Analytical and Bioanalytical Chemistry

, Volume 408, Issue 1, pp 165–176 | Cite as

Atomic force microscopy based investigations of anti-inflammatory effects in lipopolysaccharide-stimulated macrophages

  • Jiang Pi
  • Huaihong Cai
  • Fen Yang
  • Hua Jin
  • Jianxin Liu
  • Peihui Yang
  • Jiye CaiEmail author
Research Paper


A new method based on atomic force microscopy (AFM) was developed to investigate the anti-inflammatory effects of drugs on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The LPS-stimulated RAW264.7 macrophage cell line is a widely used in vitro cell model for the screening of anti-inflammatory drugs or the study of anti-inflammatory mechanisms. In this work, the inhibitory effects of dexamethasone and quercetin on LPS–CD14 receptor binding in RAW264.7 macrophages was probed by LPS-functionalized tips for the first time. Both dexamethasone and quercetin were found to inhibit LPS-induced NO production, iNOS expression, IκBα phosphorylation, and IKKα/β phosphorylation in RAW264.7 macrophages. The morphology and ultrastructure of RAW264.7 macrophages were determined by AFM, which indicated that dexamethasone and quercetin could inhibit LPS-induced cell surface particle size and roughness increase in RAW264.7 macrophages. The binding of LPS and its receptor in RAW264.7 macrophages was determined by LPS-functionalized AFM tips, which demonstrated that the binding force and binding probability between LPS and CD14 receptor on the surface of RAW264.7 macrophages were also inhibited by dexamethasone or quercetin treatment. The obtained results imply that AFM, which is very useful for the investigation of potential targets for anti-inflammatory drugs on native macrophages and the enhancement of our understanding of the anti-inflammatory effects of drugs, is expected to be developed into a promising tool for the study of anti-inflammatory drugs.


Atomic force microscopy Anti-inflammatory Lipopolysaccharide Macrophages CD14 receptor 



This work was financially supported by Macao Science and Technology Development

Fund (No. 028/2014/A1), the Overseas, Hong Kong & Macao Cooperative Research Funds of China (No. 31129002), and Jinan University’s Scientific Research Cultivation and Innovation Fund (No. 21612601).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2015_9091_MOESM1_ESM.pdf (279 kb)
ESM 1 (PDF 279 kb)


