Magnolol Inhibits iNOS, p38 Kinase, and NF-κB/Rel in Murine Macrophages

  • Li Mei Hong (Department of Pharmacology, Chosun University College of Medicine) ;
  • Chang In-Youp (Department of Anatomy, Chosun University College of Medicine) ;
  • Youn Ho-Jin (Department of Pharmacology, Chosun University College of Medicine) ;
  • Jang Dae-Sik (Korea Institute of Oriental Medicine) ;
  • Kim Jin-Sook (Korea Institute of Oriental Medicine) ;
  • Jeon Young-Jin (Department of Pharmacology, Chosun University College of Medicine)
  • Published : 2006.09.30

Abstract

We demonstrate that magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells(murine macrophage cell line). Treatment of RAW 264.7 cells with magnolol inhibited LPS-stimulated nitric oxide production in a dose-related manner. RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression. Western immunoblot analysis of phosphorylate p38 kinase showed magnolol significantly inhibited the phosphorylation of p38 kinase which is important in the regulation of iNOS gene expression. The specific p38 inhibiter SB203580 abrogated the LPS-induced NO generation and iNOS expression, whereas the selective MEK-1 inhibitor PD98059 did not affect the NO induction. Immunostaining of p65 and reporter gene assay showed that magnolol inhibited NF-${\kappa}/Rel$ nuclear translocation and transcriptional activation, respectively. Collectively, this series of experiments indicates that magnolol inhibits iNOS gene expression by blocking NF-k/Rel and p38 kinase signaling. Due to the critical role that NO release plays in mediating inflammatory responses, the inhibitory effects of magnolol or iNOS suggest that magnolol may represent a useful anti-inflammatory agent.

