Comparison of glucose derivatives effects on cartilage degradation

<p>Abstract</p> <p>Background</p> <p>Glucosamine (GlcN) is a well-recognized candidate for treatment of osteoarthritis. However, it is currently used in derivative forms, such as glucosamine-hydrochloride (GlcN-HCl) or glucosamine sulfate (GlcN-S). However, the molecular mode of action remains unclear. In this study, we compared the effects of Glucose (Glc), Glucuronic acid (GlcA), Glucosamine hydrochloride (GlcN-HCl) and Glucosamine sulfate (GlcN-S) on cartilage degradation.</p> <p>Methods</p> <p>Porcine cartilage explants were co-cultured with recombinant human IL-1β and each tested substance for 3 days. HA, s-GAG and MMP-2 releases to media were measured using ELISA, dye-binding assay and gelatin zymography, respectively. Similar studies were performed in a human articular chondrocytes (HAC) monolayer culture, where cells were co-treated with IL-1β and each reagent for 24 hours. Subsequently, cells were harvested and gene expression measured using RT-PCR. All experiments were carried out in triplicate. Student's t-tests were used for statistical analysis.</p> <p>Results</p> <p>In cartilage explants treated with IL-1β, GlcN-S had the highest chondroprotective activity of all four chemicals as shown by the inhibition of HA, s-GAG and MMP-2 released from cartilage. The anabolic (aggrecan core protein; AGG, SOX9) and catabolic (MMP-3, -13) genes in HACs treated with IL-1β and with/without chemicals were studied using RT-PCR. It was found that, GlcN-HCl and GlcN-S could reduce the expression of both MMP-3 and -13 genes. The IL-1β induced-MMP-13 gene expression was decreased maximally by GlcN-S, while the reduction of induced-MMP-3 gene expression was greatest with GlcN-HCl. Glc and GlcA reversed the effect of IL-1β on the expression of AGG and SOX9, but other substances had no effect.</p> <p>Conclusion</p> <p>This study shows that glucosamine derivatives can alter anabolic and catabolic processes in HACs induced by IL-1β. GlcN-S and GluN-HCl decreased induced MMP-3 and -13 expressions, while Glc and GlcA increased reduced-AGG and SOX9 expression. The chondroprotective study using porcine cartilage explant showed that GlcN-S had the strongest effect.</p

Sesamin stimulates osteoblast differentiation through p38 and ERK1/2 MAPK signaling pathways

BACKGROUND: Osteoporosis is a worldwide health problem predominantly affecting post-menopausal women. Therapies aimed at increasing bone mass in osteoporetic patients lag behind comparable investigation of therapeutic strategies focusing on the bone resorption process. Sesamin, a major lignan compound found in Sesamun indicum Linn., has a variety of pharmacological effects, though its activity on bone cell function is unclear. Herein we examine the effect of this lignan on osteoblast differentiation and function. METHOD: Cell cytotoxicity and proliferative in hFOB1.19 were examined by MTT and alamar blue assay up to 96 h of treatment. Gene expression of COL1, ALP, BMP-2, Runx2, OC, RANKL and OPG were detected after 24 h of sesamin treatment. ALP activity was measured at day 7, 14 and 21 of cultured. For mineralized assay, ADSCs were cultured in the presence of osteogenic media supplement with or without sesamin for 21 days and then stained with Alizarin Red S. MAPK signaling pathway activation was observed by using western blotting. RESULTS: Sesamin promoted the gene expression of COL1, ALP, OCN, BMP-2 and Runx2 in hFOB1.19. On the other hand, sesamin was able to up-regulate OPG and down-regulate RANKL gene expression. ALP activity also significantly increased after sesamin treatment. Interestingly, sesamin induced formation of mineralized nodules in adipose derived stem cells (ADSCs) as observed by Alizarin Red S staining; this implies that sesamin has anabolic effects both on progenitor and committed cell stages of osteoblasts. Western blotting data showed that sesamin activated phosphorylation of p38 and ERK1/2 in hFOB1.19. CONCLUSIONS: The data suggest that sesamin has the ability to trigger osteoblast differentiation by activation of the p38 and ERK MAPK signaling pathway and possibly indirectly regulate osteoclast development via the expression of OPG and RANKL in osteoblasts. Therefore, sesamin may be a promising phytochemical that could be developed for supplementation of osteoporotic therapy

Anti-inflammatory effect of methanol extracts of hemp leaf in IL-1β-induced synovitis

