Medicinal Chemistry Research

, Volume 26, Issue 10, pp 2547–2556 | Cite as

BMP-2 and icariin synergistically promote p38MAPK-mediated cardiomyocyte differentiation of mesenchymal stem cells via enhanced NOX4-driven ROS generation

  • Changke Jiang
  • Fang Gong
Original Research


The aim of this study was to comparatively evaluate the effects of bone morphogenetic protein-2 and the flavonoid icariin upon cardiomyocyte differentiation of bone marrow-derived mesenchymal stem cells and to investigate the mechanism(s) underlying these effects. Rat bone marrow-derived mesenchymal stem cells were isolated by fluorescence-activated cell sorting. After treatment under four experimental conditions (i.e., control, bone morphogenetic protein-2, icariin, and bone morphogenetic protein-2 + icariin), the combination of bone morphogenetic protein-2 and icariin was superior to either bone morphogenetic protein-2 or icariin monotherapy in inducing cardiomyocyte differentiation as evidenced by upregulated GATA-4, NKX2-5, cTnT, and CX-43 expression (p < 0.05). Second, the combination of bone morphogenetic protein-2 and icariin was superior to either bone morphogenetic protein-2 or icariin monotherapy in enhancing intracellular reactive oxygen species generation via synergistically promoting NADPH oxidase 4 upregulation (p < 0.05). Third, the combination of bone morphogenetic protein-2 and icariin was superior to either bone morphogenetic protein-2 or icariin monotherapy in enhancing NADPH oxidase 4-dependent, reactive oxygen species-driven p38MAPK activation (p < 0.05). Fourth, the combination of bone morphogenetic protein-2 and icariin was superior to either bone morphogenetic protein-2 or icariin monotherapy in enhancing p38MAPK-mediated bone marrow-derived mesenchymal stem cell-to-cardiomyocyte differentiation (p < 0.05). In conclusion, bone morphogenetic protein-2 and icariin combination therapy synergistically promotes p38MAPK-mediated bone marrow-derived mesenchymal stem cell-to-cardiomyocyte differentiation via enhanced NADPH oxidase 4-dependent intracellular reactive oxygen species generation. These findings provide novel insights into the NADPH oxidase 4–reactive oxygen species–p38MAPK axis-based mechanisms underlying the differentiation of bone marrow-derived mesenchymal stem cells into cardiomyocytes, which can aid in the development of superior methods for in vitro bone marrow-derived mesenchymal stem cell-to-cardiomyocyte differentiation.


Mesenchymal stem cell, MSC Bone morphogenetic protein-2, BMP-2 Icariin Cardiomyocyte 



This work was supported by the Key Project of the Chongqing Municipal Health Bureau (grant no. 2011-1-082). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