Keywords

References

  1. Beyaert, R., Cuenda, A., Vanden Berghe, W., Plaisance, S., Lee, J.C., Haegeman, G., et al. (1996): The p38/RK mitogen- activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor. EMBO J., 15, 1914-1923
  2. Chen, C.C. and Wang, J.K. (1999): p38 but not p44/42 mitogen- activated protein kinase is required for nitric oxide snthase induction mediated by lipopolysaccharide in RAW 264.7 cells. Mol. Pharmacol., 55, 481-488
  3. Chen, Y.H., Lin, S.J., Chen, J.W., Ku, H.H. and Chen, Y.L. (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. Br. J. Pharmacol., 135, 37-47 https://doi.org/10.1038/sj.bjp.0704458
  4. Chomczynski, P. and Mackey, K. (1995): Substitution of chloroform by bromo-chloropropane in the single-step method of RNA isolation. Anal. Biochem., 225, 163-164 https://doi.org/10.1006/abio.1995.1126
  5. Cuenda, A., Rouse, J., Doza, Y.N., Meier, R., Cohen, P., Gallagher, T.F., et al. (1995): SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS. Lett., 364, 229-233 https://doi.org/10.1016/0014-5793(95)00357-F
  6. Da Silva, J., Pierrat, B., Mary, J.L. and Lesslauer, W. (1997): Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes. J. Biol. Chem., 272, 28373-28380 https://doi.org/10.1074/jbc.272.45.28373
  7. Dudley, D.T., Pang, L., Decker, S.J., Bridges, A.J. and Saltiel, A.R. (1995): A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. U.S.A., 92, 7686-7689
  8. Dunnett, M. (1955) A multiple comparison procedure for comparing several treatments with a control. J. Am. Statistics Assoc., 50, 1096-1121 https://doi.org/10.2307/2281208
  9. Foey, A.D., Parry, S., Williams, L.M., Feldmann, M., Foxwel,l B.M. and Brennan, F.M. (1998): Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-$\alpha$: Role of the P38 and p442/44 mitogen-activated protein kinases. J. Immunol.,160, 920-928
  10. Fujita, S. and Taira, J. (1994): Biphenyl compounds are hydroxy radical scavengers: their effective inhibition for UV-induced mutation in Salmolella typhimurium TA102. Free Radic. Biol. Med., 17, 273-277 https://doi.org/10.1016/0891-5849(94)90083-3
  11. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S. and Tannenbaum, S.R. (1982): Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem., 126, 131-138 https://doi.org/10.1016/0003-2697(82)90118-X
  12. Hibbs, J.B. Jr., Taintor, R.R. and Vavrin, Z. (1987): Macrophage cytotoxicity: role for Larginine deiminase and imino nitrogen oxidation to nitrite. Science, 235, 473-476 https://doi.org/10.1126/science.2432665
  13. Jeon, Y.J., Han, S.H., Lee, Y.W., Yea, S.S. and Yang, K.H. (1998): Inhibition of NF-kappa B/Rel nuclear translocation by dexamethasone: mechanism for the inhibition of iNOS gene expression. Biochem. Mol. Biol. Int., 45, 435-441
  14. Jeon, Y.J., Kim, Y.G., Lee, M., Park, S.M., Han, S.B. and Kim, H.M. (2000): Radicicol suppresses expression of inducible nitric oxide synthase by blocking p38 kinase and nuclear factor-kB/Rel in lipopolysaccharide-stimulated macrophages. J. Pharmacol. Exp. Ther., 294, 548-554
  15. Lee, J.C. and Young, P.R. (1996): Role of CSB/p38/RK stress response kinase in LPS and cytokine signaling mechanisms. J. Leukoc. Biol., 59, 152-157 https://doi.org/10.1002/jlb.59.2.152
  16. Lee, J., Jung, E., Park, J., Jung, K., Lee, S., Hong, S., Park, J., Park, E., Kim, J., Park, S. and Park, D. (2005): Antiinflammatory effects of magnolol and honokiol are mediated through inhibition of the downstream pathway of MEKK-1 in NF-kappaB activation signaling. Planta. Med., 71, 338-343 https://doi.org/10.1055/s-2005-864100
  17. Lowenstein, C.J., Alley, E.W., Raval, P., Snowman, A.M., Snyder, S.H., Russell, S.W., et al. (1993): Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc. Natl. Acad. Sci. U.S.A., 90, 9730-9734
  18. Matsuda, H., Kageura, T., Oda, M., Morikawa, T., Sakamoto, Y. and Yoshikawa, M. (2001): Effects of constituents from the bark of Magnolia obovata on nitric oxide production in lipopolysaccharide-activated macrophages. Chem. Pharm. Bull, 49, 716-720 https://doi.org/10.1248/cpb.49.716
  19. Palmer, R.M., Ashton, D.S. and Moncada, S. (1988): Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature, 333, 664-666 https://doi.org/10.1038/333664a0
  20. Raingeaud, J., Gupta, S., Rogers, J.S., Dickens, M., Han, J., Ulevitch, R.J., et al. (1995): Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J. Biol. Chem., 270, 7420-7426 https://doi.org/10.1074/jbc.270.13.7420
  21. Rietschel, E.T. and Brade, H. (1992): Bacterial endotoxins. Sci. Am., 267, 54-61 https://doi.org/10.1038/scientificamerican0892-54
  22. Teng, C.M., Yu, S.M., Chen, C.C., Huang, Y.L. and Huang, T.F. (1990): EDRF-release and $Ca^{2+}$-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta. Life Sci., 47, 1153-1161 https://doi.org/10.1016/0024-3205(90)90176-R
  23. Wang, J.P., Hsu, M.F., Raung, S.L., Chang, L.C., Tsao, L.T., Lin, P.L., et al. (1999): Inhibition by magnolol of formylmethionyl- leucyl-phenylalanine-induced respiratory burst in rat neutrophils. J. Pharm. Pharmacol., 51, 285-294 https://doi.org/10.1211/0022357991772466
  24. Wang, J.P., Hsu, M.F., Raung, S.L., Chen, C.C., Kuo, J.S. and Teng, C.M. (1992): Anti-inflammatory and analgesic effects of magnolol. Naunyn Schmiedebergs Arch. Pharmacol., 346, 707-712
  25. Wang, J.P., Lin, P.L., Hsu, M.F. and Chen, C.C. (1998): Possible involvement of protein kinase C inhibition in the reduction of phorbol ester-induced neutrophil aggregation by magnolol in the rat. J. Pharm. Pharmacol., 50, 1167-1172 https://doi.org/10.1111/j.2042-7158.1998.tb03329.x
  26. Xie, Q.W., Kashiwabara, Y. and Nathan, C. (1994): Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J. Biol. Chem., 269, 4705-4708
  27. Xie, Q.W., Qhisnant, R. and Nathan, C. (1993): Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon-g and bacterial lipopolysaccharide. J. Exp. Med., 177, 1779-1784 https://doi.org/10.1084/jem.177.6.1779