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Rheumatology Advance Access originally published online on March 27, 2008
Rheumatology 2008 47(5):627-633; doi:10.1093/rheumatology/ken116
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Diacerein has a weak effect on the catabolic pathway of human osteoarthritis synovial fibroblast—comparison to its effects on osteoarthritic chondrocytes

M. A. Álvarez-Soria, G. Herrero-Beaumont, O. Sánchez-Pernaute, M. Bellido and R. Largo

Joint and Bone Research Unit, Fundación Jiménez Díaz, Autonomous University, Madrid, Spain.

Correspondence to: R. Largo, Joint and Bone Research Unit, Rheumatology Department, Fundación Jiménez Díaz, Avenida Reyes Católicos 2, 28040 Madrid, Spain. E-mail: rlargo{at}fjd.es


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. Synoviocytes play a crucial role in the inflammatory response leading to structural damage in OA. Our aim was to assess the effects of diacerein and NSAIDs on cellular responses of synoviocytes associated with inflammation and structural integrity of cartilage in OA.

Methods. The effects of diacerein, celecoxib, diclofenac, meloxicam and indomethacin on prostaglandin (PG) E2 production, cyclo-oxygenase-2 (COX-2) protein expression, nitrite levels, presence of MMP-1 and -13, and activation of nuclear factor-{kappa}B (NF-{kappa}B) were studied on stimulated OA synoviocytes and chondrocytes.

Results. Diacerein and NSAIDs inhibited IL-1β-stimulated NF-{kappa}B activation in synoviocytes and chondrocytes except indomethacin in synoviocytes. Diacerein further increased COX-2 protein expression and PGE2 synthesis in synoviocytes stimulated with IL-1β, while no effect was observed on stimulated chondrocytes. NSAIDs diminished until almost basal levels PGE2 release in both cells and, surprisingly, these drugs also diminished COX-2 protein expression both in synoviocytes and chondrocytes. With regard to structural mediators, diacerein decreased MMP-13 levels in synoviocytes but did not modify MMP-1 presence. NSAIDs induced a significant increase in MMP-1 levels in both cell types and in MMP-13 levels in chondrocytes.

Conclusions. Diacerein does not seem to reduce but rather increase inflammatory mediators in synoviocytes, while it does not overall affect chondrocyte inflammatory profile.

KEY WORDS: Osteoarthritis, Cytokines and inflammatory mediators, Extracellular matrix, Synovium, Non-steroidal anti-inflammatory drugs


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Destruction and loss of articular cartilage is a prominent feature in OA. However, pain and disability in patients with OA result from processes involving multiple tissues that contribute to joint structure and function [1]. Although synovial inflammation in OA is not as extensive as that observed in other chronic arthritis, there is mounting evidence that synovitis contributes to the symptomatic expression at least in a substantial subset of patients [2]. Moreover, synovial inflammation is an important source of pro-inflammatory mediators and plays an important role in the progression of the disease [3, 4]. IL-1β produced by chondrocytes and synovial cells is locally increased in OA and induces a large cascade of events leading to cartilage damage [5, 6]. In vitro, IL-1β is able to reproduce pro-inflammatory and extracellular matrix degradation processes that are activated in vivo during OA. Both nitric oxide (NO) and prostaglandin E2 (PGE2) produced in large concentrations in OA joints, impair the proliferation of chondrocytes and synoviocytes and enhance the activity of MMPs [7–9].

