DOI QR코드

DOI QR Code

Silibinin Inhibits LPS-Induced Macrophage Activation by Blocking p38 MAPK in RAW 264.7 Cells

  • Received : 2013.05.16
  • Accepted : 2013.05.30
  • Published : 2013.07.31

Abstract

We demonstrate herein that silibinin, a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), inhibits LPS-induced activation of macrophages and production of nitric oxide (NO) in RAW 264.7 cells. Western blot analysis showed silibinin inhibits iNOS gene expression. RT-PCR showed that silibinin inhibits iNOS, TNF-${\alpha}$, and $IL1{\beta}$. We also showed that silibinin strongly inhibits p38 MAPK phosphorylation, whereas the ERK1/2 and JNK pathways are not inhibited. The p38 MAPK inhibitor abrogated the LPS-induced nitrite production, whereas the MEK-1 inhibitor did not affect the nitrite production. A molecular modeling study proposed a binding pose for silibinin targeting the ATP binding site of p38 MAPK (1OUK). Collectively, this series of experiments indicates that silibinin inhibits macrophage activation by blocking p38 MAPK signaling.

Keywords

References

  1. Al-Anati, L., Essid, E., Reinehr, R. and Petzinger, E. (2009) Silibinin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells. Mol. Nutr. Food Res. 53, 460-466. https://doi.org/10.1002/mnfr.200800110
  2. Beyaert, R., Cuenda, A., Vanden Berghe, W., Plaisance, S., Lee, J. C., Haegeman, G., Cohen, P. and Fiers, W. (1996) The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor. EMBO J. 15, 1914-1923.
  3. Billack, B. (2006) Macrophage activation: role of toll-like receptors, nitric oxide, and nuclear factor $\kappa{B}$. Am. J. Pharm. Educ. 70, 102. https://doi.org/10.5688/aj7005102
  4. Chen, C. C. and Wang, J. K. (1999) p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 cells. Mol. Pharmacol. 55, 481-488.
  5. Cohen, J. (2002) The immunopathogenesis of sepsis. Nature 420, 885-891. https://doi.org/10.1038/nature01326
  6. Cristofalo, R., Bannwart-Castro, C. F., Magalhaes, C. G., Borges, V. T., Peracoli, J. C., Witkin, S. S. and Peracoli, M. T. (2013) Silibinin attenuates oxidative metabolism and cytokine production by monocytes from preeclamptic women. Free Radic. Res. 47, 268-275. https://doi.org/10.3109/10715762.2013.765951
  7. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R. and Lee, J. C. (1995) SB 203580 is a specifi c inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364, 229-233. https://doi.org/10.1016/0014-5793(95)00357-F
  8. Da Silva, J., Pierrat, B., Mary, J. L. and Lesslauer, W. (1997) Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes. J. Biol. Chem. 272, 28373-28380. https://doi.org/10.1074/jbc.272.45.28373
  9. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J. and Saltiel, A. R. (1995) A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. U.S.A 92, 7686-7689. https://doi.org/10.1073/pnas.92.17.7686
  10. Dunnett, C. W. (1955) A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096-1121. https://doi.org/10.1080/01621459.1955.10501294
  11. Fitzgerald, C. E., Patel, S. B., Becker, J. W., Cameron, P. M., Zaller, D., Pikounis, V. B., O'Keefe, S. J. and Scapin, G. (2003) Structural basis for p38alpha MAP kinase quinazolinone and pyridol-pyrimidine inhibitor specifi city. Nat. Struct. Biol. 10, 764-769. https://doi.org/10.1038/nsb949
  12. Foey, A. D., Parry, S. L., Williams, L. M., Feldmann, M., Foxwell, B. M. and Brennan, F. M. (1998) Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-alpha: role of the p38 and p42/44 mitogen-activated protein kinases. J. Immunol. 160, 920-928.
  13. Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S. and Tannenbaum, S. R. (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126, 131-138. https://doi.org/10.1016/0003-2697(82)90118-X
  14. Hibbs, J. B. Jr., Taintor, R. R. and Vavrin, Z. (1987) Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science 235, 473-476. https://doi.org/10.1126/science.2432665
  15. Higuchi, M., Higashi, N., Taki, H. and Osawa, T. (1990) Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and Larginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages. J. Immunol. 144, 1425-1431.
  16. Hogan, F. S., Krishnegowda, N. K., Mikhailova, M. and Kahlenberg, M. S. (2007) Flavonoid, silibinin, inhibits proliferation and promotes cell-cycle arrest of human colon cancer. J. Surg. Res. 143, 58-65. https://doi.org/10.1016/j.jss.2007.03.080
  17. Huong, P. T., Lee, M. Y., Lee, K. Y., Chang, I. Y., Lee, S. K., Yoon, S. P., Lee, D. C. and Jeon, Y. J. (2012) Synergistic induction of iNOS by IFN-$\gamma$ and glycoprotein isolated from Dioscorea batatas. Korean J. Physiol. Pharmacol. 16, 431-436. https://doi.org/10.4196/kjpp.2012.16.6.431
  18. Jeon, Y. J., Kim, Y. K., Lee, M., Park, S. M., Han, S. B. and Kim, H. M. (2000) Radicicol suppresses expression of inducible nitric-oxide synthase by blocking p38 kinase and nuclear factor-kappaB/Rel in lipopolysaccharide-stimulated macrophages. J. Pharmacol. Exp. Ther. 294, 548-554.
