Figure 1.
mRNA levels and protein expression of ARs in T/C-28a2 chondrocytes.
(A) Relative A1, A2A, A2B and A3AR mRNA levels in T/C-28a2 cells untreated or treated with PEMFs for 24 hours, normalized by using GAPDH mRNA as internal control. (B) Representative Western blotting analysis showing the immunoblot signals of A1, A2A, A2B and A3ARs in T/C-28a2 chondrocytes treated or untreated with PEMFs for 24 hours. (C) Densitometric analysis showing the ratio between ARs and β-actin. Data are reported as the mean ± SEM of six independent experiments. *, p<0.01.
Figure 2.
mRNA levels and protein expression of ARs in hFOB 1.19 osteoblasts.
(A) Relative A1, A2A, A2B and A3AR mRNA levels in hFOB 1.19 cells untreated or treated with PEMFs for 24 hours, normalized by using GAPDH mRNA as internal control. (B) Representative Western blotting analysis showing the immunoblot signals of A1, A2A, A2B and A3ARs in hFOB 1.19 osteoblasts treated or untreated with PEMFs for 24 hours. (C) Densitometric analysis showing the ratio between ARs and β-actin. Data are reported as the mean ± SEM of six independent experiments. *, p<0.01.
Figure 3.
PEMF exposure up-regulated A2A and A3ARs in T/C-28a2 chondrocytes.
Saturation curves and Scatchard plots of [3H] ZM 241385 binding to A2AARs (A, B) and of [3H] MRE 3008F20 binding to A3ARs (C, D) on membranes from T/C-28a2 chondrocyte cells before and after 24 hours of PEMF exposure. Results are reported as the mean ± SEM of six independent experiments. *, p<0.01 vs untreated T/C-28a2 chondrocytes.
Table 1.
Affinity and density of A1, A2A, A2B and A3ARs in untreated or PEMF treated T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts.
Figure 4.
PEMF exposure up-regulated A2A and A3ARs in hFOB 1.19 osteoblasts.
Saturation curves and Scatchard plots of [3H] ZM 241385 binding to A2AARs (A, B) and of [3H] MRE 3008F20 binding to A3ARs (C, D) on membranes from hFOB 1.19 osteoblast before and after 24 hours of PEMF exposure. Results are reported as the mean ± SEM of six independent experiments. *, p<0.01 vs untreated hFOB 1.19 osteoblasts.
Table 2.
Affinity and density of A2A and A3ARs in untreated or PEMF treated T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts before and after IL-1β treatment.
Figure 5.
A2A and A3AR immunofluorescence staining in T/C-28a2 and hFOB 1.19 cells.
Effect of PEMF treatment on T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts on A2A and A3AR expression, determined by immunofluorescence experiments. DAPI was used for counterstaining of nuclei. A2A (A, B) and A3AR (C, D) expression on T/C-28a2 chondrocytes in control condition (A, C) and after 24 hours exposure to PEMFs (B, D). A2A (E, F) and A3AR (G, H) expression on hFOB 1.19 osteoblasts in control condition (E, G) and after 24 hours exposure to PEMFs (F, H). Original magnification 400×.
Figure 6.
Modulation of cAMP production and cell proliferation in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts.
Effect of CGS 21680 (100 nM) and Cl-IB-MECA (100 nM) on cAMP production in the absence or in the presence of SCH 442416 (1 µM) or MRS 1334 (1 µM) before and after PEMF exposure for 24 hours in T/C-28a2 chondrocytes (A) and in hFOB 1.19 osteoblasts (B). Forskolin (1 µM) was used to better evaluate the inhibitory effect of Cl-IB-MECA. Effect of A2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 442416, 1 µM) or A3AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1334, 1 µM) on cell proliferation in the absence or in the presence of PEMF exposure for 24 hours in T/C-28a2 chondrocytes (C) and in hFOB 1.19 osteoblasts (D). Results are reported as the mean ± SEM of four independent experiments. *, p<0.05; **, p<0.01.
Figure 7.
Inhibition of IL-6, IL-8, PGE2 and VEGF by A2A or A3AR agonists and PEMFs in T/C-28a2 chondrocytes.
Effect of A2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 442416, 1 µM) or A3AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1334, 1 µM) on IL-6 (A), IL-8 (B), PGE2 (C) or VEGF (D) production in the absence or in the presence of PEMF exposure for 24 hours in T/C-28a2 chondrocytes stimulated with IL-1β (1 ng/ml). Results are reported as the mean ± SEM of four independent experiments. *, p<0.05; **, p<0.01.
Figure 8.
Modulation of IL-6, IL-8, PGE2 and OPG by A2A or A3AR agonists and PEMFs in hFOB 1.19 osteoblasts.
Effect of A2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 442416, 1 µM) or A3AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1334, 1 µM) on IL-6 (A), IL-8 (B), PGE2 (C) or OPG (D) production in the absence or in the presence of PEMF exposure for 24 hours in T/C-28a2 chondrocytes stimulated with IL-1β (1 ng/ml). Results are reported as the mean ± SEM of four independent experiments. *, p<0.05; **, p<0.01.
Figure 9.
Inhibition of NF-kB activation by A2A or A3AR agonists and PEMFs in T/C-28a2 and hFOB 1.19 cells.
Effect of A2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 442416, 1 µM) or A3AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1334, 1 µM) on NF-kB p65 subunit activation in the absence or in the presence of PEMF exposure for 24 hours in T/C-28a2 chondrocytes (A) or in hFOB 1.19 osteoblasts (B) stimulated with IL-1β (1 ng/ml). Results are reported as the mean ± SEM of four independent experiments. *, p<0.05; **, p<0.01.
Figure 10.
Proposed mechanism of anti-inflammatory effect of PEMFs through the up-regulation of A2A and A3ARs in T/C-28a2 and hFOB 1.19 cells.
PEMF exposure induce an up-regulation of A2A and A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblast. As a consequence, the anti-inflammatory effects of A2A (red arrows) and A3AR (green arrows) activation (on the right) are enhanced in the presence of PEMF exposure (on the left) resulting in a further inhibition of pro-inflammatory cytokines, NF-kB, VEGF, PGE2. PEMFs increased the effect of A2AAR stimulation on cell proliferation in both cell lines and determined a marked production of OPG in hFOB 1.19 osteoblasts (black arrow).