Until the introduction of the newest symptomatic slow-acting drugs for OA (SYSADOA), cyclooxygenase (COX) inhibitors were the most common agents used for the control of OA symptoms. In spite of the prominent role of PGE2 production in this disease, the chronic use of agents targeting the COX pathway has for the moment failed to slow OA progression in clinical practice. Compounds in the SYSADOA class include glucosamine-containing compounds, chondroitin sulphate and diacerein. Diacerein is a widely employed drug for the treatment of OA in some European countries. Diacerein, and its active metabolite, rhein, is an anthraquinone derivate that refrain the expression of IL-1 in lipopolysaccharide-activated human OA chondrocytes and synoviocytes [10]. It has been recently suggested that diacerein could have anti-inflammatory properties different from those of classical NSAIDs due to its ability to reduce acute inflammation in different animal models [11]. Rhein has been shown to reduce IL-1β induction of the inducible NO synthase (iNOS) synthesis and to enhance COX-2 production and activity in normal and osteoarthritic human chondrocytes [12]. In addition, rhein have been reported to reduce IL-1-stimulated extracellular signal-regulated kinase (ERK) signal transduction, as well as activation protein (AP1) and nuclear factor-{kappa}B (NF-{kappa}B) transcription factors activation and to enhance the synthesis of matrix components, such as type II collagen and aggrecan in bovine articular chondrocytes [13]. Most studies of the effect of diacerein on joint cells have been focused on the metabolism of chondrocytes, whereas response of synovial fibroblasts has been poorly characterized.

The effects of diacerein as symptom- and structure-modifying drug for OA have been evaluated in several clinical trials [14–16]. In a recent meta-analysis [14], it is concluded that although diacerein can have a certain benefit improving pain in patients with OA, this is a mild-size effect. Two long-term studies assessed structural progression with radiographic measurements of hip [15] and knee joint space [16] in patients with OA. Surprisingly, while the administration of diacerein was associated with a statistically significant decrease of progression at the hip level [15], no changes were demonstrated at the knee level [16].

The effect of diacerein on synovial cell response remains mainly unknown. This study was designed to assess the effects of this molecule and NSAIDs on synovial inflammatory and structural mediator's response from patients with knee OA in comparison with their effects on chondrocytes.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Cell culture
Synovial fibroblast and chondrocytes were obtained from the knee synovial membranes and the articular cartilage of patients with OA undergoing joint replacement surgery at the Orthopaedic Surgery Department of the Fundación Jiménez Díaz. The study was approved by the ethical committee from the Fundación Jiménez Díaz. Written informed consent was obtained from all patients. Negative binding with anti-Factor VIII (specific for entothelial cells; Biomeda, Foster City, CA, USA) [17] and KP-1 (anti-CD68, specific for macrophages; Dako, Glostrup, Denmark) [18] confirmed the presence of a unique fibroblast-like population in the synovial cultures. Chondrocytes were characterized by glycosaminoglycan synthesis (O-Safranin and Alcian blue/PAS) and positive staining for collagen type II and aggrecan [19]. Synoviocytes were used between passages sixth and twelfth, and chondrocytes between passages first and second. Only cartilage or synovial samples from a unique donor were employed for each culture. Thus, for each experiment, cells come from the same patient.

In each experiment, 48 h serum-deprived cells were stimulated with 10 U/ml IL-1β (Immunogenex, LA, CA, USA). Where indicated, cells were pre-incubated with diacerein (10–5 M, Sigma, St. Louis, MO, USA), celecoxib (CBX, 10–6M, Pfizer, NY, USA), diclofenac (DCF, 10–6 M, Sigma), meloxicam (MXC, 10–6M, Boehringer Ingelheim, Mannheim, Germany), and indomethacin (IND, 10–6 M, Sigma) for 60 min, and these compounds were maintained during the whole period of incubation. Diacerein and NSAIDs were tested at the concentration corresponding to the mean peak plasma concentration after oral administration of a single therapeutic dose [20–24]. Viability was checked by Trypan blue exclusion.

Prostaglandin E2 assay
Cell-conditioned media (cultured at 50 000 cells/well in 96-well plates in 150 µl media) was collected 24 h after IL-1β addition and then stored at –80°C until analysis. PGE2 content was measured by EIA (Assay Designs Inc., Michigan, USA) according to the manufacturer's instructions. The sensitivity of PGE2 EIA was 36 pg/ml.

