The spice for joint inflammation: anti-inflammatory role of curcumin in treating osteoarthritis.

Osteoarthritis is a degenerative disease of the joint affecting aging populations worldwide. It has an underlying inflammatory cause, which contributes to the loss of chondrocytes, leading to diminished cartilage layer at the affected joints. Compounds with anti-inflammatory properties are potential treatment agents for osteoarthritis. Curcumin derived from Curcuma species is an anti-inflammatory compound as such. This review aims to summarize the antiosteoarthritic effects of curcumin derived from clinical and preclinical studies. Many clinical trials have been conducted to determine the effectiveness of curcumin in osteoarthritic patients. Extracts of Curcuma species, curcuminoids and enhanced curcumin, were used in these studies. Patients with osteoarthritis showed improvement in pain, physical function, and quality of life after taking curcumin. They also reported reduced concomitant usage of analgesics and side effects during treatment. In vitro studies demonstrated that curcumin could prevent the apoptosis of chondrocytes, suppress the release of proteoglycans and metal metalloproteases and expression of cyclooxygenase, prostaglandin E-2, and inflammatory cytokines in chondrocytes. These were achieved by blocking the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) system in the chondrocytes, by preventing the activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, phosphorylation, and translocation of the p65 subunit of NF-κB complexes into the nucleus. In conclusion, curcumin is a potential candidate for the treatment of osteoarthritis. More well-planned randomized control trials and enhanced curcumin formulation are required to justify the use of curcumin in treating osteoarthritis.

Effects of Payena dasyphylla (Miq.) on hyaluronidase enzyme activity and metalloproteinases protein expressions in interleukin-1ß 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-1ß (IL-1ß) 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-1ß (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.

Effect of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose on elastase and hyaluronidase activities and its type II collagen expression.

In the current era, natural products are gaining prime attention in the fields of cosmeceuticals and pharmaceuticals due to higher safety margins and biological functions, as they have a considerable amount of potential in treating different ailments. Thus, to find effective elastase and hyaluronidase inhibitors from natural resources, fifty Korean plants were screened, and the fruit of Terminalia chebula RETZIUS (Combretaceae) was selected for further structural isolation due to its potent efficacy. The methanol crude extract of the fruits showed 80% elastase and 87% hyaluronidase enzyme inhibition activities at 1 mg/mL. The crude extract, upon bioassay-directed fractionation, led to the isolation of compound 1, whose structure was found by spectral analysis to be 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG). PGG displayed significant elastase and hyaluronidase inhibitory activities with IC50, values of 57 microg/mL and 0.86 mg/mL, respectively; also, treatment of PGG on rabbit articular chondrocytes significantly induced the type II collagen expression. Based on elastase and hyaluronidase inhibitions, and type II collagen expression, it could be suggested that PGG might have an influence on skin conditions when used cosmetically as an active anti-aging ingredient with no cytotoxicity; also, it might be beneficial in relieving painful joint conditions, and thus have relevance for treating arthritis. Therefore, it can be concluded that PGG may prove to be an active ingredient in cosmeceutical and pharmaceutical formulations, and that it definitely merits further in vivo investigations.

Proteomics role in the search for improved diagnosis, prognosis and treatment of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common rheumatic pathology. It is related to aging and is characterized primarily by cartilage degradation. Despite its high prevalence, the diagnostic methods currently available are limited and lack sensitivity. The focus of this review is the application of proteomic technologies in the search of new biomarkers for improved diagnosis, prognosis and treatment of OA. METHODS: This review focuses on the utilization of proteomics in OA biomarker research to enable early diagnosis, improved prognosis and the application of tailored treatments. RESULTS: New diagnostic tests for OA are urgently needed and would also promote the development of alternative therapeutic strategies. Considering that OA involves different tissues and complex biological processes, the most promising diagnostic approach would be the study of combinations of biomarkers. New experimental approaches for the identification and validation of OA biomarkers have recently emerged and include proteomic technologies. These techniques allow the simultaneous analysis of multiple markers and become a very powerful tool for both biomarker discovery and validation. CONCLUSIONS: Improvements in proteomics technology will undoubtedly lead to advances in characterizing new OA biomarkers and developing alternative therapies. Even so, further work is required to enhance the performance and reproducibility of proteomics tools before they can be routinely used in clinical trials and practice.

