, Volume 37, Issue 5, pp 1588–1598 | Cite as

Geniposide Plays an Anti-inflammatory Role via Regulating TLR4 and Downstream Signaling Pathways in Lipopolysaccharide-Induced Mastitis in Mice

  • Xiaojing Song
  • Wen Zhang
  • Tiancheng Wang
  • Haichao Jiang
  • Zecai Zhang
  • Yunhe Fu
  • Zhengtao Yang
  • Yongguo Cao
  • Naisheng Zhang


Geniposide is a medicine isolated from Gardenia jasminoides Ellis, which is a traditional Chinese herb that is widely used in Asia for the treatment of inflammation, brain diseases, and hepatic disorders. Mastitis is a highly prevalent and important infectious disease. In this study, we used a lipopolysaccharide (LPS)-induced mouse mastitis model and LPS-stimulated primary mouse mammary epithelial cells (mMECs) to explore the anti-inflammatory effect and the mechanism of action of geniposide. Using intraductal injection of LPS as a mouse model of mastitis, we found that geniposide significantly reduced the infiltration of inflammatory cells and downregulated the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). To further investigate the anti-inflammatory mechanism, we used LPS-stimulated mMECs as an in vitro mastitis model. The results of enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) showed that geniposide inhibited the expression of TNF-α, IL-1β, and IL-6 in a dose-dependent manner. Western blot analysis demonstrated that geniposide could suppress the phosphorylation of inhibitory kappa B (IκBα), nuclear factor-κB (NF-κB), p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Geniposide also inhibited the expression of toll-like receptor 4 (TLR4) in the LPS-stimulated mMECs. In conclusion, geniposide exerted its anti-inflammatory effect by regulating TLR4 expression, which affected the downstream NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Thus, geniposide may be a potential drug for mastitis therapy.


geniposide lipopolysaccharide (LPS) anti-inflammatory toll-like receptor nuclear factor-κB (NF-κB) mitogen-activated protein kinase (MAPK) 



This work was supported by a grant from the National Natural Science Foundation of China (Nos. 31272622, 31201925), the Research Fund for the Doctoral Program of Higher Education of China (Nos. 20110061130010, 20120061120098), and Jilin Province Science Foundation for Youths (No. 20130522087JH).

Conflict of Interest

All authors declare that they have no conflict of interest.


