Inflammation

, Volume 33, Issue 1, pp 46–57 | Cite as

Gamma-Linolenic Acid Inhibits Inflammatory Responses by Regulating NF-κB and AP-1 Activation in Lipopolysaccharide-Induced RAW 264.7 Macrophages

  • Cheng-Sue Chang
  • Hai-Lun Sun
  • Chong-Kuei Lii
  • Haw-Wen Chen
  • Pei-Yin Chen
  • Kai-Li Liu
Article

Abstract

Gamma linolenic acid (GLA) is a member of the n-6 family of polyunsaturated fatty acids and can be synthesized from linoleic acid (LA) by the enzyme delta-6-desaturase. The therapeutic values of GLA supplementation have been documented, but the molecular mechanism behind the action of GLA in health benefits is not clear. In this study, we assessed the effect of GLA with that of LA on lipopolysaccharide (LPS)-induced inflammatory responses and further explored the molecular mechanism underlying the pharmacological properties of GLA in mouse RAW 264.7 macrophages. GLA significantly inhibited LPS-induced protein expression of inducible nitric oxide synthase, pro-interleukin-1β, and cyclooxygenase-2 as well as nitric oxide production and the intracellular glutathione level. LA was less potent than GLA in inhibiting LPS-induced inflammatory mediators. Both GLA and LA treatments dramatically inhibited LPS-induced IκB-α degradation, IκB-α phosphorylation, and nuclear p65 protein expression. Moreover, LPS-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) nuclear protein–DNA binding affinity and reporter gene activity were significantly decreased by LA and GLA. Exogenous addition of GLA but not LA significantly reduced LPS-induced expression of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK)-1. Our data suggest that GLA inhibits inflammatory responses through inactivation of NF-κB and AP-1 by suppressed oxidative stress and signal transduction pathway of ERK and JNK in LPS-induced RAW 264.7 macrophages.

KEY WORDS

gamma-linolenic acid inflammation linoleic acid lipopolysaccharides mouse RAW 264.7 macrophages 

