Molecular Targets of Natural Products for Chondroprotection in Destructive Joint Diseases.

Osteoarthritis (OA) is the most common type of arthritis that occurs in an aged population. It affects any joints in the body and degenerates the articular cartilage and the subchondral bone. Despite the pathophysiology of OA being different, cartilage resorption is still a symbol of osteoarthritis. Matrix metalloproteinases (MMPs) are important proteolytic enzymes that degrade extra-cellular matrix proteins (ECM) in the body. MMPs contribute to the turnover of cartilage and its break down; their levels have increased in the joint tissues of OA patients. Application of chondroprotective drugs neutralize the activities of MMPs. Natural products derived from herbs and plants developed as traditional medicine have been paid attention to, due to their potential biological effects. The therapeutic value of natural products in OA has increased in reputation due to their clinical impact and insignificant side effects. Several MMPs inhibitor have been used as therapeutic drugs, for a long time. Recently, different types of compounds were reviewed for their biological activities. In this review, we summarize numerous natural products for the development of MMPs inhibitors in arthritic diseases and describe the major signaling targets that were involved for the treatments of these destructive joint diseases.

Gumiganghwal-tang ameliorates cartilage destruction via inhibition of matrix metalloproteinase.

ETHNOPHARMACOLOGICAL RELEVANCE: Kyung-Bang Gumiganghwal-tang tablet (GMGHT) is a standardized Korean Medicine that could treat a cold, headache, arthralgia and fever. Although GMGHT has been used for arthritis-related diseases including a sprain, arthralgia, unspecified arthritis and knee arthritis, there is no pre-clinical evidence to treat osteoarthritis (OA). This study determined the drug dosage and the mechanisms of GMGHT for OA. METHODS: OA was induced by intra-articular monoiodoacetic acid (MIA) injection in Sprague-Dawley rats. As calculated from the human equivalent dose formula, GMGHT was orally administered at the doses of 9.86, 98.6 and 986 mg/kg for 4 weeks. The arthritis score was performed by a blind test, and histological changes in articular cartilage were indicated by hematoxylin and eosin, Safranin O and toluidine blue staining. SW1353 chondrocytes were stimulated by interleukin (IL)-1ß recombinant to analyze the expressions of Type II collagen, matrix metalloproteinases (MMPs) and nuclear factor (NF)-κB. RESULTS: Rough and punctate surfaces of the femoral condyle induced by MIA, were recovered by the GMGHT treatment. The arthritis score was significantly improved in the 968 mg/kg of GMGHT-treated cartilage. Loss of chondrocytes and proteoglycan were ameliorated at the deep zone of the subchondral bone plate by the GMGHT administration in OA rats. The expression of Type II collagen was increased, while MMP-1, -3 and -13 levels were decreased in the GMGHT-treated SW1353 chondrocytes. In addition, the GMGHT treatment regulated NF-κB activation along with IL-6, transforming growth factor-ß and IL-12 production. CONCLUSIONS: GMGHT promoted the recovery of articular cartilage damage by inhibiting MMPs, accompanied with its anti-inflammatory effects in OA. GMGHT might be an alternative therapeutic treatment for OA.

Critical role for arginase II in osteoarthritis pathogenesis.

OBJECTIVE: Osteoarthritis (OA) appears to be associated with various metabolic disorders, but the potential contribution of amino acid metabolism to OA pathogenesis has not been clearly elucidated. Here, we explored whether alterations in the amino acid metabolism of chondrocytes could regulate OA pathogenesis. METHODS: Expression profiles of amino acid metabolism-regulating genes in primary-culture passage 0 mouse chondrocytes were examined by microarray analysis, and selected genes were further characterised in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The functional consequences of arginase II (Arg-II) were examined in Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg-II (Ad-Arg-II). RESULTS: The gene encoding Arg-II, an arginine-metabolising enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human patients with OA and various mouse models. Adenovirus-mediated overexpression of Arg-II in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2-/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg-II appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (matrix metalloproteinase 3 [MMP3] and MMP13) in chondrocytes via the nuclear factor (NF)-κB pathway. CONCLUSIONS: Our results indicate that Arg-II is a crucial regulator of OA pathogenesis in mice. Although chondrocytes of human and mouse do not identically, but similarly, respond to Arg-II, our results suggest that Arg-II could be a therapeutic target of OA pathogenesis.