  19. Kang, J. S., Jeon, Y. J., Kim, H. M., Han, S. H. and Yang, K. H. (2002) Inhibition of inducible nitric-oxide synthase expression by silymarin in lipopolysaccharide-stimulated macrophages. J. Pharmacol. Exp. Ther. 302, 138-144. https://doi.org/10.1124/jpet.302.1.138
  20. Kim, J. H., Kim, K., Jin, H. M., Song, I., Youn, B. U., Lee, J. and Kim, N. (2009) Silibinin inhibits osteoclast differentiation mediated by TNF family members. Mol. Cells 28, 201-207. https://doi.org/10.1007/s10059-009-0123-y
  21. Lee, J. C. and Young, P. R. (1996) Role of CSB/p38/RK stress response kinase in LPS and cytokine signaling mechanisms. J. Leukoc. Biol. 59, 152-157. https://doi.org/10.1002/jlb.59.2.152
  22. Letteron, P., Labbe, G., Degott, C., Berson, A., Fromenty, B., Delaforge, M., Larrey, D. and Pessayre, D. (1990) Mechanism for the protective effects of silymarin against carbon tetrachlorideinduced lipid peroxidation and hepatotoxicity in mice. Evidence that silymarin acts both as an inhibitor of metabolic activation and as a chain-breaking antioxidant. Biochem. Pharmacol. 39, 2027-2034. https://doi.org/10.1016/0006-2952(90)90625-U
  23. Li, M. H., Kothandan, G., Cho, S. J., Huong, P. T., Nan, Y. H., Lee, K. Y., Shin, S. Y., Yea, S. S. and Jeon, Y. J. (2010) Magnolol inhibits LPS-induced NF-kappaB/Rel activation by blocking p38 kinase in murine macrophages. Korean J. Physiol. Pharmacol. 14, 353-358. https://doi.org/10.4196/kjpp.2010.14.6.353
  24. Lowenstein, C. J., Alley, E. W., Raval, P., Snowman, A. M., Snyder, S. H., Russell, S. W. and Murphy, W. J. (1993) Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc. Natl. Acad. Sci. U.S.A 90, 9730-9734. https://doi.org/10.1073/pnas.90.20.9730
  25. Mereish, K. A., Bunner, D. L., Ragland, D. R. and Creasia, D. A. (1991) Protection against microcystin-LR-induced hepatotoxicity by silymarin: biochemistry, histopathology, and lethality. Pharm. Res. 8, 273-277. https://doi.org/10.1023/A:1015868809990
  26. Mokhtari, M. J., Motamed, N. and Shokrgozar, M. A. (2008) Evaluation of silibinin on the viability, migration and adhesion of the human prostate adenocarcinoma (PC-3) cell line. Cell Biol. Int. 32, 888-892. https://doi.org/10.1016/j.cellbi.2008.03.019
  27. Palmer, R. M., Ashton, D. S. and Moncada, S. (1988) Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333, 664-666. https://doi.org/10.1038/333664a0
  28. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. and Ferrin, T. E. (2004) UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605-1612. https://doi.org/10.1002/jcc.20084
  29. Pliskova, M., Vondracek, J., Kren, V., Gazak, R., Sedmera, P., Walterova, D., Psotova, J., Simanek, V. and Machala, M. (2005) Effects of silymarin fl avonolignans and synthetic silybin derivatives on estrogen and aryl hydrocarbon receptor activation. Toxicology 215, 80-89. https://doi.org/10.1016/j.tox.2005.06.020
  30. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J. and Davis, R. J. (1995) Pro-infl ammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J. Biol. Chem. 270, 7420-7426. https://doi.org/10.1074/jbc.270.13.7420
  31. Singh, R. P., Tyagi, A. K., Zhao, J. and Agarwal, R. (2002) Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogen-activated protein kinases and induction of apoptosis. Carcinogenesis 23, 499-510. https://doi.org/10.1093/carcin/23.3.499
  32. Stuehr, D. J. and Nathan, C. F. (1989) Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169, 1543-1555. https://doi.org/10.1084/jem.169.5.1543
  33. Su, B. and Karin, M. (1996) Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8, 402-411. https://doi.org/10.1016/S0952-7915(96)80131-2
  34. Valenzuela, A. and Garrido, A. (1994) Biochemical bases of the pharmacological action of the fl avonoid silymarin and of its structural isomer silibinin. Biol. Res. 27, 105-112.
  35. Vo, V. A., Lee, J. W., Chang, J. E., Kim, J. Y., Kim, N. H., Lee, H. J., Kim, S. S., Chun, W. and Kwon, Y. S. (2012) Avicularin inhibits lipopolysaccharide-induced infl ammatory response by suppressing ERK phosphorylation in RAW 264.7 macrophages. Biomol. Ther. 20, 532-537. https://doi.org/10.4062/biomolther.2012.20.6.532
  36. Weinstein, S. L., Sanghera, J. S., Lemke, K., DeFranco, A. L. and Pelech, S. L. (1992) Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages. J. Biol. Chem. 267, 14955-14962.
  37. Whitmarsh, A. J. and Davis, R. J. (1996) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med. 74, 589-607. https://doi.org/10.1007/s001090050063
  38. Zhao, J., Sharma, Y. and Agarwal, R. (1999) Signifi cant inhibition by the fl avonoid antioxidant silymarin against 12-O-tetradecanoylphorbol 13-acetate-caused modulation of antioxidant and infl ammatory enzymes, and cyclooxygenase 2 and interleukin-1alpha expression in SENCAR mouse epidermis: implications in the prevention of stage I tumor promotion. Mol. Carcinog. 26, 321-333. https://doi.org/10.1002/(SICI)1098-2744(199912)26:4<321::AID-MC11>3.0.CO;2-9

