, Volume 41, Issue 4, pp 1200–1214 | Cite as

Juniperonic Acid Incorporation into the Phospholipids of Murine Macrophage Cells Modulates Pro-Inflammatory Mediator Production

  • Po-Jung Tsai
  • Wen-Cheng Huang
  • Shao-Wei Lin
  • Sung-Nien Chen
  • Hung-Jing Shen
  • Hsiang Chang
  • Lu-Te Chuang


Juniperonic acid (JPA; Δ5,11,14,17-20:4), originally identified in certain gymnosperm seeds, is a rare n-3 polyunsaturated fatty acid (PUFA) with lipid-modulating effects on rats and anti-proliferative effects on fibroblast cell proliferation. However, little is known how JPA exerted its immunosuppressive effect. The objective of this study was to investigate whether JPA inhibited the production of inflammatory mediators through the modulation of cellular phospholipid fatty acid compositions. Using standard lipid chemistry techniques in conjunction with argentated column chromatography, high-purity JPA (> 98%) was extracted, isolated, and purified from Biota kernels. When murine RAW264.7 macrophages were incubated with increasing concentrations of JPA, amounts of cellular phospholipid total PUFA, JPA, and Δ7-docosatetraenoic acid (Δ7-DTA; elongation product of JPA) increased in a dose-dependent manner; however, the proportions of total monounsaturated fatty acid (MUFA) and arachidonic acid (AA) decreased. JPA suppressed the production of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) and the expression of inducible nitric oxide synthase (iNOS) up to 21, 75, 30, and 44%, respectively. The induction of cyclooxygenase-2 (COX-2) over-expression by JPA could account for the doubling of the PGE2 level. Furthermore, JPA suppressed the expression of phosphorylated mitogen-activated protein kinases (MAPK). In a separate study using the mouse ear edema model, we demonstrated that JPA also significantly suppressed inflammation, as measured by ear thickness and biopsy weight. The anti-inflammatory properties of JPA could be due, in part, to the incorporation of JPA into cellular phospholipids with subsequent modulation of membrane-mediated MAPK signaling.


Juniperonic acid (JPA) Arachidonic acid (AA) Prostaglandin E2 (PGE2Type-2 cyclooxygenase (COX-2) Macrophage 



This work was supported in part by the Ministry of Science and Technology, Executive Yuan, Taiwan under grants (MOSC103-2313-B-264-001- and MOST104-2320-B-264-002-). The authors are grateful to Professor Robert H. Glew, PhD, for editing our manuscript.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Wolff, R.L., E. Dareville, and J.-C. Martin. 1997. Positional distribution of Δ5-olefinic acids in triacylglycerols from conifer seed oils: general and specific enrichment in the sn-3 position. Journal of the American Oil Chemists' Society 74: 515–523.CrossRefGoogle Scholar
  2. 2.
    Schlenk, H., and J.L. Gellerman. 1965. Arachidonic, 5,11,14,17-eicosatetraenoic and related acids in plants—identification of unsaturated fatty acids. Journal of the American Oil Chemists' Society 42: 504–511.CrossRefGoogle Scholar
  3. 3.
    Ikeda, I., T. Oka, K. Koba, M. Sugano, and M.S.F.K. Jie. 1992. 5c,11c,14c-Eicosatrienoic acid and 5c,11c,14c,17c-eicosatetraenoic acid of Biota orientalis seed oil affect lipid metabolism in the rat. Lipids 27: 500–504.CrossRefPubMedGoogle Scholar
  4. 4.
    Smith, C.R., Jr., R. Kleiman, and I.A. Wolff. 1968. Caltha palustris L. seed oil. A source of four fatty acids with cis-5-unsaturation. Lipids 3: 37–42.CrossRefPubMedGoogle Scholar
  5. 5.
    Kawashima, H. 2005. Unusual minor nonmethylene-interrupted di-, tri-, and tetraenoic fatty acids in limpet gonads. Lipids 40: 627–630.CrossRefPubMedGoogle Scholar
  6. 6.
    Wolff, R.L., W.W. Christie, F. Pédrono, and A.M. Marpeau. 1999. Arachidonic, eicosapentaenoic, and biosynthetically related fatty acids in the seed lipids from a primitive gymnosperm, Agathis robusta. Lipids 34: 1083–1097.CrossRefPubMedGoogle Scholar
  7. 7.
