Inhibition Of Cartilage Degradation Research Articles

Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis

ObjectiveOsteoarthritis (OA) is characterized by cartilage degradation in the affected joints. Pomegranate fruit extract (PFE) inhibits cartilage degradation in vitro. The aim of this study was to determine whether oral consumption of PFE inhibits disease progression in rabbits with surgically induced OA. MethodsOA was surgically induced in the tibiofemoral joints of adult New Zealand White rabbits. In one group, animals were fed PFE in water for 8 wk postsurgery. In the second group, animals were fed PFE for 2 wk before surgery and for 8 wk postsurgery. Histologic assessment and scoring of the cartilage was per Osteoarthritis Research Society International guidelines. Gene expression and matrix metalloproteinases (MMP) activity were determined using quantitative reverse transcriptase polymerase chain reaction and fluorometric assay, respectively. Interleukin (IL)-1 β, MMP-13, IL-6, prostaglandin (PG)E2, and type II collagen (COL2A1) levels in synovial fluid/plasma/culture media were quantified using enzyme-linked immunosorbent assay. Expression of active caspase-3 and poly (ADP-ribose) polymerase p85 was determined by immunohistochemistry. Effect of PFE and inhibitors of MMP-13, mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB was studied in IL-1 β-stimulated rabbit articular chondrocytes. ResultsSafranin-O-staining and chondrocyte cluster formation was significantly reduced in the anterior cruciate ligament transaction plus PFE fed groups. Expression of MMP-3, MMP-9, and MMP-13 mRNA was higher in the cartilage of rabbits given water alone but was significantly lower in the animals fed PFE. PFE-fed rabbits had lower IL-6, MMP-13, and PGE2 levels in the synovial fluid and plasma, respectively, and showed higher expression of aggrecan and COL2A1 mRNA. Significantly higher numbers of chondrocytes were positive for markers of apoptosis in the joints of rabbits with OA given water only compared with those in the PFE-fed groups. PFE pretreatment significantly reduced IL-1 β induced IL-6 and MMPs expression in rabbit articular chondrocytes. These effects were also mimicked using MMP-13, MAPK, and NF-κB inhibitors in IL-1 β-stimulated rabbit chondrocytes. In an in vitro activity assay, PFE blocked the activity of MMP-13. Like MAPK and NF-κB inhibitors, PFE was also effective in inhibiting IL-1 β-induced PGE2 production in rabbit chondrocytes. PFE also reversed the inhibitory effect of IL-1β on COL2A1 mRNA and protein expression in IL-1 β-stimulated rabbit chondrocytes. ConclusionThe present data highlight the chondroprotective effects of PFE oral consumption in a model of posttraumatic OA and suggest that PFE-derived compounds may have potential value in the management of OA.

Acetylsalicylic acid combined with diclofenac inhibits cartilage degradation in rabbit models of osteoarthritis.

The present study aimed to investigate the effect of different concentrations of acetylsalicylic acid combined with diclofenac on the articular cartilage of a rabbit model of osteoarthritis (OA). A total of 40 New Zealand white rabbits were divided into 5 groups. Group A was a sham-operated control group, which was treated with normal saline. Groups B-E were OA models and were treated with normal saline and acetylsalicylic acid combined with diclofenac at concentrations of 5, 10 and 20 mg/kg, respectively. A cartilage macroscopic examination and a pathological observation were performed to analyze the structure of the articular cartilage in all of the treated groups. The nitric oxide (NO) content and interleukin 1β (IL-1β) levels were detected by an enzyme-linked immunosorbent assay. In addition, the protein expression of matrix metalloproteinase 3 (MMP)-3 and MMP-13 were detected by western blot analysis. The mRNA expression of tissue inhibitor of metalloproteinases 1 (TIMP1) was detected by polymerase chain reaction (PCR). The results revealed that different concentrations of the drugs significantly reduced the scores of cartilago articularis, the NO and IL-1β levels and the protein expression of MMP-3 and MMP-13. Furthermore, PCR revealed that the mRNA expression of TIMP1 was significantly upregulated, and the effects increased with increasing drug concentration. Thus, the administration of different concentrations of acetylsalicylic acid combined with diclofenac demonstrates preventive or therapeutic effects against OA progression.

