A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1ß-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes.

BACKGROUND/OBJECTIVE: Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1ß. METHODS: The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. RESULTS: BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1ß induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that BME activated autophagy in chondrocytes via inhibition of mammalian target of rapamycin (mTOR) pathway. Of importance is our finding that BME-mediated suppression of IL-1ß induced expression of IL-6, MMP-3, -9, and -13 was autophagy dependent and was abrogated by inhibition of autophagy. CONCLUSION: The above results show that the Butea monosperma (Lam.) extract has strong potential to activate autophagy and suppress IL-1ß induced expression of IL-6 and MMP-3, -9 and -13 in human OA chondrocytes. This study shows that BME or compounds derived from BME can be developed as safe and effective chondroprotective agent(s) that function by activating autophagy to suppress the expression of inflammatory and catabolic factors associated with OA pathogenesis.

Intra-articular injection of microRNA-140 (miRNA-140) alleviates osteoarthritis (OA) progression by modulating extracellular matrix (ECM) homeostasis in rats.

OBJECTIVE: Disruptions of extracellular matrix (ECM) homeostasis are key events in the pathogenesis of osteoarthritis (OA). MicroRNA-140 (miRNA-140) is expressed specifically in cartilage and regulates ECM-degrading enzymes. Our objective in this study was to determine if intra-articular injection of miRNA-140 can attenuate OA progression in rats. DESIGN: miRNA-140 levels in human normal and OA cartilage derived chondrocytes and synovial fluid were assessed by polymerase chain reaction (PCR). After primary human chondrocytes were transfected with miRNA-140 mimic or inhibitor, PCR and western blotting were performed to quantify Collagen II, MMP-13, and ADAMTS-5 expression. An OA model was induced surgically in rats, and subsequently treated with one single intra-articular injection of miRNA-140 agomir. At 4, 8, and 12 weeks after surgery, OA progression were evaluated macroscopically, histologically, and immunohistochemically in these rats. RESULTS: miRNA-140 levels were significantly reduced in human OA cartilage derived chondrocytes and synovial fluid compared with normal chondrocytes and synovial fluid. Overexpressing miRNA-140 in primary human chondrocytes promoted Collagen II expression and inhibited MMP-13 and ADAMTS-5 expression. miRNA-140 levels in rat cartilage were significantly higher in the miRNA-140 agomir group than in the control group. Moreover, behavioural scores, chondrocyte numbers, cartilage thickness and Collagen II expression levels in cartilage were significantly higher, while pathological scores and MMP-13 and ADAMTS-5 expression levels were significantly lower in the miRNA-140 agomir group than in the control group. CONCLUSION: Intra-articular injection of miRNA-140 can alleviate OA progression by modulating ECM homeostasis in rats, and may have potential as a new therapy for OA.

Gypenoside inhibits interleukin-1ß-induced inflammatory response in human osteoarthritis chondrocytes.

Gypenoside (GP), the main active ingredient of Gynostemma pentaphyllum, possesses a variety of pharmacological capacities including anti-inflammation, anti-oxidation, and anti-tumor. However, the effects of GP on IL-1ß-stimulated human osteoarthritis (OA) chondrocytes are still unknown. Therefore, this study aimed to investigate the anti-inflammatory effects of GP on IL-1ß-stimulated human OA chondrocytes and explore the possible mechanism. Our results showed that GP dose-dependently inhibited IL-1ß-induced NO and PGE2 production in human OA chondrocytes. In addition, treatment of GP inhibited the expression of MMP3 and MMP13, which was increased by IL-1ß. Finally, we found that pretreatment of GP obviously suppressed NF-κB activation in IL-1ß-stimulated human OA chondrocytes. Taken together, the results demonstrated that GP has chondro-protective effects, at least in part, through inhibiting the activation of NF-κB signaling pathway in human OA chondrocytes. Thus, these findings suggest that GP may be considered as an alternative therapeutic agent for the management of OA patients.

Ethanol extract of baked Gardeniae Fructus exhibits in vitro and in vivo anti-metastatic and anti-angiogenic activities in malignant cancer cells: Role of suppression of the NF-κB and HIF-1α pathways.

Gardeniae Fructus (GF, Zhi Zi in China), a fruit of Gardenia jasminoides Ellis, has been used in traditional medicine to reduce inflammation and headache and to treat hepatic disorders, hypertension, and icterus. In recent studies, extract of raw or stir-baked GF was shown to have pharmacological activities for viral infection, thrombosis, hyperlipidemia, convulsion, inflammation, oxidative stress, and others. In addition, baked GF extract suppressed the proteolytic activities and altered the cellular morphology of tumor cells. However, the effects of ethanol extract of baked GF (EBGF) on the metastatic and angiogenic capacities of malignant tumor cells and its detailed mechanism of action have not been reported. In this study, we found that EBGF significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 and -13 and uPA expression via suppression of PMA-induced nuclear translocation of NF-κBp65. Metastatic potential, including migration, invasion, and colonization, was substantially reduced by EBGF with no cytotoxicity. In addition, EBGF attenuated tumor-induced angiogenesis, including microvessel sprouting, migration of endothelial cells (ECs), and tube formation of ECs, by inhibiting the release of pro-angiogenic factors from tumor cells. In C57BL/6 mice, we observed that daily administration of EBGF at 50 and 100 mg/kg suppressed metastatic colonization of B16F10 melanoma cells in the lungs. Furthermore, EBGF administration did not cause adverse effects, suggesting that EBGF is safe and may be a potential herbal medicine capable of controlling metastatic malignant cancers.

Identification of Targets of a New Nutritional Mixture for Osteoarthritis Management Composed by Curcuminoids Extract, Hydrolyzed Collagen and Green Tea Extract.

OBJECTIVE: We have previously demonstrated that a mixture of curcuminoids extract, hydrolyzed collagen and green tea extract (COT) inhibited inflammatory and catabolic mediator's synthesis by osteoarthritic human chondrocytes. The objective of this study was to identify new targets of COT using genomic and proteomic approaches. DESIGN: Cartilage specimens were obtained from 12 patients with knee osteoarthritis. Primary human chondrocytes were cultured in monolayer until confluence and then incubated for 24 or 48 hours in the absence or in the presence of human interleukin(IL)-1ß (10-11M) and with or without COT, each compound at the concentration of 4 µg/ml. Microarray gene expression profiling between control, COT, IL-1ß and COT IL-1ß conditions was performed. Immunoassays were used to confirm the effect of COT at the protein level. RESULTS: More than 4000 genes were differentially expressed between conditions. The key regulated pathways were related to inflammation, cartilage metabolism and angiogenesis. The IL-1ß stimulated chemokine ligand 6, matrix metalloproteinase-13, bone morphogenetic protein-2 and stanniocalcin1 gene expressions and protein productions were down-regulated by COT. COT significantly decreased stanniocalcin1 production in basal condition. Serpin E1 gene expression and protein production were down-regulated by IL-1ß. COT reversed the inhibitory effect of IL-1ß. Serpin E1 gene expression was up-regulated by COT in control condition. CONCLUSION: The COT mixture has beneficial effect on osteoarthritis physiopathology by regulating the synthesis of key catabolic, inflammatory and angiogenesis factors. These findings give a scientific rationale for the use of these natural ingredients in the management of osteoarthritis.

Ethanol extract of Terminalia chebula fruit protects against UVB-induced skin damage.

CONTEXT: The fruit of Terminalia chebula Retz. (Combretaceae) has been used for several therapeutic purposes in Thai folk medicines. Currently, the ethanol extracts containing antioxidant compounds have shown the ability to promote collagen synthesis. OBJECTIVE: This purpose of this work was to study the effects of the ethanol extract from T. chebula fruit on the inhibition of cutaneous photodamage. MATERIALS AND METHODS: The viability of human skin fibroblasts after incubation with T. chebula at concentration 0.5-50 µg/mL for 24, 48 and 72 h was assessed by using sodium 3'-[(phenyl-amino)-carbonyl]-3,4,tetrazolium-bis(4-methoxy-6-notro)benzene-sulphonic acid hydrate (XTT). The levels of type I procollagen and matrix metalloproteinases (MMP)-1 and MMP-13 produced by UVB-irradiated fibroblasts were determined by ELISA. Skin thickness and collagen content caused by long-term UVB irradiation in male ICR mice were determined from haematoxylin and eosin stained tissue sections and spectrophotometric measurement of hydroxyproline. RESULTS: The extract (0.5-50 µg/mL) had no effect on cell viability or morphology of the human fibroblasts. In vitro studies showed that the T. chebula extract reduced the UVB-induced MMP-1 and MMP-13 expression, whereas an increased production of type I procollagen was observed. In a UVB-irradiated animal model, male ICR mice with hair shaved were chronically exposed to UVB which lead to epidermal thickness and loss of hydroxyproline. However, these effects were fully prevented by the topical application of the T. chebula ethanol extract. DISCUSSION AND CONCLUSION: These data suggested that the T. chebula ethanol fruit extract is an efficacious pharmaceutical protectant of skin against photodamage.

