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

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.

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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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google Scholar 

  3. 3.

    Stylianou, E., and J. Saklatvala. 1998. Interleukin-1. Int. J. Biochem. Cell Biol. 30: 1075–1079.

    Article  CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    Article  PubMed  Google 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.

    CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    Article  CAS  Google 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.

    Article  CAS  Google 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.

    CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    Article  CAS  Google 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.

    Article  CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    Article  CAS  Google 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.

    CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Dhillon, A. S., S. Hagan, O. Rath, and W. Kolch. 2007. MAP kinase signaling pathways in cancer. Oncogene. 26: 3279–3290.

    Article  CAS  PubMed  Google Scholar 

  33. 33.

    Witztum, J. L. 1993. Role of oxidised low density lipoprotein in atherogenesis. Br. Heart. J. 69: S12–S18.

    Article  CAS  PubMed  Google Scholar 

  34. 34.

    Richardson, S. J. 1993. Free radicals in the genesis of Alzheimer’s disease. Ann. N. Y. Acad. Sci. 695: 73–76.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google Scholar 

  36. 36.

    Borek, C. 2004. Dietary antioxidants and human cancer. Integr. Cancer Ther. 3: 333–341.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This research was funded by the National Science Council, Republic of China, under Grant NSC 95-2320-B-040-034 and by Chung Shan Medical University, under Grant CSMU 94-OM-B-003.

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Correspondence to Kai-Li Liu.

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Cheng-Shu Chang and HaiLun Sun contributed equally to this work.

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Chang, CS., Sun, HL., Lii, CK. et al. Gamma-Linolenic Acid Inhibits Inflammatory Responses by Regulating NF-κB and AP-1 Activation in Lipopolysaccharide-Induced RAW 264.7 Macrophages. Inflammation 33, 46–57 (2010). https://doi.org/10.1007/s10753-009-9157-8

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KEY WORDS

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