Sex-Specific Protection of Osteoarthritis by Deleting Cartilage Acid Protein 1.

Cartilage acidic protein 1 (CRTAC1) was recently identified as an elevated protein in the synovial fluid of patients with osteoarthritis (OA) by a proteomic analysis. This gene is also upregulated in both human and mouse OA by transcriptomic analysis. The objective of this study was to characterize the expression and function of CRTAC1 in OA. Here, we first confirm the increase of CRTAC1 in cartilage biopsies from OA patients undergoing joint replacement by real-time PCR and immunohistochemistry. Furthermore, we report that proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha upregulate CRTAC1 expression in primary human articular chondrocytes and synovial fibroblasts. Genetic deletion of Crtac1 in mice significantly inhibited cartilage degradation, osteophyte formation and gait abnormalities of post-traumatic OA in female, but not male, animals undergoing the destabilization of medial meniscus (DMM) surgery. Taken together, CRTAC1 is upregulated in the osteoarthritic joint and directly induced in chondrocytes and synovial fibroblasts by pro-inflammatory cytokines. This molecule is necessary for the progression of OA in female mice after DMM surgery and thus represents a potential therapy for this prevalent disease, especially for women who demonstrate higher rates and more severe OA.

Protective effect of thrombospondin-4 expressing mesenchymal stem cells in osteoarthritis

Background and objectives Several members of the thrombospondin (THBS) family have been shown to play a role in cartilage homeostasis and physiopathology. While mesenchymal stem cells (MSCs) express different THBS members and exert a therapeutic effect in osteoarticular diseases, the role of THBS4 has been poorly investigated. We therefore evaluated the role of THBS4 deficient MSCs in osteoarthritic mice. Methods MSCs were isolated from the bone marrow of wild type C57BL6 and THBS4 knock-out C57BL6 mice and named wt and THBS4 -/- MSC, respectively. Osteoarthritis was induced in C57BL/6 mice by collagenase injections. On day 7, 200,000 MSCs were injected in the joint and on day 42, hind paws were processed for Safranin O-Fast green staining and scored. Tibiae were incubated in 30% of Hexabrix 320 contrast agent and scanned in a microCT scanner (SkyScan). Quantification of cartilage damage was then assessed after scanning the entire articular cartilage of tibiae with a confocal laser scanning microscope (CLSM; SP5 Leica). Results Histological analysis showed a significantly lower OA score in the joints of mice injected with wt MSCs as compared to control OA mice or mice that received THBS4 -/- MSCs. These results were confirmed by CLSM analysis of tibia plateau cartilages. Both the volume and thickness of articular cartilage were significantly lower in THBS4 -/- MSCs treated mice and in control OA mice than in mice treated with wt MSCs. Furthermore after incubation of tibiae with contrast agent, microCT analysis revealed a significant depletion of GAG content in the articular cartilage. Finally, microCT analysis of subchondral bone indicated a significant increase of bone surface degradation (increase of surface/volume ratio) as well as a significant decrease of bone mineral density and subchondral bone thickness in control OA mice and mice injected with THBS4 -/- MSCs as compared to wt MSCs treated mice. Conclusions In contrast to wt MSCs, THBS4 -/- MSCs were defective in their capacity to inhibit cartilage degradation in an osteoarthritic mouse model. Altogether, the data point out the role of the THBS4 pathway in the therapeutic effect of MSCs in osteo-articular diseases.

Brief exposure to triamcinolone acetonide, but not its continous presence, strongly inhibits cartilage regeneration by chondrocytes

s / Osteoarthritis and Cartilage 24 (2016) S63eS534 S337 synovium-conditioned medium (OA-CM) or TNFa to determine IL37 expression in an inflammatory environment. The function of IL37 was investigated via overexpression of IL37 by using an adenoviral vector (Ad-IL37) and subsequent stimulation with IL-1b for 24 h to create an inflammatory environment. An adenovirus overexpressing luciferase was used as control (Ad-Luc). Gene and protein expression of the proinflammatory cytokines IL-1b, IL-6 and IL-8 and catabolic enzymes MMP1, MMP3, MMP13, ADAMTS4 and ADAMTS5 were analyzed by qPCR, Western Blot and Luminex. Results: Immunohistochemistry and qPCR analysis showed that IL37 was expressed in chondrocytes from human OA joints. Furthermore, we found increased expression of IL37 after IL-1b stimulation, both on protein and gene expression level (6.8 fold), whereas OA-CM and TNFa stimulation did not significantly induce IL37 expression. Next, we investigated the anti-inflammatory effect of IL37 in chondrocytes (n1⁄48) via overexpression of IL37 and stimulationwith IL-1b. Twenty-four hours after IL-1b stimulation, IL37 significantly reduced IL-1b (42%), IL-6 (29%) and IL-8 (49%) gene expression (Fig. 1A). In addition to gene expression analysis, Western blot analysis showed that IL37 reduced IL-1b protein expression, and Luminex analysis showed that IL37 significantly decreased IL-8 protein levels (59%), and demonstrated a trend towards a decrease in IL-6 protein expression (25%). Next, we investigated the effect of IL37 on cartilage degrading enzymes, including MMPs and ADAMTSs. Significant downregulation was found on MMP1 (35%), MMP3 (31%), MMP13 (29%) and ADAMTS5 (27%) gene expressionafter IL-1b stimulation (Fig. 1B), whereas ADATMS4 expression was not affected. Also on protein level, MMP3 and MMP13 expression were both reduced (42%) and a trend towards decreased MMP1 expression (32%) was observed. Conclusions: Pro-inflammatory cytokines and catabolic enzymes negatively affect cartilage integrity by inducing matrix loss and chondrocyte death. Blocking the production of these catabolic factors is a promising therapy for OA. In this study we show for the first time that IL37 is expressed in osteoarthritic cartilage and that IL37 expression is regulated by IL-1b in primary OA chondrocytes. Furthermore, we demonstrated that IL37 potently reduces the production and secretion of pro-inflammatory cytokines and catabolic enzymes in an inflammatory environment. These data position IL37 as a potential therapeutic agent to inhibit cartilage degradation. Figure 1. IL37 reduces IL-lb-induced pro-inflammatory cytokine and catabolic enzyme production in human OA chondrocytes. (A) IL37 significantly downregulates IL-1b (42%), IL-6 (29%) and IL-8 (48%) gene expression compared to control (Ad-Luc). (B) IL37 significantly reduced MMP1 (35%), MMP3 (31%) and MMP13 (29%) gene expression compared to control (Ad-Luc). 553 BRIEF EXPOSURE TO TRIAMCINOLONE ACETONIDE, BUT NOT ITS CONTINOUS PRESENCE, STRONGLY INHIBITS CARTILAGE REGENERATION BY CHONDROCYTES I. Jansen y, A. Tellegen z, M. Tryfonidou z, C. € Oner y, D. Saris y, N. Woike x, J. Berard x, K. Messier x, L. Creemers y. yUniv. Med. Ctr. Utrecht, Utrecht, Netherlands; zUtrecht Univ., Utrecht, Netherlands; xDSM BioMed.,

Inhibitory effects of EGb761 on the expression of matrix metalloproteinases (MMPs) and cartilage matrix destruction.

Cytokines such as tumor necrosis factor alpha (TNF-α)-induced expression of matrix metalloproteinase (MMP) play a pivotal role in the destruction of articular cartilage in patients who are suffering from osteoarthritis (OA). Collagen type II, the basis for articular cartilage, can be degraded by MMP-1, MMP-3, and 13. EGb761, the standardized extract of Ginkgo biloba produced by Dr. Willar Schwabe Pharmaceuticals, has shown its anti-inflammatory capacity. This study aimed to determine a mechanism whereby EGb761 may inhibit cartilage degradation. Our results indicated that pretreatment with EGb761 abolishes MMP-1, MMP-3, and MMP-13 gene expression and protein expression induced by TNF-α in human chondrocyte monolayer. In addition, the reduction of the tissue inhibitor of metalloproteinase-1(TIMP-1) and metalloproteinase-2 gene expression induced by TNF-α was rescued by pretreatment with EGb761. Importantly, TNF-α-induced degradation of collagen type II was ameliorated by EGb761 in a dose-dependent manner. Mechanistically, our results indicated that EGb761 treatment attenuated TNF-α-induced NF-κB activation. These actions of EGb761 suggest a mechanism by which EGb761 may act to prevent cartilage breakdown in arthritis.

