, Volume 35, Issue 3, pp 1204–1212 | Cite as

Magnolol Protects Osteoblastic MC3T3-E1 Cells Against Antimycin A-Induced Cytotoxicity Through Activation of Mitochondrial Function

  • Eun Mi ChoiEmail author


Antimycin A treatment of cells blocks the mitochondrial electron transport chain and leads to elevated ROS generation. In the present study, we investigated the protective effects of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Osteoblastic MC3T3-E1 cells were pre-incubated with magnolol before treatment with antimycin A. Cell viability and mineralization of osteoblasts were assessed by MTT assay and Alizarin Red staining, respectively. Mitochondrial dysfunction in cells was measured by mitochondrial membrane potential (MMP), complex IV activity, and ATP level. The cellular antioxidant effect of magnolol in osteoblastic MC3T3-E1 cells was assessed by measuring cardiolipin oxidation, mitochondrial superoxide levels, and nitrotyrosine content. Phosphorylated cAMP-response element-binding protein (CREB ) was evaluated using ELISA assay. Pretreatment with magnolol prior to antimycin A exposure significantly reduced antimycin A-induced osteoblast dysfunction by preventing MMP dissipation, ATP loss, and CREB inactivation. Magnolol also reduced cardiolipin peroxidation, mitochondrial superoxide, and nitrotyrosine production induced by antimycin A. These results suggest that magnolol has a protective effect against antimycin A-induced cell damage by its antioxidant effects and the attenuation of mitochondrial dysfunction. All these data indicate that magnolol may reduce or prevent osteoblast degeneration in osteoporosis or other degenerative disorders.


magnolol mitochondrial dysfunction MC3T3-E1 cells oxidative stress CREB 



This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20110005020).


