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The Anti-inflammatory Effect of Gnaphalium affine Through Inhibition of NF-κB and MAPK in Lipopolysaccharide-Stimulated RAW264.7 Cells and Analysis of Its Phytochemical Components

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Abstract

Gnaphalium affine is an annual herbaceous plant that is used as a traditional medicine in some Latin American and Asian countries. However, systematic studies on its anti-inflammatory activity and signaling pathways have not yet been reported. In this study, we investigated the anti-inflammatory effect of G. affine methanol extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells and fractioned the methanol extract into hexane, chloroform, ethyl acetate (EtOAc), butyl alcohol (BuOH), and distilled water (DW) by measuring the generation of nitric oxide (NO). G. affine inhibited the generation of NO and prostaglandin E2. The chloroform-soluble fraction most effectively inhibited LPS-stimulated NO production. We also examined the cytotoxicity of G. affine in three normal cell lines: RAW264.7, HEK293, and HaCaT. Cell viability assays showed that the methanol extract and chloroform-soluble fraction of G. affine had no cytotoxic effect on normal cell lines. The expression of pro-inflammatory mediators was also investigated. Western blotting and immunofluorescence showed that G. affine reduces the expression of iNOS, COX-2, and MAPKs, as well as activation of NF-κB in LPS-stimulated RAW264.7 cells. RT-PCR showed that G. affine also negatively regulates inflammatory cytokines at the gene expression level. Taken together, G. affine exerts its anti-inflammatory activity through inhibition of NO generation as a result of inhibiting NF-κB and MAPKs-related inflammatory signaling pathways. In addition, the result of GC–MS analysis revealed the presence of nineteen different types of constituents including guaiacol in the chloroform-soluble fraction of G. affine.

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References

  1. Goronzy, J. J., & Weyand, C. M. (2005). Rheumatoid arthritis. Immunological Reviews, 204, 55–73.

    Article  CAS  PubMed  Google Scholar 

  2. Willerson, J. T., & Ridker, P. M. (2004). Inflammation as a cardiovascular risk factor. Circulation, 109(21 suppl 1), II2.

    PubMed  Google Scholar 

  3. Neuman, M. G. (2007). Immune dysfunction in inflammatory bowel disease. Translational Research, 149, 173–186.

    Article  CAS  PubMed  Google Scholar 

  4. Shacter, E., & Weitzman, S. A. (2002). Chronic inflammation and cancer. Oncology (Williston Park), 16, 217–226.

    Google Scholar 

  5. Moncada, S., Palmer, R. M., & Higgs, E. A. (1991). Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews, 43, 109–142.

    CAS  PubMed  Google Scholar 

  6. Aktan, F. (2004). iNOS-mediated nitric oxide production and its regulation. Life Sciences, 75, 639–653.

    Article  CAS  PubMed  Google Scholar 

  7. Cho, B. O., Ryu, H. W., So, Y., Lee, C. W., Jin, C. H., Yook, H. S., et al. (2014). Anti-Inflammatory effect of mangostenone F in lipopolysaccharide-stimulated RAW264.7 macrophages by suppressing NF-κB and MAPK activation. Biomolecules and Therapeutics (Seoul), 22, 288–294.

    Article  CAS  Google Scholar 

  8. Zeng, W. C., Zhu, R. X., Jia, L. R., Gao, H., Zheng, Y., Sun, Q., et al. (2011). Chemical composition, antimicrobial and antioxidant activities of essential oil from Gnaphalium affine. Food and Chemical Toxicology, 49, 1322–1328.

    Article  CAS  PubMed  Google Scholar 

  9. Lu, Y., Li, J., Qiu, W. M., Tang, Y. Q., Zhou, X. F., Liu, Z. H., et al. (2009). Separation and purification for flavones from Gnaphlium affine D. Don by macroporous resin. Journal of Hunan Agricultural University (Natural Sciences), 35, 497–500.

    CAS  Google Scholar 

  10. Li, J., Huang, D., Chen, W., Xi, Z., Chen, C., Sun, L., et al. (2013). Two new phenolic glycosides from Gnaphalium affine D. Don and their anti-complementary activity. Molecules, 18, 7751–7760.

    Article  CAS  PubMed  Google Scholar 

  11. Xi, Z. X., Chen, W. S., Wu, Z. J., Wang, Y., Zeng, P. Y., Zhao, G. J., et al. (2012). Anti-complementary activity of flavonoids from Gnaphalium affine D. Don. Food Chem, 130, 165–170.

