Tranilast attenuates OVX-induced bone loss in mice.

<p>Bone densities of the femora were measured from vehicle-treated (sham, n = 6; OVX, n = 6), Tranilast (200 mg/kg/d)-treated (sham, n = 6; OVX, n = 6) mice 8 weeks after surgery. The representative µCT images of distal femora (1.0 mm from the growth plate of the distal femur) (A). Number of OCs in the cultures of enriched BMM (B) and whole bone marrow (C) stimulated with RANKL/M-CSF and 1,25(OH)₂D₃ (C), respectively. a, <i>p</i><0.05; a’, <i>p</i><0.01 compared with vehicle-treated SHAM. b, <i>p</i><0.05 compared with vehicle-treated OVX. No significant difference between vehicle-treated SHAM and Tranilast-treated OVX (B, C).</p

Tranilast decreases OC formation induced by RANKL.

<p>(A) BMM were incubated with Tranilast (30, 50, 70 µM) in the presence of M-CSF (20 ng/ml) and RANKL (40 ng/ml). After 3 d, cells were fixed and stained for TRAP. Means of 4 groups are significantly different (<i>P</i><0.001). ***, <i>P</i><0.001 compared with V (vehicle)-treated cells. (B) Representative photos of A. Scale bar, 200 µm. (C) BMMs were incubated with Tranilast (70 µM) in the presence of M-CSF and RANKL for 48 h, total RNA was extracted and subjected to qPCR analysis for TRAP, calcitonin receptor (CTR), and c-Fos. *, <i>P</i><0.05; **, <i>P</i><0.01 with V. (D) RANKL-induced mature OC (∼1000 cells) was incubated with or without Tranilast (70 µM) on dentine slices for 24 h, and stained for pit formation. Representative photos of the resorption pits in V- and Tranilast-treated slices are shown. Scale bar, 50 µm. There was no significant difference between them in the areas of the resorption pits as determined with the ImageJ 1.37v program. Similar results were obtained in three independent experiments.</p

Trabecular microarchitecture and biochemical markers of OVX and SHAM mice treated with vehicle or Tranilast at 8 week after surgery.

<p>Data are represented as mean±SEM. Differences between each groups were analyzed by one-way ANOVA, followed by Bonferroni post tests. a, <i>p</i><0.05; a’, <i>p</i><0.01; a”, <i>p</i><0.001 compared with vehicle-treated SHAM. b, <i>p</i><0.05 compared with vehicle-treated OVX. No significantly difference between vehicle-treated SHAM vs. Tranilast-treated SHAM.</p

Tranilast impairs a RANKL signaling.

<p>(A) BMM (5×10⁶ cells/plate) was stimulated with vehicle (V) (lane 2) or RANKL (lane 3) with Tranilast (30 µM, lane 4; 70 µM, lane 5) for 1 h. Hundred-fold excess of unlabeled probe (lane 1) was used as a negative control. NF-Y DNA binding activity was measured as an internal control. (B–F) BMM with or without Tranilast (T, 70 µM) or TGF-β (10 ng/ml) were incubated with M-CSF and RANKL for 48 h (B–D, F) and 72 h for counting TRAP-positive MNCs (E). Total RNA was extracted and subjected to qPCR analysis for NFAT2 (B, F) or TGF-β (D). The expression level before RANKL treatment was set to be 1. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 compared with V. Whole cell extracts, cytoplasmic fractions, and nuclear fractions were harvested from cultured cells and subjected to Western blot analysis with specific Abs as indicated. Abs for β-actin and lamin B1 were used for the normalization of cytoplasmic and nuclear extracts, respectively. Numbers between the panels are ratios of the intensity of NFAT2 over β-actin (total and cytosolic) or lamin B1 (nucleus) (C). ***, <i>P</i><0.001 compared with V. Numbers above the histograms are ratios of the numbers of MNC formed in the presence of Tranilast to the numbers formed in its absence (E). There was a significant difference between TGF-β and TGF-β/TL (*** <i>P</i><0.001; E, * <i>P</i><0.05; F), whereas no significant difference between V and TGF-β/TL (E, F). Similar results were obtained in three independent experiments.</p

Tranilast decreases RANKL-induced ROS level.

<p>(A) Intracellular levels of ROS upon stimulation with RANKL in the presence or absence of Tranilast (30, 60 µM) for 48 h were determined using H₂DCFDA. ROS levels were quantified by flow cytometry. (B) BMM were stimulated with M-CSF and RANKL in the presence of Tranilast (30, 50, 70 µM) with or without DPI (5 nM) for 3 d for measurement of TRAP-positive MNCs. Means of Tranilast-treated 4 groups in the presence or absence of DPI are significantly different (<i>P</i><0.001). *** <i>P</i><0.001 compared with each vehicle (V)-treated cells. Numbers above the histograms are ratios of the number of MNC formed in the presence of Tranilast to the number formed in its absence. (C) BMMs were incubated with 50 µM of Tranilast in the presence of M-CSF and RANKL for the indicted periods; total RNA was extracted and subjected to qPCR analysis for HO-1 (8 h), Gpx-1 or PRX1 (24 h). Expression level before RANKL treatment was set to be 1. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 compared to V. (D, E) BMMs were transfected with siPRX1 or scRNA. Down-regulation of PRX1 by siRNA was confirmed by RT-PCR and qPCR. The expression levels obtained from scRNA-treated cells were set to be 1. ***, <i>P</i><0.001 compared to scRNA (D). After 24 h of transfection with siRNA cells were treated with Tranilast and stimulated with RANKL for 48 h for determination of ROS level or 72 h for counting TRAP-positive MNCs. * <i>P</i><0.05, ** <i>P</i><0.01 compared with V of scRNA-transfected cells. There was a significant difference between siPRX1-treanfected V and Tranilast (* <i>P</i><0.05; ROS, ** <i>P</i><0.01; TRAP-positive MNC), whereas no significant difference between scRNA-transfected V and siPRX1-transfected Tranilast (ROS, TRAP-positive MNC). (E). (F) BMM from HO-1^+/+ and HO-1^−/− mice were incubated in the presence of M-CSF and RANKL with Tranilast (30, 50, 70 µM). After 3 d, cells were fixed and stained for TRAP. Means of Tranilast-treated 4 groups in the presence or absence of HO-1 are significantly different (<i>P</i><0.001). ** <i>P</i><0.01, ***, <i>P</i><0.001 compared with V-treated cells. Numbers above the histograms are ratios of the number of MNC formed in the presence of Tranilast to the number formed in its absence. Similar results were obtained in three independent experiments.</p