  1. 1.
    Raisz, L.G., and G.A. Rodan. 2003. Pathogenesis of osteoporosis. Endocrinology and Metabolism Clinics of North America 32: 15–24.PubMedCrossRefGoogle Scholar
  2. 2.
    Chrischilles, E.A., C.D. Butler, C.S. Davis, and R.B. Wallace. 1991. A model of lifetime osteoporosis impact. Archives of Internal Medicine 151: 2026–2032.PubMedCrossRefGoogle Scholar
  3. 3.
    Forrest, C.M., G.M. Mackay, L. Oxford, N. Stoy, T.W. Stone, and L.G. Darlington. 2005. Kynurenine pathway metabolism in patients with osteoporosis after two years of drug treatment. Clinical and Experimental Pharmacology and Physiology 33: 1078–1087.CrossRefGoogle Scholar
  4. 4.
    Yang, S., P. Madyastha, S. Bingel, W. Ries, and L. Key. 2001. A new superoxide-generating oxidase in murine osteoclasts. Journal of Biological Chemistry 276: 5452–5458.PubMedCrossRefGoogle Scholar
  5. 5.
    Muthusami, S., H. Ramachandran, B. Muthusamy, G. Vasudevan, V. Prabhu, and V. Subramaniam. 2005. Ovariectomy induces oxidative stress and impairs bone antioxidant system in adult rats. Clinica Chimica Acta 360: 81–86.CrossRefGoogle Scholar
  6. 6.
    Lee, H.C., and Y.H. Wei. 2007. Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging. Experimental Biology and Medicine 232: 592–606.PubMedGoogle Scholar
  7. 7.
    Harman, D. 1983. Free radical theory of aging: Consequences of mitochondrial aging. Age 6: 86–94.CrossRefGoogle Scholar
  8. 8.
    Krishnan, K.J., A.K. Reeve, D.C. Samuels, P.F. Chinnery, J.K. Blackwood, and R.W. Taylor. 2008. What causes mitochondrial DNA deletions in human cells? Nature Genetics 40: 275–279.PubMedCrossRefGoogle Scholar
  9. 9.
    Chomyn, A., and G. Attardi. 2003. MtDNA mutations in aging and apoptosis. Biochemical and Biophysical Research Communications 304: 519–529.PubMedCrossRefGoogle Scholar
  10. 10.
    Han, Y.H., S.H. Kim, S.Z. Kim, and W.H. Park. 2008. Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death. Chemico-Biological Interactions 171: 67–78.PubMedCrossRefGoogle Scholar
  11. 11.
    Balaban, R.S., S. Nemoto, and T. Finkel. 2005. Mitochondria, oxidants, and aging. Cell 120: 483–495.PubMedCrossRefGoogle Scholar
  12. 12.
    Choi, E.M. 2011. Kaempferol protects MC3T3-E1 cells through antioxidant effect and regulation of mitochondrial function. Food and Chemical Toxicology 49(8): 1800–1805.PubMedCrossRefGoogle Scholar
  13. 13.
    Robak, J., and E. Marcinkiewicz. 1995. Scavenging of reactive oxygen species as the mechanism of drug action. Polish Journal of Pharmacology 47: 89–98.PubMedGoogle Scholar
  14. 14.
    Nicolson, G.L. 2007. Lipid replacement and antioxidant supplements to prevent membrane oxidation and restore mitochondrial function in metabolic syndrome and fatiguing illnesses. Townsend Letters 286: 112–120.Google Scholar
  15. 15.
    Farris, M.W., C.B. Chan, M. Patel, B. Van Houten, and S. Orrenius. 2005. Role of mitochondria in toxic oxidative stress. Molecular Interventions 5: 94–111.CrossRefGoogle Scholar
  16. 16.
    Wang, J.P., M.F. Hsu, S.L. Raung, C.C. Chen, J.S. Kuo, and C.M. Teng. 1992. Anti-inflammatory and analgesic effects of magnolol. Naunyn-Schmiedeberg's Archives of Pharmacology 346: 707–712.PubMedCrossRefGoogle Scholar
  17. 17.
    Teng, C.M., S.M. Yu, C.C. Chen, Y.L. Huang, and T.F. Huang. 1990. EDRF-release and Ca2+-channel blockade by magnolol, an anti-platelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta. Life Sciences 47: 1153–1161.PubMedCrossRefGoogle Scholar
  18. 18.
    Fujita, S., and J. Taira. 1994. Biphenyl compounds are hydroxy radical scavengers: Their effective inhibition for UV induced mutation in Salmolella typhimurium TA102. Free Radical Biology & Medicine 17: 273–277.CrossRefGoogle Scholar
  19. 19.