Nitrite assay
The quantity of nitrites in the culture medium was measured by the Griess method. Quiescent cells were incubated with 10 U/ml IL-1β with or without drugs for 24 h, and equal volumes of supernatant and Griess reagent (1% sulphanilamide, 0.1% N-1-naphthylethylenediamide dihydrochloride in 5% H3PO4) were incubated at room temperature for 10 min. The absorbance was measured with a spectrophotometer at 570 nm. The concentration of nitrites was calculated using a standard curve made by successive dilutions of a solution of sodium nitrite (Fluka, Buchs, Switzerland).

Western blot analysis
For protein studies, quiescent cells were incubated with 10 U/ml IL-1β with or without drugs for 24 h. The COX-2 determinations were carried out as previously described [25]. Equal amount of total cell proteins (for COX-2) or cell culture supernatants (for MMP-1 and -13) were run on SDS–PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Molsheim, France). Membranes were blocked in 0.1 mmol/l Tris, pH 7.4, and 0.1 mmol/l NaCl containing 0.3% Tween-20 and 6% dry skimmed milk for 60 min at room temperature and incubated overnight with anti-COX-2 (Santa Cruz Biotech, Heidelberg, Germany), anti-MMP-1 (Chemicon, CA, USA), and anti-MMP-13 (Calbiochem, EMD Biosciences, La Jolla, CA, USA) antibodies at 4°C. Both MMP antibodies specifically react with precursor and active forms of human MMP-1 and -13. After washing, detections were made by incubation with peroxidase-conjugated secondary antibodies and developed by an enhanced chemiluminiscence kit (ECL, Amersham, Buckinghamshire, UK). For total cell protein experiments, membranes were incubated with anti-{alpha}-tubulin (Sigma) as control.

Electrophoretic mobility shift assay
For nuclear factor studies, cells were pre-incubated with the different drugs tested for 1 h and then stimulated with IL-1β for another 1 h. Nuclear extracts were obtained as previously described [25].

For NF{kappa}B binding assay, consensus oligonucleotide (Promega, Madison, WI, USA) was [32P] end-labelled with g-[32P]ATP and T4 polynucleotide kinase (Promega). Nuclear extracts were equilibrated for 10 min in a binding buffer [25] and then the labelled probe was added and incubated for 20 min at room temperature. The reaction was stopped by adding gel-loading buffer (250 mmol/l Tris–HCl, 0.2% bromophenol blue, 0.2% xylene cyanol and 40% glycerol) and run on a non-denaturing 4% acrylamide gel in Tris–Borate. The gel was dried and exposed to X-ray film.

Statistical analysis
Densitometric results, expressed as arbitrary units (AU) as n-fold over control, PGE2 concentration (as picograms/ml) and nitrite levels (as micromolar) are expressed as the mean ± S.E.M. All statistical analyses were performed using SPSS 8.0 for Windows (SPSS Inc). Data from multiple groups were compared using Kruskal–Wallis and Mann–Whitney non-parametric analyses as appropriate. Statistical significance was set at P-value < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Direct effect of incubating cells with drugs tested
Viability studies were carried out for all the experimental conditions of the study. Previous studies have demonstrated that at concentrations of 10–5 M rhein did not significantly affect the cell death score after 3 days of culture [26]. In our experimental conditions, none of the molecules were found to alter cell survival as tested by Trypan blue exclusion (viability in all cases being >90%) (data not shown). In all experiments, cells were exposed to each of the drugs in the absence of IL-1β treatment. Except otherwise stated, none of the compounds induced a direct effect upon basal cell expression of the molecules that were the purpose of this study at the concentrations and incubation periods employed.