Purpose: To examine the effectiveness of some hemp (Canabis sativa) leaf extracts as an antiinflammatory agent on synovitis in vitro.Methods: Synovial fibroblast cell line SW982 was induced with 5 ng/mL of interleukin 1-beta (IL-1β) to trigger cellular inflammation. The cells were then treated with prepared extracts of hemp (Canabis sativa) leaf originating from three different cultivation sites with varying proportions of terpenoids and cannabinoids, especially cannabidiol (CBD) and tetrahydrocannabinol (THC). Nitric oxide (NO) and prostaglandin E2 (PGE2) production as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and IL-1β genes were determined.Results: All hemp extracts reduced the production of NO and PGE2 in a dose-dependent manner. The expressions of iNOS, COX-2 and IL-1β genes were significantly decreased by the hemp extracts. This effect was likely related to the amount of sesquiterpenoids and THC. The extract from Huai Hoi (HH) cultivar showed the most promising results for further studies.Conclusion: The extracts of hemp leaf substantially reduces the level of biomarkers for inflammation in vitro. Therefore, the extracts have a potential application as an inflammatory counteractant in synovitis.Keywords: Canabis sativa, Hemp, Synovitis, Cannabinoids, Nitric oxide, Interleukin-1 bet

In vitro suppression of the MMP-3 gene in normal and cytokine-treated human chondrosarcoma using small interfering RNA

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinase (MMPs) synthesized and secreted from connective tissue cells have been thought to participate in degradation of the extracellular matrix. Increased MMPs activities that degrade proteoglycans have been measured in osteoarthritis cartilage. This study aims to suppress the expression of the <it>MMP-3 </it>gene in <it>in vitro </it>human chondrosarcoma using siRNA.</p> <p>Methods</p> <p>Cells were categorized into four groups: control (G.1); transfection solution treated (G.2); negative control siRNA treated (G.3); and <it>MMP-3 </it>siRNA treated (G.4). All four groups were further subdivided into two groups - treated and non-treated with IL-1β- following culture for 48 and 72 h. We observed the effects of gene suppression according to cell morphology, glycosaminoglycan (GAG) and hyaluronan (HA) production, and gene expression by using real-time polymerase chain reaction (PCR).</p> <p>Results</p> <p>In IL-1β treated cells the apoptosis rate in G.4 was found to be lower than in all other groups, while viability and mitotic rate were higher than in all other groups (<it>p </it>< 0.05). The production of GAG and HA in G.4 was significantly higher than the control group (<it>p </it>< 0.05). <it>MMP-3 </it>gene expression was downregulated significantly (<it>p </it>< 0.05).</p> <p>Conclusion</p> <p><it>MMP-3 </it>specific siRNA can inhibit the expression of <it>MMP-3 </it>in chondrosarcoma. This suggests that <it>MMP-3 </it>siRNA has the potential to be a useful preventive and therapeutic agent for osteoarthritis.</p

A sulfated glycosaminoglycan array for molecular interactions between glycosaminoglycans and growth factors or anti-glycosaminoglycan antibodies

Glycosaminoglycans (GAGS) take part in numerous biological processes by binding to protein molecules and functionally regulating protein-ligand interactions; therefore, molecular interactions of GAGs have been studied by several methods, including surface plasmon resonance, enzyme-linked immunosorbent assays (ELISAs), and GAG microarrays. To achieve rapid, sensitive, and high-throughput screening of GAG interactions, we have developed a novel microarray in which GAGs, including chondroitin sulfate, heparan sulfate, and heparin, were immobilized. The microarray is made from cyclic polyolefin substrate coated with metacrylate polymers, which have phospholipid groups as side chains. The polymer also has aminooxy groups that react specifically with aldehyde groups at the reducing termini of GAG chains, whereas the phospholipid groups prevent nonspecific adsorption of proteins. Thus, minute amounts of GAGs can be chemically immobilized on the surface with low nonspecific binding of proteins. Using this array, interactions between GAGs and antibodies against chondroitin or heparan sulfate and heparin-binding growth factors were examined. The results were in agreement with previously reported specificities, suggesting that the GAG array is useful for high-throughput interaction analyses between GAGs and functional proteins in miniscule amounts and can be applied to both basic studies of GAGs and the development of diagnostic methods for metabolic diseases involving GAGs. (c) 2013 Elsevier Inc. All rights reserved