Zinc Protects Articular Chondrocytes through Changes in Nrf2-Mediated Antioxidants, Cytokines and Matrix Metalloproteinases.

Osteoarthritis (OA) is an age-related degenerative joint disease characterized by high oxidative stress, chondrocyte death and cartilage damage. Zinc has been implicated in the antioxidant capacity of the cell, and its deficiency might inhibit chondrocyte proliferation. The present study examined the potential of zinc as a preventive supplement against OA using the in vitro chondrosarcoma cell line SW1353 and an in vivo Wistar rat model to mimic OA progress induced by monosodium iodoacetate (MIA). The results demonstrated that, in SW1353 cells, 5 µM MIA exposure increased oxidative stress and decreased the expression of GPx1 and Mn-SOD but still increased GSH levels and HO-1 expression and enhanced the expression of interleukin (IL)-10, IL-1ß, and matrix metalloproteinase (MMP)-13. Zinc addition could block these changes. Besides, the expression of Nrf2 and phosphorylated (p)-Akt was dramatically increased, implicating the p-Akt/Nrf2 pathway in the effects of zinc on MIA-treated cells. A rat model achieved similar results as those of cell culture, and 1.6 mg/kg/day of zinc supplementation is sufficient to prevent OA progress, while 8.0 mg/kg/day of zinc supplementation does not have a better effect. These findings indicate that zinc supplementation exerts a preventive effect with respect to MIA-induced OA progress.

Tyrosine kinase Fyn promotes osteoarthritis by activating the ß-catenin pathway.

OBJECTIVES: To investigate the role of tyrosine kinase Fyn in the development of osteoarthritis (OA) and the underlying mechanisms, and to define whether targeting Fyn could prevent OA in mice. METHODS: Cartilage samples from normal and aged mice were analysed with proteome-wide screening. Fyn expression was examined with immunofluorescence in human and age-dependent or experimental mouse OA cartilage samples. Experimental OA in Fyn-knockout mice was induced by destabilisation of the medial meniscus. Primary cultured mouse chondrocytes were treated with proinflammatory cytokine interleukin-1ß. The inhibitor of Src kinase family, AZD0530 (saracatinib), and inhibitor of Fyn, PP1, were used to treat experimental OA in mice. RESULTS: Fyn expression was markedly upregulated in human OA cartilage and in cartilage from aged mice and those with post-traumatic OA. Fyn accumulates in articular chondrocytes and interacts directly with and phosphorylates ß-catenin at Tyr142, which stabilises ß-catenin and promotes its nuclear translocation. The deletion of Fyn effectively delayed the development of post-traumatic and age-dependent OA in mice. Fyn inhibitors AZD0530 and PP1 significantly attenuated OA progression by blocking the ß-catenin pathway and reducing the levels of extracellular matrix catabolic enzymes in the articular cartilage. CONCLUSIONS: Fyn accumulates and activates ß-catenin signalling in chondrocytes, accelerating the degradation of the articular cartilage and OA development. Targeting Fyn is a novel and potentially therapeutic approach to the treatment of OA.

Anti-arthritic activity of 11-O-(4'-O-methyl galloyl)-bergenin and Crassula capitella extract in rats.