Abbreviations

AP-1

activator protein-1

COX-2

cyclooxygenase-2

EMSA

electrophoretic mobility shift assay

ERK1/2

extracellular signal-regulated kinase1/2

GAPDH

glyceraldehydes-3-phosphate dehydrogenase

GLA

gamma-linolenic acid

GSH

glutathione

IL-1β

interleukin-1β

iNOS

inducible form of NOS

IS

internal standard

JNK

c-Jun NH2-terminal kinase

LA

linoleic acid

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinase

MTT

3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide

NF-κB

nuclear factor-κB

NO

nitric oxide

NOS

nitric oxide synthase

PGE2

prostaglandin E2

PMSF

phenylmethylsulfonyl fluoride

rcRNA

recombinant RNA

RT-PCR

reverse transcriptase polymerase chain reaction

SEAP

secretory alkaline phosphatase

References

  1. 1.
    Korhonen, R., A. Lahti, H. Kankaanranta, and E. Moilanen. 2005. Nitric oxide production and signaling in inflammation. Curr. Drug Targets Inflamm. Allergy 4: 471–479.CrossRefPubMedGoogle Scholar
  2. 2.
    Park, J. Y., M. H. Pillinger, and S. B. Abramson. 2006. Prostaglandin E2 synthesis and secretion: the role of PGE2 synthases. Clin. Immunol. 119: 229–240.CrossRefPubMedGoogle Scholar
  3. 3.
    Stylianou, E., and J. Saklatvala. 1998. Interleukin-1. Int. J. Biochem. Cell Biol. 30: 1075–1079.CrossRefPubMedGoogle Scholar
  4. 4.
    Chen, F., V. Castranova, X. Shi, and L. M. Demers. 1999. New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. Clin. Chem. 45: 7–17.PubMedGoogle Scholar
  5. 5.
    Xie, Q. W., Y. Kashiwabara, and C. Nathan. 1994. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269: 4705–4808.PubMedGoogle Scholar
  6. 6.
    Appleby, S. B., A. Ristimaki, K. Neilson, K. Narko, and T. Hla. 1994. Structure of the human cyclo-oxygenase-2 gene. Biochem. J. 302: 723–727.PubMedGoogle Scholar
  7. 7.
    Zenz, R., R. Eferl, C. Scheinecker, K. Redlich, J. Smolen, H. B. Schonthaler, L. Kenner, E. Tschachler, and E. F. Wagner. 2008. Activator protein 1 (Fos/Jun) functions in inflammatory bone and skin disease. Arthritis Res. Ther. 10: 201–210.CrossRefPubMedGoogle Scholar
  8. 8.
    Das, U. N. 2007. Gamma-linolenic acid therapy of human glioma—a review of in vitro, in vivo, and clinical studies. Med. Sci. Monit. 13: RA119–RA131.PubMedGoogle Scholar
  9. 9.
    Kapoor, R., and Y. S. Huang. 2006. Gamma linolenic acid: an antiinflammatory omega-6 fatty acid. Curr. Pharm. Biotechnol. 7: 531–534.CrossRefPubMedGoogle Scholar
  10. 10.
    Fan, Y. Y., and R. S. Chapkin. 1998. Importance of dietary gamma-linolenic acid in human health and nutrition. J. Nutr. 128: 1411–1414.PubMedGoogle Scholar
  11. 11.
    Pham, H., K. Vang, and V. A. Ziboh. 2006. Dietary gamma-linolenate attenuates tumor growth in a rodent model of prostatic adenocarcinoma via suppression of elevated generation of PGE2 and 5S-HETE. Prostaglandins Leukot. Essent. Fat. Acids. 74: 271–282.CrossRefGoogle Scholar
  12. 12.
    Kavanagh, T., P. E. Lonergan, and M. A. Lynch. 2004. Eicosapentaenoic acid and gamma-linolenic acid increase hippocampal concentrations of IL-4 and IL-10 and abrogate lipopolysaccharide-induced inhibition of long-term potentiation. Prostaglandins Leukot. Essent. Fat. Acids. 70: 391–397.CrossRefGoogle Scholar
  13. 13.
    Furse, R. K., R. G. Rossetti, and R. B. Zurier. 2001. Gamma linolenic acid, an unsaturated fatty acid with anti-inflammatory properties, blocks amplification of IL-1 beta production by human monocytes. J. Immunol. 167: 490–496.PubMedGoogle Scholar
  14. 14.
    Johnson, M. M., D. D. Swan, M. E. Surette, J. Stegner, T. Chilton, A. N. Fonteh, and F. H. Chilton. 1997. Dietary supplementation with gamma-linolenic acid alters fatty acid content and eicosanoid production in healthy humans. J. Nutr. 127: 1435–1444.PubMedGoogle Scholar
