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DARU Journal of Pharmaceutical Sciences

, Volume 26, Issue 2, pp 215–227 | Cite as

Differential regulation of CD4+ T cell subsets by Silymarin in vitro and in ovalbumin immunized mice

  • Haideh Namdari
  • Maryam Izad
  • Farhad Rezaei
  • Zahra Amirghofran
Research Article
  • 76 Downloads

Abstract

CD4+ T cell subsets including regulatory T cells (Tregs), Th1 and Th17 are critical for control and development of inflammation and autoimmunity. We investigated the in vitro and in vivo effects of silymarin, a well-known herbal medicine on differentiation and function of Tregs and Th1 and Th17 responses. For in vitro study, mice splenocytes treated with 20–30 μg/ml silymarin were evaluated for gene expressions of specific transcription factors and cytokines of CD4+ T cell subsets using real-time PCR. Induction of Treg cell development in the presence of silymarin was performed on isolated naïve CD4+ T cells. Effect of silymarin-induced Tregs on T cell suppression was determined by CFSE labeling method. Results of this part showed that silymarin significantly decreased IFNγ, RORγt and IL-17 gene expressions and upregulated Foxp3, TGF-β and IL-10 mRNA. More silymarin-enhanced naïve CD4+ T cells differentiated to Tregs (67%) than the control (47%). Silymarin-induced Tregs reduced proliferation of naïve activated T cells (<50%). For in vivo study, mice were immunized with ovalbumin (Ova) on days 1 and 14. Silymarin (100 mg/Kg) was intraperitoneally administered two days before the first Ova challenge followed by on every day for two weeks. Splenocytes were then isolated for assessment of CD4+ T cell subsets and ex vivo analysis using flow cytometry. Treatment of Ova-immunized mice with silymarin increased Tregs (11.24 ± 1.2%, p < 0.01(but decreased Th1 (1.72 ± 0.4%, p < 0.001) and Th17 (1.07 ± 0.04%, p < 0.001) cells. Ex vivo Ova challenge of splenocytes from Ova-immunized mice treated with silymarin decreased proliferation of splenocytes, IFNγ (2.76% of control) and IL-17 (<8%) along with increased TGF-β (59.7%) expressions in CD4+T-bet+, CD4+RORγt+ and CD4+Foxp3+ T cells, respectively. In conclusion, silymarin promoted Treg differentiation and function and decreased Th1 and Th17 cells. Silymarin may differentially regulate CD4+ T cell responses which can provide potential benefits for its use as treatment of immune-related diseases.

Graphical abstract

Keywords

Silymarin Regulatory T cells Th1 Th17 Ovalbumin 

Notes

Acknowledgments

We would like to express our specific thanks to the Deputy of Research Affairs of Shiraz University of Medical Sciences (grant no. 7611) for financial support. This work was extracted from thesis written by one of the authors H. Namdari.

Authors’ contributions

HN and ZA wrote this manuscript, designed this study and analyzed the data; HN performed the experiments. MI and FR help in flow cytometry analysis. All authors read and approved the final manuscript.

Compliance with ethical standards

Consent for publication

All authors agree to publish our manuscript.

Competing interests

The authors declare that they have no conflict of interest in this work.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors. All protocols for animal care and treatment were approved by the local ethics committee.

Supplementary material

40199_2018_229_MOESM1_ESM.docx (1.1 mb)
ESM 1 (DOCX 1091 kb)