Tenuigenin Prevents IL-1ß-induced Inflammation in Human Osteoarthritis Chondrocytes by Suppressing PI3K/AKT/NF-κB Signaling Pathway.

Tenuigenin (TEN), the main active component of Polygala tenuifolia, has been reported to have anti-inflammatory effects. However, the effects of TEN on IL-1ß-stimulated osteoarthritis chondrocytes have not been reported. The purpose of this study was to investigate the anti-inflammatory effects and mechanism of TEN on IL-1ß-stimulated human osteoarthritis chondrocytes. Human osteoarthritis chondrocytes were pretreated with or without TEN for 1 h and then stimulated with IL-1ß. The production of NO and PGE2 were detected by the Griess reagent and ELISA. The expression of NF-κB and MAPKs (p38, JNK, ERK) were measured by Western blot analysis. The production of MMP-1, MMP3, and MMP13 were measured by ELISA. The results showed that treatment of TEN significantly inhibited IL-1ß-induced NO and PGE2 production. TEN also suppressed IL-1ß-induced MMP-1, MMP3, and MMP13 expression. Furthermore, TEN was found to inhibit IL-1ß-induced NF-κB activation, PI3K, and AKT phosphorylation. In conclusion, these results suggest that TEN inhibits IL-1ß-induced inflammation in human osteoarthritis chondrocytes by inhibiting PI3K/AKT/NF-κB signaling pathway.

Suppressive Effect of Dietary Fucoidan on Proinflammatory Immune Response and MMP-1 Expression in UVB-Irradiated Mouse Skin.

It is well known that ultraviolet B irradiation leads to dermal inflammation. In this study, we found that Mekabu fucoidan suppressed edema, decreased the thickness of the prickle cell layer, and decreased matrix metalloproteinase 1 in the skin of mice irradiated with ultraviolet B. Moreover, we found that the mean level of interferon gamma of Mekabu fucoidan-treated, ultraviolet B-irradiated mice (approximately 2.2 ng/mL) was not significantly different from that in normal mice (approximately 2.5 ng/mL). In contrast, a significant decrease in the mean level of interferon gamma (approximately 1.3 ng/mL) in ultraviolet B-irradiated control mice was observed compared with that in Mekabu fucoidan-treated, ultraviolet B-irradiated mice. The mean thickness of the prickle cell layer in the skin of Mekabu fucoidan-treated, ultraviolet B-irradiated mice was less than that in the ultraviolet B-irradiated control mice. Metalloproteinase 1 activity was significantly higher in the skin of ultraviolet B-irradiated mice than in the skin of untreated, nonirradiated normal mice. Metalloproteinase 1 in the skin of ultraviolet B-irradiated, Mekabu fucoidan- or L(+)-ascorbic acid (vitamin C)-treated mice was significantly lower than that in the ultraviolet B-irradiated control mice. Mitigation of the morphological changes in Mekabu fucoidan-treated mice was correlated with a decrease in metalloproteinase 1 levels. These data indicate that Mekabu fucoidan is an effective suppressor of inflammation in an ultraviolet B-irradiated mouse model.

