polymerase chain reaction. J. Comput. Biol. 12(8): 1047-1064.

Zhuo, Z., Zhang, L., Mu, Q., Lou, Y., Gong, Z., Shi, Y., Ouyang, G. & Zhang, Y. 2009. The effect of combination treatment with docosahexaenoic acid and 5-fluorouracil on the MRNA expression of apoptosis-related genes, including the novel gene BCL2L12, in gastric cancer cells. In Vitro Cellular and Developmental Biology - Animal 45(1-2): 69-74. https://doi.org/10.1007/s11626-008-9154-5

 

*Corresponding author; email: nurfariha@usim.edu.my

 

 

 

 

 

 

 

 

 

 

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