Proteomics makes progress in cartilage and arthritis research.

Understanding biology at the systems level is a powerful method for discovery of previously unrecognized molecular pathways and mechanisms in human disease. The application of proteomics to arthritis research has lagged behind many other clinical targets, partly due to the unique biochemical properties of cartilage and associated biological fluids such as synovial fluid. In recent years, however, proteomic-based studies in cartilage and arthritis research have risen sharply and have started to make a significant impact on our understanding of joint disease, including the discovery of new and promising biomarkers of cartilage degeneration, a hallmark of arthritis. In this review we will make the case for the ongoing proteomic analysis of cartilage and other tissues affected by joint disease, overview some of the core proteomic techniques and discuss how the challenge of cartilage proteomics has been met through technical innovation. The major outcomes and information obtained from recent proteomic analysis of synovial fluid, cartilage and chondrocytes will also be described. In addition, we present some novel insights into post-translational regulation of cartilage proteins, through proteomic identification of proteolytic fragments in mouse cartilage extracts and explant culture media. We conclude with our prediction of how emerging proteomic technologies that have yet to be applied in arthritis research are likely to contribute further important information.

[Joint resurfacing using allograft chondrocytes embedded in alginate gel].

OBJECTIVE: To explore the feasibility of repairing the articular cartilage with allo-articular chondrocytes embedded in alginate gel. METHODS: Allo-articular chondrocytes were isolated from three adult New zealand rabbits. The cells were cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS). Chondrocytes of 2nd - 3rd passage were harvested and were diluted to 5.0 x 10(7) cells/ml with 1.2% alginate. Then alginate gel was formed by 102 mM CaCl(2). The gels were cultured subsequently for 1 week and then transferred to the full-thickness defects in the femoral condyles of adult rabbits. In control group the defects were left untreated. The animals were sacrificed in the 3rd and 6th month after operation respectively. The specimens were decalcified with 50% formic acid. The histologic sections were stained with safranin O-fast green, hematoxylin-eosin (H&E) and picric acid-Sirius red and immunohisto-stained for type II collagen and aggrecan. The repairing efficiency was evaluated according to Wakitani scoring. RESULT: In the experiment group all 8 defects acquired repair, 7/8 were repaired with mature hyaline cartilage tissue, and 1/8 was with fibrocartilage tissue for less cell-gel inputted. The thick of repaired tissues were closed to the normal and the tissue integrated smoothly with cartilage around the defects. Safranin O staining of the matrix acted in accordance with the normal and immunostaining for type II collagen and aggrecan showed positive. Picric acid-Sirius red staining showed that the chondrocytes lined in lines and the collagen aligned like Gothic architecture structure by polarization microscopy. There was no evidence of residue of alginate and inflammation in 3rd month specimens and no obvious deterioration at 6th month. But in control group, only a small amount of fibrous, fibrocartilage, or hyaline-like tissue was seen on the surface of the defects. Wakitani scoring showed 1.75 points for the experiment group and 7.65 for the control group. CONCLUSION: It is a promising way to repair the articular cartilage with homogeneous articular chondrocytes embedded in alginate gel.

Detection of apoptosis in cartilage in situ and in isolated chondrocytes.

Apoptosis is a form of programmed cell death conceptually opposed to necrosis. In view of the inherent difficulty in accurately detecting apoptosis in chondrocytes, this chapter describes complementary techniques that may be used in combination. During apoptotic death, protein and DNA breakdown is accomplished by caspases (cysteine proteases) in a highly regulated manner. Activation of caspases occurs in the initiation and/or the execution phase of certain apoptotic programs and represents an early physiologic marker of apoptosis. Here we present an immunoblotting technique that allows the detection of caspase-3 processing in cultured human chondrocytes. Apoptosis leads to plasma membrane asymmetry and to externalization of phosphatidylserine residues, which are bound with high affinity by annexin V. In the early stages of apoptosis, cells typically have an intact cell membrane. Apoptotic cells will not stain positive with propidium iodide, whereas externalization of phosphatidylserine will be detected by annexin V. Terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) works on the principle that DNA strand breaks (single or double) that occur during apoptosis can be identified by labeling free 3'-hydroxyl termini. Labeled nucleotides are polymerized to these termini in a reaction catalyzed by TdT. The tissue can then be examined histologically for identification of TUNEL-positive cells in situ.