Alterative effects of an oral alginate extract on experimental rabbit osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a common joint disease that causes disabilities in elderly. However, few agents with high efficacy and low side effects have been developed to treat OA. In this study, we evaluated the effects of the alginate extract named CTX in OA cell and rabbit models. RESULTS: CTX was formulated by hydrolyzing sodium alginate polymers with alginate lyase and then mixing with pectin. HPLC was used to analyze the CTX content. Human chondrosarcoma SW1353 cells treated with interleukin-1ß were used as OA model cells to investigate the effects of CTX on chondrocyte inflammation and anabolism. CTX at concentrations up to 1000 µg/ml exerted low cytotoxicity. It inhibited the gene expression of proinflammatory matrix metalloproteinases (MMPs) including MMP1, MMP3 and MMP13 in a dose-dependent manner and increased the mRNA level of aggrecan, the major proteoglycan in articular cartilage, at 1000 µg/ml. Thirteen-week-old New Zealand White rabbits underwent a surgical anterior cruciate ligament transection and were orally treated with normal saline, glucosamine or CTX for up to 7 weeks. Examinations of the rabbit femur and tibia samples demonstrated that the rabbits taking oral CTX at a dosage of 30 mg/kg/day suffered lesser degrees of articular stiffness and histological cartilage damage than the control rabbits. CONCLUSIONS: The gene expression profiles in the cell and the examinations done on the rabbit cartilage suggest that the alginate extract CTX is a pharmaco-therapeutic agent applicable for OA therapy.

Inhibitory effects of EGb761 on the expression of matrix metalloproteinases (MMPs) and cartilage matrix destruction.

Cytokines such as tumor necrosis factor alpha (TNF-α)-induced expression of matrix metalloproteinase (MMP) play a pivotal role in the destruction of articular cartilage in patients who are suffering from osteoarthritis (OA). Collagen type II, the basis for articular cartilage, can be degraded by MMP-1, MMP-3, and 13. EGb761, the standardized extract of Ginkgo biloba produced by Dr. Willar Schwabe Pharmaceuticals, has shown its anti-inflammatory capacity. This study aimed to determine a mechanism whereby EGb761 may inhibit cartilage degradation. Our results indicated that pretreatment with EGb761 abolishes MMP-1, MMP-3, and MMP-13 gene expression and protein expression induced by TNF-α in human chondrocyte monolayer. In addition, the reduction of the tissue inhibitor of metalloproteinase-1(TIMP-1) and metalloproteinase-2 gene expression induced by TNF-α was rescued by pretreatment with EGb761. Importantly, TNF-α-induced degradation of collagen type II was ameliorated by EGb761 in a dose-dependent manner. Mechanistically, our results indicated that EGb761 treatment attenuated TNF-α-induced NF-κB activation. These actions of EGb761 suggest a mechanism by which EGb761 may act to prevent cartilage breakdown in arthritis.

Shikonin inhibits invasiveness of osteosarcoma through MMP13 suppression.

Osteosarcoma (OS) is the most common primary malignant bone tumor, notorious for its metastasis. We have recently shown that shikonin, an effective constituent extracted from Chinese medicinal herb, induces necroptosis in OS cells. Nevertheless, the effects of low-dose shikonin on the invasiveness of OS cells are unknown. Here, we showed that shikonin dose-dependently decreased OS cell invasiveness in both scratch wound healing assay and transwell cell migration assay. Moreover, the direct target of shikonin on cell invasiveness was found to be matrix metalloproteinase (MMP)-13. Further, the inhibitory effects of shikonin on cell invasiveness were completely abolished in MMP13-overexpressing OS cells. Together, these data suggest that shikonin may suppress OS invasiveness through MMP13 suppression. Thus, our data highlight a previous unappreciated role for shikonin in suppressing OS cell metastasis.

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.

Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1ß induced matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human chondrocytes.

OBJECTIVE: Interleukin-1ß (IL-1ß) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. METHODS: Effects of WIN-55 were studied on IL-1ß stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. RESULTS: Treatment with WIN-55 alone or in combination with IL-1ß, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1ß, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. CONCLUSION: Cannabinoid WIN-55 can reduce both basal and IL-1ß stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.

[Effect of laminarin polysaccharide on activity of matrix metalloproteinase in photoaging skin].