PGE2 production
The release of PGE2 was measured after 24 h in supernatants of IL-1β-activated cells. In both cell types, IL-1β stimulation caused a significant increase in PGE2 concentration (Fig. 1A and B). In synoviocytes, diacerein not only did not diminish PGE2 levels but also elicited a further increase in the IL-1β-induced release of this pro-inflammatory mediator to the cell supernatants (Fig. 1A). In contrast, the four different NSAIDs were able to abolish completely PGE2 release in synovial cells exposed to IL-1β (100% inhibition for all of them). In chondrocytes, diacerein did not produce changes in the IL-1β-induced PGE2 release (Fig. 1B), whereas in the presence of NSAIDs, there was a significant decrease in PGE2 levels in IL-1β-treated chondrocytes (100% inhibition in all cases) compared with IL-1β-stimulated cells (Fig. 1B).


Figure 1
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  		FIG. 1. Effect of the different compounds on PGE2 (A, B) and nitrite (C) presence in cell supernatants. Cells were pre-incubated for 60 min with diacerein (DC, 10–5 M), CBX (10–6 M), DCF (10–6 M), MXC (10–6 M) and IND (10–6 M) and then stimulated with 10 U/ml IL-1β for 24 h. Conditioned media was collected after 24 h for PGE2 EIA and nitrite measurement. (A) PGE2 in synoviocyte culture, (B) PGE2 in chondrocyte culture and (C) nitrite levels in chondrocyte culture (grey bars correspond to stimulated treated cells; white bars correspond to treated cells). Concentrations are shown as mean ± S.E.M. of three independent experiments. In each of them, PGE2 and nitrites were measured in triplicate; *P < 0.05 vs basal, #P < 0.05 vs IL-1β alone.

 
Nitrite levels
Neither quiescent nor IL-1β-stimulated synoviocytes rendered detectable nitrite levels at supernatants of cell culture after 24 h of incubation. Chondrocyte stimulation with IL-1β resulted in a 25-fold increase of nitrite levels to supernatants at 24 h of stimulation. The incubation of cells with diacerein inhibited the effect of IL-1β on nitrite release, while none of the NSAID tested altered IL-1β-induced nitrite release (Fig. 1C).

Cox-2 protein expression
At 24 h of incubation, IL-1β-induced 55-fold and 29-fold increases in COX-2 protein levels in synoviocytes and chondrocytes, respectively (Fig. 2). In synoviocytes, consistent with the increase observed in PGE2 production, diacerein did not diminish COX-2 levels but induced a further increase in the up-regulation of COX-2 protein expression with IL-1β (P = 0.013). NSAIDs produced a significant inhibition of IL-1β-enhanced COX-2 protein expression in synoviocytes (P = 0.008 in all cases) (Fig. 2A). Diacerein was not able to inhibit the up-regulation of COX-2 protein expression in IL-1β-stimulated chondrocytes, whereas all NSAIDs reduced the levels of COX-2 protein in IL-1β-stimulated chondrocytes as had been found in synoviocytes (P = 0.004 in all cases, Fig. 2B).


Figure 2
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  		FIG. 2. Effect of drugs in cells stimulated with IL-1β on COX-2 protein expression. Cells were pre-incubated for 60 min with diacerein (DC, 10–5 M), CBX (10–6 M), DCF (10–6 M), MXC (10–6 M) and IND (10–6 M) and then stimulated with 10 U/ml IL-1β for 24 h. Upper panels: representative western blots corresponding to hybridization with anti-COX-2 and anti-{alpha}-tubulin antibodies for synoviocytes (A) and chondrocytes (B). Lower panels: densitometric analyses of COX-2 levels in six different experiments, corrected to those of {alpha}-tubulin for synoviocytes (A) and chondrocytes (B) (grey bars correspond to stimulated treated cells; white bars correspond to treated cells) (Mean ± S.E.M.); *P < 0.05 vs basal, #P < 0.05 vs IL-1β alone.

