Effect of oleocanthal and its derivatives on inflammatory response induced by lipopolysaccharide in a murine chondrocyte cell line.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 62, No. 6, June 2010, pp 1675–1682 DOI 10.1002/art.27437 © 2010, American College of Rheumatology Effect of Oleocanthal and Its Derivatives on Inflammatory Response Induced by Lipopolysaccharide in a Murine Chondrocyte Cell Line Anna Iacono,1 Rodolfo Gómez,2 Jeffrey Sperry,3 Javier Conde,2 Giuseppe Bianco,4 Rosaria Meli,5 Juan J. Gómez-Reino,2 Amos B. Smith, III,3 and Oreste Gualillo2 Objective. In joint diseases, cartilage homeostasis is disrupted by mechanisms that are driven by combinations of biologic factors that vary according to the disease process. In osteoarthritis (OA), biomechanical stimuli predominate, with up-regulation of both catabolic and anabolic factors. Likewise, OA progression is characterized by increased nitric oxide (NO) production, which has been associated with cartilage degradation. Given the relevance of cartilage degenerative diseases in our society, the development of a novel pharmacologic intervention is a critically important public health goal. Recently, oleocanthal isolated from extra virgin olive oil was found to display nonsteroidal antiinflammatory drug activity similar to that of ibuprofen, a drug widely used in the therapeutic management of joint inflammatory diseases. We undertook this study to evaluate the effect of oleocanthal and its derivatives on the modulation of NO production in chondrocytes. Methods. Cultured ATDC-5 chondrocytes were tested with different doses of oleocanthal and its derivatives. Cell viability was evaluated using the MTT assay. Nitrite accumulation was determined in culture supernatant using the Griess reaction. Inducible NO synthase (NOS2) protein expression was examined using Western blotting analysis. Results. Oleocanthal and its derivatives decreased lipopolysaccharide-induced NOS2 synthesis in chondrocytes without significantly affecting cell viability at lower concentrations. Among the derivatives we examined, derivative 231 was the most interesting, since its inhibitory effect on NOS2 was devoid of cytotoxicity even at higher concentrations. Conclusion. This class of molecules shows potential as a therapeutic weapon for the treatment of inflammatory degenerative joint diseases. Supported in part by the Instituto de Salud Carlos III (RETICS Program, RD08/0075 [RIER]), within the VI NP of R⫹D⫹I 2008–2011. Mr. Gómez is recipient of a predoctoral fellowship from the University of Santiago de Compostela (program for consolidated research groups [GI-1957]). Dr. Bianco is recipient of a predoctoral fellowship from the University of Salerno through the International Mobility Programme of the Italian Ministry of Education, Research and University. Dr. Gualillo’s work was funded by the Instituto de Salud Carlos III and the Xunta de Galicia (SERGAS) through a research contract and grants PI08/0040 and PGIDIT07PXIB918090PR. 1 Anna Iacono, PhD: Santiago University Clinical Hospital, Santiago de Compostela, Spain, and University of Naples Federico II, Naples, Italy; 2Rodolfo Gómez, BS, Javier Conde, BS, Juan J. GómezReino, MD, PhD, Oreste Gualillo, PharmD, PhD: Santiago University Clinical Hospital, Santiago de Compostela, Spain; 3Jeffrey Sperry, PhD, Amos B. Smith, III, PhD: University of Pennsylvania, Philadelphia; 4Giuseppe Bianco, PhD: Santiago University Clinical Hospital, Santiago de Compostela, Spain, and University of Salerno, Salerno, Italy; 5Rosaria Meli, PhD: University of Naples Federico II, Naples, Italy. Dr. Iacono and Mr. Gómez contributed equally to this work. Dr. Smith has applied for an international patent (PCT/ US2007/067393) for the use of oleocanthal and structurally and functionally similar compounds in the therapeutic management of joint inflammatory diseases. Address correspondence and reprint requests to Oreste Gualillo, PharmD, PhD, Santiago University Clinical Hospital, Research Laboratory 9 (NEIRID LAB, Laboratory of