Inhibitory Effect of Esculetin on the Inducuble Nitric Oxide Synthase Expression in TNF-stimulated 3T3-L1 Adipocytes

  • Yang, Jeong-Yeh (Department of Oral Biochemistry, School of Dentistry and institute of Oral Bioscience, Chonbuk National University)
  • Published : 2003.10.21


While nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is beneficial for host survival, it is also detrimental to the host. Thus, regulation of iNOS gene expression may be an effective therapeutic strategy for the prevention of unwanted reactions at various pathologic conditions. During the screening process for the possible iNOS regulators, we observed that esculetin is a potent inhibitor of cytokine-induced iNOS expression. The treatment of 3T3-L1 adipocytes with the tumor necrosis factor-${\alpha}$ (TNF) induced iNOS expression, leading to enhanced NO production. TNF-induced NO production was inhibited by esculetin in a dose-dependent manner. Esculetin inhibited the TNF-induced NO production at the transcriptional level through suppression of iNOS mRNA and subsequent iNOS protein expression. These results suggest esculetin, a component of natural products, as a naturally occurring, nontoxic means to attenuate iNOS expression and NO-mediated cytotoxicity.


  1. Flatman S, Hurst JS, McDonald-Gibson RG, Jonas GE, Slater TF. Biochemical studies on a 12-lipoxygenase in human uterine cervix. Biochim Biophys Acta 883: 7 14, 1986
  2. Fruhbeck G, Gomez-Ambrosi J, Muruzabal FJ, Burrell MA. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. Am J Physiol Endocrinol Metab 280: E827 847, 2001
  3. Gainsford T, Willson TA, Metcalf D, Handman E, McFarlane C, Ng A, Nicola NA, Alexander WS, Hilton DJ. Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells. Proc Natl Acad Sci USA 93: 14564 14568, 1996
  4. Gray DS. Diagnosis and prevalence of obesity. Med Clin North Am 73: 1 13, 1989
  5. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126: 131 138, 1982
  6. Grunfeld C, Feingold KR. Tumor necrosis factor, interleukin, and interferon induced changes in lipid metabolism as part of host defense. Proc Soc Exp Biol Med 200: 224 7, 1992
  7. Hashimoto T, Kihara M, Yokoyama K, Fujita T, Kobayashi S, Matsushita K, Tamura K, Hirawa N, Toya Y, Umemura S. Lipoxygenase products regulate nitric oxide and inducible nitric oxide synthase production in interleukin-1beta stimulated vascular smooth muscle cells. Hypertens Res 26: 177 184, 2003
  8. Hecht SS, Kenney PM, Wang M, Trushin N, Agarwal S, Rao AV, Upadhyaya P. Evaluation of butylated hydroxyanisole, myoinositol, curcumin, esculetin, resveratrol and lycopene as inhibitors of benzo[a]pyrene plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice. Cancer Lett 137: 123-130, 1999
  9. Hoshi S, Goto M, Koyama N, Nomoto K, Tanaka H. Regulation of vascular smooth muscle cell proliferation by nuclear factorkappaB and its inhibitor, I-kappaB. J Biol Chem 275: 883 889, 2000
  10. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259: 87 91, 1993
  11. Huang HC, Lai MW, Wang HR, Chung YL, Hsieh LM, Chen CC. Antiproliferative effect of esculetin on vascular smooth muscle cells: possible roles of signal transduction pathways. Eur J Pharmacol 237: 39 44, 1993
  12. Jaffrey SR, Snyder SH. Nitric oxide: a neural messenger. Annu Rev Cell Dev Biol 11: 417 440, 1995
  13. Kapur S, Marcotte B, Marette A. Mechanism of adipose tissue iNOS induction in endotoxemia. Am J Physiol 276: E635 641, 1999
  14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680 685, 1970
  15. Lin WL, Wang CJ, Tsai YY, Liu CL, Hwang JM, Tseng TH. Inhibitory effect of esculetin on oxidative damage induced by t-butyl hydroperoxide in rat liver. Arch Toxicol 74: 467 472, 2000
  16. Martin-Aragon S, Benedi JM, Villar AM. Effects of the antioxidant (6,7-dihydroxycoumarin) esculetin on the glutathione system and lipid peroxidation in mice. Gerontology 44: 21 25, 1998
  17. Matsunaga K, Yoshimi N, Yamada Y, Shimizu M, Kawabata K, Ozawa Y, Hara A, Mori H. Inhibitory effects of nabumetone, a cyclooxygenase-2 inhibitor, and esculetin, a lipoxygenase inhibitor, on N-methyl-N-nitrosourea-induced mammary carcinogenesis in rats. Jpn J Cancer Res 89: 496 501, 1998
  18. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109 142, 1991
  19. Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest 100: 2417 2423, 1997
  20. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915 918, 1994
  21. Neichi T, Koshihara Y, Murota S. Inhibitory effect of esculetin on 5-lipoxygenase and leukotriene biosynthesis. Biochim Biophys Acta 753: 130 132, 1983
  22. Pan SL, Huang YW, Guh JH, Chang YL, Peng CY, Teng CM. Esculetin inhibits Ras-mediated cell proliferation and attenuates vascular restenosis following angioplasty in rats. Biochem Pharmacol 65: 1897 1905, 2003
  23. Pond CM. Interactions between adipose tissue and the immune system. Proc Nutr Soc 55: 111 126, 1996
  24. Ribiere C, Jaubert AM, Gaudiot N, Sabourault D, Marcus ML, Boucher JL, Denis-Henriot D, Giudicelli Y. White adipose tissue nitric oxide synthase: a potential source for NO production. Biochem Biophys Res Commun 222: 706 712, 1996
  25. Rosen BS, Cook KS, Yaglom J, Groves DL, Volanakis JE, Damm D, White T, Spiegelman BM. Adipsin and complement factor D activity: an immune-related defect in obesity. Science 244: 1483 1487, 1989
  26. Shuldiner AR, Yang R, Gong DW. Resistin, obesity and insulin resistance--the emerging role of the adipocyte as an endocrine organ. N Engl J Med 345: 1345 1346, 2001
  27. Tracey KJ. Cerami A. Tumor necrosis factor and regulation of metabolism in infection: role of systemic versus tissue levels. Proc Soc Exp Biol Med 200: 233 239, 1992
  28. Tubaro A, Del Negro P, Ragazzi E, Zampiron S, Della Loggia R. Anti-inflammatory and peripheral analgesic activity of esculetin in vivo. Pharmacol Res Commun 20 Suppl 5: 83 85, 1988
  29. Xie Q, Nathan C. The high-output nitric oxide pathway: role and regulation. J Leukoc Biol 56: 576 582, 1994
  30. Yang JY, Koo BS, Kang MK, Rho HW, Sohn HS, Jhee EC, Park JW. Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes. Exp Mol Med 34: 353 360, 2002