OBJECTIVES: Isolation and identification of phytochemicals of Crassula capitella (Thunberg), evaluation of the anti-arthritic potential of the extract and the major isolated compound; 11-O-(4'-O-methyl galloyl)-bergenin and underlying their mechanism on rat model of rheumatoid arthritis (RA). METHODS: Different fractions were subjected to column chromatography giving fourteen compound identified by mass and NMR spectroscopic techniques. RA was induced by intraplantar injection of complete Freund's adjuvant into the right hind paw of rats. Influence of tested samples in comparable to methotrexate on paw oedema, body weight gain, serum diagnostic markers, cartilage and bone degeneration enzymes, pro-inflammatory mediators and oxidative stress biomarkers in arthritic rats. KEY FINDINGS: Fourteen phenolic compounds were isolated and identified for the first time from C. capitella. The major compound identified as 11-O-(4'-O-methyl galloyl)-bergenin. Treatment of arthritic rats with extract or 11-O-(4'-O-methyl galloyl)-bergenin with the tested doses can reduce the progression and severity of RA. CONCLUSION: Crassula capitella is a new natural and abundant source for 11-O-(4'-O-methyl galloyl)-bergenin for resolving chronic inflammatory diseases as RA through antioxidant, anti-inflammatory and membrane stabilizing mechanism.

Electroacupuncture protects against articular cartilage erosion by inhibiting mitogen-activated protein kinases in a rat model of osteoarthritis.

OBJECTIVE: The therapeutic effects of electroacupuncture (EA) on osteoarthritis (OA) are well documented; however, the precise mechanisms of action have not yet been fully elucidated. The present study aimed to investigate the effect of EA on cartilage in an experimental animal model of OA induced by anterior cruciate ligament transection (ACLT) and to examine for concomitant changes in the expression of mitogen-activated protein kinases (MAPKs) in the articular cartilage. METHODS: Thirty-three-month-old male Sprague Dawley rats were randomly divided into the following three groups (n=10 each): sham operated group (Control group), ACLT without treatment (ACLT group), and ACLT with EA treatment (ACLT+EA group). One week after ACLT, rats in the ACLT+EA group received 12 weeks of EA treatment. Histological analysis and quantitative real-time PCR were used to investigate the effects of EA on cartilage morphology (quantified using modified Mankin scores) and expression of MAPKs (p38, c-Jun N-terminal kinase (c-Jun), and extracellular signal-regulated kinase (ERK)1), respectively. RESULTS: ACLT produced coarse cartilage surfaces, fibrous degeneration, and fissuring, all of which were suppressed by EA treatment. Although Mankin scores in the ACLT+EA group were significantly higher compared to the Control group (p<0.01), they were significantly lower than the (untreated) ACLT group (p<0.01). The increase in mRNA expression of p38, c-Jun, ERK1, and matrix metalloproteinase (MMP)-13 observed in cartilage after ACLT was significantly inhibited by EA. CONCLUSIONS: EA appears to prevent the degeneration of articular cartilage, at least partly through regulation of MMP-13 and inhibition of MAPKs in the cartilage of rats with ACLT-induced OA.

Alarmins S100A8/S100A9 aggravate osteophyte formation in experimental osteoarthritis and predict osteophyte progression in early human symptomatic osteoarthritis.

OBJECTIVE: Alarmins S100A8 and S100A9 are major products of activated macrophages regulating cartilage damage and synovial activation during murine and human osteoarthritis (OA). In the current study, we investigated whether S100A8 and S100A9 are involved in osteophyte formation during experimental OA and whether S100A8/A9 predicts osteophyte progression in early human OA. METHODS: OA was elicited in S100A9-/- mice in two experimental models that differ in degree of synovial activation. Osteophyte size, S100A8, S100A9 and VDIPEN neoepitope was measured histologically. Chondrogenesis was induced in murine mesenchymal stem cells in the presence of S100A8. Levels of S100A8/A9 were determined in plasma of early symptomatic OA participants of the Cohort Hip and Cohort Knee (CHECK) cohort study and osteophytes measured after 2 and 5 years. RESULTS: Osteophyte size was drastically reduced in S100A9-/- mice in ligaments and at medial femur and tibia on days 21 and 42 of collagenase-induced OA, in which synovial activation is high. In contrast, osteophyte size was not reduced in S100A9-/- mice during destabilised medial meniscus OA, in which synovial activation is scant. S100A8 increased expression and activation of matrix metalloproteinases during micromass chondrogenesis, thereby possibly increasing cartilage matrix remodelling allowing for larger osteophytes. Interestingly, early symptomatic OA participants of the CHECK study with osteophyte progression after 2 and 5 years had elevated S100A8/A9 plasma levels at baseline, while C-reactive protein, erythrocyte sedimentation rate and cartilage oligomeric matrix protein were not elevated at baseline. CONCLUSIONS: S100A8/A9 aggravate osteophyte formation in experimental OA with high synovial activation and may be used to predict osteophyte progression in early symptomatic human OA.