Cited by

  1. Anti-allergic effect of α-cubebenoate isolated from Schisandra chinensis using in vivo and in vitro experiments vol.173, 2015, https://doi.org/10.1016/j.jep.2015.07.049
  2. Scutellarein Reduces Inflammatory Responses by Inhibiting Src Kinase Activity vol.19, pp.5, 2015, https://doi.org/10.4196/kjpp.2015.19.5.441
  3. Adenosine dialdehyde suppresses MMP-9-mediated invasion of cancer cells by blocking the Ras/Raf-1/ERK/AP-1 signaling pathway vol.86, pp.9, 2013, https://doi.org/10.1016/j.bcp.2013.08.022
  4. Fisetin Suppresses Macrophage-Mediated Inflammatory Responses by Blockade of Src and Syk vol.23, pp.5, 2015, https://doi.org/10.4062/biomolther.2015.036
  5. Anti-Allergic Effect of Oroxylin A from Oroxylum indicum Using in vivo and in vitro Experiments vol.24, pp.3, 2016, https://doi.org/10.4062/biomolther.2016.071
  6. Kaempferol, a dietary flavonoid, ameliorates acute inflammatory and nociceptive symptoms in gastritis, pancreatitis, and abdominal pain vol.59, pp.7, 2015, https://doi.org/10.1002/mnfr.201400820
  7. Therapeutic effects of silibinin on LPS-induced acute lung injury by inhibiting NLRP3 and NF-κB signaling pathways vol.108, 2017, https://doi.org/10.1016/j.micpath.2017.05.011
  8. Anti-Inflammatory Effects of Chloranthalactone B in LPS-Stimulated RAW264.7 Cells vol.17, pp.11, 2016, https://doi.org/10.3390/ijms17111938
  9. IKKβ-Targeted Anti-Inflammatory Activities of a Butanol Fraction of Artificially CultivatedCordyceps pruinosaFruit Bodies vol.2014, 2014, https://doi.org/10.1155/2014/562467
  10. NF-κB/AP-1-Targeted Inhibition of Macrophage-Mediated Inflammatory Responses by Depigmenting Compound AP736 Derived from Natural 1,3-Diphenylpropane Skeleton vol.2014, 2014, https://doi.org/10.1155/2014/354843
  11. Aspergillus fumigatus -induced early inflammatory response in pulmonary microvascular endothelial cells: Role of p38 MAPK and inhibition by silibinin vol.49, 2017, https://doi.org/10.1016/j.intimp.2017.05.038
  12. Synergistic anti-tumor actions of luteolin and silibinin prevented cell migration and invasion and induced apoptosis in glioblastoma SNB19 cells and glioblastoma stem cells vol.1629, 2015, https://doi.org/10.1016/j.brainres.2015.10.010
  13. Anti-inflammatory Activities ofGouania leptostachyaMethanol Extract and its Constituent Resveratrol vol.29, pp.3, 2015, https://doi.org/10.1002/ptr.5262
  14. Sphingosine 1-phosphate induced anti-atherogenic and atheroprotective M2 macrophage polarization through IL-4 vol.26, pp.10, 2014, https://doi.org/10.1016/j.cellsig.2014.07.009
  15. The Dietary Flavonoid Kaempferol Mediates Anti-Inflammatory Responses via the Src, Syk, IRAK1, and IRAK4 Molecular Targets vol.2015, 2015, https://doi.org/10.1155/2015/904142
  16. Effect of silibinin and vitamin E on the ASK1-p38 MAPK pathway in D-galactosamine/lipopolysaccharide induced hepatotoxicity vol.241, pp.11, 2016, https://doi.org/10.1177/1535370216636719
  17. ATP-Binding Pocket-Targeted Suppression of Src and Syk by Luteolin Contributes to Its Anti-Inflammatory Action vol.2015, 2015, https://doi.org/10.1155/2015/967053
