Advertisement

Journal of Natural Medicines

, Volume 71, Issue 2, pp 349–356 | Cite as

Shikonin inhibits TNF-α production through suppressing PKC-NF-κB-dependent decrease of IL-10 in rheumatoid arthritis-like cell model

  • Wen-Xiao Sun
  • Yan Liu
  • Wei Zhou
  • He-Wei Li
  • Jian Yang
  • Zhen-Bing ChenEmail author
Original Paper

Abstract

Shikonin, a major effective component in the Chinese herbal medicine Lithospermum erythrorhizon Sieb., exhibits an anti-inflammatory property towards rheumatoid arthritis (RA), but the potential mechanism is unclear. Our aim was to investigate the mechanism of shikonin on the lipopolysaccharide (LPS)-induced fibroblast-like synoviocyte (LiFLS) inflammation model. Fibroblast-like synoviocytes (FLSs) were treated with 200 μg/ml of LPS for 24 h to establish the RA-like model, LiFLS. FLSs were pretreated with shikonin (0.1–1 μM) for 30 min in the treatment groups. Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays were used to detect mRNA and protein levels of interleukin (IL)-10 and tumor necrosis factor (TNF)-α. Signal proteins involved in IL-10 production were analyzed by Western blotting. Shikonin significantly reversed the inhibitory effects of LPS on IL-10 expression in FLSs by inactivating the PKC-NF-κB pathway. In addition, shikonin inhibited LPS-induced TNF-α expression in FLSs, and this effect was markedly diminished by IL-10-neutralizing antibody. The IL-10-mediated suppression of TNF-α transcription was demonstrated by no response to the protein synthesis inhibitor cyclohexamide and no mRNA decay. Shikonin inhibits LPS-induced TNF-α production in FLSs through suppressing the PKC-NF-κB-dependent decrease in IL-10, and this study also highlights the potential application of shikonin in the treatment of RA.

Keywords

Rheumatoid arthritis Inflammation Lithospermum erythrorhizon Protein kinase C Protein synthesis mRNA stability 

Notes

Acknowledgements

This work was supported by grants from the Science and Technology Planning Natural Scientific Foundation of Hubei Province of China (2010CDB07501).

Compliance with ethical standards

Conflict of interest

The authors declare that no conflict of interests exists.

Supplementary material

11418_2016_1064_MOESM1_ESM.tif (556 kb)
Supplementary Fig. 1. Identification of FLSs by microscope and flow cytometry. LiFLSs was identified by (a) microscopy (200× and 400×) or (b) flow cytometry using antibodies anti-vimentin and anti-CD68. (TIFF 556 kb)