Bushen Zhuangjin Decoction promotes chondrocyte proliferation by stimulating cell cycle progression.

Bushen Zhuangjin Decoction (BZD), a well-known formulation in Traditional Chinese Medicine, has been widely used for the treatment of osteoarthritis (OA). Due to the poor intrinsic repair capacity of chondrocytes, promoting the proliferation of chondrocytes is an efficient treatment to delay the progression of cartilage degradation. The present study, therefore, focused on the effect of BZD on chondrocyte proliferation, exploring the mechanism of BZD on the inhibition of cartilage degradation. Chondrocytes isolated from the knee articular cartilage of Sprague Dawley rats were cultured and identified by type II collagen immunohistochemistry. It was found that BZD promoted chondrocyte viability in a dose- and time-dependent manner. To investigate if BZD promoted the chondrocyte viability by stimulating the cell cycle progression a flow cytometer was used, and the results showed that the percentage proportion of G0/G1 cells was significantly lower, and the percentage proportion of S cells was significantly higher, in treated cells compared with that in untreated cells. To gain insight into the mechanism underlying the effect of BZD on the cell cycle progression, the mRNA and protein expression of cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and p21 was measured by reverse transcription-polymerase chain reaction and western blotting, respectively. The mRNA and protein expression of cyclin D1, CDK4 and CDK6 in the BZD-treated chondrocytes was significantly upregulated, while the mRNA and protein expression of p21 was significantly downregulated, compared with that in the untreated chondrocytes. These results suggested that BZD promoted chondrocyte proliferation by accelerating G1/S transition, indicating that BZD is a potential therapeutic agent for the treatment of OA.

Chondroprotective and anti-inflammatory effects of S-methylisothiourea, an inducible nitric oxide synthase inhibitor in cartilage and synovial explants model of osteoarthritis

To study the chondroprotective and anti-inflammatory potential of inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea (SMT) in in-vitro model. Rabbit cartilage explants were stimulated with recombinant human interleukin 1β (rhIL-1β), and the chondroprotective and anti-inflammatory effects of SMT were investigated. Rat synovial explants were stimulated with LPS, and the anti-inflammatory effect of SMT on synovium was studied. To examine the role of SMT in synovial inflammation mediated cartilage damage, LPS stimulated synovial explants were cultured with dead cartilage with or without SMT for 72 h. The culture medium was analysed for sulfated glycosaminoglycans (GAGs) and hydroxyproline as measure of proteoglycans and collagen degradation, respectively. SMT significantly reduced GAGs, hydroxyproline, matrix metalloproteinase (MMP)-13, tumour necrosis factor alpha (TNF-α), prostaglindin E2 (PGE2 ) and nitrite release in stimulated rabbit cartilage media indicating chondroprotective and anti-inflammatory effects of SMT in osteoarthritis (OA). Stimulated synovial explants caused release of nitrite, PGE2 , IL-1β and TNF-α in the medium which were significantly reduced by SMT indicating its anti-inflammatory action. SMT significantly reduced GAGs and hydroxyproline in medium and shown protective effect against synovium-mediated cartilage damage. SMT inhibited cartilage degradation, synovial inflammation and synovium-mediated cartilage damage, suggesting that SMT may be an agent for pharmacological intervention in OA.

Collagen type III deposition in articular cartilage may be a distinctive biomarker of hip osteoarthritis

Collagen type III deposition in articular cartilage may be a distinctive biomarker of hip osteoarthritis

Xanthan gum inhibits cartilage degradation by down-regulating matrix metalloproteinase-1 and -3 expressions in experimental osteoarthritis

We previously reported that intra-articular injection of xanthan gum protected the joint cartilage and reduced osteoarthritis progression. In this study, the effects of xanthan gum on chondrocytes apoptosis were evaluated using the labeling assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, to determine the protein expression of matrix metalloproteinase-1, 3, and tissue inhibitors of metalloproteinase-1 using immunohistochemistry and Western blot assay in cartilage of papain-induced rabbit osteoarthritis model. Compared to the negative control group, intra-articular injection of xanthan gum, once every 2 weeks for 5 weeks significantly inhibited chondrocytes apoptosis and matrix metalloproteinase-1 and -3 protein expression levels and also enhanced the tissue inhibitors of metalloproteinase-1 production in cartilage. No significant differences between the xanthan gum-treated group and the sodium hyaluronate-treated group (intra-articular injection of sodium hyaluronate only once a week for 5 weeks) were observed.

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes

Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. OA chondrocytes were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. After 3, 7, 14 and 21 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I, II, and X collagen (COL1A1, COL2A1, COl10A1), matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and the serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1). After 3 weeks, DNA content and proteoglycan and collagen content and release were determined. Type II collagen was analyzed at the protein level by Western blot. Overexpression of hsa-miR-148a had no effect on ACAN, COL1A1 and SERPINH1 gene expression, but increased COL2A1 and decreased COL10A1, MMP13 and ADAMTS5 gene expression. Luciferase reporter assay confirmed direct interaction of miR-148a and COL10A1, MMP13 and ADAMTS5. The matrix deposited by the miR-148a overexpressing cells contained more proteoglycans and collagen, in particular type II collagen. Proteoglycan and collagen release into the culture medium was inhibited, but total collagen production was increased. Overexpression of hsa-miR-148a inhibits hypertrophic differentiation and increases the production and deposition of type II collagen by OA chondrocytes, which is accompanied by an increased retention of proteoglycans. Hsa-miR-148a might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production.

Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1β induced matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human chondrocytes

Interleukin-1β (IL-1β) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. Treatment with WIN-55 alone or in combination with IL-1β, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.

In vitro evaluation of S-(+)-ibuprofen as drug candidate for intra-articular drug delivery system

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locally release non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen to reduce pain in patients with osteoarthritis. The present study examines the efficacy of S-(+)-ibuprofen on cartilage degradation as drug candidate for DDS loading. Humeral cartilage and joint capsule explants were collected from healthy sheep shoulder joints and they were cultured in mono- or in co-culture for 13 days with LPS in combination with S-(+)-ibuprofen at 50 µM and 1 mM. S-(+)-ibuprofen (50 µM) blocked prostaglandins production in LPS-activated explants but did not reduce cartilage degradation. By contrast, 1 mM S-(+)-ibuprofen treatment of cartilage explants reduced nitric oxide synthesis by 51% (p = 0.0072), proteoglycans degradation by 35% (p = 0.0114) and expression of serum amyloid protein – the main protein induced upon LPS challenge – by 44% (p < 0.0001). On contrary, in presence of synovial membrane, the protective effects of S-(+)-ibuprofen on cartilage damages were significantly diminished. At 1mM, S-(+)-ibuprofen reduced the cell lysis during culture of cartilage and joint capsule either in mono- or in co-culture. This study performed on sheep explants shows that 1 mM S-(+)-ibuprofen inhibited cartilage degradation via a mechanism independent of cyclooxygenase inhibition. Reduction of prostaglandins synthesis at 50 µM in all treatment groups and reduction of cartilage degradation observed at 1 mM suggest that S-(+)-ibuprofen could be considered as a promising drug candidate for the loading of intra-articular DDS.

FRI0231 Atrosab, a humanized antibody directed against tnf-receptor 1, hold great promises for the treatment of rheumatoid arthritis

BackgroundTo date, the five anti-TNFα biologics on the market are firmly established as therapeutics across several inflammatory and autoimmune disorders including rheumatoid arthritis (RA), inflammatory bowel diseases (IBD), psoriatic arthritis,...

OP0127 Identification of serological biomarker profiles associated with early response to tocilizumab in rheumatoid arthritis

BackgroundRA characterized by poly-articular and synovial inflammation, cartilage loss and erosion of subchondral bone. It is critical to diagnose and effectively treat the disease early to suppress inflammation and prevent...