[Effects of acupotomy intervention on regional pathological changes and expression of carti- lage-mechanics related proteins in rabbits with knee osteoarthritis].

OBJECTIVE: To observe the effect of acupotomy (needle-knife) therapy on local pathological changes and cartilage-mechanics related protein expression in rabbits with knee osteoarthritis (KOA) so as to study its mechanisms underlying improving KOA. METHODS: Forty New Zealand rabbits were randomly divided into normal control group, model group, acupotomy group, and electroacupuncture (EA) group (n = 10 in each group). The KOA model was established by immobilization of the left knee-joint (modified Videman method) for 6 weeks. After modeling, acupotomy relaxing was applied to the lateral collateral ligament and patellar ligament of the left knee-joint, once a week for 3 times, and EA (2 Hz/100 Hz, 3 mA) was applied to the left "Yanglingquan" (GB 34), "Yinlingquan" (SP 9), "Neixiyan" (EX-LE 4) and "Waixiyan" (ST 35) for 20 min, 3 times a week for three weeks. The expression levels of Integrin ß1, type II collagen (Col-II), matrix metalloproteinase (MMP)-3 and Aggrecan proteins of the cartilage tissue of the left femoral medial and external condyles were observed by Western blot. Pathological changes of the knee-joint by X-ray scanning and those of the femoral condyle tissue were evaluated by Mankin's scores under light microscope after H. E. staining. RESULTS: X-ray showed successful modeling, and pathological changes of the articular cartilage belonged to the early and moderate lesion of knee osteoarthritis. The Mankin's score was significantly higher in the model group than in the control group (P < 0.01) , and after the treatment, the Mankin's scores were significantly decreased in the acupotomy. group (P < 0.01), rather than in the EA group (P > 0.05). The results of Western blot showed that after modeling, the expression levels of Integrin ß 1, Col-II and Aggrecan proteins of the femoral articular cartilage were considerably decreased (P < 0.01), while that of MMP-3 protein was significantly increased (P < 0.01). Compared with the model group, the decreased expression levels of Integrin ß 1, Col-II and Aggrecan proteins in the acupotomy group and Integrin ß 1 protein in the EA group were notably up-regulated (P < 0.01 , P < 0.05), and MMP-3 expression in the acupotomy group was significantly down-regulated (P < 0.01). No significant changes were found in the EA group in the expression levels of Col-II , Aggrecan and MMP-3 proteins compared with the model group (P > 0.05). CONCLUSION: Acupotomy intervention can relieve KOA-induced injury of the knee-joint in KOA rats, which is associated with its actions in raising expression levels of Integrin ß 1, Col-II and Aggrecan proteins and in lowering the expression of MMP-3 proteins in the articular cartilage, probably by adjusting the mechanics-related signal pathway of the articular chondrocytes.

Inhibition of matrix metalloproteinase-13 expression in IL-1ß-treated articular chondrocytes by a steroidal saponin, spicatoside A, and its cellular mechanisms of action.

