Protective effects of biochanin A on articular cartilage: in vitro and in vivo studies.

BACKGROUND: Increased production of matrix metalloproteinases (MMPs) is closely related to the progression of osteoarthritis (OA). The present study was performed to investigate the potential value of biochanin A in inhibition of MMP expression in both rabbit chondrocytes and an animal model of OA. METHODS: MTT assay was performed to assess chondrocyte survival in monolayers. The mRNA and protein expression of MMPs (including MMP-1, MMP-3, and MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in interleukin-1 < beta > (IL-1ß)-induced rabbit chondrocytes were determined by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The involvement of the NF-kappaB (NF-κB) pathway activated by IL-1ß was determined by western blotting. The in vivo effects of biochanin A were evaluated by intra-articular injection in an experimental OA rabbit model induced by anterior cruciate ligament transection (ACLT). RESULTS: Biochanin A downregulated the expression of MMPs and upregulated TIMP-1 at both the mRNA and protein levels in IL-1ß-induced chondrocytes in a dose-dependent manner. In addition, IL-1ß-induced activation of NF-κB was attenuated by biochanin A, as determined by western blotting. Moreover, biochanin A decreased cartilage degradation as determined by both morphological and histological analyses in vivo. CONCLUSIONS: Taken together, these findings suggest that biochanin A may be a useful agent in the treatment and prevention of OA.

Inhibition of matrix metalloproteinases and inducible nitric oxide synthase by andrographolide in human osteoarthritic chondrocytes.

OBJECTIVE: The aim of this study was to investigate the effects of andrographolide on matrix metalloproteinases (MMP) 1, 3, and 13 and inducible nitric oxide synthase (iNOS) in human articular chondrocytes from osteoarthritic cartilage. METHODS: Passaged chondrocytes were pretreated with or without andrographolide for 2 h, followed by coincubation with interleukin-1 beta (IL-1ß) 1 ng/ml for 24 h. Expression levels of MMP-1, 3, and 13, tissue inhibitor of metalloproteinase-1 (TIMP-1), and iNOS were evaluated using real-time-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Nitric oxide (NO) was analyzed using the Griess reaction assay. Involvement of nuclear factor kappa B (NF-κB) was assessed by Western blotting, transient transfection, and luciferase reporter assay. RESULTS: Andrographolide tested in these in vitro studies was found be an effective antiarthritic agent, as evidenced by potent inhibition of MMP-1, 3, and 13 and iNOS expression, as well as upregulation of TIMP-1 in IL-1ß-stimulated human articular chondrocytes (p < 0.05). The mechanism of andrographolide's inhibitory effects was mediated by attenuating the activation of NF-κB in human chondrocytes in the presence of IL-1ß. CONCLUSIONS: Andrographolide was a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes, suggesting that this natural compound may merit consideration as a therapeutic agent for treating and preventing osteoarthritis.

Knockdown of KIAA1199 suppresses IL-1ß-induced cartilage degradation and inflammatory responses in human chondrocytes through the Wnt/ß-catenin signalling pathway.

The overproduction of proteolytic enzymes and dysregulation of extracellular matrix (ECM) metabolism have been shown to accelerate the degradation process of articular cartilage. The purpose of this study was to investigate the role of KIAA1199 and its association with the pathophysiology of osteoarthritis (OA). We found that the expression of KIAA1199 was significantly upregulated in OA cartilage compared with normal tissues. Serum levels of KIAA1199 were higher in OA patients than in non-OA patients. Furthermore, knockdown of KIAA1199 inhibited interleukin-1 beta (IL-1ß)-induced ECM metabolic imbalance by regulating the expression of A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5; matrix metallopeptidase-13; aggrecan; and COL2A1. In addition, silencing of KIAA1199 significantly decreased the expression of inflammatory mediators such as prostaglandin E2, IL-6, and TNF-α. Mechanistic analyses further revealed that IL-1ß-induced activation of the Wnt/ß-catenin pathway was suppressed during KIAA1199 knockdown. Moreover, KIAA1199 expression was also upregulated in an in vivo rat OA model. Together, these results increase our understanding of the emerging role of KIAA1199 in the process of OA degeneration, and may lead to a novel molecular target to prevent cartilage degradation.

Emodin ameliorates cartilage degradation in osteoarthritis by inhibiting NF-κB and Wnt/ß-catenin signaling in-vitro and in-vivo.

The overproduction of MMPs (matrix metalloproteinases) and members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) protein family plays an important role in the pathogenesis of osteoarthritis (OA). The potential of selective MMPs or ADAMTS inhibitors as chemopreventive agents for OA has been demonstrated in several studies. In this study, we investigated the protective effects of emodin (1,3,8-trihydroxy-6-methylanthaquinone), isolated from the root of Rheum palmatum L., in the inhibition of MMP and ADAMTS expression in both rat chondrocytes and an animal model of OA. The expression of MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, aggrecan, and collagen II mRNA and protein in interleukin-1beta (IL-1ß)-induced rat chondrocytes was followed by quantitative real-time PCR and western blot. The activation of the NF-κB and Wnt/ß-catenin pathways by IL-1ß was assessed by western blot. The in vivo effects of emodin were evaluated by intra-articular injection in rats in an experimental model of OA induced by anterior cruciate ligament transection. Emodin dose-dependently down-regulated the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 at both the mRNA and protein level in IL-1ß-stimulated rat chondrocytes. In addition, the IL-1ß-induced activation of NF-κB and Wnt signals was attenuated by emodin, as determined by western blotting. The intra-articular injection of emodin in a rat OA model ameliorated OA progression, as determined in morphological and histological analyses in vivo. Taken together, our findings demonstrate that emodin is a promising therapeutic agent for the prevention and treatment of OA.

A possible mechanism in DHEA-mediated protection against osteoarthritis.

Dehydroepiandrosterone (DHEA) and its ester form, DHEA-S, are the most abundant steroids in human plasma. Our previous studies showed that DHEA protects against osteoarthritis (OA). The aim of this paper was to explore the possible mechanisms that underlie DHEA-mediated protection against OA. We tested the expression of ß-catenin, it was increased significantly in OA. Rabbit cartilage was treated with various concentrations of DHEA in both IL-1ß-induced rabbit chondrocytes and in rabbit cartilage from the anterior cruciate ligament transaction-induced OA model. We found DHEA decreased the expression of ß-catenin. Then we further activated Wnt/ß-catenin signaling by ß-catenin transfection and inactivated it by the inhibitor Dickkopf1 in chondrocytes to reveal its role in the pathogenesis of OA. It turns out the protective effect of DHEA was significantly decreased when Wnt/ß-catenin signaling was activated, while inactivating Wnt/ß-catenin signaling enhanced the effects of DHEA. Therefore, we hypothesize that DHEA probably exerted its chondroprotective effect by regulating Wnt/ß-catenin signaling. Our findings demonstrate the critical role of Wnt/ß-catenin signaling in DHEA-mediated protection against OA.

Celastrol, an inhibitor of heat shock protein 90ß potently suppresses the expression of matrix metalloproteinases, inducible nitric oxide synthase and cyclooxygenase-2 in primary human osteoarthritic chondrocytes.

Overexpression of matrix metalloproteinases (MMPs), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have long been suggested to play crucial roles in the progression of osteoarthritis. Studies have showed that selective MMPs, iNOS and COX-2 inhibitors possess great potential as chondroprotective agents for osteoarthritis. Therefore, there have been intensive efforts to develop novel natural compounds that target MMPs, iNOS and COX-2 activation. As interleukin-1ß (IL-1ß) is one of the key proinflammatory cytokines contributing to the progression in osteoarthritis, we investigated the effect of celastrol, a triterpenoid compound extracted from the Chinese herb Tript erygium wilfordii Hook F, in neutralizing the inflammatory effects of IL-1ß on MMPs, iNOS and COX-2 expression as well as nitric oxide (NO) and prostaglandin E2 (PGE2) production. Protein expression was detected by Western blotting or by enzyme-linked immunosorbent assay (ELISA); messenger RNA (mRNA) expression was examined by real-time reverse transcription-polymerase chain reaction analysis and the involvement of signal pathway was assessed by transient transfection and luciferase activity assay. We found that treatment of primary human osteoarthritic chondrocytes with various concentrations of celastrol resulted in striking decrease in the expression of MMP-1, MMP-3, MMP-13, iNOS-2 and COX-2. In addition, celastrol treatment of cells also inhibited the activation of nuclear factor-kappa B (NF-kappaB). Taken together, we provide evidence that celastrol can protect human chondrocytes by downregulating the expression of MMPs, iNOS and COX-2. We suggest that celastrol could be a useful agent for prevention and treatment of osteoarthritis.

Vorinostat, a HDAC inhibitor, showed anti-osteoarthritic activities through inhibition of iNOS and MMP expression, p38 and ERK phosphorylation and blocking NF-κB nuclear translocation.

Overproduction of nitric oxide (NO) and matrix metalloproteinases (MMPs) plays an important role in the pathogenesis of osteoarthritis (OA). In present study, we investigated whether vorinostat can inhibit the catabolic effects of IL-1ß in vitro, especially the inhibition of MMPs and inducible nitric oxide synthase (iNOS) through the attenuation of nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase (MAPK) pathways in human chondrocytes. Human OA chondrocytes were either left untreated or treated with various concentrations of vorinostat followed by incubation with IL-1ß (5ng/mL). Effects of vorinostat on IL-1ß-induced gene and protein expression of iNOS, MMP-1, MMP-13 and tissue inhibitors of metalloproteinase-1 (TIMP-1) were verified by quantitative real time-PCR and Western blot analysis. Production of NO, MMP-1, MMP-13 and TIMP-1 released in culture supernatant was estimated using commercially available kits. The roles of NF-κB and MAPK pathways in the regulation of targeted genes and the mechanism involved in vorinostat mediated modulation of these genes were determined by Western blot using specific antibodies. We found that vorinostat down-regulated iNOS, MMP-1 and MMP-13 expression and up-regulated TIMP-1 expression in human OA chondrocytes. In addition, the release of NO, MMP-1 and MMP-13 secreted from IL-1ß stimulated chondrocytes was also suppressed by vorinostat. Interestingly, vorinostat selectively inhibited IL-1ß-induced p38 and ERK1/2 activation without affecting JNK activation. Furthermore, we observed that vorinostat inhibited NF-κB pathway by suppressing the degradation of I-κBα and attenuating NF-κB p65 translocation to the nucleus. These results suggest that vorinostat may be a promising therapeutic agent for the prevention and treatment of OA.