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes.

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.

Expression and signal transduction of calcium-sensing receptors in cartilage and bone.

We previously showed that Ca2+-sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca2+]([Ca2+]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (approximately 140-190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca2+]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca2+]o reduced PTH-induced cAMP formation. Raising [Ca2+]o also increased intracellular free [Ca2+] in SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca2+]o may couple to signaling pathways important in skeletal metabolism.

Evidence for stanniocalcin gene expression in mammalian bone.

Stanniocalcin (STC) acts as a regulator of calcium and phosphate homeostasis in an endocrine manner in bony fish. Recently, complementary DNAs encoding human and mouse STC have been characterized, and the messenger RNA (mRNA) expression was identified in various tissues, such as kidney, small intestine, prostate, thyroid, and ovary. Because previous studies concerning the effects of fish STC on mammalian bone have been discussed, there is a good possibility that mammalian STC is a local factor in bone. Here, we demonstrated STC mRNA expression in neonatal mouse calvaria, the primary cultured mouse osteoblast-rich fractions, and human and mouse osteoblastic cell lines. We also mapped the cellular distribution of the STC mRNA in femur and calvaria in developing mice. Several transcripts with a major 4-kb band were detected in all samples. The cellular distribution of the mRNA expression corresponded closely to osteoblasts in both femur and calvaria. Significant labeling of the STC mRNA was also identified in chondrocytes but not in osteoclasts and other bone marrow elements. These results are the first evidence that hormone may be actually expressed in osteoblasts and chondrocytes, and they strongly implicate the involvement of local STC in both endochondral and membrane bone as an autocrine/paracrine factor.

Reduced cartilage proteoglycan loss during zymosan-induced gonarthritis in NOS2-deficient mice and in anti-interleukin-1-treated wild-type mice with unabated joint inflammation.

OBJECTIVE: To investigate the role of nitric oxide (NO) and interleukin-1 in (IL-1) joint inflammation and cartilage destruction during zymosan-induced gonarthritis (ZIA). METHODS: Monarticular arthritis was elicited by intraarticular injection of zymosan. The effect of NO deficiency on arthritis was studied in mice with genetically disrupted NOS2. The role of IL-1 was examined by treating wild-type mice with neutralizing anti-murine IL-1(alpha+beta) antibodies. Joint swelling was measured externally by the increased uptake of circulating 99mtechnetium pertechnetate. Proteoglycan (PG) synthesis was assessed using 35S-sulfate incorporation into patellae ex vivo. Histology evaluated exudation and infiltration of leukocytes and the extent of cartilage destruction. RESULTS: The proinflammatory mediators NO, IL-1, and IL-6 were released by the articular tissues during the first hours of inflammation. Interestingly, anti-IL-1 treatment moderately reduced, and NOS2 deficiency moderately enhanced, joint swelling. However, the influx of neutrophils into the joint occurred independently of IL-1 and NOS2 activities. In the first week of inflammation, chondrocyte PG synthesis was significantly suppressed and chondrocytes became unresponsive to their essential anabolic factor, insulin-like growth factor 1 (IGF-1). Anti-IL-1 treatment or NOS2 deficiency prevented the inhibition of PG synthesis, and the chondrocytes remained IGF-1 responsive. Intraarticular injections of IL-1alpha into NOS2-deficient mice did not affect PG synthesis, thus proving that NO mediated this IL-1 effect in vivo. Furthermore, histology showed that cartilage PG loss was markedly ameliorated in NOS2-deficient and anti-IL-1-treated mice. Intermediate cartilage pathology was found in mice that were heterozygous for disrupted NOS2. CONCLUSION: IL-1 and NO play a minor role in edema and neutrophil influx, but a major role in cartilage destruction of ZIA. In this model of murine arthritis, cartilage destruction was, for the most part, caused by pronounced suppression of PG synthesis and IGF-1 unresponsiveness of the chondrocytes, which were induced by de novo-synthesized IL-1 and were mediated by NOS2 activation.