 
MMP-1 and -13 protein expression
After 24 h of incubation, IL-1β elicited a significant increase of the presence of MMP-1 in the culture medium both in synoviocytes and chondrocytes (Fig. 3A and B). Diacerein did not modify MMP-1 release in IL-1β-stimulated synoviocytes (Fig. 3A), while all NSAIDs induced a significant increase in MMP-1 release as compared with IL-1β-stimulated synoviocytes (P = 0.001 for CBX, P = 0.003 for DCF, P = 0.018 for MXC, and P = 0.005 for IND) (Fig. 3A). In chondrocytes, diacerein did not induce a statistically significant change of collagenase protein expression induced by IL-1β in these cells, whereas all NSAIDs caused a significant enhancement of the enzyme release (P = 0.006 for CBX, P = 0.008 for DCF and MXC and P = 0.004 for IND) (Fig. 3B).


Figure 3
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  		FIG. 3. MMP-1 levels in cell supernatants. Cells were pre-incubated for 60 min with diacerein (DC, 10–5 M), CBX (10–6 M), DCF (10–6 M), MXC (10–6 M) and IND (10–6 M) and then stimulated with 10 U/ml IL-1β for 24 h. Conditioned media was collected after 24 h for MMP-1 measurement. Upper panels: representative western blots for MMP-1 protein in synoviocytes (A) and chondrocytes (B). Lower panels: densitometric analyses of MMP-1 levels in four different experiments for synoviocytes (A) and chondrocytes (B) (grey bars correspond to stimulated treated cells; white bars correspond to treated cells) (mean ± S.E.M.); *P < 0.05 vs basal, #P < 0.05 vs IL-1β alone.

 
IL-1β induced a significant up-regulation of the presence of MMP-13 presence in both cell types (Fig. 4). In synoviocytes, diacerein prevented the increase in MMP-13 elicited by IL-1β (Fig. 4A), while DCF, MXC and IND did not show a significant effect upon MMP-13. Finally, CBX significantly increased the presence of MMP-13 compared with IL-1β-stimulated synoviocytes (Fig. 4A). In chondrocytes, diacerein did not reverse the increase in MMP-13 protein expression induced by IL-1β, whereas the presence of NSAIDs produced a statistically significant increase in MMP-13 protein expression as compared with IL-1β-stimulated chondrocytes (Fig. 4B).


Figure 4
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  		FIG. 4. MMP-13 levels in cell supernatants. Cells were pre-incubated for 60 min with diacerein (DC, 10–5 M), CBX (10–6 M), DCF (10–6 M), MXC (10–6 M) and IND (10–6 M) and then stimulated with 10 U/ml IL-1β for 24 h. Conditioned media was collected after 24 h for MMP-13 measurement. Upper panels: representative western blots for MMP-13 protein in synoviocytes (A) and chondrocytes (B). Lower panels: densitometric analyses of MMP-13 levels in four different experiments for synoviocytes (A) and chondrocytes (B) (grey bars correspond to stimulated treated cells; white bars correspond to treated cells) (mean ± S.E.M.); *P < 0.05 vs basal, #P < 0.05 vs IL-1β alone.

 
IL-1β-induced NF-{kappa}B activation
NF-{kappa}B-binding sites are present in the promoter regions of many genes involved in the pathophysiology of OA, such as COX-2, iNOS and some MMP [27]. The activation of NF-{kappa}B in synoviocytes and chondrocytes was studied at 60 min after incubation with IL-1β (10 U/ml), as previously reported [25]. At this time, IL-1β caused a marked increase in NF-{kappa}B binding to its consensus oligonucleotide sequences in both cell types (Fig. 5). In synoviocytes, diacerein significantly inhibited NF-{kappa}B binding in comparison with IL-1β-stimulated cells (P = 0.004). CBX, DCF and MXC significantly diminished NF-{kappa}B-binding activity of IL-1β-stimulated human synoviocytes (P = 0.001 in all cases), whereas IND did not show any effect (Fig. 5A). In chondrocytes, diacerein showed a statistically significant inhibition of IL-1β-induced NF-{kappa}B binding (P = 0.001) (Fig. 5B). Interestingly, all NSAIDs significantly diminished NF-{kappa}B binding induced by IL-1β (P = 0.015 for CBX, P = 0.001 for DCF, P = 0.035 for MXC, and P = 0.012 for IND) (Fig. 5B).


