Molecular and Cellular Biochemistry

, Volume 407, Issue 1–2, pp 89–95 | Cite as

Anti-inflammatory effect of procyanidin B1 on LPS-treated THP1 cells via interaction with the TLR4–MD-2 heterodimer and p38 MAPK and NF-κB signaling

  • Jing Xing
  • Rui Li
  • Nan Li
  • Jian Zhang
  • Yueqing Li
  • Ping Gong
  • Dongna Gao
  • Hui Liu
  • Yu Zhang


Anti-inflammatory effects of procyanidin B1 have been documented; however, the molecular mechanisms that are involved have not been fully elucidated. Molecular docking models were applied to evaluate the binding capacity of lipopolysaccharide (LPS) and procyanidin B1 with the toll-like receptor (TLR)4/myeloid differentiation factor (MD)-2 complex. LPS-induced production of the proinflammatory cytokine tumor necrosis factor (TNF)-α in a human monocyte cell line (THP1) was measured by ELISA. mRNA expression of MD-2, TLR4, TNF receptor-associated factor (TRAF)-6, and nuclear factor (NF)-κB was measured by real-time PCR with or without an 18-h co-treatment with procyanidin B1. In addition, protein expression of phosphorylated p38 mitogen-activated protein kinase (MAPK) and NF-κB was determined by Western blotting. Structural modeling studies identified Tyr296 in TLR4 and Ser120 in MD-2 as critical sites for hydrogen bonding with procyanidin B1, similar to the sites occupied by LPS. The production of TNF-α was significantly decreased by procyanidin B1 in LPS-treated THP1 cells (p < 0.05). Procyanidin B1 also significantly suppressed levels of phosphorylated p38 MAPK and NF-κB protein, as well as mRNA levels of MD-2, TRAF-6, and NF-κB (all p < 0.05). Procyanidin B1 can compete with LPS for binding to the TLR4–MD-2 heterodimer and suppress downstream activation of p38 MAPK and NF-κB signaling pathways.


Anti-inflammatory LPS Procyanidin B1 THP1 cells TLR4–MD-2 heterodimer 



This study was supported by the Young Starting Foundation of the First Affiliated Hospital, Dalian Medical University (QN2012008) and the 2013 Dalian Science and Technology Planning Project (guidance project).

Conflict of interest

The authors do not have any conflicts of interest to declare.