  18. Immunotoxicological Effects of Aripiprazole:In vivoandIn vitroStudies vol.19, pp.4, 2015, https://doi.org/10.4196/kjpp.2015.19.4.365
  19. (E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone displays suppression of inflammatory responses via inhibition of Src, Syk, and NF-κB vol.20, pp.1, 2016, https://doi.org/10.4196/kjpp.2016.20.1.91
  20. The Regulatory Role of Activating Transcription Factor 2 in Inflammation vol.2014, 2014, https://doi.org/10.1155/2014/950472
  21. Silybin reduces obliterated retinal capillaries in experimental diabetic retinopathy in rats vol.740, 2014, https://doi.org/10.1016/j.ejphar.2014.07.033
  22. Molecular and biochemical evidence on the protective role of ellagic acid and silybin against oxidative stress-induced cellular aging 2017, https://doi.org/10.1007/s11010-017-3172-0
  23. 3-deoxysilybin exerts anti-inflammatory effects by suppressing NF-κB activation in lipopolysaccharide-stimulated RAW264.7 macrophages vol.78, pp.12, 2014, https://doi.org/10.1080/09168451.2014.948377
  24. Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro vol.6, pp.10, 2014, https://doi.org/10.3390/toxins6102962
  25. Silibinin Inhibits Neutrophilic Inflammation and Mucus Secretion Induced by Cigarette Smoke via Suppression of ERK-SP1 Pathway vol.30, pp.12, 2016, https://doi.org/10.1002/ptr.5686
  26. Identification of a novel anti-inflammatory compound, α-cubebenoate from Schisandra chinensis vol.153, pp.1, 2014, https://doi.org/10.1016/j.jep.2014.02.027
  27. Silibinin alleviates inflammation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes and has a therapeutic effect on arthritis in rats vol.8, pp.1, 2018, https://doi.org/10.1038/s41598-018-21674-6
  28. Hydroxysafflor yellow A (HSYA) targets the NF-κB and MAPK pathways and ameliorates the development of osteoarthritis vol.9, pp.8, 2018, https://doi.org/10.1039/C8FO00732B
  29. ‐induced reactive oxygen species/reactive nitrogen species generation pp.1099-1573, 2019, https://doi.org/10.1002/ptr.6293
  30. Troxerutin suppresses the inflammatory response in advanced glycation end-product-administered chondrocytes and attenuates mouse osteoarthritis development vol.10, pp.8, 2013, https://doi.org/10.1039/c9fo01089k
  31. Anti-inflammatory effect of Barringtonia angusta methanol extract is mediated by targeting of Src in the NF-κB signalling pathway vol.59, pp.1, 2013, https://doi.org/10.1080/13880209.2021.1938613
  32. α-Cyperone (CYP) down-regulates NF-κB and MAPKs signaling, attenuating inflammation and extracellular matrix degradation in chondrocytes, to ameliorate osteoarthritis in mice vol.13, pp.13, 2013, https://doi.org/10.18632/aging.203259
  33. Molecular insight into isoform specific inhibition of PI3K-α and PKC-η with dietary agents through an ensemble pharmacophore and docking studies vol.11, pp.1, 2021, https://doi.org/10.1038/s41598-021-90287-3
  34. Understanding immune-modulatory efficacy in vitro vol.352, pp.None, 2013, https://doi.org/10.1016/j.cbi.2021.109776