Aciculatin inhibits IL-1β-induced G-CSF production in a concentration-dependent manner.

<p>(A) 1×10⁶ fibroblast-like synoviocytes (FLS) were incubated with or without 10 µM aciculatin for 30 min and then for 24 h with or without 10 ng/mL of IL-1β in the continued presence of aciculatin; thereafter, cell culture supernatants were assayed for cytokine levels by using a Milliplex® assay. (B) FLS were incubated with 0–30 ng/mL of IL-1β for 24 h, and then the culture supernatants were assayed for G-CSF by using ELISA. (C) FLS were incubated with 0, 1, or 10 µM aciculatin for 30 min, and then for 5 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. The G-CSF mRNA levels in the cells were measured using RT-PCR, and a control with only 10 µM aciculatin was included. (D) Cells were incubated for 30 min with 0–10 µM aciculatin and then for 24 h with 10 ng/mL of IL-1β in the continued presence of aciculatin, before the G-CSF in the culture supernatants were measured using ELISA. (E) The viability of the FLS was determined after 24 h treatment with 1–10 µM of aciculatin compared to the control group by using the MTT assay. Data are represented as mean ± SEM, with n = 3. *<i>p</i><0.05 and **<i>p</i><0.01 compared with the control group; #<i>p</i><0.05 and ##<i>p</i><0.01 for the comparisons of the groups indicated.</p

Aciculatin inhibits the phosphorylation of JAK, STAT, and Akt in FLS and the differentiation of 32Dcl3 cells.

<p>(A, C, and D) FLS were incubated with 0, 1, 3, or 10 µM aciculatin (A1, A3, and A10) for 30 min, and then for 24 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. Whole cell extracts were then prepared for western blot analysis for the indicated proteins (A and C); equal amounts of cell culture media (“conditioned medium”) were collected and concentrated 10-fold (v/v) (lanes 1–4) or PBS only (lane 5), and then immunoprecipitated with 1 µg of anti-G-CSF antibody, followed by immunoblot analysis using anti-G-CSF antibody or anti-β-actin antibody (as an internal control) (D). (B) FLS were incubated with 0 or 10 µM aciculatin for 30 min, and then for 1 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. The DNA binding activity of the nuclear extracts was then examined in an electrophoretic mobility shift assay using a specific STAT3 DNA probe. (E) Ten-fold concentrated conditioned medium was prepared from FLS incubated with or without aciculatin, and then with IL-1β as in (D), or with IL-1β plus an anti-G-CSF antibody. 32Dcl3 cells were incubated for 10 days with a medium containing 50% of these different conditioned mediums. The cells were then were subjected to Wright-Giemsa staining to detect neutrophils (top row) or washed twice with PBS, incubated at 4°C for 45 min with anti-CD11b FITC-conjugated and anti-CD11a/CD18 PE-conjugated antibodies, and their fluorescence was analyzed by FACScan flow cytometry (bottom row). Magnification  = ×100; scale bar  = 20 µm. In (A) and (C), the extents of indicated proteins expression were quantitated using a densitometer with the Image-Pro plus software, and the relative levels were calculated as the ratios of proteins to GAPDH or β-actin protein levels. The results are expressed as the mean ± SEM, with n = 3. *<i>p</i><0.05 and **<i>p</i><0.01 compared with the control group; #<i>p</i><0.05 and ##<i>p</i><0.01 for the comparisons of the groups indicated.</p

Aciculatin inhibits MAPK pathways.

<p>(A) Cells were incubated for 30 min with 0–10 µM aciculatin or 20 µM SP600125 (a JNK inhibitor), SB203580 (a p38 inhibitor), or PD98059 (an ERK inhibitor), and then for 30 min with 10 ng/mL of IL-1β in the continued presence of aciculatin or inhibitor; a control with only 10 µM aciculatin was used. The cells were then harvested, and whole cell extracts were prepared for western blot analysis of the indicated proteins. The extents of indicated proteins expression were quantitated using a densitometer with a scientific imaging system, and the relative levels were calculated as the ratios of indicated proteins to GAPDH protein levels. (B) The cells were incubated with 0 or 10 µM aciculatin for 30 min, and then for 1 h with 10 ng/mL IL-1β in the continued presence of aciculatin; a control with only 10 µM aciculatin was used. Nuclear extracts were then subjected to a DNA-binding reaction with biotinylated oligonucleotides specific for AP-1. The DNA binding activity of the AP-1 complex is indicated by an arrow. (C) Cells (1×10⁵ cells) were transiently transfected with 1 µg of p5xATF6-GL3 for 24 h, and then incubated with 0–10 µM aciculatin for 30 min. The cells were then incubated for 6 h with 10 ng/mL of IL-1β in the continued presence of aciculatin, following which luciferase activity was measured. (D) FLS were incubated for 30 min with a vehicle, 10 µM aciculatin, or different inhibitors as indicated (20 µM), and then for 24 h with 10 ng/mL of IL-1β in the continued presence of aciculatin or inhibitor, before the supernatants were assayed for G-CSF by ELISA. In (A), (C), and (D), the results are expressed as the mean ± SEM, with n = 3. *<i>p</i><0.05 and **<i>p</i><0.01 compared with the control group; #<i>p</i><0.05 and ##<i>p</i><0.01 for the comparisons of the groups indicated.</p

Aciculatin inhibits NF-κB activation in IL-1β-stimulated FLS.