References

  1. 1.
    Filippin LI, Vercelino R, Marroni N, Xavier RM (2008) Redox signalling and the inflammatory response in rheumatoid arthritis. Clin Exp Immunol 152:415–422CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Cheon H, Yu SJ, Yoo D, Chae I, Song G, Sohn J (2002) Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-β1 in synovial fibroblasts from rheumatoid arthritis and normal individuals. Clin Exp Immunol 127:547–552CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Xu H, He Y, Yang X, Liang L, Zhan Z, Ye Y, Sun L (2007) Anti-malarial agent artesunate inhibits TNF-α-induced production of proinflammatory cytokines via inhibition of NF-κB and PI3 kinase/Akt signal pathway in human rheumatoid arthritis fibroblast-like synoviocytes. Rheumatology 46:920–926CrossRefPubMedGoogle Scholar
  4. 4.
    Bartok B, Firestein GS (2010) Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev 233:233–255CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kawashima M, Ogura N, Akutsu M, Ito K, Kondoh T (2013) The anti-inflammatory effect of cyclooxygenase inhibitors in fibroblast-like synoviocytes from the human temporomandibular joint results from the suppression of PGE2 production. J Oral Pathol Med 42:499–506CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Henry D, Lim LL, Rodriguez LAG, Gutthann SP, Carson JL, Griffin M, Savage R, Logan R, Moride Y, Hawkey C, Hill S, Fries JT (1996) Variability in risk of gastrointestinal complications with individual non-steroidal anti-inflammatory drugs: results of a collaborative meta-analysis. BMJ 312:1563–1566CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    MacDonald T, Morant S, Robinson GC, Shield MJ, McGilchrist MM, Murray FE, McDevitt DG (1997) Association of upper gastrointestinal toxicity of non-steroidal anti-inflammatory drugs with continued exposure: cohort study. BMJ 315:1333–1337CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Staniforth V, Wang SY, Shyur LF, Yang NS (2004) Shikonins, phytocompounds from Lithospermum erythrorhizon, inhibit the transcriptional activation of human tumor necrosis factor α promoter in vivo. J Biol Chem 279:5877–5885CrossRefPubMedGoogle Scholar
  9. 9.
    Chiu SC, Yang NS (2007) Inhibition of tumor necrosis factor-α through selective blockade of pre-mRNA splicing by shikonin. Mol Pharmacol 71:1640–1645CrossRefPubMedGoogle Scholar
  10. 10.
    Lu L, Qin A, Huang H, Zhou P, Zhang C, Liu N, Li S, Wen G, Zhang C, Dong W, Wang X, Dou P, Liu J (2011) Shikonin extracted from medicinal Chinese herbs exerts anti-inflammatory effect via proteasome inhibition. Eur J Pharmacol 658:242–247CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Liang D, Sun Y, Shen Y, Li F, Song X, Zhou E, Zhao F, Liu Z, Fu Y, Guo M, Zhang N, Yang Z, Cao Y (2013) Shikonin exerts anti-inflammatory effects in a murine model of lipopolysaccharide-induced acute lung injury by inhibiting the nuclear factor-kappaB signaling pathway. Int Immunopharmacol 16:475–480CrossRefPubMedGoogle Scholar
  12. 12.
    Kim YO, Hong SJ, Yim SV (2010) The efficacy of shikonin on cartilage protection in a mouse model of rheumatoid arthritis. Korean J Physiol Pharmacol 14:199–204CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Dai Q, Fang J, Zhang FS (2009) Dual role of shikonin in early and late stages of collagen type II arthritis. Mol Biol Rep 36:1597–1604CrossRefPubMedGoogle Scholar
  14. 14.
    Asadullah K, Sterry W, Volk H (2003) Interleukin-10 therapy-review of a new approach. Pharmacol Rev 55:241–269CrossRefPubMedGoogle Scholar
  15. 15.
    Katsikis PD, Chu CQ, Brennan FM, Maini RN, Feldmann M (1994) Immunoregulatory role of interleukin 10 in rheumatoid arthritis. J Exp Med 179:1517–1527CrossRefPubMedGoogle Scholar
  16. 16.
    Llorente L, Richaud-Patin Y, Fior R, Alcocer-Varela J, Wijdenes J, Fourrier BM, Galanaud P, Emilie D (1994) In vivo production of interleukin-10 by non-T cells in rheumatoid arthritis, Sjögren’s syndrome, and systemic lupus erythematosus. Arthritis Rheum 37:1647–1655CrossRefPubMedGoogle Scholar