  15. 15.
    Fan, Y. Y., and R. S. Chapkin. 1993. Phospholipid sources of metabolically elongated gammalinolenic acid: conversion to prostaglandin E1 in stimulated mouse macrophages. J. Nutr. Biochem. 4: 602–607.CrossRefGoogle Scholar
  16. 16.
    Zhao, G., T. D. Etherton, K. R. Martin, J. P. Vanden Heuvel, P. J. Gillies, S. G. West, and P. M. Kris-Etherton. 2005. Anti-inflammatory effects of polyunsaturated fatty acids in THP-1 cells. Biochem. Biophys. Res. Commun. 336: 909–917.CrossRefPubMedGoogle Scholar
  17. 17.
    Toborek, M., Y. W. Lee, R. Garrido, S. Kaiser, and B. Hennig. 2002. Unsaturated fatty acids selectively induce an inflammatory environment in human endothelial cells. Am. J. Clin. Nutr. 75: 119–125.PubMedGoogle Scholar
  18. 18.
    Denizot, F., and R. Lang. 1986. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods. 89: 271–277.CrossRefPubMedGoogle Scholar
  19. 19.
    Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [15N]-nitrate in biological fluids. Anal. Biochem. 126: 131–138.CrossRefPubMedGoogle Scholar
  20. 20.
    Reed, D. J., J. R. Babson, P. W. Beatty, A. E. Brodie, W. W. Ellis, and D. W. Potter. 1980. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106: 55–62.CrossRefPubMedGoogle Scholar
  21. 21.
    Lowry, O. H., N. J. Rosebrough, A. I. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275.PubMedGoogle Scholar
  22. 22.
    Belury, M. A., S. Y. Moya-Camarena, K. L. Liu, and J. P. Vanden Heuvel. 1997. Dietary conjugated linoleic acid induced peroxisome-specific enzyme accumulation and ornithine decarboxylase activity in mouse liver. J. Nutr. Biochem. 8: 579–584.CrossRefGoogle Scholar
  23. 23.
    Vanden Heuvel, J. P., G. C. Clark, M. C. Kohn, A. M. Tritscher, W. F. Greenlee, G. W. Lucier, and D. A. Bell. 1994. Dioxin-responsive genes: examination of dose–response relationships using quantitative reverse transcriptase-polymerase chain reaction. Cancer Res. 54: 62–68.PubMedGoogle Scholar
  24. 24.
    Liu, K. L., W. C. Chen, R. Y. Wang, Y. P. Lei, L. Y. Sheen, and C. K. Lii. 2006. DATS reduces LPS-induced iNOS expression, NO production, oxidative stress and NF-κB activation in RAW 264.7 macrophages. J. Agric. Food Chem. 54: 3472–3478.CrossRefPubMedGoogle Scholar
  25. 25.
    de Lima, T. M., L. de Sa Lima, C. Scavone, and R. Curi. 2006. Fatty acid control of nitric oxide production by macrophages. FEBS Lett. 580: 3287–3295.CrossRefPubMedGoogle Scholar
  26. 26.
    Tak, P. P., and G. S. Firestein. 2001. NF-kappa B: a key role in inflammatory diseases. J. Clin. Invest. 107: 7–11.CrossRefPubMedGoogle Scholar
  27. 27.
    Fuhrmann, H., E. A. Miles, A. L. West, and P. C. Calder. 2007. Membrane fatty acids, oxidative burst and phagocytosis after enrichment of P388D1 monocyte/macrophages with essential 18-carbon fatty acids. Ann. Nutr. Metab. 51: 155–162.CrossRefPubMedGoogle Scholar
  28. 28.
    Kaminska, B. 2005. MAPK signaling pathways as molecular targets for anti-inflammatory therapy—from molecular mechanisms to therapeutic benefits. Biochim. Biophys. Acta. 1754: 253–262.PubMedGoogle Scholar
  29. 29.
    Kang, J. S., Y. D. Yoon, K. H. Lee, S. K. Park, and H. M. Kim. 2004. Costunolide inhibits interleukin-1beta expression by down-regulation of AP-1 and MAPK activity in LPS-stimulated RAW 264.7 cells. Biochem. Biophys. Res. Commun. 313: 171–177.CrossRefPubMedGoogle Scholar
  30. 30.
    Kim, H. G., D. H. Yoon, W. H. Lee, S. K. Han, B. Shrestha, C. H. Kim, M. H. Lim, W. Chang, S. Lim, S. Choi, W. O. Song, J. M. Sung, K. C. Hwang, and T. W. Kim. 2007. Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-kappaB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage. J. Ethnopharmacol. 114: 307–315.CrossRefPubMedGoogle Scholar