  1. 1.
    Byun EB, Sung NY, Byun EH, Song DS, Kim JK, Park JH, Song BS, Park SH, Lee JW, Byun MW et al (2013) The procyanidin trimer C1 inhibits LPS-induced MAPK and NF-kB signaling through TLR4 in macrophages. Int Immunopharmacol 15:450–456. doi: 10.1016/j.intimp.2012.11.021 CrossRefPubMedGoogle Scholar
  2. 2.
    Hwang SJ, Yoon WB, Lee OH, Cha SJ, Kim JD (2014) Radical-scavenging-linked antioxidant activities of extracts from black chokeberry and blueberry cultivated in Korea. Food Chem 146:71–77. doi: 10.1016/j.foodchem.2013.09.035 CrossRefPubMedGoogle Scholar
  3. 3.
    Serra AT, Rocha J, Sepodes B, Matias AA, Feliciano RP, de Carvalho A, Bronze MR, Duarte CM, Figueira ME (2012) Evaluation of cardiovascular protective effect of different apple varieties—correlation of response with composition. Food Chem 135:2378–2386. doi: 10.1016/j.foodchem.2012.07.067 CrossRefPubMedGoogle Scholar
  4. 4.
    Zhao J, Wang J, Chen Y, Agarwal R (1999) Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of procyanidin B5-3′-gallate as the most effective antioxidant constituent. Carcinogenesis 20:1737–1745CrossRefPubMedGoogle Scholar
  5. 5.
    Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynski CA, Joshi SS, Pruess HG (2000) Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology 148:187–197CrossRefPubMedGoogle Scholar
  6. 6.
    Moini H, Rimbach G, Packer L (2000) Molecular aspects of procyanidin biological activity: disease preventative and therapeutic potentials. Drug Metab Drug Interact 17:237–259CrossRefGoogle Scholar
  7. 7.
    Terra X, Valls J, Vitrac X, Merrillon JM, Arola L, Ardevol A, Blade C, Fernandez-Larrea J, Pujadas G, Salvado J et al (2007) Grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated RAW 264.7 macrophages by inhibiting NFkB signaling pathway. J Agric Food Chem 55:4357–4365. doi: 10.1021/jf0633185 CrossRefPubMedGoogle Scholar
  8. 8.
    Prasain JK, Peng N, Dai Y, Moore R, Arabshahi A, Wilson L, Barnes S, Michael Wyss J, Kim H, Watts RL (2009) Liquid chromatography tandem mass spectrometry identification of proanthocyanidins in rat plasma after oral administration of grape seed extract. Phytomedicine 16:233–243. doi: 10.1016/j.phymed.2008.08.006 CrossRefPubMedGoogle Scholar
  9. 9.
    Terra X, Palozza P, Fernandez-Larrea J, Ardevol A, Blade C, Pujadas G, Salvado J, Arola L, Blay MT (2011) Procyanidin dimer B1 and trimer C1 impair inflammatory response signalling in human monocytes. Free Radic Res 45:611–619. doi: 10.3109/10715762.2011 CrossRefPubMedGoogle Scholar
  10. 10.
    Jung M, Triebel S, Anke T, Richling E, Erkel G (2009) Influence of apple polyphenols on inflammatory gene expression. Mol Nutr Food Res 53:1263–1280. doi: 10.1002/mnfr.200800575 CrossRefPubMedGoogle Scholar
  11. 11.
    Brikos C, O’Neill LA (2008) Signalling of toll-like receptors. Handb Exp Pharmacol 183:21–50CrossRefPubMedGoogle Scholar
  12. 12.
    Coll RC, O’Neill LA (2010) New insights into the regulation of signalling by toll-like receptors and nod-like receptors. J Innate Immun 2:406–421. doi: 10.1159/000315469 CrossRefPubMedGoogle Scholar
  13. 13.
    Takeda K, Akira S (2005) Toll-like receptors in innate immunity. Int Immunol 17:1–14. doi: 10.1093/intimm/dxh186 CrossRefPubMedGoogle Scholar
  14. 14.
    Medzhitov R, Preston-Hurlburt P, Janeway CA Jr (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394–397CrossRefPubMedGoogle Scholar
  15. 15.
    Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, Kimoto M (1999) MD-2, a molecule that confers lipopolysaccharide responsiveness on toll-like receptor 4. J Exp Med 189:1777–1782PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Erridge C, Bennett-Guerrero E, Poxton IR (2002) Structure and function of lipopolysaccharides. Microbes Infect 4:837–851CrossRefPubMedGoogle Scholar
  17. 17.
    Raetz CR, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635–700. doi: 10.1146/annurev.biochem.71.110601.135414 PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K (2002) Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 196:1645–1651PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Kolek MJ, Carlquist JF, Muhlestein JB, Whiting BM, Horne BD, Bair TL, Anderson JL (2004) Toll-like receptor 4 gene Asp299Gly polymorphism is associated with reductions in vascular inflammation, angiographic coronary artery disease, and clinical diabetes. Am Heart J 148:1034–1040CrossRefPubMedGoogle Scholar
  20. 20.