Matrix metalloproteinase-13 (MMP-13) plays a critical role in degrading major collagens in human cartilage under some pathological conditions such as osteoarthritis. To establish the therapeutic potential against cartilage degradation, the effects of 12 naturally-occurring triterpenoids and steroids on MMP-13 induction were examined in the human chondrocyte cell line, SW1353. They included coreanoside F1, suavissimoside R1, spicatoside A, 25(S)-ruscogenin, methyl protogracillin, hederagenin, loniceroside A, loniceroside B, loniceroside C, smilaxin A, smilaxin C, and ursolic acid. Among these, only spicatoside A and 25(S)-ruscogenin were found to inhibit MMP-13 expression in IL-1ß-treated SW1353 cells at a pharmacologically-relevant concentration of 10 µM. These effects were also supported by the finding that spicatoside A (20 µM) reduced glycosaminoglycan release from IL-1α-treated rabbit joint cartilage culture to some degree. When the cellular mechanisms of action of spicatoside A in MMP-13 inhibition were investigated, the blocking point was not found among the MMP-13 signaling molecules examined such as mitogen-activated protein kinases, activator protein-1, and nuclear transcription factor-κB. Instead, spicatoside A was found to reduce MMP-13 mRNA stability. All of these findings suggest that spicatoside A and 25(S)-ruscogenin have a therapeutic potential for protecting against cartilage breakdown in arthritic disorders.

Effect of Ermiao Recipe with medicinal guide Angelicae Pubescentis Radix on promoting the homing of bone marrow stem cells to treat cartilage damage in osteoarthritis rats.

OBJECTIVE: To investigate the effect of Ermiao Recipe (, EMR) with medicinal guide Angelicae Pubescentis Radix (APR) on the homing of bone marrow stem cells (BMSCs) to focal zone in osteoarthritis (OA) rats. METHODS: Forty-eight Sprague-Dawley rats were randomly assigned to the sham-operated, model, EMR, and EMR plus APR groups (12 rats in each group). The OA rat model was induced by anterior cruciate ligament transection and medial meniscus resection. All rats were injected with recombinant human granulocyte colonystimulating factor [rhG-CSF, 30 µg/(kg·d) for continuous 7 days], and rats in the EMR and EMR plus APR groups were treated with EMR or EMR plus APR at 1.6 or 1.9 g/(kg·d) for 3 or 6 weeks, respectively. Cartilage histopathologic changes were observed by hematoxylin and eosin staining. Chondrocytes apoptosis and cartilage matrix components were tested by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay and special staining. Interleukin-1ß (IL-1 ß), tumor necrosis factor α (TNF-α), bone morphogenetic protein 2 (BMP-2), and transforming growth factor beta-1 (TGF-ß1) in serum were detected by enzyme-linked immunosorbent assay or radioimmunoassay assay. Matrix metalloproteinase (MMP)-13, tissue inhibitors of metalloproteinase (TIMP)-1, bromodeoxyuridine (BrdU), cluster of differentiation 34 (CD34), and stromal cell derived factor 1 (SDF-1) were measured by immunohistochemistry assay. RESULTS: EMR and EMR plus APR significantly inhibited articular cartilage damage and synovium inflammation in OA rats at 3 or 6 weeks of treatment, the most obvious changes in these parameters were found in the EMR plus APR group. At 6 weeks, compared with EMR treatment, EMR plus APR remarkably inhibited chondrocytes apoptosis and the release of IL-1ß and TNF-α, obviously decreased MMP-13 expression, and significantly increased expressions of proteoglycan, collagen, type II collagen and TIMP-1, serum levels of BMP-2 and TGF-ß1 as well as expressions of BrdU, CD34 and SDF-1 in cartilage articular (P<0.01 or P<0.05). CONCLUSION: The medicinal guide APR improved the therapeutic effects of EMR on OA rats by promoting directional homing of BMSCs to focal zone.

Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes.

BACKGROUND: WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes. METHODS: The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1ß-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed. RESULTS: WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1ß-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1ß. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic acid or mangiferin in IL-1ß-stimulated chondrocytes. CONCLUSIONS: WIN-34B is potentially valuable as a treatment for OA by virtue of its suppression of MMPs, ADAMTSs, and inflammatory mediators, and it's up-regulation of TIMP-1 and TIMP-3 involved in the MAPK pathway.