    Huang, Y.S., S.L. Pereira, and A.E. Leonard. 2004. Enzymes for transgenic biosynthesis of long-chain polyunsaturated fatty acids. Biochimie 86: 793–798.CrossRefPubMedGoogle Scholar
  8. 8.
    Sugano, M., I. Ikeda, K. Wakamatsu, and Y. Oka. 1994. Influence of Korean pine (Pinus koraiensis)-seed oil containing cis-5,cis-9,cis-12-octadecatrienoic acid on polyunsaturated fatty acid metabolism, eicosanoid production and blood pressure. British Journal of Nutrition 72: 775–783.CrossRefPubMedGoogle Scholar
  9. 9.
    Morishige, J., N. Amano, K. Hirano, H. Nishio, T. Tanaka, and K. Satouchi. 2008. Inhibitory effect of juniperonic acid (delta-5c,11c,14c,17c-20:4, omega-3) on bombesin-induced proliferation of Swiss 3T3 cells. Biological and Pharmaceutical Bulletin 31: 1786–1789.CrossRefPubMedGoogle Scholar
  10. 10.
    Calder, P.C. 2006. N-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. American Journal of Clinical Nutrition 83: 1505S–1519S.CrossRefPubMedGoogle Scholar
  11. 11.
    Halade, G.V., M.M. Rahman, A. Bhattacharya, J.L. Barnes, B. Chandrasekar, and G. Fernandes. 2010. Docosahexaenoic acid-enriched fish oil attenuates kidney disease and prolongs median and maximal life span of autoimmune lupus-prone mice. Journal of Immunology 184: 5280–5286.CrossRefGoogle Scholar
  12. 12.
    Huang, W.-C., P.-J. Tsai, Y.-L. Huang, S.-N. Chen, and L.-T. Chuang. 2014. PGE2 production suppressed by chemically-synthesized Δ7-eicosatrienoic acid in macrophages through the competitive inhibition of COX-2. Food Chemistry and Toxicology 66: 122–133.CrossRefGoogle Scholar
  13. 13.
    Chen, S.-J., L.-T. Chuang, and S.-N. Chen. 2015. Incorporation of eicosatrienoic acid exerts mild anti-inflammatory properties in murine RAW264.7 cells. Inflammation 38: 534–545.CrossRefPubMedGoogle Scholar
  14. 14.
    Tanaka, T., T. Hattori, M. Kouchi, K. Hirano, and K. Satouchi. 1998. Methylene-interrupted double bond in polyunsaturated fatty acid is an essential structure for metabolism by the fatty acid chain elongation system of rat liver. Biochimica et Biophysica Acta 1393: 299–306.CrossRefPubMedGoogle Scholar
  15. 15.
    Tanaka, T., T. Takimoto, J.-I. Morishige, Y. Kikuta, T. Sugiura, and K. Satouchi. 1999. Non-methylene-interrupted polyunsaturated fatty acids: effective substitute for arachidonate of phosphatidylinositol. Biochemical and Biophysical Research Communications 264: 683–688.CrossRefPubMedGoogle Scholar
  16. 16.
    Berger, A., I. Monnard, M. Baur, C. Charbonnet, I. Safonova, and A. Jomard. 2002. Epidermal anti-inflammatory properties of 5,11,14 20:3: effects on mouse ear edema, PGE2 levels in cultured keratinocytes, and PPAR activation. Lipids in Health and Diseases 1: 5.CrossRefGoogle Scholar
  17. 17.
    Chen, S.-J., W.-C. Huang, T.-T. Yang, J.-H. Lu, and L.-T. Chuang. 2012. Incorporation of sciadonic acid into cellular phospholipids reduces pro-inflammatory mediators in murine macrophages through NK-κB and MAPK signaling pathways. Food Chemistry and Toxicology 50: 3687–3695.CrossRefGoogle Scholar
  18. 18.
    Chuang, L.-T., P.-J. Tsai, C.-L. Lee, and Y.-S. Huang. 2009. Uptake and incorporation of pinolenic acid reduces n-6 polyunsaturated fatty acid and downstream prostaglandin formation in murine macrophage. Lipids 44: 217–224.CrossRefPubMedGoogle Scholar
  19. 19.