    Guo J, Friedman SL (2010) Toll-like receptor 4 signaling in liver injury and hepatic fibrogenesis. Fibrogenesis Tissue Repair 3:21. doi: 10.1186/1755-1536-3-21 PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Iwami KI, Matsuguchi T, Masuda A, Kikuchi T, Musikacharoen T, Yoshikai Y (2000) Cutting edge: naturally occurring soluble form of mouse toll-like receptor 4 inhibits lipopolysaccharide signaling. J Immunol 165:6682–6686CrossRefPubMedGoogle Scholar
  22. 22.
    Janssens S, Burns K, Tschopp J, Beyaert R (2002) Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88. Curr Biol 12:467–471CrossRefPubMedGoogle Scholar
  23. 23.
    Ohta S, Bahrun U, Tanaka M, Kimoto M (2004) Identification of a novel isoform of MD-2 that downregulates lipopolysaccharide signaling. Biochem Biophys Res Commun 323:1103–1108CrossRefPubMedGoogle Scholar
  24. 24.
    Palsson-McDermott EM, Doyle SL, McGettrick AF, Hardy M, Husebye H, Banahan K, Gong M, Golenbock D, Espevik T, O’Neill LA (2009) TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88-independent TLR4 pathway. Nat Immunol 10:579–586. doi: 10.1038/ni.1727 CrossRefPubMedGoogle Scholar
  25. 25.
    Hosoi S, Shimizu E, Arimori K, Okumura M, Hidaka M, Yamada M, Sakushima A (2008) Analysis of CYP3A inhibitory components of star fruit (Averrhoa carambola L.) using liquid chromatography-mass spectrometry. J Nat Med 62:345–348. doi: 10.1007/s11418-008-0239-y CrossRefPubMedGoogle Scholar
  26. 26.
    Lima Mdos S, Silani Ide S, Toaldo IM, Correa LC, Biasoto AC, Pereira GE, Bordignon-Luiz MT, Ninow JL (2014) Phenolic compounds, organic acids and antioxidant activity of grape juices produced from new Brazilian varieties planted in the Northeast Region of Brazil. Food Chem 161:94–103. doi: 10.1016/j.foodchem.2014.03.109 CrossRefPubMedGoogle Scholar
  27. 27.
    Shimada T, Tokuhara D, Tsubata M, Kamiya T, Kamiya-Sameshima M, Nagamine R, Takagaki K, Sai Y, Miyamoto K, Aburada M (2012) Flavangenol (pine bark extract) and its major component procyanidin B1 enhance fatty acid oxidation in fat-loaded models. Eur J Pharmacol 677:147–153. doi: 10.1016/j.ejphar.2011.12.034 CrossRefPubMedGoogle Scholar
  28. 28.
    Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, Lee H, Lee JO (2007) Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130:1071–1082. doi: 10.1016/j.cell.2007.09.008 CrossRefPubMedGoogle Scholar
  29. 29.
    Ohto U, Fukase K, Miyake K, Satow Y (2007) Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa. Science 316:1632–1634CrossRefPubMedGoogle Scholar
  30. 30.
    Park BS, Song DH, Kim HM, Choi BS, Lee H, Lee JO (2009) The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 458:1191–1195. doi: 10.1038/nature07830 CrossRefPubMedGoogle Scholar
  31. 31.
    Sung NY, Yang MS, Song DS, Kim JK, Park JH, Song BS, Park SH, Lee JW, Park HJ, Kim JH et al (2013) Procyanidin dimer B2-mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages. Biochem Biophys Res Commun 438:122–128. doi: 10.1016/j.bbrc.2013.07.038 CrossRefPubMedGoogle Scholar
  32. 32.
    Chacon MR, Ceperuelo-Mallafre V, Maymo-Masip E, Mateo-Sanz JM, Arola L, Guitierrez C, Fernandez-Real JM, Ardevol A, Simon I, Vendrell J (2009) Grape-seed procyanidins modulate inflammation on human differentiated adipocytes in vitro. Cytokine 47:137–142. doi: 10.1016/j.cyto.2009.06.001 CrossRefPubMedGoogle Scholar
  33. 33.
    Gray P, Michelsen KS, Sirois CM, Lowe E, Shimada K, Crother TR, Chen S, Brikos C, Bulut Y, Latz E et al (2010) Identification of a novel human MD-2 splice variant that negatively regulates lipopolysaccharide-induced TLR4 signaling. J Immunol 184:6359–6366. doi: 10.4049/jimmunol.0903543 PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Sung NY, Yang MS, Song DS, Byun EB, Kim JK, Park JH, Song BS, Lee JW, Park SH, Park HJ et al (2013) The procyanidin trimer C1 induces macrophage activation via NF-kB and MAPK pathways, leading to Th1 polarization in murine splenocytes. Eur J Pharmacol 714:218–228. doi: 10.1016/j.ejphar.2013.02.059 CrossRefPubMedGoogle Scholar
  35. 35.
    Diya Z, Lili C, Shenglai L, Zhiyuan G, Jie Y (2008) Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS. Innate Immun 14:99–107. doi: 10.1177/1753425907088244 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Jing Xing
    • 1
  • Rui Li
    • 1
  • Nan Li
    • 1
  • Jian Zhang
    • 1
  • Yueqing Li
    • 2
  • Ping Gong
    • 1
  • Dongna Gao
    • 1
  • Hui Liu
    • 1
  • Yu Zhang
    • 1
  1. 1.Emergency DepartmentFirst Affiliated Hospital of Dalian Medical UniversityDalianChina
  2. 2.College of Pharmaceutical Science and TechnologyDalian University of TechnologyDalianChina

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