Action mechanisms of complementary and alternative medicine therapies for rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized as a chronic inflammatory disease in joints and concomitant destruction of cartilage and bone. Cartilage extracellular matrix components, such as type II collagen and aggrecan are enzymatically degraded by matrix metalloproteinases (MMPs) and aggrecanases in RA. Currently, treatments targeting cytokines, including anti-tumor necrosis factor (TNF) α antibodies, soluble TNF receptor, anti-interleukin (IL)-6 receptor antibody, and IL-1 receptor antagonist, are widely used for treating RA in addition to antiantiinflammatory agents and disease-modifying antirheumatic drugs (DMARDs), such as inflmethotrexate, but these treatments have some problems, especially in terms of cost and the increased susceptibility of patients to infection in addition to the existence of low-responders to these treatments. Therefore, therapeutics that can be safely used for an extended period of time would be preferable. Complementary and alternative medicines including traditional Chinese medicines (TCM) have been used for the arthritic diseases through the ages. Recently, there are many reports concerning the anti-arthritic action mechanisms of TCM-based herbal formulas and crude herbal extracts or isolated ingredients. These natural herbal medicines are thought to moderately improve RA, but they exert various actions for the treatment of RA. In this review, the current status of the mechanism exploration of natural compounds and TCM-based herbal formulas are summarized, focusing on the protection of cartilage destruction in arthritic diseases including RA and osteoarthritis.

Effect of dehydroepiandrosterone on aggrecanase expression in articular cartilage in a rabbit model of osteoarthritis.

To investigate the in vivo effect of dehydroepiandrosterone (DHEA) on the expression of aggrecanases and their endogenous inhibitor in a rabbit model of OA. Ten New Zealand white rabbits underwent bilateral anterior cruciate ligament transection (ACLT). One knee of each rabbit was randomly assigned to receive 100 µM DHEA dissolved in dimethylsulphoxide (DMSO) and the other was treated with DMSO only. The treatment was given once a week for 5 weeks, starting 4 weeks after transection. All rabbits were euthanized 9 weeks after ACLT treatment, and the knee joints were evaluated by gene expression analysis. Intra-articular administration of DHEA significantly reduced the gene expression of aggrecanases, while markly increasing that of tissue inhibitor of metalloproteinase-3 (TIMP-3), an endogenous inhibitor of aggrecanases. DHEA may have beneficial effects on OA by influencing the balance between aggrecanases and TIMP-3 through which DHEA may protect against OA.

Superoxide dismutase downregulation in osteoarthritis progression and end-stage disease.

BACKGROUND: Oxidative stress is proposed as an important factor in osteoarthritis (OA). OBJECTIVE: To investigate the expression of the three superoxide dismutase (SOD) antioxidant enzymes in OA. METHODS: SOD expression was determined by real-time PCR and immunohistochemistry using human femoral head cartilage. SOD2 expression in Dunkin-Hartley guinea pig knee articular cartilage was determined by immunohistochemistry. The DNA methylation status of the SOD2 promoter was determined using bisulphite sequencing. RNA interference was used to determine the consequence of SOD2 depletion on the levels of reactive oxygen species (ROS) using MitoSOX and collagenases, matrix metalloproteinase 1 (MMP-1) and MMP-13, gene expression. RESULTS: All three SOD were abundantly expressed in human cartilage but were markedly downregulated in end-stage OA cartilage, especially SOD2. In the Dunkin-Hartley guinea pig spontaneous OA model, SOD2 expression was decreased in the medial tibial condyle cartilage before, and after, the development of OA-like lesions. The SOD2 promoter had significant DNA methylation alterations in OA cartilage. Depletion of SOD2 in chondrocytes increased ROS but decreased collagenase expression. CONCLUSION: This is the first comprehensive expression profile of all SOD genes in cartilage and, importantly, using an animal model, it has been shown that a reduction in SOD2 is associated with the earliest stages of OA. A decrease in SOD2 was found to be associated with an increase in ROS but a reduction of collagenase gene expression, demonstrating the complexities of ROS function.

Inhibition of cyclooxygenase-2 expression and prostaglandin E2 production in chondrocytes by avocado soybean unsaponifiables and epigallocatechin gallate.