References

  1. 1.
    Raphael I, Nalawade S, Eagar TN, Forsthuber TG. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine. 2015;74(1):5–17.CrossRefPubMedGoogle Scholar
  2. 2.
    Huber S, Gagliani N, O'Connor W Jr, Geginat J, Caprioli F. CD4(+) T Helper Cell Plasticity in Infection, Inflammation, and Autoimmunity. Mediat Inflamm. 2017;2017:7083153.  https://doi.org/10.1155/2017/7083153. CrossRefGoogle Scholar
  3. 3.
    Rezaei N, Amirghofran Z, Nikseresht A, Ashjazade N, Zoghi S, Tahvili S, et al. In Vitro effects of sodium benzoate on Th1/Th2 deviation in patients with multiple sclerosis. Immunol Investig. 2016;45(7):679–91.  https://doi.org/10.1080/08820139.2016.1208216.CrossRefGoogle Scholar
  4. 4.
    Namdari H, Izad M, Amirghofran Z. Modulation of CD4+ T cell subsets by Euphorbia microciadia and Euphorbia osyridea plant extracts.IJI . 2017;14(2):134–50.Google Scholar
  5. 5.
    Krebs CF, Schmidt T, Riedel JH, Panzer U. T helper type 17 cells in immune-mediated glomerular disease. Nat Rev Nephrol. 2017;13(10):647–59.  https://doi.org/10.1038/nrneph.2017.112.CrossRefPubMedGoogle Scholar
  6. 6.
    Miossec P, Korn T, Kuchroo VK. Interleukin-17 and type 17 helper T cells. N Engl J Med. 2009;361(9):888–98.CrossRefPubMedGoogle Scholar
  7. 7.
    McGovern JL, Wright GP, Stauss HJ. Engineering specificity and function of therapeutic regulatory T cells. Front Immunol. 2017;8:1517.  https://doi.org/10.3389/fimmu.2017.01517.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Li J, Dong X, Zhao L, Wang X, Wang Y, Yang X, et al. Natural killer cells regulate Th1/Treg and Th17/Treg balance in chlamydial lung infection. J Cell Mol Med. 2016;20(7):1339–51.  https://doi.org/10.1111/jcmm.12821.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lee GR. The balance of Th17 versus Treg cells in autoimmunity. Int J Mol Sci. 2018;19(3):730.CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Lee W, Lee GR. Transcriptional regulation and development of regulatory T cells. Exp Mol Med. 2018;50(3):e456.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Schmidt A, Eriksson M, Shang MM, Weyd H, Tegner J. Comparative analysis of protocols to induce human CD4+Foxp3+ regulatory T cells by combinations of IL-2, TGF-beta, retinoic acid, rapamycin and butyrate. PLoS One. 2016;11(2):e0148474.  https://doi.org/10.1371/journal.pone.0148474.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Mohr A, Malhotra R, Mayer G, Gorochov G, Miyara M. Human FOXP 3+ T regulatory cell heterogeneity. Clin Transl Immunology. 2018;7(1):e1005.CrossRefGoogle Scholar
  13. 13.
    Karimi G, Vahabzadeh M, Lari P, Rashedinia M, Moshiri M. "Silymarin", a promising pharmacological agent for treatment of diseases.IJBMS . 2011;14(4):308–17.Google Scholar
  14. 14.
    Hellerbrand C, Schattenberg JM, Peterburs P, Lechner A, Brignoli R. The potential of silymarin for the treatment of hepatic disorders. Clin Phytosci. 2017;2(1):7.Google Scholar
  15. 15.
    Federico A, Dallio M, Loguercio C. Silymarin/silybin and chronic liver disease: a marriage of many years. Molecules. 2017;22(2):191.CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Ferenci P. Silymarin in the treatment of liver diseases: what is the clinical evidence? CLD. 2016;7(1):8–10.CrossRefGoogle Scholar
  17. 17.
    Gharagozloo M, Amirghofran Z. Effects of silymarin on the spontaneous proliferation and cell cycle of human peripheral blood leukemia T cells. J Cancer Res Clin Oncol. 2007;133(8):525–32.  https://doi.org/10.1007/s00432-007-0197-x.CrossRefPubMedGoogle Scholar
  18. 18.
    Tamayo C, Diamond S. Review of clinical trials evaluating safety and efficacy of milk thistle (Silybum marianum [L.] Gaertn.). Integrative Cancer Therapies. 2007;6(2):146–57.CrossRefPubMedGoogle Scholar
  19. 19.
    Morishima C, Shuhart MC, Wang CC, Paschal DM, Apodaca MC, Liu Y et al. Silymarin inhibits in vitro T-cell proliferation and cytokine production in hepatitis C virus infection. Gastroenterology. 2010;138(2):671–81, 81 e1–2.  https://doi.org/10.1053/j.gastro.2009.09.021.CrossRefGoogle Scholar