    Article  CAS  Google Scholar 

  12. Aritomi, M., Shimojoe, M., & Mazaki, T. (1964). Chemical constituents in flowers of Gnaphalium affine D. Don. Yakugaku Zasshi, 84, 895–896.

    CAS  PubMed  Google Scholar 

  13. Aoshima, Y., Hasegawa, Y., Hasegawa, S., Nagasaka, A., Kimura, T., Hashimoto, S., et al. (2003). Isolation of GnafC, a polysaccharide constituent of Gnaphalium affine, and synergistic effects of GnafC and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells. Bioscience, Biotechnology, and Biochemistry, 67, 2068–2074.

    Article  CAS  PubMed  Google Scholar 

  14. Zeng, W. C., Zhang, W. C., Zhang, W. H., He, Q., & Shi, B. (2013). The antioxidant activity and active component of Gnaphalium affine extract. Food and Chemical Toxicology, 58, 311–317.

    Article  CAS  PubMed  Google Scholar 

  15. Morimoto, M., Tanimoto, K., Nakano, S., Ozaki, T., Nakano, A., & Komai, K. (2003). Insect antifeedant activity of flavones and chromones against Spodoptera litura. Journal of Agriculture and Food Chemistry, 51, 389–393.

    Article  CAS  Google Scholar 

  16. Morimoto, M., Kumeda, S., & Komai, K. (2000). Insect antifeedant flavonoids from Gnaphalium affine D. Don. Journal of Agricultural and Food Chemistry, 48, 1888–1891.

    Article  CAS  PubMed  Google Scholar 

  17. Cirino, G., Distrutti, E., & Wallace, J. L. (2006). Nitric oxide and inflammation. Inflammation & Allergy: Drug Targets, 5, 115–119.

    Article  CAS  PubMed  Google Scholar 

  18. Ghosh, S., May, M. J., & Kopp, E. B. (1998). NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annual Review of Immunology, 16, 225–260.

    Article  CAS  PubMed  Google Scholar 

  19. Xie, Q. W., Kashiwabara, Y., & Nathan, C. (1994). Role of transcription factor NFkappaB/Rel in induction of nitric oxide synthase. Journal of Biological Chemistry, 269, 4705–4708.

    CAS  PubMed  Google Scholar 

  20. 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. Proceedings of the National Academy of Sciences of the United States of America, 90, 9730–9734.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Collart, M. A., Baeuerle, P., & Vassalli, P. (1990). Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B. Molecular and Cellular Biology, 10, 1498–1506.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Libermann, T. A., & Baltimore, D. (1990). Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Molecular and Cellular Biology, 10, 2327–2334.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hiscott, J., Marois, J., Garoufalis, J., D’Addario, M., Roulston, A., Kwan, I., et al. (1993). Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter: evidence for a positive autoregulatory loop. Molecular and Cellular Biology, 13, 6231–6240.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Siebenlist, U., Franzoso, G., & Brown, K. (1994). Structure, regulation and function of NF-kappa B. Annual Review of Cell Biology, 10, 405–455.

    Article  CAS  PubMed  Google Scholar 

  25. Hwang, D., Jang, B. C., Yu, G., & Boudreau, M. (1997). Expression of mitogen-inducible cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kappaB signaling pathways in macrophages. Biochemical Pharmacology, 54, 87–96.

    Article  CAS  PubMed  Google Scholar 

  26. Martin, M., Schifferle, R. E., Cuesta, N., Vogel, S. N., Katz, J., & Michalek, S. M. (2003). Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide. The Journal of Immunology, 171, 717–725.

    Article  CAS  PubMed  Google Scholar 

  27. Androulidaki, A., Iliopoulos, D., Arranz, A., Doxak, C., Schworer, S., Zacharioudakiet, V., et al. (2009). The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs. Immunity, 31, 220–231.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nathan, C., & Xie, Q. W. (1994). Regulation of biosynthesis of nitric oxide. Journal of Biological Chemistry, 269, 13725–13728.

    CAS  PubMed  Google Scholar 

  29. Tracey, K. J., Fong, Y., Hesse, D. G., Manogue, K. R., Lee, A. T., Kuo, G. C., et al. (1987). Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature, 330, 662–664.

    Article  CAS  PubMed  Google Scholar 

  30. Akira, S., Taga, T., & Kishimoto, T. (1993). Interleukin-6 in biology and medicine. Advances in Immunology, 54, 1–78.

    Article  CAS  PubMed  Google Scholar 

  31. Zhang, X., & Morrison, D. C. (1993). Lipopolysaccharide-induced selective priming effects on tumor necrosis factor alpha and nitric oxide production in mouse peritoneal macrophages. Journal of Experimental Medicine, 177, 511–516.