OBJECTIVE: To evaluate the anti-inflammatory effect of the combination of avocado soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in cytokine-activated equine chondrocytes. METHODS: Production of type II collagen and aggrecan was verified by immunohistochemistry and Western blot. Chondrocytes were incubated with: (1) control media alone, (2) ASU (4 microg/ml; 8.3 microg/ml), (3) EGCG (4, 40, 400 ng/ml), or (4) the combination of ASU and EGCG for 24h. Cells were next incubated with control medium alone or with IL-1beta (10 ng/ml) and TNF-alpha (1 ng/ml). COX-2 gene expression by real-time PCR analysis and NF-kappaB nuclear translocation by immunohistochemistry were performed after 1h of incubation. PGE(2) production was determined by immunoassay after 24h of incubation. RESULTS: Equine chondrocytes responded to cytokine activation by up-regulated gene expression of COX-2 and increased PGE(2) production. Activation was associated with NF-kappaB translocation. Individually, ASU and EGCG marginally inhibited COX-2 expression and PGE(2) production in activated chondrocytes. In contrast, the combination of ASU and EGCG reduced COX-2 expression close to non-activated control levels and significantly inhibited PGE(2) production. These reductions were statistically greater than those of ASU or EGCG alone. The inhibition of COX-2 expression and PGE(2) production was associated with inhibition of NF-kappaB translocation. CONCLUSION: The present study demonstrates that the anti-inflammatory activity of ASU and EGCG is potentiated when used in combination. This combination may offer an attractive supplement or alternative to non-steroidal anti-inflammatory drugs (NSAIDs) in the management of osteoarthritis.

Contrasting effects of n-3 and n-6 fatty acids on cyclooxygenase-2 in model systems for arthritis.

Cyclooxygenase-2 (COX-2) is intimately involved in symptoms of arthritis while dietary n-3 polyunsaturated fatty acids (PUFA) are thought to be beneficial. In these experiments, using both bovine and human in vitro systems that mimic features of arthritis, we show that the n-3 PUFA eicosapentaenoic acid (EPA) is able to reduce mRNA and protein levels of COX-2. Activity, as assessed through prostaglandin E(2) formation, was also reduced in a dose-dependent manner. These effects of EPA contrasted noticeably with the n-6 PUFA, arachidonic acid. The data provide direct evidence for a molecular mechanism by which dietary n-3 PUFA, such as EPA, can reduce inflammation and, hence, associated symptoms in arthritis.

Panax ginseng C.A. Meyer modulates the levels of MMP3 in S12 murine articular cartilage cell line.

AIM OF THE STUDY: The destruction of cartilage in patients with osteoarthritis occurs due to an imbalance between matrix synthesis and degradation. Cartilage degradation is induced by the activation of matrix metalloproteinases (MMPs). Therefore, this study was conducted to evaluate the cartilage protective effect of Panax ginseng C.A. Meyer (PG). MATERIALS AND METHODS: S12 cells were treated with various concentrations of extract of PG and gensenosides Rd and Rb(3) for 3h, after which 10 ng/ml interleukin-1beta (IL-1beta) was added to the culture media. The levels of MMP3 in the conditioned media were then evaluated using an enzyme-linked immunosorbent assay (ELISA). In addition, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of Type II Collagen and Pro-collagenase. Furthermore, Western blot analysis was performed to identify the roles that PG played in the ERK and p38 signaling pathways. RESULTS: The MMP3 secretion levels of S12 cells were significantly lowered in response to treatment with PG and gensenosides Rd and Rb(3) at a concentration of 100 microg/ml when compared to cells that were treated with IL-1beta. In addition, PG induced the mRNA expression of Type II Collagen dose dependently. Furthermore, phosphorylated p38 and ERK were detected in S12 articular cartilage cell line that was treated with IL-1beta. PG decreased the phosphorylation of p38, but PG did not exert any effect on phospho-ERK. CONCLUSIONS: These findings indicate that PG and gensenosides Rd and Rb(3) suppress MMP3 secretion and that gensenosides Rd and Rb(3) are the major elements involved in the suppression of MMP3 by PG. Furthermore, the suppression of MMP3 by PG occurs via the inhibition of phospho-p38 activation. Therefore, PG may exert a protective effect against the cartilage degradation of OA.

