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Tetracycline and its analogs have been shown to be of therapeutic benefit in several animal models of disease and in human arthritis The collagenase subfamily of MMP consists of 3 different enzymes: fibroblast collagenase (MMP-1) 3 , neutrophil collagenase (MMP-8) Because of the discrepancy between the ability of doxycycline to prevent the development of experimental osteoarthritis (OA) and the level required to directly inhibit collagenase, we have investigated other modes by which doxycycline could inhibit collagenase activity. We have reported 10 that doxycycline at concentrations as low as 1 µg/ml (2.08 µM) are capable of downregulating the production of MMP-1 and MMP-13. It has also been shown by Jonat and co-workers that 1 µM doxycycline (0.48 µg/ml) will inhibit stromelysin transcriptio

Effects of Payena dasyphylla (Miq.) on hyaluronidase enzyme activity and metalloproteinases protein expressions in interleukin-1beta stimulated human chondrocytes cells

Background: Hyaluronidases have been found as the target enzymes in the development of osteoarthritis (OA) disease. While there is still no curative treatment for this disease, recent studies on the treatment of OA were focused on the effectiveness of natural products which are expected to improve the symptoms with minimal side effects. The aim of this study was to screen selected Malaysian plants on their anti-hyaluronidase activity as well as to evaluate the active plant and its derived fractions on its potential anti-arthritic and antioxidant activities.Methods: A total of 20 methanolic crude extracts (bark and leaf) from ten different plants were screened using a colorimetric hyaluronidase enzymatic assay. The active plant extract (Payena dasyphylla) was then studied for its hyaluronidase inhibitory activity in the interleukin-1β (IL-1β) stimulated human chondrocytes cell line (NHAC-kn) using zymography method. The Payena dasyphylla methanolic bark extract was then fractionated into several fractions in where the ethyl acetate (EA) fraction was evaluated for its inhibitory effects on the HYAL1 and HYAL2 gene expressions using reverse transcription-polymerase chain reaction (RT-PCR) technique. While the MMP-3 and MMP-13 protein expressions were evaluated using western blot method. The phenolic and flavonoid contents of the three fractions as well as the antioxidant property of the EA fraction were also evaluated.Results: Bark extract of Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 91.63%. The plant extract also inhibited hyaluronidase expression in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml). Similarly, treatment with Payena dasyphylla ethyl acetate (EA) fraction (100 μg/ml) inhibited the HYAL1 and HYAL2 mRNA gene expressions as well as MMP-3 and MMP-13 protein expression in a dose dependent manner. Payena dasyphylla EA fraction has demonstrated the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively as compared to water and hexane fractions. In addition, the Payena dasyphylla EA fraction showed strong antioxidant activity with IC50 value of 11.64 ± 1.69 μg/mL.Conclusion: These findings have shown that Payena dasyphylla might contained potential phenolic compounds that inhibiting the key enzyme in osteoarthritis development, which is the hyaluronidase enzyme through interruption of HYAL1 and HYAL1 gene expressions. The degradation of cartilage could also be inhibited by the plant through suppression of MMP-3 and MMP-13 protein expressions. We also reported that the inhibitory effect of Payena dasyphylla on hyaluronidase activity and expression might be due to its anti-oxidant property

Melanocortin peptides protect chondrocytes from mechanically induced cartilage injury

Introduction Mechanical injury can greatly influence articular cartilage, propagating inflammation, cell injury and death – risk factors for the development of osteoarthritis. Melanocortin peptides and their receptors mediate anti-inflammatory and pro-resolving mechanisms in chondrocytes. This study aimed to investigate the potential chondroprotective properties of α-MSH and [DTRP8]-γ-MSH in mechanically injured cartilage explants, their ability to inhibit pro-inflammatory and stimulate anti-inflammatory cytokines in in situ and in freshly isolated articular chondrocytes. Methods The effect of melanocortins on in situ chondrocyte viability was investigated using confocal laser scanning microscopy of bovine articular cartilage explants, subjected to a single blunt impact (1.14 N, 6.47 kPa) delivered by a drop tower. Chondroprotective effects of α-MSH, [DTRP8]-γ-MSH and dexamethasone on cytokine release by TNF-α-activated freshly isolated articular chondrocytes/mechanically injured cartilage explants were investigated by ELISA. Results A single impact to cartilage caused discreet areas of chondrocyte death, accompanied by pro-inflammatory cytokine release; both parameters were modulated by α-MSH, [DTRP8]-γ-MSH and dexamethasone. Melanocortin pre-treatment of TNF-α-stimulated freshly isolated chondrocytes resulted in a bell-shaped inhibition in IL-1β, IL-6 and IL-8, and elevation of IL-10 production. The MC3/4 antagonist, SHU9119, abrogated the effect of [DTRP8]-γ-MSH but not α-MSH on cytokine release. Conclusion Melanocortin peptide pre-treatment prevented chondrocyte death following mechanical impact to cartilage and led to a marked reduction of pro-inflammatory cytokines, whilst prompting the production of anti-inflammatory/pro-resolving cytokine IL-10. Development of small molecule agonists towards melanocortin receptors could thus be a viable approach for preventing chondrocyte inflammation and death within cartilage and represent an alternative approach for the treatment of osteoarthritis

Cellular senescence in osteoarthritis pathology

Cellular senescence is a state of stable proliferation arrest of cells. The senescence pathway has many beneficial effects and is seen to be activated in damaged/stressed cells, as well as during embryonic development and wound healing. However, the persistence and accumulation of senescent cells in various tissues can also impair function and have been implicated in the pathogenesis of many age‐related diseases. Osteoarthritis (OA), a severely debilitating chronic condition characterized by progressive tissue remodeling and loss of joint function, is the most prevalent disease of the synovial joints, and increasing age is the primary OA risk factor. The profile of inflammatory and catabolic mediators present during the pathogenesis of OA is strikingly similar to the secretory profile observed in ‘classical’ senescent cells. During OA, chondrocytes (the sole cell type present within articular cartilage) exhibit increased levels of various senescence markers, such as senescence‐associated beta‐galactosidase (SAβGal) activity, telomere attrition, and accumulation of p16ink4a. This suggests the hypothesis that senescence of cells within joint tissues may play a pathological role in the causation of OA. In this review, we discuss the mechanisms by which senescent cells may predispose synovial joints to the development and/or progression of OA, as well as touching upon various epigenetic alterations associated with both OA and senescence

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage