Effect of CORM-2 on oxidative stress levels in OA synoviocytes.

<p>Cells were incubated with IL-1β (10 ng/ml) in the presence or absence of CORM-2 (100, 200 µM) or RuCl₃ (200 µM) for 30 min. Oxidative stress was measured by the oxidation of dihydrorhodamine 123. Rhodamine positive cells were counted in 6–8 microscopic power fields. Data are expressed as % of oxidative stress. Left panels: oxidative stress, right panels: DAPI. Duplicate samples from 3 patients were used. +<i>P</i><0.05 with respect to nonstimulated cells. *<i>P</i><0.05 with respect to IL-1β. Original magnification: x200.</p

Effect of CORM-2 on synoviocyte proliferation and migration rate.

<p>Cells were stimulated with IL-1β (10 ng/ml) for 24 h in the presence or absence of CORM-2 (50, 100, 200 µM) or RuCl₃ (200 µM). (A) Cell proliferation was determined by the MTT assay. (B) Transwell chambers were kept in culture for 24 h. Upper compartment was detached and cells migrated to the lower side were counted in 6–8 microscopic power fields. Data (A) are expressed as mean±S.E.M. of % proliferating cells, considering 100% the proliferation induced in basal conditions whereas data (B) are expressed as % of cells migrated to the lower compartment, considering 100% the migration induced by IL-1β. Duplicate samples from 6 (A) and 4 (B) patients were used. +<i>P</i><0.05, ++<i>P</i><0.01 with respect to nonstimulated cells. **<i>P</i><0.01 with respect to IL-1β.</p

Effect of CORM-2 on MMP mRNA levels in OA synoviocytes.

<p>(A) MMP-1, (B) MMP-3 mRNA levels. Cells were stimulated with IL-1β (10 ng/ml) for 16 h in the presence or absence of CORM-2 (100, 200 µM) or RuCl₃ (200 µM). mRNA expression was measured by real-time PCR. Data are expressed as mean±S.E.M. Duplicate samples from 6 patients were used. ++<i>P</i><0.01 with respect to nonstimulated cells. *<i>P</i><0.05, **<i>P</i><0.01 with respect to IL-1β.</p

Effect of CORM-2 on HO-1 protein (A) and mRNA expression (B) in OA synoviocytes.

<p>Cells were stimulated with IL-1β (10 ng/ml) for 24 h (A) and 16 h (B) in the presence or absence of CORM-2 (50, 100, 200 µM) or RuCl₃ (200 µM). Protein expression was determined in cell lysates by Western blotting and mRNA levels were determined by real-time PCR. Relative expression of HO-1 and β-actin protein bands was calculated after densitometric analysis. Data are expressed as mean±S.E.M. Samples from 4 patients were used. ++<i>P</i><0.01 with respect to nonstimulated cells. **<i>P</i><0.01 with respect to IL-1β.</p

Effect of CORM-2 on chemokine mRNA levels in OA synoviocytes.

<p>(A) IL-8, (B) CCL2 and (C) CCL20 mRNA levels. Cells were stimulated with IL-1β (10 ng/ml) for 16 h in the presence or absence of CORM-2 (100, 200 µM) or RuCl₃ (200 µM). mRNA expression was determined by real-time PCR. Data are expressed as mean±S.E.M. Duplicate samples from 3 patients were used. ++<i>P</i><0.01 with respect to nonstimulated cells. *<i>P</i><0.05, **<i>P</i><0.01 with respect to IL-1β.</p

Effect of CORM-2 on NF-κB and AP-1 activation in OA synoviocytes.

<p>(A) NF-κB binding to DNA, (B) NF-κB p65 translocation (left panels: p65, right panels: DAPI), (C) IκBα phosphorylation and (D) AP-1 binding to DNA. Cells were stimulated with IL-1β (10 ng/ml) for 30 min (C) or 1 h (A, B, D) with IL-1β (10 ng/ml) in the presence or absence of CORM-2 (100, 200 µM). IκBα phosphorylation and NF-κB/AP-1 binding to DNA were determined by ELISA in cytosolic and nuclear fractions respectively, whereas p65 translocation was analyzed by immunofluorescence. Original magnification x400. Data are expressed as mean±S.E.M. of independent cultures with cells from 3 different donors. AU = arbitrary units. +<i>P</i><0.05, ++<i>P</i><0.01 with respect to nonstimulated cells. *<i>P</i><0.05, **<i>P</i><0.01 with respect to IL-1β.</p

Effect of CORM-2 on MMP activity and MMP levels released into the culture medium in OA synoviocytes.

<p>(A) MMP activity, (B) MMP-1 protein, (C) MMP-3 protein levels in the culture medium. Cells were stimulated with IL-1β (10 ng/ml) for 24 h in the presence or absence of CORM-2 (50, 100, 200 µM) or RuCl₃ (200 µM). MMP activity was analyzed by fluorometric procedures in cell supernatants (A) and protein levels were determined by ELISA in cell supernatants (B–C), Data are expressed as mean±S.E.M. Duplicate samples from 6 patients were used. ++<i>P</i><0.01 with respect to nonstimulated cells. *<i>P</i><0.05 with respect to IL-1β.</p

Effects of specific inhibitors of MAPK, Akt and NF-κB on IL-1β induced cell proliferation and chemokine and MMP mRNA expression in OA synoviocytes.

<p>(A) Cell proliferation, (B) IL-8, (C) CCL2, (D) CCL20, (E) MMP-1, (F) MMP-3 mRNA expression. Cells were treated with UO126 (10 µM), SP600125 (25 µM), SB202190 (10 µM), MK2206 (20 µM) or PS1145 (10 µM), specific inhibitors of MEK/ERK1/2, JNK1/2, p38, Akt and IκB kinase, respectively, 1 h before adding IL-1β for a total incubation time of 24 h (A) or 16 h (B–F). Cell proliferation was determined by the MTT method whereas mRNA expression was analyzed by real-time PCR. Data are expressed as mean±S.E.M. Duplicate samples from 3 patients were used. +<i>P</i><0.05, ++<i>P</i><0.01 with respect to nonstimulated cells. *<i>P</i><0.05, **<i>P</i><0.01 with respect to IL-1β.</p

Effect of CORM-2 and IL-1β on Akt and MAPK phosphorylation in OA synoviocytes.

<p>Cells were stimulated with IL-1β (10 ng/ml) for 5 min in the presence or absence of CORM-2 (100, 200 µM). Protein expression was determined in cell lysates by Western blotting using specific antibodies against phosphorylated or total proteins. Relative expression of phosphorylated and total protein bands was calculated after densitometric analysis. AU = arbitrary units. Data are expressed as mean±S.E.M. (samples from 3 patients). ++<i>P</i><0.01 with respect to nonstimulated cells. *<i>P</i><0.05, **<i>P</i><0.01 with respect to IL-1β.</p