<p>(A) FLS (1×10⁶ cells) were treated with 0–10 µM aciculatin or 20 µM PDTC for 30 min, and were then incubated for 30 min with 10 ng/mL of IL-1β in the continued presence of aciculatin or PDTC. The cells were then harvested, and whole cell extracts were subjected to western blot analysis for the indicated proteins. The bar graphs represent the ratio of phosphorylation of IKKα/β and of p65 proteins expression to the relative levels of GAPDH protein that were quantitated using a densitometer scanner and Image-Pro plus software. (B) FLS were incubated with 0 or 10 µM aciculatin for 30 min, and then for 1 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. The DNA binding activity of the nuclear extracts was then examined in an electrophoretic mobility shift assay using a biotinylated NF-κB DNA probe; controls included 10 µM aciculatin alone and the use of the unlabeled probe (“cold”) to compete for the binding of the labeled probe. (C) Cells (1×10⁵ cells) were transiently transfected with 1 µg of pGL4.32[<i>luc2P/</i>NF-κB-RE<i>/</i>Hygro] for 24 h and incubated with 0–10 µM aciculatin for 30 min prior to stimulation for 6 h with 10 ng/mL of IL-1β in the continued presence of aciculatin, and then luciferase activity was measured. A control with only 10 µM aciculatin was used. In (A) and (C), the results are expressed as the mean ± SEM, with n = 3. *<i>p</i><0.05 and **<i>p</i><0.01 compared with the control group; #<i>p</i><0.05 and ##<i>p</i><0.01 for the comparisons of the groups indicated.</p

Magnolol inhibits MAPK pathways and c-fos nuclear translocation.

<p>Cells were treated with 10 ng/mL of IL-1β for different time periods (A) or were treated with magnolol at the indicated concentration for 30 min, then stimulated with IL-1β in the continued presence of magnolol for 30 min (B). The cells were then harvested and whole cell extracts prepared for Western blot analysis for the indicated proteins. (C) Cells (1×10⁵ cells) were transiently transfected with 1 µg of p5xATF6-GL3 for 24 h, then incubated with 0–25 µg/mL magnolol for 30 min before activation with 10 ng/mL of IL-1β in the continued presence of magnolol for another 6 h, when luciferase activity was measured as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031368#s4" target="_blank">Materials and Methods</a>”. The results are expressed as the mean ± standard error of the mean (SEM), with n = 3. * <i>p</i><0.05 compared to the control group; #<i>p</i><0.05 for the comparison of the indicated groups. (D) Cells were incubated with vehicle or IL-1β (10 ng/mL) for 1 h or were pretreated with 25 µg/mL of magnolol for 30 min, then stimulated with IL-1β in the continued presence of magnolol for 1 h. Nuclear extracts were then subjected to a DNA-binding reaction with oligonucleotides specific for AP-1. The specific DNA-binding activity of the AP-1 complex is indicated by an arrow. (E) Cells (1×10⁵ cells) were incubated with or without 25 µg/mL of magnolol for 30 min, then stimulated with 10 ng/mL of IL-1β in the continued presence of magnolol for 1 h, when samples were prepared for confocal microscopy analysis. Scale bar = 20 µm.</p

Magnolol suppresses arthritis development in an adjuvant-induced arthritis (AIA) model.

<p>After onset of arthritis, rats were treated with 100 mg/kg magnolol or vehicle (on day 2) for a total of 14 days. (A) Swelling of the ankle joints of both paws were markedly suppressed by treatment of arthritic rats with 100 mg/kg magnolol, as compared to the vehicle group. (B) Both hind paw volumes of non-treated (basal) rats, vehicle-treated, and magnolol-treated rats were measured using a digital plethysmometer on the indicated day after AIA induction. (C) Hematoxylin and eosin staining of tissue specimens from the left ankle joints of vehicle-treated rats and magnolol-treated rats. The specimen from a vehicle-treated rat exhibits severe synovitis (arrowhead) and leukocyte (arrow) infiltration. Magnolol treatment inhibited both effects. (D) Body weight of non-treated (basal) rats, vehicle-treated, and magnolol-treated rats measured on the indicated day after AIA induction. (E) Quantification of cytokines in sera by ELISA on day 16. In (B) and (D), the results are expressed as the mean ± standard error of the mean (SEM), with n = 5. * <i>p</i><0.05 compared to the control group; #<i>p</i><0.05 for the comparison of the indicated groups.</p

Magnolol inhibits NF-κB activation in IL-1β-stimulated FLS.

<p>FLS (1×10⁶ cells) were treated with or without 10 ng/mL of IL-1β for the indicated times (A) or were treated with 0–25 µg/mL of magnolol for 30 min, then stimulated with 10 ng/mL IL-1β for 30 min in the continued presence of magnolol (B), then the cells were harvested and whole cell extracts subjected to Western blot analysis for the indicated proteins. (C) Cells (1×10⁵ cells) were transiently transfected with 1 µg of pGL4.32[<i>luc2P/</i>NF-κB-RE<i>/</i>Hygro] for 24 h and treated with 0–25 µg/mL magnolol for 30 min prior to stimulation with 10 ng/mL of IL-1β in the continued presence of magnolol for a further 6 h, then luciferase activity was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031368#s4" target="_blank">Materials and Methods</a>. The results are expressed as the mean ± standard error of the mean (SEM), with n = 3. * <i>p</i><0.05 compared to the control group; #<i>p</i><0.05 for comparison of indicated groups. (D) FLS were incubated with vehicle or 10 ng/mL of IL-1β or were pretreated with 25 µg/mL of magnolol for 30 min, then incubated with IL-1β in the continued presence of magnolol for 1 h, then the DNA binding activity of the nuclear extracts was examined in an electrophoretic mobility shift assay using a specific NF-κB DNA probe. (E) Cells (1×10⁵ cells) were left untreated or were treated with 25 µg/mL of magnolol for 30 min, then stimulated with 10 ng/mL of IL-1β in the continued presence of magnolol for 1 h, when samples were prepared for confocal microscopy analysis. Scale bar = 20 µm.</p

Chemical structure of magnolol.

<p>Chemical structure of magnolol.</p