Modulation of cytokine-induced prostaglandin E2 production in cultures of articular chondrocytes obtained from carpal joints of camels (Camelus dromedarius).

OBJECTIVE: To determine whether camel articular chondrocytes can be maintained in tissue culture without phenotype loss and whether the response to cytokine stimulation can be modulated. SAMPLE POPULATION: Cartilage from 4 carpal joints of healthy adult dromedary camels (Camelus dromedarius). PROCEDURES: Chondrocytes were evaluated for type II collagen and aggrecan production They were incubated with control media or with 2 test mixtures (alone and then in combination) that have anti-inflammatory activity (avocado-soybean unsaponifiables, glucosamine, and chondroitin sulfate [ie, ASU + GLU + CS] and pentosan polysulfate and N-acetyl glucosamine [ie, PPS + NG]). Cells were then stimulated with interleukin-1ß and tumor necrosis factor-α to determine prostaglandin (PG) E2 production and nuclear factor (NF)-κB activation. RESULTS: Chondrocytes proliferated in media used for propagating equine chondrocytes; they produced type II collagen and aggrecan. Cytokine stimulation induced PGE2 production and translocation of NF-κB. Incubation with each test mixture significantly inhibited PGE2 production. The combination of ASU + GLU + CS and PPS + NG significantly potentiated PGE2 inhibition and disrupted NF-κB translocation, compared with effects for either mixture alone. CONCLUSIONS AND CLINICAL RELEVANCE: Chondrocytes proliferated without loss of the cartilage phenotype. Responses to cytokines were significantly inhibited by the mixtures of ASU + GLU + CS and PPS + NG, which indicated that this response can be modulated. This culture technique can be used to study the functional properties of camel chondrocytes and identify agents that may potentially be used to treat and manage joint inflammation.

Anti-Inflammatory Effect of Carprofen Is Enhanced by Avocado/Soybean Unsaponifiables, Glucosamine and Chondroitin Sulfate Combination in Chondrocyte Microcarrier Spinner Culture.

OBJECTIVE: Osteoarthritis is a painful, chronic joint disease affecting man and animals with no known curative therapies. Palliative nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used but they cause adverse side effects prompting the search for safer alternatives. To address this need, we evaluated the anti-inflammatory activity of avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS) with or without the NSAID carprofen. DESIGN: Canine chondrocytes were propagated in microcarrier spinner culture and incubated with (1) control medium, (2) ASU (8.3 µg/mL) + GLU (11 µg/mL) + CS (20 µg/mL) combination for 24 hours; and/or carprofen (40 ng/mL). Cultures were next incubated with control medium alone or IL-1ß (10 ng/mL) for another 24 hours. Production of PGE2, IL-6, IL-8, and MCP-1 (also known as CCL-2) were measured by ELISA. RESULTS: Chondrocytes proliferated in microcarrier spinner culture and produced type II collagen and aggrecan. Stimulation with IL-1ß induced significant increases in PGE2, IL-6, IL-8, and MCP-1 production. The increases in production were suppressed by carprofen as well as [ASU+GLU+CS]. The combination of carprofen and [ASU+GLU+CS] reduced PGE2 production significantly more than either preparation alone. The inhibitory effect of carprofen on IL-6, IL-8, and MCP-1 production was significantly less than that of [ASU+GLU+CS], whereas the combination did not reduce the production of these molecules significantly more than [ASU+GLU+CS] alone. CONCLUSIONS: The potentiating effect of [ASU+GLU+CS] on low-dose carprofen was identified in chondrocyte microcarrier spinner cultures. Our results suggest that the combination of low-dose NSAIDs like carprofen with [ASU+GLU+CS] could offer a safe, effective management for joint pain.

Avocado/Soybean Unsaponifiables, Glucosamine and Chondroitin Sulfate Combination Inhibits Proinflammatory COX-2 Expression and Prostaglandin E2 Production in Tendon-Derived Cells.

Tendinopathy, a common disorder in man and horses, is characterized by pain, dysfunction, and tendon degeneration. Inflammation plays a key role in the pathogenesis of tendinopathy. Tendon cells produce proinflammatory molecules that induce pain and tissue deterioration. Currently used nonsteroidal anti-inflammatory drugs are palliative but have been associated with adverse side effects prompting the search for safe, alternative compounds. This study determined whether tendon-derived cells' expression of proinflammatory cyclooxygenase (COX)-2 and production of prostaglandin E2 (PGE2) could be attenuated by the combination of avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS). ASU, GLU, and CS have been used in the management of osteoarthritis-associated joint inflammation. Tenocytes in monolayer and microcarrier spinner cultures were incubated with media alone, or with the combination of ASU (8.3 µg/mL), GLU (11 µg/mL), and CS (20 µg/mL). Cultures were next incubated with media alone, or stimulated with interleukin-1ß (IL-1ß; 10 ng/mL) for 1 h to measure COX-2 gene expression, or for 24 h to measure PGE2 production, respectively. Tenocyte phenotype was analyzed by phase-contrast microscopy, immunocytochemistry, and Western blotting. Tendon-derived cells proliferated and produced extracellular matrix component type I collagen in monolayer and microcarrier spinner cultures. IL-1ß-induced COX-2 gene expression and PGE2 production were significantly reduced by the combination of (ASU+GLU+CS). The suppression of IL-1ß-induced inflammatory response suggests that (ASU+GLU+CS) may help attenuate deleterious inflammation in tendons.

α-Lipoic Acid Potentiates the Anti-Inflammatory Activity of Avocado/Soybean Unsaponifiables in Chondrocyte Cultures.

