M.W. Grzanna

Inhibition of cyclooxygenase-2 expression and prostaglandin E2 production in chondrocytes by avocado soybean unsaponifiables and epigallocatechin gallate

OBJECTIVE To evaluate the anti-inflammatory effect of the combination of avocado soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in cytokine-activated equine chondrocytes. METHODS Production of type II collagen and aggrecan was verified by immunohistochemistry and Western blot. Chondrocytes were incubated with: (1) control media alone, (2) ASU (4 microg/ml; 8.3 microg/ml), (3) EGCG (4, 40, 400 ng/ml), or (4) the combination of ASU and EGCG for 24h. Cells were next incubated with control medium alone or with IL-1beta (10 ng/ml) and TNF-alpha (1 ng/ml). COX-2 gene expression by real-time PCR analysis and NF-kappaB nuclear translocation by immunohistochemistry were performed after 1h of incubation. PGE(2) production was determined by immunoassay after 24h of incubation. RESULTS Equine chondrocytes responded to cytokine activation by up-regulated gene expression of COX-2 and increased PGE(2) production. Activation was associated with NF-kappaB translocation. Individually, ASU and EGCG marginally inhibited COX-2 expression and PGE(2) production in activated chondrocytes. In contrast, the combination of ASU and EGCG reduced COX-2 expression close to non-activated control levels and significantly inhibited PGE(2) production. These reductions were statistically greater than those of ASU or EGCG alone. The inhibition of COX-2 expression and PGE(2) production was associated with inhibition of NF-kappaB translocation. CONCLUSION The present study demonstrates that the anti-inflammatory activity of ASU and EGCG is potentiated when used in combination. This combination may offer an attractive supplement or alternative to non-steroidal anti-inflammatory drugs (NSAIDs) in the management of osteoarthritis.

Inhibition of cytokine expression and prostaglandin E2 production in monocyte/macrophage-like cells by avocado/soybean unsaponifiables and chondroitin sulfate.

Osteoarthritis is characterized by inflammation and increased production of pro-inflammatory mediators including cytokines and prostaglandin E2 (PGE2). Macrophage-like cells in synovial tissue produce these mediators which induce degradative enzymes that break down cartilage. We determined whether avocado/soybean unsaponifiables (ASU) and chondroitin sulfate (CS) can inhibit cytokine expression and PGE2 production using monocyte/macrophage-like cell models. Cells were incubated for 24 hours with either control media alone, ASU alone (NMX1000; 8.3 ?g/ml), CS alone (TRH122; 20 ?g/ml), or a combination of both preparations. Cells were activated with cytokines or lipopolysaccharide for 1 or 24 hours to determine cytokine gene expression by RT-PCR and PGE2 production by immunoassay, respectively. In response to activation, THP-1 cells exhibited increased expression of TNF-? and IL-1?, while RAW cells increased synthesis of PGE2. Cytokine expression and PGE2 synthesis were significantly decreased by the combination of ASU and CS compared to the individual treatments alone (P<0.05). Our finding supports the potential of the ASU and CS combination to suppress the synthesis of pro-inflammatory mediators in the joint.

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-1β (IL-1β) 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-1β (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-1β 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.

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-1β 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.

Fluoride-Substituted Apatites Support Proliferation and Expression of Human Osteoblast Phenotype In Vitro

This study aimed to test the hypothesis that fluoride (F) ions rele as d from F-substituted apatite (FAp) modulate osteoblast behavior in vitro. FAp samples of varying F concentrations (0.01, 0.08, 2.01 and 3.34 wt% F) were used in this study. Human osteoblasts (1x10 /well) were incubated in the presence or absence of Fap (5mg/well) for four days at 37C, 5% CO2. Viability, proliferative capacity by radiolabeled thymidine uptake and RNA were d t rmined. Results showed the following: viability and proliferative capacity of cells expos ed to FAp particle suspension were similar to control cells; collagen Type 1 expression was compara ble in all groups whereas alkaline phosphatase expression was variable; and osteocalcin expression was e nhanced in osteoblasts cells exposed to FAp with F concentrations 0.08 wt% and higher. These results de monstrated that F ions released from F-substituted apatites support proliferative capacit y of human osteoblast cells and enhance osteocalcin expression. These findings suggest that fluoride m ay play a role in bone formation and therefore F-releasing bone graft materials may provide added advanta ge.

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 1β (IL-1β) 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-1β. Results LPS, IL-1β, or H2O2 significantly increased PGE2 production. ASU or LA alone suppressed PGE2 production in LPS and IL-1β 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-1β 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.

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-1β (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-1β 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.

431 INHIBITION OF CYTOKINE-INDUCED INFLAMMATORY GENE EXPRESSION IN CHONDROCYTES FROM HORSES AFFLICTED WITH HEREDITARY EQUINE REGIONAL DERMAL ASTHENIA (HERDA) BY THE COMBINATION OF AVOCADO/SOYBEAN UNSAPONIFIABLES, GLUCOSAMINE, AND CHONDROITIN SULFATE

TLR-6), cytokines (IL-1b, IL-6, TNF-a) and metalloproteases (MMP-13, MMP-3 and MMP-9) were determinate in normal, OA rats and OA rats treated with 100ng/d of 1,25(OH)2D3 (4IU). Results: TLR-1, TLR-2 and TLR-6 were detected along the progression of OA but their expression did not differ significantly with the control group. Remarkable, a significant increased (p < 0.05; p < 0.0001) of TLR4 mRNA expression was observed since day 8 (2.5 fold) of OA induction. By immunohistofluorescence, TLR-4 was detected in the superficial and middle zones of cartilage from day 3 to 20 with highest expression at day 8. In another hand, a notably level of IL-1b expression (19 fold) was observed at day 8 of OA induction (p < 0.0001), while TNFa and IL-6 did not show a significant difference within the experimental groups. Among the MMPs, the MMP-3 showed the highest expression at the 8 (2.8 fold; p < 0.05) and 10 (3 fold; p < 0.0001) days of OA. Vitamin D treatment showed a clear reduction of TLR4, IL-1b and MMP-3 during the progression (3, 10 and 20 days) of OA. Conclusions: TLR4 is expressed in rat articular cartilage and it is up regulated during the induction of OA. Signaling through TLR-4 might be the pro-inflammatory mechanism in osteoarthritis that results in the expression of IL-1b and MMP-3. Interfering this signaling pathway by the use of vitamin D reduce the progression of OA in the rat model.