Diacerein inhibits the synthesis of resorptive enzymes and reduces osteoclastic differentiation/survival in osteoarthritic subchondral bone: a possible mechanism for a protective effect against subchondral bone remodelling

Abstract

Introduction: Subchondral bone alterations represent an essential component of osteoarthritis (OA). Modifying the abnormal subchondral bone metabolism may be indicated to treat OA. We investigated the effect of diacerein and rhein on the changes occurring in subchondral bone during OA. To this end, we determined the drugs' effects on metalloprotease-13 (MMP-13) synthesis on subchondral bone and on the osteoblast signalling pathways. In osteoclasts, we studied MMP-13 and cathepsin K production as well as cell differentiation, proliferation, and survival.

Methods: The effect of diacerein/rhein on the production of subchondral bone MMP-13 was determined by enzyme-linked immunosorbent assay. Signalling pathways were evaluated on osteoblasts by Western blot. Osteoclast experiments were performed using cells from the pre-osteoclastic murine cell line Raw 264.7. Osteoclast MMP-13 and cathepsin K activities were determined by specific bioassays and differentiation of these cells quantified by tartrate-resistant acid phosphatase staining.

Results: Diacerein and rhein reduced, in a dose-dependent manner, the interleukin-1-beta (IL-1beta)-induced MMP-13 production in OA subchondral bone. This effect occurred through the inhibition of ERK1/2 (extracellular signal-regulated kinase-1/2) and p38. In osteoclasts, they significantly reduced the activity of MMP-13 and cathepsin K. Moreover, these drugs effectively blocked the IL-1beta effect on the osteoclast differentiation process and the survival of mature osteoclasts.

Conclusion: Altogether, these data suggest that diacerein/rhein could impact the abnormal subchondral bone metabolism in OA by reducing the synthesis of resorptive factors and osteoclast formation.

Figure 1

Representative immunohistochemical staining section for (a) metalloprotease-13 (MMP-13) and (b) cathepsin K in human osteoarthritis subchondral bone. MMP-13 was detected in the osteoblasts (Ob) as well as in the osteoclasts (Oc). Cathepsin K was detected only in osteoclasts. Original magnification, ×100.

Figure 2

Effect of diacerein and rhein on metalloprotease-13 (MMP-13) production in human osteoarthritis subchondral bone. Subchondral bone explants were incubated for 5 days with or without interleukin-1-beta (IL-1β) (5 ng/mL) and diacerein or rhein (10 or 20 μg/mL). Data are expressed as fold changes compared with IL-1β-treated control, which was assigned a value of 1. Statistical analysis was performed versus IL-1β-treated control.

Figure 3

Effect of diacerein and rhein on subchondral bone osteoblast intracellular mitogen-activated protein (MAP) kinase pathways. Subchondral bone osteoblasts were pre-incubated for 2 hours with diacerein or rhein at 20 μg/mL and incubated for 30 minutes in the presence or absence of interleukin-1-beta (IL-1β) (100 pg/mL). Levels of phosphorylated (a) extracellular signal-regulated kinase-1/2 (ERK1/2), (b) p38, and (c) stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) (p46 and p54) MAP kinases were studied by Western blot and quantified by densitometry as described in Materials and methods. Data are expressed as fold changes compared with IL-1β-treated control, which was assigned a value of 1. Statistical analysis was performed versus IL-1β-treated control.

Figure 4

Effect of diacerein and rhein on the osteoclastic levels of (a) metalloprotease-13 (MMP-13) and (b) cathepsin K. Determination was performed in the conditioned medium for MMP-13 and on cell lysates for cathepsin K. Raw 264.7 cells were incubated for 5 days with RANKL (100 ng/mL), allowing the cells to differentiate into osteoclasts. After this period, the cells were incubated for 2 days together with RANKL in the presence or absence of interleukin-1-beta (IL-1β) (100 pg/mL) and diacerein or rhein (10 or 20 μg/mL). Data are expressed as fold changes compared with IL-1β-treated control, which was assigned a value of 1. Statistical analysis was performed versus IL-1β-treated control. RANKL, receptor activator of nuclear factor-κB ligand.

Figure 5

Effect of diacerein and rhein on osteoclast survival. Raw 264.7 cells were incubated for 5 days with RANKL (100 ng/mL) and for an additional 2 days together with RANKL in the presence or absence of interleukin-1-beta (IL-1β) (100 pg/mL) and diacerein or rhein (20 μg/mL). The number of differentiated osteoclasts was determined by the tartrate-resistant acid phosphatase staining assay. Data are expressed as fold changes compared with IL-1β-treated control, which was assigned a value of 1. Statistical analysis was performed versus IL-1β-treated control. RANKL, receptor activator of nuclear factor-κB ligand.

Figure 6

Effect of diacerein and rhein on osteoclast (a) proliferation/differentiation and (b) total cells. Raw 264.7 cells were incubated for 7 days with RANKL (100 ng/mL) in the presence or absence of interleukin-1-beta (IL-1β) (100 pg/mL) and diacerein or rhein (20 μg/mL). The number of differentiated osteoclasts was determined by the tartrate-resistant acid phosphatase staining assay. Data are expressed as fold changes compared with IL-1β-treated control, which was assigned a value of 1. Statistical analysis was performed versus IL-1β-treated control. RANKL, receptor activator of nuclear factor-κB ligand.