    Wang, J.P., P.L. Lin, M.F. Hsu, and C.C. Chen. 1998. Possible involvement of protein kinase C inhibition in the reduction of phorbol ester-induced neutrophil aggregation by magnolol in the rat. Journal of Pharmacy and Pharmacology 50: 1167–1172.PubMedCrossRefGoogle Scholar
  20. 20.
    Wang, J.P., M.F. Hsu, S.L. Raung, L.C. Chang, L.T. Tsao, P.L. Lin, and C.C. Chen. 1999. Inhibition by agnolol of formylmethionyl-leucyl-phenylalanine-induced respiratory burst in rat neutrophils. Journal of Pharmacy and Pharmacology 51: 285–294.PubMedCrossRefGoogle Scholar
  21. 21.
    Chen, Y.H., S.J. Lin, J.W. Chen, H.H. Ku, and Y.L. Chen. 2002. Magnolol attenuates VCAM-1 expression in vitro in TNF-a-treated human aortic endothelial cells and in vivo in the aorta of cholesterol-fed rabbits. British Journal of Pharmacology 135: 37–47.PubMedCrossRefGoogle Scholar
  22. 22.
    Matsuda, H., T. Kageura, M. Oda, T. Morikawa, Y. Sakamoto, and M. Yoshikawa. 2001. Effects of constituents from the bark of Magnolia obovata on nitric oxide production in lipopolysaccharide-activated macrophages. Chemical and Pharmaceutical Bulletin 49: 716–720.CrossRefGoogle Scholar
  23. 23.
    Kong, C.W., K. Tsai, J.H. Chin, W.L. Chang, and C.Y. Hong. 2000. Magnolol attenuates peroxidative damage and improves survival of rats with sepsis. Shock 13: 24–28.PubMedCrossRefGoogle Scholar
  24. 24.
    Shih, H.C., Y.H. Wei, and C.H. Lee. 2003. Magnolol alters cytokine response after hemorrhagic shock and increases survival in subsequent intraabdominal sepsis in rats. Shock 20: 264–268.PubMedCrossRefGoogle Scholar
  25. 25.
    Shih, H.C., Y.H. Wei, and C.H. Lee. 2004. Magnolol alters the course of endotoxin tolerance and provides early protection against endotoxin challenge following sublethal hemorrhage in rats. Shock 22: 358–363.PubMedCrossRefGoogle Scholar
  26. 26.
    Quarles, L.D., D.A. Yohay, L.W. Lever, R. Caton, and R.J. Wenstrup. 1992. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development. Journal of Bone and Mineral Research 7: 683–690.PubMedCrossRefGoogle Scholar
  27. 27.
    Stambough, J.L., C.T. Brigghton, J.P. Iannotti, and B.T. Storey. 1984. Characterization of growth plate mitochondria. Journal of Orthopaedic Research 45: 235–246.CrossRefGoogle Scholar
  28. 28.
    Wuthier, R.E., J.E. Chin, J.E. Hale, T.C. Register, L.V. Hale, and Y. Ishikawa. 1985. Isolation and characterization of calcium accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture. Journal of Biological Chemistry 260: 15972–15979.PubMedGoogle Scholar
  29. 29.
    An, J.H., J.Y. Yang, B.Y. Ahn, S.W. Cho, J.Y. Jung, H.Y. Cho, Y.M. Cho, S.W. Kim, K.S. Park, S.Y. Kim, H.K. Lee, and C.S. Shin. 2010. Enhanced mitochondrial biogenesis contributes to Wnt induced osteoblastic differentiation of C3H10T1/2 cells. Bone 47: 140–150.PubMedCrossRefGoogle Scholar
  30. 30.
    Scarpulla, R.C. 2008. Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiological Reviews 88: 611–638.PubMedCrossRefGoogle Scholar
  31. 31.
    Handschin, C., J. Rhee, J. Lin, P.T. Tarr, and B.M. Spiegelman. 2003. An autoregulatory loop controls peroxisome proliferator-activated receptor gamma coactivator 1alpha expression in muscle. Proceedings of the National Academy of Sciences of the United States of America 100: 7111–7116.PubMedCrossRefGoogle Scholar
  32. 32.
    Cammarota, M., G. Paratcha, L.R. Bevilaqua, M. Levi de Stein, M. Lopez, and A. Pellegrinode Iraldi. 1999. Cyclic AMP-responsive element binding protein in brain mitochondria. Journal of Neurochemistry 72: 2272–2277.PubMedCrossRefGoogle Scholar
  33. 33.
    Ryu, H., J. Lee, S. Impey, R.R. Ratan, and R.J. Ferrante. 2005. Antioxidants modulate mitochondrial PKA and increase CREB binding to D-loop DNA of the mitochondrial genome in neurons. Proceedings of the National Academy of Sciences of the United States of America 102: 13915–13920.PubMedCrossRefGoogle Scholar