  17. 17.
    Cush JJ, Splawski JB, Thomas R, Mcfarlin JE, Schulze-Koops H, Schulze-Koops H, Davis LS, Fujita K, Lipsky PE (1995) Elevated interleukin-10 levels in patients with rheumatoid arthritis. Arthritis Rheum 38:96–104CrossRefPubMedGoogle Scholar
  18. 18.
    Moelants EA, Mortier A, Van Damme J, Proost P (2013) Regulation of TNF-α with a focus on rheumatoid arthritis. Immunol Cell Biol 91:393–401CrossRefPubMedGoogle Scholar
  19. 19.
    Jobin C, Haskill S, Mayer L, Panja A, Sartor RB (1997) Evidence for altered regulation of I kappa B alpha degradation in human colonic epithelial cells. J Immunol 158:226–234PubMedGoogle Scholar
  20. 20.
    Song SS, Huang B, Wang QT, Wu YJ, Fu JJ, Zhang YF, Chang Y, Chen ZY, Wu HX, Wang D, Zhang LL, Wei W (2013) BF02, a recombinant TNFR2 fusion protein, alleviates adjuvant arthritis by regulating T lymphocytes in rats. Acta Pharmacol Sin 34:414–423CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Matsuda S, Uchikawa R, Yamada M, Arizono N (1995) Cytokine mRNA expression profiles in rats infected with the intestinal nematode Nippostrongylus brasiliensis. Infect Immun 63:4653–4660PubMedPubMedCentralGoogle Scholar
  22. 22.
    Padegimas L, Kowalczyk TH, Adams S, Gedeon CR, Oette SM, Dines K, Hyatt SL, Sesenoglu-Laird O, Tyr O, Moen RC, Cooper MJ (2012) Optimization of hCFTR lung expression in mice using DNA nanoparticles. Mol Ther 20:63–72CrossRefPubMedGoogle Scholar
  23. 23.
    Matsuno H, Yudoh K, Katayama R, Nakazawa F, Uzuki M, Sawai T, Yonezawa T, Saeki Y, Panayi GS, Pitzalis C (2002) The role of TNF-α in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera. Rheumatology 41:329–337CrossRefPubMedGoogle Scholar
  24. 24.
    Noh KT (2014) Protein kinase C delta (PKCδ)- extracellular-signal regulated kinase 1/2 (ERK1/2) signaling cascade regulates glycogen synthase kinase-3 (GSK-3) inhibition- mediated interleukin-10 (IL-10) expression in lipopolysaccharide (LPS)-induced endotoxemia (IRM5P. 704). J Immunol 192:62–65Google Scholar
  25. 25.
    La Porta C, Comolli R (1997) PKC-dependent modulation of IkB alpha-NFkB pathway in low metastatic B16F1 murine melanoma cells and in highly metastatic BL6 cells. Anticancer Res 18:2591–2597Google Scholar
  26. 26.
    Lee JW, Cheong IY, Kim HS, Lee JJ, Lee YS, Kwon YS, Kim MJ, Lee HJ, Kim SS, Chun W (2011) Anti-inflammatory activity of 1-docosanoyl cafferate isolated from Rhus verniciflua in LPS-stimulated BV2 microglial cells. Korean J Physiol Pharmacol 15:9–15CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Armstrong L, Jordan N, Millar A (1996) Interleukin 10 (IL-10) regulation of tumour necrosis factor alpha (TNF-alpha) from human alveolar macrophages and peripheral blood monocytes. Thorax 51:143–149CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Smallie T, Ricchetti G, Horwood NJ, Feldmann M, Clark AR, Williams LM (2010) IL-10 inhibits transcription elongation of the human TNF gene in primary macrophages. J Exp Med 207:2081–2088CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Schneider-Poetsch T, Ju J, Eyler DE, Jewett JC, Bhat S, Rawal VH, Green R, Liu JO (2010) Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6:209–217CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Leclerc GJ, Leclerc GM, Barredo JC (2002) Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines. Cancer Cell Int 2:1CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Weidensdorfer D, Stöhr N, Baude A, Lederer M, Köhn M, Schierhorn A, Buchmeier S, Wahle E, Hüttelmaier S (2009) Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs. RNA 15:104–115CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Hur GM, Hwang YB, Lee JH, Bae SH, Park JS, Lee CJ, Seok JH (2003) Caffeic acid phenethyl ester inhibits the PKC-induced IL-6 gene expression in the synoviocytes of rheumatoid arthritis patients. Korean J Physiol Pharmacol 7:363–368Google Scholar
  33. 33.
    Chen X, Yang L, Oppenheim JJ, Howard O (2002) Cellular pharmacology studies of shikonin derivatives. Phytother Res 16:199–209CrossRefPubMedGoogle Scholar