Figure 5
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  		FIG. 5. Effect of drugs on NF-{kappa}B binding induced by IL-1β. Cells were pre-incubated for 60 min with diacerein (DC, 10–5 M), CBX (10–6 M), DCF (10–6 M), MXC (10–6 M) and IND (10–6 M) and then stimulated with 10 U/ml IL-1β for 60 min. Upper panels: representative autoradiograms of five different experiments with similar results for synoviocytes (A) and chondrocytes (B). Lower panels: densitometric analyses for specific NF-{kappa}B complexes in four different experiments for synoviocytes (A) and chondrocytes (B) (grey bars correspond to stimulated treated cells; white bars correspond to treated cells) (mean ± S.E.M.); *P < 0.05 vs basal, #P < 0.05 vs IL-1β alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
During the OA process, chondrocytes and synovial fibroblasts show independent capacities to initiate and respond to injury, ultimately resulting in degeneration of all joint structures. Although metabolic factors are probably dominant in the chondrocyte transformation during OA, there is accumulating evidence suggesting that synoviocytes play crucial roles in the propagation of inflammation leading to structural damage [28]. Synovial inflammation and local concentration of pro-inflammatory mediators seem to be directly involved in pain generation in OA joints [4, 29]. Thus, it seems reasonable to consider the synoviocyte and not only the chondrocyte as a potential therapeutic target in OA, as well as to assess the potential mechanisms of actions of different drugs for OA on these two cells. Some in vitro studies have suggested that the administration of diacerein may prevent cartilage damage, although the results seem contradictory [10, 12, 26, 30]. However, the effect of this compound on isolated synovial cells or the synovial membrane has been poorly examined.

In our experiments, IL-1β stimulation significantly increased all the parameters studied in chondrocytes. Concerning synoviocytes the cytokine had the same effect except for nitrite levels. Although there is a general agreement that chondrocyte stimulation increases NO synthesis during OA, the literature is unclear about the role of synoviocytes. In our conditions, IL-1β-stimulated synoviocytes produced extremely low nitrite levels, frequently not detectable, which is consistent with other studies [31].

Diacerein inhibited NF-{kappa}B activation in synoviocytes and chondrocytes and reduced nitrite release in chondrocytes. Inhibitors of NF-{kappa}B activation have been shown to have anti-inflammatory effects both in cultured cells and in animal models of inflammatory diseases [32]. Diacerein has been shown to reduce the signs of inflammation in various experimental models of acute inflammation [11], an effect that could be related to its ability to inhibit NF-{kappa}B activation. However, diacerein not only did not reverse the IL-1β-induced increase in COX-2 protein expression and PGE2 release in chondrocytes, but also increased the levels of these mediators in synoviocytes. NF-{kappa}B does not seem to be the only transcription factor that regulates the expression of COX-2, and its promoter does contain binding sequences for several transcription regulatory elements, such a TATA box, a NF-IL-6 motif, two AP-2 sites, three Sp1 sites, two NF-{kappa}B sites, a CRE motif and an E-box [33]. NF-{kappa}B fulfils an important role in regulating COX-2 expression although the contribution of the other transcription factors is also relevant [34, 35]. In fact, diacerein had been shown to rather stimulate prostaglandin synthesis in vivo and in vitro [12, 26, 36], which in turn may account for the poor symptomatic effect of the drug in clinical trials [14–16].