  34. 34.
    Simbula, G., P.A. Glascott, S. Jr Akita, J.B. Hoek, and J.L. Farber. 1997. Two mechanisms by which ATP depletion potentiates induction of the mitochondrial permeability transition. American Journal of Physiology 273: C479–C488.PubMedGoogle Scholar
  35. 35.
    Kim, C.H., S.U. Kang, J. Pyun, M.H. Lee, H.S. Hwang, and H. Lee. 2008. Epicatechin protects auditory cells against cisplatin-induced death. Apoptosis 13: 1184–1194.PubMedCrossRefGoogle Scholar
  36. 36.
    Paradies, G., G. Petrosillo, M. Pistolese, and F.M. Ruggiero. 2002. Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage. Gene 286: 135–141.PubMedCrossRefGoogle Scholar
  37. 37.
    Paradies, G., G. Petrosillo, M. Pistolese, N. Di Venosa, A. Federici, and F.M. Ruggiero. 2004. Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin. Circulation Research 94: 53–59.PubMedCrossRefGoogle Scholar
  38. 38.
    Zhang, L., L. Yu, and C.A. Yu. 1998. Generation of superoxide anion by succinate-cytochrome c reductase from bovine heart mitochondria. Journal of Biological Chemistry 273: 33972–33976.PubMedCrossRefGoogle Scholar
  39. 39.
    Forquer, I., R. Covian, M.K. Bowman, B.L. Trumpower, and D.M. Kramer. 2006. Similar transition states mediate the Q-cycle and superoxide production by the cytochrome bc1 complex. Journal of Biological Chemistry 281: 38459–38465.PubMedCrossRefGoogle Scholar
  40. 40.
    Yin, Y., S. Yang, L. Yu, and C.A. Yu. 2010. Reaction mechanism of superoxide generation during ubiquinol oxidation by the cytochrome bc1 complex. Journal of Biological Chemistry 285: 17038–17045.PubMedCrossRefGoogle Scholar
  41. 41.
    Radi, R. 2004. Nitric oxide, oxidants, and protein tyrosine nitration. Proceedings of the National Academy of Sciences of the United States of America 101: 4003–4008.PubMedCrossRefGoogle Scholar
  42. 42.
    Bartesaghi, S., J. Wenzel, M. Trujillo, M. Lopez, J. Joseph, B. Kalyanaraman, and R. Radi. 2010. Lipid peroxyl radicals mediate tyrosine dimerization and nitration in membranes. Chemical Research in Toxicology 23: 821–835.PubMedCrossRefGoogle Scholar
  43. 43.
    Cadenas, E., and K.J. Davies. 2000. Mitochondrial free radical generation, oxidative stress, and aging. Free Radical Biology & Medicine 29: 222–230.CrossRefGoogle Scholar
  44. 44.
    Mao, G.D., and M.J. Poznansky. 1992. Electron spin resonance study on the permeability of superoxide radicals in lipid bilayers and biological membranes. FEBS Letters 305: 233–236.PubMedCrossRefGoogle Scholar
  45. 45.
    Hsu, J.L., Y. Hsieh, C. Tu, D. O’Connor, H.S. Nick, and D.N. Silverman. 1996. Catalytic properties of human manganese superoxide dismutase. Journal of Biological Chemistry 271: 17687–17691.PubMedCrossRefGoogle Scholar
  46. 46.
    Hausladen, A., and I. Fridovich. 1994. Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not. Journal of Biological Chemistry 269: 29405–29408.PubMedGoogle Scholar
  47. 47.
    Zhou, L., and D.Y. Zhu. 2009. Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications. Nitric Oxide 20: 223–230.PubMedCrossRefGoogle Scholar
  48. 48.
    Lacza, Z., E. Pankotai, and D.W. Busija. 2009. Mitochondrial nitric oxide synthase: current concepts and controversies. Frontiers in Bioscience 14: 4436–4443.PubMedCrossRefGoogle Scholar
  49. 49.
    Carr, A., J. Miller, J.A. Eisman, and D.A. Cooper. 2001. Osteopenia in HIV-infected men: Association with asymptomatic lactic acidemia and lower weight pre-antiretroviral therapy. AIDS 15: 703–709.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Food and Nutrition, Education Graduate SchoolKyung Hee UniversitySeoulSouth Korea

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