  1. 1.
    Khan SA, Everest P, Servos S, Foxwell N, Zahringer U, Brade H, Rietschel ET, Dougan G, Charles IG, Maskell DJ (1998) A lethal role for lipid A in Salmonella infections. Mol Microbiol 29(2):571–579CrossRefGoogle Scholar
  2. 2.
    Raschke WC, Baird S, Ralph P, Nakoinz I (1978) Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 15(1):261–267CrossRefGoogle Scholar
  3. 3.
    Lu CL, Zhu YF, Hu MM, Wang DM, Xu XJ, Lu CJ, Zhu W (2015) Optimization of astilbin extraction from the rhizome of Smilax glabra, and evaluation of its anti-inflammatory effect and probable underlying mechanism in lipopolysaccharide-induced RAW264.7 macrophages. Molecules 20(1):625–644CrossRefGoogle Scholar
  4. 4.
    Chen WC, Yen CS, Huang WJ, Hsu YF, Ou G, Hsu MJ (2015) WMJ-S-001, a novel aliphatic hydroxamate derivative, exhibits anti-inflammatory properties via MKP-1 in LPS-stimulated RAW264.7 macrophages. Br J Pharmacol 172(7):1894–1908CrossRefGoogle Scholar
  5. 5.
    Yang JH, Kim KM, Kim MG, Seo KH, Han JY, Ka SO, Park BH, Shin SM, Ku SK, Cho IJ, Hwan Ki S (2015) Role of sestrin2 in the regulation of proinflammatory signaling in macrophages. Free Radic Biol Med 78:156–167CrossRefGoogle Scholar
  6. 6.
    Akira S, Takeda K, Kaisho T (2001) Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2(8):675–680CrossRefGoogle Scholar
  7. 7.
    Triantafilou M, Triantafilou K (2002) Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends Immunol 23(6):301–304CrossRefGoogle Scholar
  8. 8.
    Zhang GL, Ghosh S (2001) Toll-like receptor-mediated NF-kappa B activation: a phylogenetically conserved paradigm in innate immunity. J Clin Investig 107(1):13–19CrossRefGoogle Scholar
  9. 9.
    Park YC, Rimbach G, Saliou C, Valacchi G, Packer L (2000) Activity of monomeric, dimeric, and trimeric flavonoids on NO production, TNF-alpha secretion, and NF-kappa B-dependent gene expression in RAW 264.7 macrophages. Febs Lett 465(2–3):93–97CrossRefGoogle Scholar
  10. 10.
    Kim HG, Shrestha B, Lim SY, Yoon DH, Chang WC, Shin DJ, Han SK, Park SM, Park JH, Park HI, Sung JM, Jang Y, Chung N, Hwang KC, Kim TW (2006) Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappa B through Akt and P38 inhibition in RAW 264.7 macrophage cells. Eur J Pharmacol 545(2–3):192–199CrossRefGoogle Scholar
  11. 11.
    Ji GY, Zhang YP, Yang QH, Cheng SB, Hao J, Zhao XH, Jiang ZQ (2012) Genistein suppresses LPS-induced inflammatory response through inhibiting NF-kappa B following AMP kinase activation in RAW 264.7 macrophages. PLoS One 7(12), e53101CrossRefGoogle Scholar
  12. 12.
    Dupres V, Menozzi FD, Locht C, Clare BH, Abbott NL, Cuenot S, Bompard C, Raze D, Dufrene YF (2005) Nanoscale mapping and functional analysis of individual adhesins on living bacteria. Nat Methods 2(7):515–520CrossRefGoogle Scholar
  13. 13.
    Cross SE, Jin YS, Rao J, Gimzewski JK (2007) Nanomechanical analysis of cells from cancer patients. Nat Nanotech 2(12):780–783CrossRefGoogle Scholar
  14. 14.
    Pi J, Jin H, Yang F, Chen ZW, Cai JY (2014) In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine. Nanoscale 6(21):12229–12249CrossRefGoogle Scholar
  15. 15.
    Yang XH, Yang WJ, Wang Q, Li HM, Wang KM, Yang L, Liu W (2010) Atomic force microscopy investigation of the characteristic effects of silver ions on Escherichia coli and Staphylococcus epidermidis. Talanta 81(4–5):1508–1512CrossRefGoogle Scholar
  16. 16.
    Li M, Liu LQ, Xi N, Wang YC (2015) Nanoscale monitoring of drug actions on cell membrane using atomic force microscopy. Acta Pharmacol Sin 36(7):769–782. doi: 10.1038/aps.2015.28 CrossRefGoogle Scholar
  17. 17.
    Wang M, Ruan Y, Chen Q, Li S, Wang Q, Cai J (2011) Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca(2+) concentration. Eur J Pharmacol 650(1):41–47CrossRefGoogle Scholar
  18. 18.
    Jin H, Zhong X, Wang ZY, Huang X, Ye HY, Ma SY, Chen Y, Cai JY (2011) Sonodynamic effects of hematoporphyrin monomethyl ether on CNE-2 cells detected by atomic force microscopy. J Cell Biochem 112(1):169–178CrossRefGoogle Scholar
  19. 19.
    Kim KS, Cho CH, Park EK, Jung MH, Yoon KS, Park HK (2012) AFM-detected apoptotic changes in morphology and biophysical property caused by paclitaxel in Ishikawa and HeLa cells. PLoS One 7(1), e30066CrossRefGoogle Scholar
  20. 20.
    Zhang X, Shi X, Xu L, Yuan J, Fang X (2013) Atomic force microscopy study of the effect of HER 2 antibody on EGF mediated ErbB ligand-receptor interaction. Nanomedicine 9(5):627–635CrossRefGoogle Scholar
  21. 21.
    Zhang L, Yang F, Cai JY, Yang PH, Liang ZH (2014) In-situ detection of resveratrol inhibition effect on epidermal growth factor receptor of living MCF-7 cells by atomic force microscopy. Biosens Bioelectron 56:271–277CrossRefGoogle Scholar
  22. 22.
    Bunim JJ, Black RL, Lutwak L, Peterson RE, Whedon GD (1958) Studies on dexamethasone, a new synthetic steroid, in rheurheumatoid arthritis: a preliminary report; adrenal cortical, metabolic and early clinical effects. Arthritis Rheum 1(4):313–331CrossRefGoogle Scholar
  23. 23.
    Li LC, Scudds RA, Heck CS, Harth M (1996) The efficacy of dexamethasone iontophoresis for the treatment of rheumatoid arthritic knees: a pilot study. Arthritis Care Res 9(2):126–132CrossRefGoogle Scholar