  1. 1.
    Bradley, A. 2002. Bovine mastitis: an evolving disease. Veterinary Journal 164(2): 116–128.CrossRefGoogle Scholar
  2. 2.
    Viguier, C., S. Arora, N. Gilmartin, K. Welbeck, and R. O’Kennedy. 2009. Mastitis detection: current trends and future perspectives. Trends in Biotechnology 27(8): 486–493.PubMedCrossRefGoogle Scholar
  3. 3.
    Bannerman, D.D., M.J. Paape, J.W. Lee, X. Zhao, J.C. Hope, and P. Rainard. 2004. Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clinical and Diagnostic Laboratory Immunology 11(3): 463–472.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Opal, S.M., P.J. Scannon, J.L. Vincent, M. White, S.F. Carroll, J.E. Palardy, N.A. Parejo, J.P. Pribble, and J.H. Lemke. 1999. Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock. Journal of Infectious Diseases 180(5): 1584–1589.PubMedCrossRefGoogle Scholar
  5. 5.
    Oliver, S., and L. Calvinho. 1995. Influence of inflammation on mammary gland metabolism and milk composition. Journal of Animal Science 73(suppl 2): 18–33.Google Scholar
  6. 6.
    De Schepper, S., A. De Ketelaere, D.D. Bannerman, M.J. Paape, L. Peelman, and C. Burvenich. 2008. The toll-like receptor-4 (TLR-4) pathway and its possible role in the pathogenesis of Escherichia coli mastitis in dairy cattle. Veterinary Research 39(1): 1–23.CrossRefGoogle Scholar
  7. 7.
    J-h, Liu, F. Yin, L.-x. Guo, X.-h. Deng, and Y.-h. Hu. 2009. Neuroprotection of geniposide against hydrogen peroxide induced PC12 cells injury: involvement of PI3 kinase signal pathway. Acta Pharmacologica Sinica 30(2): 159–165.CrossRefGoogle Scholar
  8. 8.
    Peng, C.H., C.N. Huang, S.P. Hsu, and C.J. Wang. 2007. Penta-acetyl geniposide-induced apoptosis involving transcription of NGF/p75 via MAPK-mediated AP-1 activation in C6 glioma cells. Toxicology 238(2–3): 130–139.PubMedCrossRefGoogle Scholar
  9. 9.
    Wu, S.Y., G.F. Wang, Z.Q. Liu, J.J. Rao, L. Lu, W. Xu, S.G. Wu, and J.J. Zhang. 2009. Effect of geniposide, a hypoglycemic glucoside, on hepatic regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin. Acta Pharmacologica Sinica 30(2): 202–208.PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Liu, H.T., J.L. He, W.M. Li, Z. Yang, Y.X. Wang, J. Yin, Y.G. Du, and C. Yu. 2010. Geniposide inhibits interleukin-6 and interleukin-8 production in lipopolysaccharide-induced human umbilical vein endothelial cells by blocking p38 and ERK1/2 signaling pathways. Inflammation Research : Official Journal of the European Histamine Research Society 59(6): 451–461.CrossRefGoogle Scholar
  11. 11.
    Zhang, G., J.-L. He, X.-Y. Xie, and C. Yu. 2012. LPS-induced iNOS expression in N9 microglial cells is suppressed by geniposide via ERK, p38 and nuclear factor-κB signaling pathways. International Journal of Molecular Medicine 30(3): 561–568.PubMedGoogle Scholar
  12. 12.
    Fu, Y., B. Liu, J. Liu, Z. Liu, D. Liang, F. Li, D. Li, Y. Cao, X. Zhang, N. Zhang, et al. 2012. Geniposide, from Gardenia jasminoides Ellis, inhibits the inflammatory response in the primary mouse macrophages and mouse models. International Immunopharmacology 14(4): 792–798.PubMedCrossRefGoogle Scholar
  13. 13.
    Barham W, Sherrill T, Connelly L, Blackwell T, Yull F. 2012. Intraductal injection of LPS as a mouse model of mastitis: signaling visualized via an NF-κB reporter transgenic. Journal of visualized experiments: JoVE (67).Google Scholar
  14. 14.
    Chandler, R.L. 1970. Experimental bacterial mastitis in the mouse. Journal of Medical Microbiology 3(2): 273–282.PubMedCrossRefGoogle Scholar
  15. 15.
    Anderson, J.C. 1974. Experimental staphylococcal mastitis in the mouse: effects of extracellular products and whole bacterial cells from a high-virulence and a low-virulence strain of Staphylococcus aureus. Journal of Medical Microbiology 7(2): 205–212.PubMedCrossRefGoogle Scholar
  16. 16.
    Smalley, M.J., J. Titley, and M.J. O’Hare. 1998. Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting. In Vitro Cellular & Developmental Biology Animal 34(9): 711–721.CrossRefGoogle Scholar
  17. 17.
    Fu, Y., L. Bo, X. Feng, Z. Liu, D. Liang, F. Li, D. Li, Y. Cao, S. Feng, and X. Zhang. 2012. Lipopolysaccharide increases Toll-like receptor 4 and downstream Toll-like receptor signaling molecules expression in bovine endometrial epithelial cells. Veterinary Immunology and Immunopathology 151: 20–27.PubMedCrossRefGoogle Scholar
  18. 18.
    Li, F., D. Liang, Z. Yang, T. Wang, W. Wang, X. Song, M. Guo, E. Zhou, D. Li, Y. Cao, et al. 2013. Astragalin suppresses inflammatory responses via down-regulation of NF-kappaB signaling pathway in lipopolysaccharide-induced mastitis in a murine model. International Immunopharmacology 17(2): 478–482.PubMedCrossRefGoogle Scholar
  19. 19.
    Liang, D., Y. Sun, Y. Shen, F. Li, X. Song, E. Zhou, F. Zhao, Z. Liu, Y. Fu, M. Guo, et al. 2013. Shikonin exerts anti-inflammatory effects in a murine model of lipopolysaccharide-induced acute lung injury by inhibiting the nuclear factor-kappaB signaling pathway. International Immunopharmacology 16(4): 475–480.PubMedCrossRefGoogle Scholar
  20. 20.