OBJECTIVE: To study the effect of laminarin polysaccharide (LP) on the activity of matrix metalloproteinase of photoaging skins. METHOD: Kunming SPF mice were prepared with back hair shaved, and randomly divided into the control group, the model group, the LP low does group (LP-L, 1 mg x kg(-1)), the LP high dose group (LP-H, 5 mg x kg(-1)) and the Vit E (100 mg x kg(-1)) group. They were abdominally injected with drugs twice on a daily basis. Except for the control group, all groups were exposed to ultraviolet rays for 1 hour every day, five times on a weekly basis, with accumulated exposure dose of UVB being 21.60 J x cm(-2) and accumulated exposure dose of UVA being 84.02 J x cm(-2). Eight weeks later, exposed back skins were collected to detect thickness of dermis by HE stain, content of hydroxyproline (Hyp) by chemical colorimetry, and serum MMP-1 and TIMP-1 content by ELISA. In addition, matrix metalloproteinase-1 (MMP-1) mRNA and relative content of tissue inhibitor of metalloproteinase-1 (TIMP1) mRNA was analyzed with Real-time PCR. RESULT: Compared with the model group, the LP-H group could significantly increase the thickness of dermis, skin Hyp content and serum TIMP-1 level, and decrease relative content of MMP-1 mRNA in skin and MMP-1 content in serum. CONCLUSION: LP can regulate the metabolism of collagen photoaging skins by adjusting the activity of matrix metalloproteinase.

Salubrinal reduces expression and activity of MMP13 in chondrocytes.

OBJECTIVE: Stress to the endoplasmic reticulum (ER) and inflammatory cytokines induce expression and activity of matrix metalloproteinase 13 (MMP13). Since a synthetic agent, salubrinal, is known to alleviate ER stress and attenuate nuclear factor kappa B (NFκB) signaling, we addressed a question whether upregulation of MMP13 by ER stress and cytokines is suppressed by administration of salubrinal. METHODS: Using C28/I2 human chondrocytes, we applied ER stress with tunicamycin and inflammatory distress with tumor necrosis factor α (TNFα) and interleukin 1ß (IL1ß). RNA interference with siRNA specific to NFκB p65 (RelA) was employed to examine a potential involvement of NFκB signaling in salubrinal's action in regulation of MMP13. We also employed primary human chondrocytes and evaluated MMP13 activity. RESULTS: The result showed that tunicamycin activated p38 mitogen-activated protein kinase (MAPK), while inflammatory cytokines activated p38 MAPK and NFκB. In both cases, salubrinal significantly reduced expression and activity of MMP13. Silencing NFκB reduced inflammatory cytokine-driven upregulation of MMP13 activity. CONCLUSIONS: The results demonstrate that salubrinal downregulates expression and activity MMP13 through p38 and NFκB signaling, suggesting its potential usage to treat degenerative diseases such as osteoarthritis.

Yeast hydrolysate protects cartilage via stimulation of type II collagen synthesis and suppression of MMP-13 production.

Type II collagen (COL II) is one of the primary components of hyaline cartilage and plays a key role in maintaining chondrocyte function. COL II is the principal target of destruction, and matrix metalloproteases (MMPs) have a major role in arthritis. In the present study, we investigated the chondroctye protection effects of specific fraction of yeast hydrolysate ((10-30 kDa molecular weight peptides). The mRNA expression of COL II was significantly increased in the YH-treated group compared to the control at concentrations above 50 µg/ml, respectively. The 200 µg/ml YH-treated group (3.43 ± 0.23 µg/ml) showed significantly reduced glycosaminoglycan (GAG) degradation relative to that in the interleukin-1ß (IL-1ß)-treated control group (4.72 ± 0.05 µg/ml). In the YH-treated group, MMP-13 level was significantly decreased in a dose-dependent manner compared to the IL-1ß-treated group without YH treatment. However, MMP-1 and MMP-3 level were not different from that of control. Under the same conditions, we also examined mRNA levels of COL II. The mRNA expression of COL II was significantly higher in the YH-treated group than in the IL-1ß-treated control group at concentrations above 100 µg/ml. In conclusion, YH stimulated COL II synthesis and significantly inhibited MMP-13 and GAG degradation caused by IL-1ß treatment.

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.

Boswellia frereana (frankincense) suppresses cytokine-induced matrix metalloproteinase expression and production of pro-inflammatory molecules in articular cartilage.