  34. 34.
    Bai GZ, Yu HT, Ni YF, Li XF, Zhang ZP, Su K, Lei J, Liu BY, Ke CK, Zhong DX (2013) Shikonin attenuates lipopolysaccharide-induced acute lung injury in mice. J Surg Res 182:303–311CrossRefPubMedGoogle Scholar
  35. 35.
    Alsaleh G, Suffert G, Semaan N, Juncker T, Frenzel L, Gottenberg JE, Sibilia J, Pfeffer S, Wachsmann D (2009) Bruton’s tyrosine kinase is involved in miR-346-related regulation of IL-18 release by lipopolysaccharide-activated rheumatoid fibroblast-like synoviocytes. J Immunol 182:5088–5097CrossRefPubMedGoogle Scholar
  36. 36.
    Philippe L, Alsaleh G, Pichot A, Ostermann E, Zuber G, Frisch B, Sibilia J, Pfeffer S, Bahram S, Wachsmann D (2013) MiR-20a regulates ASK1 expression and TLR4-dependent cytokine release in rheumatoid fibroblast-like synoviocytes. Ann Rheum Dis 72:1071–1079CrossRefPubMedGoogle Scholar
  37. 37.
    Chomarat P, Vannier E, Dechanet J, Rissoan MC, Banchereau J, Dinarello CA, Miossec P (1995) Balance of IL-1 receptor antagonist/IL-1 beta in rheumatoid synovium and its regulation by IL-4 and IL-10. J Immunol 154:1432–1439PubMedGoogle Scholar
  38. 38.
    Hart P, Ahern M, Smith M, Finlay-Jones J (1995) Comparison of the suppressive effects of interleukin-10 and interleukin-4 on synovial fluid macrophages and blood monocytes from patients with inflammatory arthritis. Immunology 84:536PubMedPubMedCentralGoogle Scholar
  39. 39.
    Hart PH, Hunt EK, Bonder CS, Watson CJ, Finlay-Jones JJ (1996) Regulation of surface and soluble TNF receptor expression on human monocytes and synovial fluid macrophages by IL-4 and IL-10. J Immunol 157:3672–3680PubMedGoogle Scholar
  40. 40.
    Oswald IP, Wynn TA, Sher A, James SL (1992) Interleukin 10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor alpha required as a costimulatory factor for interferon gamma-induced activation. Proc Natl Acad Sci USA 89:8676–8680CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Chernoff AE, Granowitz EV, Shapiro L, Vannier E, Lonnemann G, Angel JB, Kennedy JS, Rabson AR, Wolff SM, Dinarello CA (1995) A randomized, controlled trial of IL-10 in humans. Inhibition of inflammatory cytokine production and immune responses. J Immunol 154:5492–5499PubMedGoogle Scholar
  42. 42.
    St Clair EW (1999) Interleukin 10 treatment for rheumatoid arthritis. Ann Rheum Dis 58:I99–I102CrossRefPubMedGoogle Scholar
  43. 43.
    Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishmoto T, Akira S (1993) Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA 90:10193–10197CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Tak PP, Firestein GS (2001) NF-κB: a key role in inflammatory diseases. J Clin Invest 107:7–11CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Leghmari K, Bennasser Y, Bahraoui E (2008) HIV-1 Tat protein induces IL-10 production in monocytes by classical and alternative NF-κB pathways. Eur J Cell Biol 87:947–962CrossRefPubMedGoogle Scholar
  46. 46.
    Correa F, Hernangomez M, Mestre L, Loria F, Spagnolo A, Docagne F, Di Marzo V, Guaza C (2010) Anandamide enhances IL-10 production in activated microglia by targeting CB(2) receptors: roles of ERK1/2, JNK, and NF-kappaB. Glia 58:135–147CrossRefPubMedGoogle Scholar
  47. 47.
    Zhang J, Ping P, Vondriska TM, Tang XL, Wang GW, Cardwell EM, Bolli R (2003) Cardioprotection involves activation of NF-kappa B via PKC-dependent tyrosine and serine phosphorylation of I kappa B-alpha. Am J Physiol Heart Circ Physiol 285:H1753–H1758CrossRefPubMedGoogle Scholar

Copyright information

© The Japanese Society of Pharmacognosy and Springer Japan 2016

Authors and Affiliations

  • Wen-Xiao Sun
    • 1
  • Yan Liu
    • 1
  • Wei Zhou
    • 1
  • He-Wei Li
    • 1
  • Jian Yang
    • 1
  • Zhen-Bing Chen
    • 2
    Email author
  1. 1.Department of Orthopedic and Hand Surgery, Liyuan HospitalHuazhong University of Science and TechnologyWuhanChina
  2. 2.Department of Orthopedic and Hand Surgery, Union Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina

Personalised recommendations