Objective Pro-inflammatory mediators such as prostaglandin E-2 (PGE2) play major roles in the pathogenesis of osteoarthritis (OA). Although current pharmacologic treatments reduce inflammation, their prolonged use is associated with deleterious side effects prompting the search for safer and effective alternative strategies. The present study evaluated whether chondrocyte production of PGE2 can be suppressed by the combination of avocado/soybean unsaponifiables (ASU) and α-lipoic acid (LA). Design Chondrocytes from articular cartilage of equine joints were incubated for 24 hours with: (1) control media, (2) ASU, (3) LA, or (4) ASU + LA combination. Cells were activated with lipopolysaccharide (LPS), interleukin 1ß (IL-1ß) or hydrogen peroxide (H2O2) for 24 hours and supernatants were immunoassayed for PGE2. Nuclear factor-kappa B (NF-κB) analyses were performed by immunocytochemistry and Western blot following 1 hour of activation with IL-1ß. Results LPS, IL-1ß, or H2O2 significantly increased PGE2 production. ASU or LA alone suppressed PGE2 production in LPS and IL-1ß activated cells. Only LA alone at 2.5 µg/mL was inhibitory in H2O2-activated chondrocytes. ASU + LA inhibited more than either agent alone in all activated cells. ASU + LA also inhibited the IL-1ß induced nuclear translocation of NF-κB. Conclusions The present study provides evidence that chondrocyte PGE2 production can be inhibited by the combination of ASU + LA more effectively than either ASU or LA alone. Inhibition of PGE2 production is associated with the suppression of NF-κB translocation. The potent inhibitory effect of ASU + LA on PGE2 production could offer a potential advantage for a combination anti-inflammatory/antioxidant approach in the management of OA.

Expression of pro-inflammatory mediators is inhibited by an avocado/soybean unsaponifiables and epigallocatechin gallate combination.

BACKGROUND: Osteoarthritis (OA) is characterized by inflammation, joint immobility, and pain. Non-pharmacologic agents modulating pro-inflammatory mediator expression offer considerable promise as safe and effective treatments for OA. We previously determined the anti-inflammatory effect of an avocado/soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) combination on prostaglandin E2 (PGE2) production and nuclear factor-kappa B (NF-κB) translocation. The aim of this study was to evaluate the effects of ASU + EGCG on pro-inflammatory gene expression. FINDINGS: Articular chondrocytes from carpal joints of mature horses were pre-incubated for 24 hours with control media alone or ASU (8.3 µg/mL) + EGCG (40 ng/mL), followed by one hour activation with interleukin-1 beta (IL-1ß, 10 ng/mL) and tumor necrosis factor-alpha (TNF-α, 1 ng/mL). Total cellular RNA was isolated and real-time PCR performed to measure IL-1ß, TNF-α, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and interleukin-8 (IL-8) gene expression. Intracellular localization of NF-κB was analyzed by immunohistochemistry and Western blot. Pre-treatment with ASU + EGCG significantly (P < 0.001) decreased gene expression of IL-1ß, TNF-α, IL-6, COX-2, and IL-8 in cytokine-activated chondrocytes. Western blot and immunostaining confirmed NF-κB translocation inhibition. CONCLUSIONS: We demonstrate that ASU + EGCG inhibits cytokine-induced gene expression of IL-1ß, TNF-α, IL-6, COX-2, and IL-8 through modulation of NF-κB. Our results indicate that the activity of ASU + EGCG affects a wide array of inflammatory molecules in addition to decreasing PGE2 synthesis in activated chondrocytes. The responsiveness of chondrocytes to this combination supports its potential utility for the inhibition of joint inflammation.

Modulation of inflammation and oxidative stress in canine chondrocytes.

OBJECTIVE: To determine whether oxidative stress could be induced in canine chondrocytes in vitro. SAMPLE: Chondrocytes obtained from healthy adult mixed-breed dogs. PROCEDURES: Harvested chondrocytes were maintained at 37°C with 5% CO2 for 24 hours. To assess induction of oxidative stress, 2 stimuli were used: hydrogen peroxide and a combination of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). To determine the effect of hydrogen peroxide, a set of chondrocyte-seeded plates was incubated with control medium alone or hydrogen peroxide (100, 200, or 300µM) for 24 hours. For inhibition of oxidative stress, cells were incubated for 24 hours with N-acetylcysteine (NAC; 10mM) before exposure to hydrogen peroxide. Another set of chondrocyte-seeded plates was incubated with control medium alone or with IL-1ß (10 ng/mL) and TNF-α (1 ng/mL) for 24 hours. Supernatants were obtained for measurement of prostaglandin E2 production, and cell lysates were used for measurement of superoxide dismutase (SOD) activity and reduced-glutathione (GSH) concentration. RESULTS: Chondrocytes responded to the oxidative stressor hydrogen peroxide with a decrease in SOD activity and GSH concentration. Exposure to the antioxidant NAC caused an increase in SOD activity in hydrogen peroxide-stressed chondrocytes to a degree comparable with that in chondrocytes not exposed to hydrogen peroxide. Similarly, NAC exposure induced significant increases in GSH concentration. Activation with IL-1ß and TNF-α also led to a decrease in SOD activity and increase in prostaglandin E2 production. CONCLUSIONS AND CLINICAL RELEVANCE: Canine chondrocytes responded to the oxidative stress caused by exposure to hydrogen peroxide and cytokines. Exposure to oxidative stress inducers could result in perturbation of chondrocyte and cartilage homeostasis and could contribute to the pathophysiology of osteoarthritis. Use of antioxidants, on the other hand, may be helpful in the treatment of arthritic dogs.