  31. 31.
    Hennig, B., W. Lei, X. Arzuaga, D. D. Ghosh, V. Saraswathi, and M. Toborek. 2006. Linoleic acid induces proinflammatory events in vascular endothelial cells via activation of PI3K/Akt and ERK1/2 signaling. J. Nutr. Biochem. 17: 766–772.CrossRefPubMedGoogle Scholar
  32. 32.
    Dhillon, A. S., S. Hagan, O. Rath, and W. Kolch. 2007. MAP kinase signaling pathways in cancer. Oncogene. 26: 3279–3290.CrossRefPubMedGoogle Scholar
  33. 33.
    Witztum, J. L. 1993. Role of oxidised low density lipoprotein in atherogenesis. Br. Heart. J. 69: S12–S18.CrossRefPubMedGoogle Scholar
  34. 34.
    Richardson, S. J. 1993. Free radicals in the genesis of Alzheimer’s disease. Ann. N. Y. Acad. Sci. 695: 73–76.CrossRefPubMedGoogle Scholar
  35. 35.
    Rahman, I., J. Marwick, and P. Kirkham. 2004. Redox modulation of chromatin remodeling: impact on histone acetylation and deacetylation, NF-kappaB and pro-inflammatory gene expression. Biochem. Pharmacol. 68: 1255–1267.CrossRefPubMedGoogle Scholar
  36. 36.
    Borek, C. 2004. Dietary antioxidants and human cancer. Integr. Cancer Ther. 3: 333–341.CrossRefPubMedGoogle Scholar
  37. 37.
    Gloire, G., S. Legrand-Poels, and J. Piette. 2006. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem. Pharmacol. 72: 1493–1505.CrossRefPubMedGoogle Scholar
  38. 38.
    Haddad, J. J., and H. L. Harb. 2005. L-gamma-Glutamyl-L-cysteinyl-glycine (glutathione; GSH) and GSH-related enzymes in the regulation of pro- and anti-inflammatory cytokines: a signaling transcriptional scenario for redox(y) immunologic sensor(s)? Mol. Immunol. 42: 987–1014.CrossRefPubMedGoogle Scholar
  39. 39.
    Sun, S., H. Zhang, B. Xue, Y. Wu, J. Wang, Z. Yin, and L. Luo. 2006. Protective effect of glutathione against lipopolysaccharide-induced inflammation and mortality in rats. Inflamm. Res. 55: 504–510.CrossRefPubMedGoogle Scholar
  40. 40.
    Komatsu, W., K. Ishihara, M. Murata, H. Saito, and K. Shinohara. 2003. Docosahexaenoic acid suppresses nitric oxide production and inducible nitric oxide synthase expression in interferon-gamma plus lipopolysaccharide-stimulated murine macrophages by inhibiting the oxidative stress. Free Radic. Biol. Med. 34: 1006–1016.CrossRefPubMedGoogle Scholar
  41. 41.
    Devi, M. A., and N. P. Das. 1994. Antiproliferative effect of polyunsaturated fatty acids and interleukin-2 on normal and abnormal human lymphocytes. Experientia. 50: 489–492.CrossRefPubMedGoogle Scholar
  42. 42.
    Colquhoun, A., and R. I. Schumacher. 2001. gamma-Linolenic acid and eicosapentaenoic acid induce modifications in mitochondrial metabolism, reactive oxygen species generation, lipid peroxidation and apoptosis in Walker 256 rat carcinosarcoma cells. Biochim. Biophys. Acta. 1533: 207–219.PubMedGoogle Scholar
  43. 43.
    McGeer, P. L., and E. G. McGeer. 2004. Inflammation and the degenerative diseases of aging. Ann. N.Y. Acad. Sci. 1035: 104–116.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Cheng-Sue Chang
    • 1
  • Hai-Lun Sun
    • 2
    • 3
  • Chong-Kuei Lii
    • 4
  • Haw-Wen Chen
    • 4
  • Pei-Yin Chen
    • 5
  • Kai-Li Liu
    • 5
    • 6
  1. 1.Department of NeurologyChanghua Christian HospitalChanghua CityTaiwan
  2. 2.Department of AllergyChung Shan Medical University HospitalTaichungTaiwan
  3. 3.School of MedicineChung Shan Medical UniversityTaichungTaiwan
  4. 4.Department of NutritionChina Medical UniversityTaichungTaiwan
  5. 5.Department of NutritionChung Shan Medical UniversityTaichungTaiwan
  6. 6.Department of DietitianChung Shan Medical University HospitalTaichungTaiwan

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