    Notebaert, S., and E. Meyer. 2006. Mouse models to study the pathogenesis and control of bovine mastitis. A review. The Veterinary quarterly 28(1): 2–13.PubMedCrossRefGoogle Scholar
  21. 21.
    Rainard, P., and C. Riollet. 2006. Innate immunity of the bovine mammary gland. Veterinary Research 37(3): 369–400.PubMedCrossRefGoogle Scholar
  22. 22.
    Schmitz, S., M.W. Pfaffl, H.H.D. Meyer, and R.M. Bruckmaier. 2004. Short-term changes of mRNA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis. Domestic Animal Endocrinology 26(2): 111–126.PubMedCrossRefGoogle Scholar
  23. 23.
    Copray, J.C.V.M., I. Mantingh, N. Brouwer, K. Biber, B.M. Kust, R.S.B. Liem, I. Huitinga, F.J.H. Tilders, A.M. Van Dam, and H.W.G.M. Boddeke. 2001. Expression of interleukin-1 beta in rat dorsal root ganglia. Journal of Neuroimmunology 118(2): 203–211.PubMedCrossRefGoogle Scholar
  24. 24.
    Heinrich, P.C., I. Behrmann, S. Haan, H.M. Hermanns, G. Muller-Newen, and F. Schaper. 2003. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochemical Journal 374(Pt 1): 1–20.PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Boulanger, D., E. Brouillette, F. Jaspar, F. Malouin, J. Mainil, F. Bureau, and P. Lekeux. 2007. Helenalin reduces Staphylococcus aureus infection in vitro and in vivo. Veterinary Microbiology 119(2–4): 330–338.PubMedCrossRefGoogle Scholar
  26. 26.
    Notebaert, S., D. Demon, T. Vanden Berghe, P. Vandenabeele, and E. Meyer. 2008. Inflammatory mediators in Escherichia coli-induced mastitis in mice. Comparative Immunology, Microbiology and Infectious Diseases 31(6): 551–565.PubMedCrossRefGoogle Scholar
  27. 27.
    Lu, Y.C., W.C. Yeh, and P.S. Ohashi. 2008. LPS/TLR4 signal transduction pathway. Cytokines 42(2): 145–151.CrossRefGoogle Scholar
  28. 28.
    Chen, Z., J. Hagler, V.J. Palombella, F. Melandri, D. Scherer, D. Ballard, and T. Maniatis. 1995. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes & Development 9(13): 1586–1597.CrossRefGoogle Scholar
  29. 29.
    Guo, M.Y., N.S. Zhang, D.P. Li, D.J. Liang, Z.C. Liu, F.Y. Li, Y.H. Fu, Y.G. Cao, X.M. Deng, and Z.T. Yang. 2013. Baicalin plays an anti-inflammatory role through reducing nuclear factor-kappa B and p38 phosphorylation in S. aureus-induced mastitis. International Immunopharmacology 16(2): 125–130.PubMedCrossRefGoogle Scholar
  30. 30.
    Notebaert, S., L. Duchateau, and E. Meyer. 2005. NF-kappaB inhibition accelerates apoptosis of bovine neutrophils. Veterinary Research 36(2): 229–240.PubMedCrossRefGoogle Scholar
  31. 31.
    Demeyere, K., Q. Remijsen, D. Demon, K. Breyne, S. Notebaert, F. Boyen, C.J. Guérin, P. Vandenabeele, and E. Meyer. 2013. Escherichia coli induces bovine neutrophil cell death independent from caspase-3/-7/-1, but with phosphatidylserine exposure prior to membrane rupture. Veterinary Immunology and Immunopathology 153(1): 45–56.PubMedCrossRefGoogle Scholar
  32. 32.
    Zhong, W.T., G.F. Chi, L.X. Jiang, L.W. Soromou, N. Chen, M.X. Huo, W.X. Guo, X.M. Deng, and H.H. Feng. 2013. p-Cymene modulates in vitro and in vivo cytokine production by inhibiting MAPK and NF-kappa B activation. Inflammation 36(3): 529–537.PubMedCrossRefGoogle Scholar
  33. 33.
    Chang, L., and M. Karin. 2001. Mammalian MAP kinase signalling cascades. Nature 410(6824): 37–40.PubMedCrossRefGoogle Scholar
  34. 34.
    Muzio, M., J. Ni, P. Feng, and V.M. Dixit. 2013. IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling (reprinted from Science, vol 278, pg 1612-1615, 1997). Journal of Immunology 190(1): 16–19.Google Scholar
  35. 35.
    McDermott, E.P., and L.A.J. O’Neill. 2002. Ras participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1. Journal of Biological Chemistry 277(10): 7808–7815.PubMedCrossRefGoogle Scholar
  36. 36.
    Miller, S.I., R.K. Ernst, and M.W. Bader. 2005. LPS, TLR4 and infectious disease diversity. Nature Reviews Microbiology 3(1): 36–46.PubMedCrossRefGoogle Scholar
  37. 37.
    Triantafilou, M., and K. Triantafilou. 2005. The dynamics of LPS recognition: complex orchestration of multiple receptors. Journal of Endotoxin Research 11(1): 5–11.PubMedGoogle Scholar
  38. 38.
    Goldammer, T., H. Zerbe, A. Molenaar, H.-J. Schuberth, R. Brunner, S. Kata, and H.-M. Seyfert. 2004. Mastitis increases mammary mRNA abundance of β-defensin 5, toll-like-receptor 2 (TLR2), and TLR4 but not TLR9 in cattle. Clinical and Diagnostic Laboratory Immunology 11(1): 174–185.PubMedPubMedCentralGoogle Scholar
  39. 39.
    Wang, J., J. Hou, P. Zhang, D. Li, C. Zhang, and J. Liu. 2012. Geniposide reduces inflammatory responses of oxygen-glucose deprived rat microglial cells via inhibition of the TLR4 signaling pathway. Neurochemical Research 37(10): 2235–2248.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Xiaojing Song
    • 1
  • Wen Zhang
    • 1
  • Tiancheng Wang
    • 1
  • Haichao Jiang
    • 1
  • Zecai Zhang
    • 1
  • Yunhe Fu
    • 1
  • Zhengtao Yang
    • 1
  • Yongguo Cao
    • 1
  • Naisheng Zhang
    • 1
    • 2
  1. 1.College of Veterinary MedicineJilin UniversityChangchunPeople’s Republic of China
  2. 2.Department of Clinical Veterinary Medicine, College of Veterinary MedicineJilin UniversityChangchunPeople’s Republic of China

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