    Haagsma, N., C.M. van Gent, J.B. Luten, R.W. de Jong, and E. van Doorn. 1982. Preparation of an ω3 fatty acid concentrate from cod liver oil. Journal of the American Oil Chemists' Society 59: 117–118.CrossRefGoogle Scholar
  20. 20.
    Christie, W.W. 1993. Preparation of ester derivatives of fatty acids for chromatographic analysis. In Advances in lipid methodology – Two, ed. W.W. Christie, 69–111. Dundee: Oily Press.Google Scholar
  21. 21.
    Folch, J., M. Lees, and G.H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226: 497–509.PubMedGoogle Scholar
  22. 22.
    Svetashev, V.I. 2011. Mild method for preparation of 4,4-dimethyloxazoline derivatives of polyunsaturated fatty acids for GC-MS. Lipids 46: 463–467.CrossRefPubMedGoogle Scholar
  23. 23.
    Green, L.C., D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, and S.R. Tannenbaum. 1982. Analysis of nitrate, nitritie and [15N]nitrate in biological fluids. Analytical Biochemistry 126: 131–138.CrossRefPubMedGoogle Scholar
  24. 24.
    Tsai, T.-H., L.-T. Chuang, T.-J. Lien, Y.-R. Liing, W.-Y. Chen, and P.-J. Tsai. 2013. Rosmarinus officinalis extract suppresses Propionibacterium acnes-induced inflammatory responses. Journal of Medicinal Food 16: 324–333.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Tanaka, T., J. Morishige, D. Iwawaki, T. Fukuhara, N. Hamamura, K. Hirano, T. Osumi, and K. Satouchi. 2007. Metabolic pathway that produces essential fatty acids from polymethylene-interrupted polyunsaturated fatty acids in animal cells. FEBS Journal 274: 2728–2737.CrossRefPubMedGoogle Scholar
  26. 26.
    Nieves, D., and J.J. Moreno. 2006. Effect of arachidonic and eicosapentaenoic acid metabolism on RAW 264.7 macrophage proliferation. Journal of Cell Physiology 208: 428–434.CrossRefGoogle Scholar
  27. 27.
    Jaudszus, A., M. Gruen, B. Watzl, C. Ness, A. Roth, A. Lochner, D. Barz, H. Gabriel, M. Rothe, and G. Jahreis. 2013. Evaluation of suppressive and pro-resolving effects of EPA and DHA in human primary monocytes and T-helper cells. Journal of Lipid Research 54: 923–935.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Grønn, M., E. Christensen, T.A. Hagve, and B.O. Christophersen. 1991. Peroxisomal retroconversion of docosahexaenoic acid (22:6(n-3)) to eicosapentaenoic acid (20:5(n-3)) studied in isolated rat liver cells. Biochimica et Biophysica Acta 1081: 85–91.CrossRefPubMedGoogle Scholar
  29. 29.
    Sprecher, H., and C.-J. Lee. 1975. The absence of an 8-desaturases in rat liver: a reevaluation of optional pathways for the metabolism of linoleic and linolenic acids. Biochimica et Biophysica Acta 388: 113–125.CrossRefPubMedGoogle Scholar
  30. 30.
    Kelder, B., P. Mukeji, S. Kirchner, G. Hovanec, A.E. Leonard, L.-T. Chuang, J.J. Kopchick, and Y.S. Huang. 2001. Expression of fungal desaturase genes in cultured mammalian cells. Molecular and Cellular Biochemistry 219: 7–11.CrossRefPubMedGoogle Scholar
  31. 31.
    Awad, A.B., A.L. Young, and C.S. Fink. 1996. The effect of unsaturated fatty acids on membrane composition and signal transduction in HT-29 human colon cancer cells. Cancer Letters 108: 25–33.CrossRefPubMedGoogle Scholar
  32. 32.
    Burns, C.P., D.G. Luttenegger, D.T. Dudley, G.R. Buettner, and A.A. Spector. 1979. Effect of modification of plasma membrane fatty acid composition on fluidity and methotrexate transport in L1210 murine leukemia cells. Cancer Research 39: 1726–1732.PubMedGoogle Scholar
  33. 33.
