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Identification of Baicalin as an immunoregulatory compound by controlling TH17 cell differentiation

PLoS ONE (2011) 6(2)
DOI: 10.1371/journal.pone.0017164
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Abstract

TH17 cells have been implicated in a growing list of inflammatory disorders. Antagonism of TH17 cells can be used for the treatment of inflammatory injury. Currently, very little is known about the natural compound controlling the differentiation of TH17 cells. Here, we showed that Baicalin, a compound isolated from a Chinese herb, inhibited TH17 cell differentiation both in vitro and in vivo. Baicalin might inhibit newly generated TH17 cells via reducing RORγt expression, and together with up-regulating Foxp3 expression to suppress RORγt-mediated IL-17 expression in established TH17 cells. In vivo treatment with Baicalin could inhibit TH17 cell differentiation, restrain TH17 cells infiltration into kidney, and protect MRL/lpr mice against nephritis. Our findings not only demonstrate that Baicalin could control TH17 cell differentiation but also suggest that Baicalin might be a promising therapeutic agent for the treatment of TH17 cells-mediated inflammatory diseases. © 2011 Yang et al.

Figures

  • Figure 1. Baicalin inhibits TH17 cell differentiation in vitro. (A) CD4 +CD252 T cells from B6 mice were stimulated with anti-CD3, anti-CD28, and the indicated cytokines in the presence (open columns) or absence (filled columns) of Baicalin. IL-17 mRNA was analyzed by real-time RT-PCR at the indicated times. Results were expressed as mean 6 SD, and fold induction compared with vehicle control (expression in vehicle control was set as 1.0). (B) IL-17 was examined by ELISA in culture supernatants after 3 days activation. Results were expressed as mean 6 SD, and compared with vehicle control. (C) CD4+CD252 T cells were cultured under TH17 conditions in the presence of indicated doses of Baicalin for 3 days, and then restimulated with PMA and Ionomycin in the presence of Brefeldin A for 5 hours. The percentages of IL-17+ cells among CD4+ T cells were examined by flow cytometry. These experiments were performed three times with similar results. doi:10.1371/journal.pone.0017164.g001
  • Figure 2. Baicalin inhibits IL-6R and RORct mRNA expression. (A) CD4+CD252 T cells from B6 mice were stimulated with anti-CD3, anti-CD28, and the indicated cytokines in the presence (open columns) or absence (filled columns) of Baicalin. IL-6R mRNA expression was analyzed by real-time RT-PCR at the indicated times. Results were expressed as mean 6 SD, and fold induction compared with vehicle control (expression in vehicle control was set as 1.0). (B) CD4+CD252 T cells were cultured under the indicated cytokines with or without 20 mM Baicalin for 2 days, IL-6R protein was measured by western blot analysis. (C) RORct mRNA expression was analyzed by real-time RT-PCR at the indicated times. Results were expressed as mean 6 SD, and fold induction compared with vehicle control (expression in vehicle control was set as 1.0). These experiments were performed three times with similar results. doi:10.1371/journal.pone.0017164.g002
  • Figure 3. Baicalin up-regulates Foxp3 and down-regulates RORct expression. (A) CD4+CD252 T cells from B6 mice were stimulated with anti-CD3, anti-CD28, and the indicated cytokines, after 2 days stimulation, Baicalin was added for additional 2 days. The percentages of Foxp3+ and IL17+ cells among CD4+ T cells were determined by flow cytometry. (B) CD4+CD252 T cells from B6 mice were stimulated with anti-CD3, anti-CD28, and the indicated cytokines, after 2 days stimulation, Baicalin was added for additional 2 days. Foxp3 and RORct mRNA expression were examined by RTPCR. (C) CD4+CD252 T cells from B6 mice were cultured under TH17 conditions for 2 days and then transiently transfected with control plasmids (Control) or Foxp3 expression plasmids (Foxp3) in the presence or absence of Baicalin. 2 days later, RORct, IL-17, and Foxp3 mRNA were examined by RT-PCR. doi:10.1371/journal.pone.0017164.g003
  • Figure 4. Baicalin inhibits TH17 cell differentiation in vivo. (A) MRL/lpr mice were treated with Baicalin or vehicle for 9 weeks, B6 mice (control) were treated with vehicle. Relative urine protein increases = urine protein (mg/L) at indicated time point - urine protein (mg/L) of week 0 (n = 6 for each group). In the comparison between Baicalin- and vehicle-treated mice, the asterisk indicates p,0.001. (B) Histopathology of kidneys. Hematoxylin and eosin staining (H&E, left) and periodic acid-Sciff-staining (PAS, right). Scale bar, 100 mm. (C) The renal score of control and MRL/lpr mice (n = 6 for each group). (D) Spleen index of control and MRL/lpr mice (n = 6 for each group). (E) IL-6R and RORct mRNA levels in spleen cells of MRL/lpr mice were analyzed by RT-PCR. (F) The percentages of IL-17+ and Foxp3+ cells among CD4+ T cells of spleen cells isolated from MRL/lpr mice were determined by flow cytometry analysis. (n = 6 for each group). Results were expressed as mean 6 SD. Spleens were shown. doi:10.1371/journal.pone.0017164.g004
  • Table 1. Survival data in MRL/lpr mice and B6 control.
  • Figure 5. Baicalin inhibits IL-17+ lymphocyte infiltration into kidney. (A) IL-17 immunohistochemical staining in kidneys from MRL/lpr mice treated with Baicalin or vehicle for 9 weeks, arrow shows the typical IL-17+ lymphocytes. Scale bar, 100 mm. Five independent microscopic fields were selected randomly for each sample to ensure representativeness and homogeneity. Right panel shows the IL-17+ cells counted under 6100 magnification. (B) CCL20 mRNA expression in kidney. (C) CD4+CD252 T cells from B6 mice were cultured under TH17 conditions with or without Baicalin for 3 days, CCR6 mRNA expression in TH17 cells was examined by RT-PCR. These experiments were performed three times with similar results. doi:10.1371/journal.pone.0017164.g005
  • Figure 6. Baicalin inhibits IL-17 mediated gene expression of inflammatory molecules. (A) HUVEC was cultured in the presence of 50 ng/ml IL-17 in the presence or absence of Baicalin for 24 hours. Expression of the indicated genes was examined by RT-PCR. (B) HUVEC was stimulated in the presence of IL-17 with or without Baicalin for 24 hours. Jurkat cells were added for an additional 24 hours. After washing twice, the adhesive cells were counted under 6200 magnifications. Five independent microscopic fields were selected randomly for each sample to ensure representativeness and homogeneity. Results were expressed as mean 6 SD. Arrows indicate the adhesive cells. Scale bar, 100 mm. These experiments were performed three times with similar results. doi:10.1371/journal.pone.0017164.g006
  • Table 2. The following primer pairs were used.

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Yang, J., Yang, X., Chu, Y., & Li, M. (2011). Identification of Baicalin as an immunoregulatory compound by controlling TH17 cell differentiation. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0017164

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