  20. 20.
    Polyak SJ, Morishima C, Shuhart MC, Wang CC, Liu Y, Lee DYW. Inhibition of T-cell inflammatory cytokines, hepatocyte NF-κB signaling, and HCV infection by standardized silymarin. Gastroenterology. 2007;132(5):1925–36.CrossRefPubMedGoogle Scholar
  21. 21.
    Schümann J, Prockl J, Kiemer AK, Vollmar AM, Bang R, Tiegs G. Silibinin protects mice from T cell-dependent liver injury☆. J Hepatol. 2003;39(3):333–40.CrossRefPubMedGoogle Scholar
  22. 22.
    Almasi E, Gharagozloo M, Eskandari N, Almasi A, Sabzghabaee AM. Inhibition of Apoptosis and Proliferation in T Cells by Immunosuppressive Silymarine. Iranian Journal of Allergy, Asthma and Immunology. 2017;16(2):107.Google Scholar
  23. 23.
    Gharagozloo M, Javid EN, Rezaei A, Mousavizadeh K. Silymarin inhibits cell cycle progression and mTOR activity in activated human T cells: therapeutic implications for autoimmune diseases. Basic Clin Pharmacol Toxicol. 2013;112(4):251–6.  https://doi.org/10.1111/bcpt.12032.CrossRefPubMedGoogle Scholar
  24. 24.
    Gharagozloo M, Karimi M, Amirghofran Z. Immunomodulatory effects of silymarin in patients with beta-thalassemia major. Int Immunopharmacol. 2013;16(2):243–7.  https://doi.org/10.1016/j.intimp.2013.04.016.CrossRefPubMedGoogle Scholar
  25. 25.
    Riccardi C, Nicoletti I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc. 2006;1(3):1458–61.  https://doi.org/10.1038/nprot.2006.238.CrossRefPubMedGoogle Scholar
  26. 26.
    Long AE, Tatum M, Mikacenic C, Buckner JH. A novel and rapid method to quantify Treg mediated suppression of CD4 T cells. J Immunol Methods. 2017;449:15–22.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Gharagozloo M, Velardi E, Bruscoli S, Agostini M, Di Sante M, Donato V, et al. Silymarin suppress CD4+ T cell activation and proliferation: effects on NF-κB activity and IL-2 production. Pharmacol Res. 2010;61(5):405–9.CrossRefPubMedGoogle Scholar
  28. 28.
    Kuwabara T, Ishikawa F, Kondo M, Kakiuchi T. The role of IL-17 and related cytokines in inflammatory autoimmune diseases. Mediat Inflamm. 2017;2017:1–11.  https://doi.org/10.1155/2017/3908061.CrossRefGoogle Scholar
  29. 29.
    Adeyemo O, Doi H, Rajender Reddy K, Kaplan DE. Impact of oral silymarin on virus- and non-virus-specific T-cell responses in chronic hepatitis C infection. J Viral Hepat. 2013;20(7):453–62.  https://doi.org/10.1111/jvh.12050.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Lovelace ES, Maurice NJ, Miller HW, Slichter CK, Harrington R, Magaret A, et al. Silymarin suppresses basal and stimulus-induced activation, exhaustion, differentiation, and inflammatory markers in primary human immune cells. PLoS One. 2017;12(2):e0171139.  https://doi.org/10.1371/journal.pone.0171139.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Dardalhon V, Korn T, Kuchroo VK, Anderson AC. Role of Th1 and Th17 cells in organ-specific autoimmunity. J Autoimmun. 2008;31(3):252–6.  https://doi.org/10.1016/j.jaut.2008.04.017.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Sonar SA, Lal G. Differentiation and transmigration of CD4 T cells in Neuroinflammation and autoimmunity. Front Immunol. 2017;8:1695.  https://doi.org/10.3389/fimmu.2017.01695.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Arellano B, Graber DJ, Sentman CL. Regulatory T cell-based therapies for autoimmunity. Discov Med. 2016;22(119):73–80.PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Immunology, Medical SchoolShiraz University of Medical SciencesShirazIran
  2. 2.Department of Immunology, Faculty of MedicineTehran University of Medical SciencesTehranIran
  3. 3.Department of Virology, School of Public HealthTehran University of Medical SciencesTehranIran
  4. 4.Autoimmune Disease Research CenterShiraz University of Medical SciencesShirazIran

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