    Kim, W., N.A. Khan, D.N. McMurray, I.A. Prior, N. Wang, and R.S. Chapkin. 2010. Regulatory activity of polyunsaturated fatty acids in T-cell signaling. Progress in Lipid Research 49: 250–261.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Marinelli, C., R. Di Liddo, L. Facci, T. Bertalot, M.T. Conconi, M. Zusso, S.D. Skaper, and P. Giusti. 2015. Ligand engagement of toll-like receptors regulates their expression in cortical microglia and astrocytes. Journal of Neuroinflammation 12: 244.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wang, J., A.V. Grishin, and H.R. Ford. 2016. Experimental anti-inflammatory drug Semapimod inhibits toll-like receptor signaling by targeting the TLR chaperone gp96. Journal of Immunology 196: 5130–5137.CrossRefGoogle Scholar
  36. 36.
    Lo, C., K.C. Chiu, M. Fu, R. Lo, and S. Helton. 1999. Fish oil augments macrophage cyclooxygenase II (COX-2) gene expression induced by endotoxin. Journal of Surgical Research 86: 103–107.CrossRefPubMedGoogle Scholar
  37. 37.
    Maldve, R.E., Y. Kim, S.J. Muga, and S.M. Fischer. 2000. Prostaglandin E2 regulation of cyclooxygenase expression in keratinocytes is mediated via cyclic nucleotide-linked prostaglandin receptors. Journal of Lipid Research 41: 873–881.PubMedGoogle Scholar
  38. 38.
    Yang, P., D. Chan, E. Felix, C. Cartwright, D.G. Menter, T. Madden, R.D. Klein, S.M. Fischer, and R.A. Newman. 2004. Formation and antiproliferative effect of prostaglandin E(3) from eicosapentaenoic acid in human lung cancer cells. Journal of Lipid Research 45: 1030–1039.CrossRefPubMedGoogle Scholar
  39. 39.
    Harris, W.S., B.-E. Hustvedt, E. Hagen, M.H. Green, G. Lu, and C.A. Drevon. 1997. N-3 fatty acids and chylomicron metabolism in the rat. Journal of Lipid Research 38: 503–515.PubMedGoogle Scholar
  40. 40.
    Calder, P.C. 2006. Polyunsaturated fatty acids and inflammation. Prostaglandins, Leukotrienes and Essential Fatty Acids 75: 197–202.CrossRefGoogle Scholar
  41. 41.
    Jump, D.B., and S.D. Clarke. 1999. Regulation of gene expression by dietary fat. Annual Review of Nutrition 19: 63–90.CrossRefPubMedGoogle Scholar
  42. 42.
    Duplus, E., M. Glorian, and C. Forest. 2000. Fatty acid regulation of gene transcription. Journal of Biological Chemistry 275: 30749–30752.CrossRefPubMedGoogle Scholar
  43. 43.
    Shahbakhti, H., R.E. Watson, R.M. Azurdia, C.Z. Ferreira, M. Garmyn, and L.E. Rhodes. 2004. Influence of eicosapentaenoic acid, an omega-3 fatty acid, on ultraviolet-B generation of prostaglandin-E2 and proinflammatory cytokines interleukin-1 beta, tumor necrosis factor-alpha, interleukin-6 and interleukin-8 in human skin in vivo. Photochemistry and Photobiology 80: 231–235.CrossRefPubMedGoogle Scholar
  44. 44.
    Wang, C., W.S. Harris, M. Chung, A.H. Lichtenstein, E.M. Balk, B. Kupelnick, H.S. Jordan, and J. Lau. 2006. N-3 fatty acids from fish or fish-oil supplements, but not α-linolenic acid, benefit cardiovascular disease outcomes in primary- and secondary-prevention studies: a systematic review. American Journal of Clinical Nutrition 84: 5–17.CrossRefPubMedGoogle Scholar
  45. 45.
    Halade, G.V., and V. Kain. 2017. Obesity and cardiometabolic defects in heart failure pathology. Comprehensive Physiology 7: 1463–1477.CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Human Development and Family StudiesNational Taiwan Normal UniversityTaipeiTaiwan
  2. 2.Program of Nutritional Science, School of Life ScienceNational Taiwan Normal UniversityTaipeiTaiwan
  3. 3.Department of Biotechnology and Pharmaceutical TechnologyYuanpei University of Medical TechnologyHsinchuTaiwan

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