Five to ten year follow up of matrix associated autologous chondrocyte transplantation (MACT) in the knee

s / Osteoarthritis and Cartilage 20 (2012) S10–S53 S27 In this study, we evaluate the RNAi effect against MMP13 in preventing cartilage degradation by directly injecting chemically modulated siRNAs to surgically induced murine OA knee joints. Our hypothesis was that effective RNAi could be achieved by injection of highly concentrated siRNA in the knee joint, resulting in reduced development of osteoarthritis. Methods: Knee OAs were surgically induced to C57BL/6 mice by destabilization of the medial meniscus (DMM) method, by transecting the medial meniscotibial ligament under general anesthesia. To determine the timing of protease expressions in the murine DMM induced OA model, messenger RNA (mRNA) of MMP13 from knee cartilage were analyzed at 3 days, 1 week, 2 weeks, 4 weeks, and 6 weeks after surgery by quantitative polymerase chain reaction (qPCR). One week after DMM induction, chemically modulated siRNAs were injected in the knee joint. Two different content of injection were evaluated. Treatment groups consisted of a group which MMP13 siRNA was injected, and another group in which control siRNA was injected. The control siRNAwas confirmed not to affect MMP13 in vitro. The RNAi effects were assessed by quantifying MMP13 mRNA in knee cartilage at 48 hours after siRNA injection. Histological assessment of articular cartilage at 8 weeks post DMM operation was conducted to assess the effect of siRNA injection at one week after DMM in osteoarthritis progression, according to the OARSI recommendation for OA knee scoring in mice. Results: Surgically induced OAs were successfully achieved in the DMM model of mice. MMP13 expressions elevated from the third post operative day, and peaked in a week after DMM surgery (Fig. 1). Expression level of MMP13 mRNA was significantly decreased at 48 hours after injection of MMP13 siRNA to the knee joint, compared to negative control siRNA injection (Fig. 2). Significant improvement was observed in the histological score of MMP siRNA treated group, compared to the control siRNA injected group. Conclusions: Direct injection of siRNAs targeting MMP13 at the early phase of OA development has the potential to inhibit protease expression and inhibits cartilage degradation in vivo.

Follistatin alleviates synovitis and articular cartilage degradation induced by carrageenan in mice

Purpose: Osteoarthritis (OA), a chronic degenerative joint disorder characterized by articular cartilage destruction and osteophyte formation, is prevalent in our society as a major cause of disability. Molecular pathogenesis of OA is not fully understood, it is considered that acute joint inflammation plays significant roles in the onset of OA. This idea is supported by various animal experiments, since articular cartilage degradation is induced by the intra-articular injection of carrageenan or zymosan. Previously we showed that intra-articular injection of recombinant human BMP7 (rhBMP7) inhibited cartilage degradation induced by zymosan partly through the inhibition of inflammatory cytokine expression such as IL1-beta in the joint. From these data we hypothesized that TGF-βeta/BMP signal maintains joint homeostasis by controlling inflammatory status. Here we report that follistatin, an endogenous inhibitor for Activin which belongs to TGF-beta/BMP family and works as a proinflammatory cytokine, effectively alleviates synovitis and articular cartilage degradation induced by the intra-articular injection of carrageenan in mice. Methods: This study was approved and conducted in accordance with the guideline of the animal committee of Tokyo Medical and Dental University. Male C57Bl/6J mice (12weeks old) were purchased from ORIENTAL YEAST co.,Ltd (Tokyo, Japan). They were housed under a 12-h light-dark cycle and allowed food and water ad libitum. Twelve mice were randomly divided into two groups (n=6/group). Mice were anesthetized by the inhalation of 5% isoflurane in oxygen. Under deep anesthesia, a solution of 30μg lamda-carrageenan (Sigma-Aldrich) in 5μL saline was injected into the left knee joint through the lateral margin of the patella tendon. Recombinant mouse follistatin (25ng in 5μl in physiological saline, Sigma-Aldrich) was injected into the left knee at 30 minutes before carrageenan challenge. Mice were maintained in cage ad libitum for 3 days after the challenge. Knee joints were dissected, fixed in 4% paraformaldehyde, decalcified, embedded, and 5μm sagittal sections were prepared for histology. Integrity of articular cartilage and synovium was assessed by Hematoxylin and Eosin staining. To assess articular cartilage damage, three sections (apart from 150μm respectively) were stained by Safranin O and 400 μm in width of articular cartilage between anterior and posterior edge of medial meniscus was contoured into 3 areas according to the dyeability: Grade I; intact cartilage, Grade II; mildly denatured cartilage with reduced safranin O staining, and Grade III; severely denatured cartilage with no Safranin O staining. Each area was measured using Zeiss Axio Vision Image Analysis system. Kruskal-Wallis test followed by Tukey-Kramer methods was used for statistical analysis. Results: Image analyses indicated that dyeability of articular cartilage by Safranin O was significantly reduced by the single intra-articular injection of carrageenan at 3 days (Grade I: 21.4%, Grade II: 33.8%, Grade III: 44.8%), although we did not observe any obvious alteration in the articular surface structure at this stage. In contrast, the loss of dyeability after carrageenan injection was significantly improved by the pre-injection of follistatin (Grade I: 26.4%, Grade II: 73.6%, Grade III: 0%, p<0.05). Hematoxylin and Eosin staining showed that the cellularity of synovial tissue is greatly increased in carrageenan-injected mice. In contrast, these inflammatory responses were greatly alleviated by the pre-injection of follistatin. Conclusions: Carrageenan-induced arthritis is a well-established experimental model to investigate inflammation-mediated articular cartilage degradation in rodents. Here we report that follistatin effectively inhibits the loss of Safranin O dyeability of articular cartilage induced by carrageenan. Our data strongly suggest that the Activin signal pathway is involved in the process of joint inflammation and proteoglycan loss in the cartilage matrix. We believe that our experimental system will be of great use for analyzing the molecular events undergoing the onset of OA.