The four NSAIDs used in the study yielded very uniform results. With the exception of IND in synoviocytes, these compounds inhibited IL-1β-stimulated NF-{kappa}B activation, prevented PGE2 production by synovial cells and chondrocytes, and intriguingly diminished COX-2 protein expression in synovial fibroblasts and chondrocytes. None of them modified nitrite generation induced by IL-1β. The ability of these compounds to inhibit NF-{kappa}B activation after cell stimulation as well as the capacity to reduce COX-2 protein expression has been already described in leukaemia cells [37]. With regard to joint cells, contradictory in vitro results have been reported [7, 38]. We have previously demonstrated that both selective and non-selective COX-2 inhibitors decreased gene and protein expression of COX-2 in the synovial membrane of patients with knee OA [4]. In the present study, it is also shown that PGE2 synthesis inhibition evokes a direct effect on COX-2 gene and protein presence both in synoviocytes and chondrocytes. The mechanisms underlying this effect remains unknown, although PGE2-dependent and -independent mechanisms may contribute [4, 39].

Progressive degradation of the extracellular matrix that comprises joint tissues is a major feature in OA leading to permanent loss of function. Major enzymes involved in the process are MMPs that are able to degrade some of the components of the extracellular matrix. Collagenase 1 (MMP-1) and collagenase 3 (MMP-13) are involved in cartilage collagen II degradation in OA [40]. In our experiments, diacerein diminished MMP-13 levels in synoviocytes, although it did not modify MMP-1 levels in both the cells tested and MMP-13 protein expression in chondrocytes.

Previous studies have shown that rhein inhibited the induction in the expression of several MMPs in healthy bovine chondrocytes using higher doses of rhein than were employed here [41]. Other reports have demonstrated that rhein down-regulates the expression of different pro-MMP genes in healthy rabbit chondrocytes, although higher doses of diacerein were employed to completely reverse the increase in MMP gene expression induced by IL-1β [30]. Moreover, these experiments were carried out on healthy bovine or rabbit chondrocytes and it would not be surprising to observe differences in the responses of healthy bovine or rabbit cells and human OA cells [42, 43]. However, it is possible that diacerein could partially modify some MMP synthesis since in human chondrocytes it has been shown that rhein produced a statistically significant decrease of MMP-3/TIMP-1 ratio in the presence of IL-1β [26]. These results could partially explain the structure-modifying effect of diacerein on OA found in some studies [15].

NSAIDs induced a statistically significant increase in MMP-1 levels in synovial fibroblasts and chondrocytes, and in MMP-13 levels in chondrocytes. Previous in vitro studies have demonstrated that PGE2 depletion could activate cellular catabolism by enhancing MMP synthesis in synovial fibroblasts [44, 45]. These results may explain the lack of retardation of cartilage destruction despite the favourable profile of NSAIDs in controlling pro-inflammatory pathogenetic routes [46]. According to our data, MMPs could be induced even in the presence of a significant inhibition in NF-{kappa}B activation. Although several NF-{kappa}B-binding sites are present in the promoter regions of MMP genes, other transcription factors and signalling pathways are implicated in the regulation of the gene expression of these enzymes [47, 48].

These findings clearly indicate that NSAIDs and diacerein act by different pathways on pain and inflammation in OA. However, further investigation is needed to clarify the clinical impact of the PGE2 over expression induced by diacerein. The release of PGE2 at high concentrations to the inflammatory site would contribute to increase inflammation-related pain. However, diacerein has been shown to relieve chronic pain in knee and hip OA [14–16]. PGE2 may act as a dual mediator, on one hand supporting disease progression by enhancing the proteoglycan degradation [7], and on the other hand by down-regulating the expression and synthesis of cytokines [49]. On the whole, these actions could indicate that either excessive or sub-physiological PGE2 levels are undesirable, a hypothesis that has been already approached by our group and other authors [49–51].

Therefore, diacerein does not seem to reduce but rather increase the release of inflammatory mediators in synoviocytes, while it does not overall affect chondrocyte inflammatory profile. The present finding could account for the weak response in pain control, which has been reported after the use of diacerein in patients with knee OA.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Marta Pulido for editing the manuscript and for editorial assistance.

Funding: This work has been supported by research grants from the Spanish Ministry of Education (SAF2006/2704) and Fondo de Investigaciones Sanitarias (CP03/00011, PI06/0032).

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 2 November 2007; revised version accepted 15 February 2008.
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