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Supplementary Information


  1. Aouadi M, Bost F, Caron L, Laurent K, Le Marchand Brustel Y, Binetruy B (2006) p38 mitogen-activated protein kinase activity commits embryonic stem cells to either neurogenesis or cardiomyogenesis. Stem Cells 24(5):1399–1406CrossRefPubMedGoogle Scholar
  2. Asumda FZ, Chase PB (2012) Nuclear cardiac troponin and tropomyosin are expressed early in cardiac differentiation of rat mesenchymal stem cells. Differentiation 83(3):106–115CrossRefPubMedGoogle Scholar
  3. Atashi F, Modarressi A, Pepper MS (2015) The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: a review. Stem Cells Dev 24(10):1150–1163CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bhandari DR, Seo K-W, Roh K-H, Jung J-W, Kang S-K, Kang K-S (2010) REX-1 expression and p38 MAPK activation status can determine proliferation/differentiation fates in human mesenchymal stem cells. PloS One 5(5):e10493CrossRefPubMedPubMedCentralGoogle Scholar
  5. Crespo FL, Sobrado VR, Gomez L, Cervera AM, McCreath KJ (2010) Mitochondrial reactive oxygen species mediate cardiomyocyte formation from embryonic stem cells in high glucose. Stem Cells 28(7):1132–1142PubMedGoogle Scholar
  6. Csiszar A, Ahmad M, Smith KE, Labinskyy N, Gao Q, Kaley G et al. (2006) Bone morphogenetic protein-2 induces proinflammatory endothelial phenotype. Am J Pathol 168(2):629–638CrossRefPubMedPubMedCentralGoogle Scholar
  7. Dikalov SI, Dikalova AE, Bikineyeva AT, Schmidt HH, Harrison DG, Griendling KK (2008) Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production. Free Radic Biol Med 45(9):1340–1351CrossRefPubMedPubMedCentralGoogle Scholar
  8. Ding L, Liang X-G, Hu Y, Zhu D-Y, Lou Y-J (2008) Involvement of p38MAPK and reactive oxygen species in icariin-induced cardiomyocyte differentiation of murine embryonic stem cells in vitro. Stem Cells Dev 17(4):751–760CrossRefPubMedGoogle Scholar
  9. Heusch G (2013) Cardioprotection: chances and challenges of its translation to the clinic. Lancet 381(9861):166–175CrossRefPubMedGoogle Scholar
  10. Hou J, Lü AL, Liu BW, Xing YJ, Da J, Hou ZL, Ai SY (2013) Combination of BMP-2 and 5-AZA is advantageous in rat bone marrow-derived mesenchymal stem cells differentiation into cardiomyocytes. Cell Biol Int 37(12):1291–1299CrossRefPubMedGoogle Scholar
  11. Jin M-s, Shi S, Zhang Y, Yan Y, Sun X-d, Liu W, Liu H-w (2010) Icariin-mediated differentiation of mouse adipose-derived stem cells into cardiomyocytes. Mol Cell Biochem 344(1–2):1–9CrossRefPubMedGoogle Scholar
  12. Kanda Y, Hinata T, Kang SW, Watanabe Y (2011) Reactive oxygen species mediate adipocyte differentiation in mesenchymal stem cells. Life Sci 89(7):250–258CrossRefPubMedGoogle Scholar
  13. Li H, Yu B, Zhang Y, Pan Z, Xu W, Li H (2006a) Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes. Biochem Biophys Res Commun 341(2):320–325CrossRefPubMedGoogle Scholar
  14. Li J, Stouffs M, Serrander L, Banfi B, Bettiol E, Charnay Y et al. (2006b) The NADPH oxidase NOX4 drives cardiac differentiation: role in regulating cardiac transcription factors and MAP kinase activation. Mol Biol Cell 17(9):3978–3988CrossRefPubMedPubMedCentralGoogle Scholar
  15. Meng D, Lv D-D, Fang J (2008) Insulin-like growth factor-I induces reactive oxygen species production and cell migration through Nox4 and Rac1 in vascular smooth muscle cells. Cardiovasc Res 80(2):299–308CrossRefPubMedGoogle Scholar
  16. Murray TV, Smyrnias I, Shah AM, Brewer AC (2013) NADPH oxidase 4 regulates cardiomyocyte differentiation via redox activation of c-Jun protein and the cis-regulation of GATA-4 gene transcription. J Biol Chem 288(22):15745–15759CrossRefPubMedPubMedCentralGoogle Scholar
  17. Parikh A, Wu J, Blanton RM, et al. (2015) Signaling pathways and gene regulatory networks in cardiomyocyte differentiation[J]. Tissue Engineering Part B: Reviews 21(4): 377–392Google Scholar
  18. Qin S, Zhou W, Liu S, Chen P, Wu H (2015) Icariin stimulates the proliferation of rat bone mesenchymal stem cells via ERK and p38 MAPK signaling. Int J Clin Exp Med 8(5):7125PubMedPubMedCentralGoogle Scholar
  19. Reczek CR, Chandel NS (2015) ROS-dependent signal transduction. Curr Opin Cell Biol 33:8–13CrossRefPubMedGoogle Scholar
  20. Schmelter M, Ateghang B, Helmig S, Wartenberg M, Sauer H (2006) Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation. FASEB J 20(8):1182–1184CrossRefPubMedGoogle Scholar
  21. 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. Cardiovasc Dis 22:24Google Scholar
  22. Sirokmány G, Donkó Á, Geiszt M (2016) Nox/Duox family of NADPH oxidases: lessons from knockout mouse models. Trends Pharmacol Sci 37(4):318–327CrossRefPubMedGoogle Scholar
  23. Son Y, Kim S, Chung H-T, Pae H-O (2013) Reactive oxygen species in the activation of MAP kinases. Methods Enzymol 528:27–48CrossRefPubMedGoogle Scholar
  24. Takac I, Schröder K, Zhang L, Lardy B, Anilkumar N, Lambeth JD et al. (2011) The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4. J Biol Chem 286(15):13304–13313CrossRefPubMedPubMedCentralGoogle Scholar
  25. Vieira RDO, Hueb W, Hlatky M, Favarato D, Rezende PC, Garzillo CL et al. (2012) Cost-effectiveness analysis for surgical, angioplasty, or medical therapeutics for coronary artery disease 5-year follow-up of medicine, angioplasty, or surgery study (MASS) II trial. Circulation 126(11 suppl 1):S145–S150CrossRefPubMedGoogle Scholar
  26. Wagner K-H, Brath H (2012) A global view on the development of non communicable diseases. Prev Med 54:S38–S41CrossRefPubMedGoogle Scholar
  27. Wei H, Li Z, Hu S, Chen X, Cong X (2010) Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases, p38 and JNK. J Cell Biochem 111(4):967–978CrossRefPubMedGoogle Scholar
  28. Wo Y, Zhu D, Yu Y, Lou Y (2008) Involvement of NF-κB and AP-1 activation in icariin promoted cardiac differentiation of mouse embryonic stem cells. Eur J Pharmacol 586(1):59–66CrossRefPubMedGoogle Scholar
  29. Wu J, Kubota J, Hirayama J, Nagai Y, Nishina S, Yokoi T et al. (2010) p38 Mitogen-activated protein kinase controls a switch between cardiomyocyte and neuronal commitment of murine embryonic stem cells by activating myocyte enhancer factor 2C-dependent bone morphogenetic protein 2 transcription. Stem Cells Dev 19(11):1723–1734CrossRefPubMedGoogle Scholar
  30. Yang K, Wang XQ, He YS, Lu L, Chen QJ, Liu J, Shen WF (2010) Advanced glycation end products induce chemokine/cytokine production via activation of p38 pathway and inhibit proliferation and migration of bone marrow mesenchymal stem cells. Cardiovasc Diabetol 9(1):66CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.Department of PediatricsYongchuan Hospital Affiliated to Chongqing Medical UniversityChongqingChina

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