Evaluation of a magnetic resonance biomarker of osteoarthritis disease progression: doxycycline slows tibial cartilage loss in the Dunkin Hartley guinea pig.

The objective was to assess the effect of doxycycline treatment on a magnetic resonance imaging (MRI) biomarker of cartilage volume loss, and on matrix metalloproteinase (MMP) activity in a guinea pig osteoarthritis model. Guinea pigs (9 months old) were dosed with vehicle or doxycycline, 0.6, 3.0 mg/kg/day for 66 days. Fat-suppressed 3D gradient-echo MRI of the left knee was acquired pre- and post dosing. Change in medial tibial plateau (MTP) cartilage volume (MT.VC) was determined using image analysis. At termination, MTP cartilage was removed from knees and proteolytic MMP activity determined using a fluorescent peptide substrate assay. Vehicle-treated animals lost 20.5% (95% CI mean 25.6-15.1) MT.VC. The doxycycline (0.6 mg/kg/day) group lost 8.6% (P < 0.05, 95% CI 20.6 to -5.3) whilst the 3.0 mg/kg/day group lost 10.0% (P < 0.05, 95% CI 13.9-6.0%). Endogenous levels of active MMPs were below limits of detection in all samples. However, doxycycline treatment ablated amino phenyl mercuric acid activated MMP-13 and MMP-8 levels, reduced MMP-9 levels by 65% and MMP-1 levels by 24%. Doxycycline treatment resulted in partial protection from MT.VC loss and was associated with complete reduction in MMP-13 and MMP-8, and partial reduction in MMP-9 activity. These data imply a role of MMPs in cartilage degeneration but incomplete protection suggests that additional doxycycline insensitive mechanisms are important in this model. The protective effect of doxycycline correlates with the clinical finding of lessened joint space narrowing, strengthens the utility of this animal model in identifying disease-modifying osteoarthritic drugs and supports the use of MRI biomarkers of cartilage loss.

Inhibition of cyclooxygenase 2 expression by diallyl sulfide on joint inflammation induced by urate crystal and IL-1beta.

OBJECTIVE: Investigation of the effects of diallyl sulfide (DAS), a garlic sulfur compound, on joint tissue inflammatory responses induced by monosodium urate (MSU) crystals and interleukin-1beta (IL-1beta). DESIGN: The HIG-82 synovial cell line was used to establish the experimental model and DAS regime. Primary cultures of articular chondrocytes and synovial fibroblasts obtained from patients undergoing joint replacement for osteoarthritis were used in experimental studies. Cyclooxygenase (COX) expression following MSU crystal and IL-1beta stimulation with/without DAS co-incubation was assessed by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunocytochemistry and nuclear factor-kappa B (NF-kappaB) activation determined by electrophoretic mobility shift assay. Prostaglandin E2 (PGE(2)) production was measured by enzyme-linked immunosorbent assay (ELISA). DAS effects on COX gene expression in an MSU crystal-induced acute arthritis in rats were assessed by RT-PCR. RESULTS: MSU crystals upregulated COX-2 expression in HIG-82 cells and this was inhibited by co-incubation with DAS. DAS inhibited MSU crystal and IL-1beta induced elevation of COX-2 expression in primary synovial cells and chondrocytes. Production of PGE(2) induced by crystals was suppressed by DAS and celecoxib. MSU crystals had no effect on expression of COX-1 in synovial cells. NF-kappaB was activated by MSU crystals and this was blocked by DAS. Increased expression of COX-2 in synovium following intraarticular injection of MSU crystals in a rat model was inhibited by co-administration of DAS. CONCLUSIONS: DAS prevents IL-1beta and MSU crystal induced COX-2 upregulation in synovial cells and chondrocytes and ameliorates crystal induced synovitis potentially through a mechanism involving NF-kappaB. Anti-inflammatory actions of DAS may be of value in treatment of joint inflammation.