Polyoxypregnane glycoside from Dregea volubilis extract inhibits IL-1β-induced expression of matrix metalloproteinase via activation of NF-κB in human chondrocytes

Interleukin-1β (IL-1β) induces the expression of matrix metalloproteinases (MMPs) implicated in cartilage and joint degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). Polyoxypregnane glycoside (PPG), active compound was identified from Dregea volubilis extract by chemical analysis, shown to exert chondroprotective effects in cartilage explant models. However, no studies have been undertaken for the molecular investigation of whether PPG constituents protect the human articular chondrocyte (HAC). In the present studies, HAC was co-treated with IL-1β and PPG. The expression of MMPs, type II collagen, phosphorylation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathway were determined by Western immunoblotting. PPG (6.25-25μM) decreased the IL-1β-induced HA release from chondrocyte to culture medium. The mode of action of PPG was likely mediated through inhibiting expression of MMP-1, -3 and -13 in the medium, which was associated with the inhibition of mRNA expression. PPG had no effect on IL-1β-induced phosphorylation of MAPK pathway. Conversely, PPG decreased phosphorylation of IκB kinase and IκBα degradation. Taken together, these results indicate that PPG may inhibit cartilage degradation in OA and may also be used as nutritional supplement for maintaining joint integrity and function.

Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-α and matrix metalloproteinase-13 in human chondrocytes

IntroductionThe major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) can activate chondrocytes and induce the production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG) on AGE-modified-BSA (AGE-BSA)-induced activation and production of TNFα and MMP-13 in human OA chondrocytes.MethodsHuman chondrocytes were derived from OA cartilage by enzymatic digestion and stimulated with in vitro-generated AGE-BSA. Gene expression of TNFα and MMP-13 was measured by quantitative RT-PCR. TNFα protein in culture medium was determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the MMP-13 production in the culture medium, phosphorylation of mitogen-activated protein kinases (MAPKs), and the activation of NF-κB. DNA binding activity of NF-κB p65 was determined using a highly sensitive and specific ELISA. IκB kinase (IKK) activity was determined using an in vitro kinase activity assay. MMP-13 activity in the culture medium was assayed by gelatin zymography.ResultsEGCG significantly decreased AGE-stimulated gene expression and production of TNFα and MMP-13 in human chondrocytes. The inhibitory effect of EGCG on the AGE-BSA-induced expression of TNFα and MMP-13 was mediated at least in part via suppression of p38-MAPK and JNK activation. In addition, EGCG inhibited the phosphorylating activity of IKKβ kinase in an in vitro activity assay and EGCG inhibited the AGE-mediated activation and DNA binding activity of NF-κB by suppressing the degradation of its inhibitory protein IκBα in the cytoplasm.ConclusionsThese novel pharmacological actions of EGCG on AGE-BSA-stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG-derived compounds may inhibit cartilage degradation by suppressing AGE-mediated activation and the catabolic response in human chondrocytes.

Bone morphogenetic protein 7 inhibits cartilage degradation in a rabbit model of osteoarthritis

Bone morphogenetic protein 7 inhibits cartilage degradation in a rabbit model of osteoarthritis