The aim of this study was to assess the anti-inflammatory efficacy of Boswellia frereana extracts in an in vitro model of cartilage degeneration and determine its potential as a therapy for treating osteoarthritis. Cartilage degradation was induced in vitro by treating explants with 5 ng/ml interleukin1alpha (IL-1alpha) and 10 ng/ml oncostatin M (OSM) over a 28-day period, in the presence or absence of 100 microg/ml B. frereana. Treatment of IL-1alpha/OSM stimulated cartilage explants with B. frereana inhibited the breakdown of the collagenous matrix. B. frereana reduced MMP9 and MMP13 mRNA levels, inhibited MMP9 expression and activation, and significantly reduced the production of nitrite (stable end product of nitric oxide), prostaglandin E2 and cycloxygenase-2. Epi-lupeol was identified as the principal constituent of B. frereana. This is the first report on the novel anti-inflammatory properties of Boswellia frereana in an in vitro model of cartilage degradation. We have demonstrated that B. frereana prevents collagen degradation, and inhibits the production of pro-inflammatory mediators and MMPs. Due to its efficacy we propose that B. frereana should be examined further as a potential therapeutic agent for treating inflammatory symptoms associated with arthritis.

Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-alpha and matrix metalloproteinase-13 in human chondrocytes.

INTRODUCTION: The major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) can activate chondrocytes and induce the production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG) on AGE-modified-BSA (AGE-BSA)-induced activation and production of TNFalpha and MMP-13 in human OA chondrocytes. METHODS: Human chondrocytes were derived from OA cartilage by enzymatic digestion and stimulated with in vitro-generated AGE-BSA. Gene expression of TNFalpha and MMP-13 was measured by quantitative RT-PCR. TNFalpha protein in culture medium was determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the MMP-13 production in the culture medium, phosphorylation of mitogen-activated protein kinases (MAPKs), and the activation of NF-kappaB. DNA binding activity of NF-kappaB p65 was determined using a highly sensitive and specific ELISA. IkappaB kinase (IKK) activity was determined using an in vitro kinase activity assay. MMP-13 activity in the culture medium was assayed by gelatin zymography. RESULTS: EGCG significantly decreased AGE-stimulated gene expression and production of TNFalpha and MMP-13 in human chondrocytes. The inhibitory effect of EGCG on the AGE-BSA-induced expression of TNFalpha and MMP-13 was mediated at least in part via suppression of p38-MAPK and JNK activation. In addition, EGCG inhibited the phosphorylating activity of IKKbeta kinase in an in vitro activity assay and EGCG inhibited the AGE-mediated activation and DNA binding activity of NF-kappaB by suppressing the degradation of its inhibitory protein IkappaBalpha in the cytoplasm. CONCLUSIONS: These novel pharmacological actions of EGCG on AGE-BSA-stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG-derived compounds may inhibit cartilage degradation by suppressing AGE-mediated activation and the catabolic response in human chondrocytes.

[Study on Yiqi Huayu Bushen Recipe and its disassembled recipes in regulating mRNA expression of collagens and metabolic enzymes in extracellular matrix of cervical disc].

OBJECTIVE: To study the effects of Yiqi Huayu Bushen Recipe (YHBR) and its disassembled recipes on mRNA expressions of collagen I, III, X, matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMP) in extracellular matrix of cervical disc in model rats of cervical vertebral disc degeneration. METHODS: The mRNA expressions of collagens, MMP-13 and TIMP-1 were detected by RT-PCR. The strips were scanned by gel imaging system scanner, and the optical density was autocalculated by computer. RESULTS: Compared with those of the sham-operative group, the mRNA expressions of collagen I , Ill and X and MMP-13 of the model rats increased markedly (P < 0.01), which were lowered by YHBR and its disassembled recipes (P < 0.01 or P < 0.05), and the levels after YHBR treatment were significantly different to those after Western medicine treatment. However, no remarkable change was found in TIMP-1 mRNA expression in the model rats (P > 0.05). CONCLUSION: In the degenerated intervertebral disc the mRNA expressions of collagen I , III, X and MMP-13 increased, TIMP-1 mRNA expression decreased and the proportion of MMPs/TIMP was in imbalance. YHBR and its disassembled recipes could postpone the degeneration of intervertebral disc through regulating mRNA expressions of collagens and their correlated metabolic enzymes.