  24. 24.
    Huang H, Hu G, Wang C, Xu H, Chen X, Qian A (2014) Cepharanthine, an alkaloid from Stephania cepharantha Hayata, inhibits the inflammatory response in the RAW264.7 cell and mouse models. Inflammation 37(1):235–246CrossRefGoogle Scholar
  25. 25.
    Zhao L, Tao JY, Zhang SL, Pang R, Jin F, Dong JH, Guo YJ (2007) Inner anti-inflammatory mechanisms of petroleum ether extract from Melilotus suaveolens ledeb. Inflammation 30(6):213–223CrossRefGoogle Scholar
  26. 26.
    Rogerio AP, Kanashiro A, Fontanari C, da Silva EV, Lucisano-Valim YM, Soares EG, Faccioli LH (2007) Anti-inflammatory activity of quercetin and isoquercitrin in experimental murine allergic asthma. Inflamm Res 56(10):402–408CrossRefGoogle Scholar
  27. 27.
    Valerio DA, Georgetti SR, Magro DA, Casagrande R, Cunha TM, Vicentini FTMC, Vieira SM, Fonseca MJV, Ferreira SH, Cunha FQ, Verri WA (2009) Quercetin reduces inflammatory pain: inhibition of oxidative stress and cytokine production. J Nat Prod 72(11):1975–1979CrossRefGoogle Scholar
  28. 28.
    Jung WJ, Sung MK (2004) Effects of major dietary antioxidants on inflammatory markers of RAW 264.7 macrophages. Biofactors 21(1–4):113–117CrossRefGoogle Scholar
  29. 29.
    Nakamura M, Omura S (2008) Quercetin regulates the inhibitory effect of monoclonal non-specific suppressor factor beta on tumor necrosis factor-alpha production in LPS-stimulated macrophages. Biosci Biotechnol Biochem 72(7):1915–1920CrossRefGoogle Scholar
  30. 30.
    Lin HY, Juan SH, Shen SC, Hsu FL, Chen YC (2003) Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1. Biochem Pharmacol 66(9):1821–1832CrossRefGoogle Scholar
  31. 31.
    Kao FS, Ger W, Pan YR, Yu HC, Hsu RQ, Chen HM (2012) Chip-based protein-protein interaction studied by atomic force microscopy. Biotechnol Bioeng 109(10):2460–2467CrossRefGoogle Scholar
  32. 32.
    Oh GS, Pae HO, Lee BS, Kim BN, Kim JM, Kim HR, Jeon SB, Jeon WK, Chae HJ, Chung HT (2006) Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide. Free Radic Biol Med 41(1):106–119CrossRefGoogle Scholar
  33. 33.
    Li B, Lee DS, Choi HG, Kim KS, Kang DG, Lee HS, Jeong GS, Kim YC (2011) Sauchinone suppresses pro-inflammatory mediators by inducing heme oxygenase-1 in RAW264.7 macrophages. Biol Pharm Bull 34(10):1566–1571CrossRefGoogle Scholar
  34. 34.
    Su KY, Yu CY, Chen YP, Hua KF, Chen YL (2014) 3,4-Dihydroxytoluene, a metabolite of rutin, inhibits inflammatory responses in lipopolysaccharide-activated macrophages by reducing the activation of NF-kappaB signaling. BMC Complement Altern Med 14:21CrossRefGoogle Scholar
  35. 35.
    Kim KS, Cui X, Lee DS, Ko W, Sohn JH, Yim JH, An RB, Kim YC, Oh H (2014) Inhibitory effects of benzaldehyde derivatives from the marine fungus Eurotium sp. SF-5989 on inflammatory mediators via the induction of heme oxygenase-1 in lipopolysaccharide-stimulated RAW264.7 macrophages. Int J Mol Sci 15(12):23749–23765CrossRefGoogle Scholar
  36. 36.
    Bonizzi G, Karin M (2004) The two NF-kappa B activation pathways and their role in innate and adaptive immunity. Trends Immunol 25(6):280–288CrossRefGoogle Scholar
  37. 37.
    Pang HY, Liu G, Liu GT (2009) Compound FLZ inhibits lipopolysaccharide-induced inflammatory effects via down-regulation of the TAK-IKK and TAK-JNK/p38MAPK pathways in RAW264.7 macrophages. Acta Pharmacol Sin 30(2):209–218CrossRefGoogle Scholar
  38. 38.
    Rim HK, Cho W, Sung SH, Lee KT (2012) Nodakenin suppresses lipopolysaccharide-induced inflammatory responses in macrophage cells by inhibiting tumor necrosis factor receptor-associated factor 6 and nuclear factor-kappa B pathways and protects mice from lethal endotoxin shock. J Pharmacol Exp Ther 342(3):654–664CrossRefGoogle Scholar
  39. 39.
    Pi J, Li T, Liu JX, Su XH, Wang R, Yang F, Bai HH, Jin H, Cai JY (2014) Detection of lipopolysaccharide induced inflammatory responses in RAW264.7 macrophages using atomic force microscope. Micron 65:1–9CrossRefGoogle Scholar
  40. 40.
    Jin Y, Tachibana I, Takeda Y, He P, Kang S, Suzuki M, Kuhara H, Tetsumoto S, Tsujino K, Minami T, Iwasaki T, Nakanishi K, Kohmo S, Hirata H, Takahashi R, Inoue K, Nagatomo I, Kida H, Kijima T, Ito M, Saya H, Kumanogoh A (2013) Statins decrease lung inflammation in mice by upregulating tetraspanin CD9 in macrophages. PLoS One 8(9), e73706CrossRefGoogle Scholar
  41. 41.
    Duan W, Zhou J, Zhang S, Zhao K, Zhao L, Ogata K, Sakaue T, Mori A, Wei T (2011) ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts. Biochim Biophys Acta 1813(5):772–783CrossRefGoogle Scholar
  42. 42.
    Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249(4975):1431–1433CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Jiang Pi
    • 1
  • Huaihong Cai
    • 2
  • Fen Yang
    • 1
  • Hua Jin
    • 1
  • Jianxin Liu
    • 1
  • Peihui Yang
    • 2
  • Jiye Cai
    • 1
    • 2
    Email author
  1. 1.State Key Laboratory of Quality Research in Chinese MedicinesMacau University of Science and TechnologyMacauChina
  2. 2.Department of ChemistryJinan UniversityGuangzhouChina

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