    Article  CAS  PubMed  Google Scholar 

  32. Funk, C. D. (2001). Prostaglandins and leukotrienes: advances in eicosanoid biology. Science, 294, 1871–1875.

    Article  CAS  PubMed  Google Scholar 

  33. D’Acquisto, F., Iuvone, T., Rombolà, L., Sautebin, L., Di Rosa, M., & Carnuccio, R. (1997). Involvement of NF-kappaB in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Letters, 418, 175–178.

    Article  PubMed  Google Scholar 

  34. Xie, Q. W., Whisnant, R., & Nathan, C. (1993). Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. Journal of Experimental Medicine, 177, 1779–1784.

    Article  CAS  PubMed  Google Scholar 

  35. Caamaño, J., & Hunter, C. A. (2002). NF-kappaB family of transcription factors: central regulators of innate and adaptive immune functions. Clinical Microbiology Reviews, 15, 414–429.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Tanaka, H., Fujita, N., & Tsuruo, T. (2005). 3-Phosphoinositide-dependent protein kinase-1-mediated IkappaB kinase beta (IkkB) phosphorylation activates NF-kappaB signaling. Journal of Biological Chemistry, 280, 40965–40973.

    Article  CAS  PubMed  Google Scholar 

  37. Ghosh, S., & Karin, M. (2002). Missing pieces in the NF-kappaB puzzle. Cell, 109, S81–S96.

    Article  CAS  PubMed  Google Scholar 

  38. Hayden, M. S., & Ghosh, S. (2008). Shared principles in NF-kappaB signaling. Cell, 132, 344–362.

    Article  CAS  PubMed  Google Scholar 

  39. Chang, L., & Karin, M. (2001). Mammalian MAP kinase signaling cascades. Nature, 410, 37–40.

    Article  CAS  PubMed  Google Scholar 

  40. Feng, G. J., Goodridge, H. S., Harnett, M. M., Wei, X. Q., Nikolaev, A. V., & Higson, A. P. (1999). Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase. The Journal of Immunology, 163, 6403–6412.

    CAS  PubMed  Google Scholar 

  41. Raymond, B., Crepin, V. F., Collins, J. W., & Frankel, G. (2011). The WxxxE effector EspT triggers expression of immune mediators in an Erk/JNK and NF-kappaB- dependent manner. Cellular Microbiology, 13, 1881–1893.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Xagorari, A., Roussos, C., & Papapetropoulos, A. (2002). Inhibition of LPS-stimulated pathways in macrophages by the flavonoid luteolin. British Journal of Pharmacology, 136, 1058–1064.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yang, H. H., Hwangbo, K., Zheng, M. S., Cho, J. H., Son, J. K., Kim, H. Y., et al. (2015). Inhibitory effects of (-)-loliolide on cellular senescence in human dermal fibroblasts. Archives of Pharmacal Research, 38, 876–884.

    Article  CAS  PubMed  Google Scholar 

  44. Koeduka, T., Fridman, E., Gang, D. R., Vassão, D. G., Jackson, B. L., Kish, C. M., et al. (2006). Eugenol and isoeugenol, characteristic aromatic constituents of spices, are biosynthesized via reduction of a coniferyl alcohol ester. Proceedings of the National Academy of Sciences of the United States of America, 103, 10128–10133.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Murakami, Y., Hirata, A., Ito, S., Shoji, M., Tanaka, S., Yasui, T., et al. (2007). Re-evaluation of cyclooxygenase-2-inhibiting activity of vanillin and guaiacol in macrophages stimulated with lipopolysaccharide. Anticancer Research, 27, 801–807.

    CAS  PubMed  Google Scholar 

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    Authors and Affiliations

    1. Department of Microbiology, College of Natural Sciences, Pukyong National University, 45, Yongso-ro, Nam-Gu, Busan, 608-737, Korea

      Yeong-Ae Seong, Dukhyun Hwang & Gun-Do Kim

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    1. Yeong-Ae Seong
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    2. Dukhyun Hwang
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    3. Gun-Do Kim
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    Correspondence to Gun-Do Kim.

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    Yeong-Ae Seong and Dukhyun Hwang have contributed equally to this study.

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    Seong, YA., Hwang, D. & Kim, GD. The Anti-inflammatory Effect of Gnaphalium affine Through Inhibition of NF-κB and MAPK in Lipopolysaccharide-Stimulated RAW264.7 Cells and Analysis of Its Phytochemical Components. Cell Biochem Biophys 74, 407–417 (2016). https://doi.org/10.1007/s12013-016-0744-7

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