Neuro Endocrine Interactions in Rheumatology and Inflammatory Diseases), Building C, Level 2, Calle Choupana s/n, 15706 Santiago de Compostela, Spain. E-mail: [email protected]; [email protected] Submitted for publication October 7, 2009; accepted in revised form February 23, 2010. The use of nonsteroidal antiinflammatory drugs (NSAIDs) has been the major pharmacologic approach to treating the symptoms of degenerative and inflammatory arthropathies, even though these drugs fail to modify the degenerative processes as the diseasemodifying antirheumatic drugs do (1,2). The search for alternative therapies that might influence the joint degenerative processes and the resulting lesions is thus of paramount importance. Toward this goal, recent studies 1675 1676 suggest that food and beverages that are rich in antioxidant and antiinflammatory compounds might contribute to the prevention of inflammatory diseases such as osteoarthritis (OA) (3–5). Indeed, many phenolic compounds extracted from extra virgin olive oil have attracted considerable attention recently, given their antioxidant (6), antiinflammatory (7), and antithrombotic activities (8). In addition, olive oil has been suggested to alleviate a variety of disorders, including cognitive decline due to neurodegeneration (i.e., Alzheimer’s disease) (9). In 2005, Beauchamp et al (10) isolated and identified an olive oil phenolic compound, (–)-decarboxymethyl ligstroside aglycone, also known as oleocanthal (oleo for olive, canth for sting, and al for aldehyde). Similarly to NSAIDs, oleocanthal induces a strong stinging sensation in the throat and has a potency and pharmacodynamic profile strikingly similar to that of ibuprofen; both compounds inhibit the cyclooxygenase 1 (COX-1) and COX-2 enzymes (10). Among the well-known inflammatory mediators, nitric oxide (NO) plays an important role in the regulation of many physiologic functions (i.e., vasodilatation, neurotransmission, and inflammation) (11,12). NO produced from the constitutive forms of NO synthase (NOS), namely, endothelial and neuronal NOS, functions principally as a vasodilator and neurotransmitter, respectively (13). The third form of NOS, known as inducible NOS (iNOS; or NOS2), is generally not present in resting cells but is induced by various stimuli, which include challenge by bacterial lipopolysaccharide (LPS), tumor necrosis factor ␣ (TNF␣), interleukin-1␤ (IL-1␤), picolinic acid, lipoarabinomannan, phorbol ester, interferon-␥, and hypoxia (14–18). In contrast to the constitutive forms of NOS, iNOS is regulated primarily at the level of transcription, because calmodulin is already tightly bound and, therefore, iNOS expression is largely independent of intracellular calcium (19,20). Accumulating evidence supports a role for NO in the cartilage degenerative process and the resulting lesions (21). Previous studies demonstrated that nitrite (NO2–), the stable end product of NO, was found in elevated concentrations in the synovial fluid and serum of patients with rheumatoid arthritis (RA) and OA (22). In addition, OA cartilage spontaneously produces NO, probably reflecting the in vivo stimulation of chondrocytes by cytokines (23–25). It appears that the high amounts of NO released in arthritic joints are mainly of chondrocyte origin (23,26,27). Moreover, chondrocytes in superficial cartilage layers are a particularly rich source of this reactive molecule (28). Therefore, NO IACONO ET AL production by iNOS may reflect the degree of inflammation and, as such, may now provide a measure to assess the effect of potential antiinflammatory agents on the joint degenerative inflammatory process. To date, no data have been reported that relate to the effects of oleocanthal on LPS-induced NO production in chondrocytes. We have now investigated the effects of oleocanthal and some related synthetic derivatives on NO production and iNOS expression in the murine chondrogenic cell line ATDC-5, which we have challenged with Escherichia coli LPS in order to mimic an inflammatory response in chondrocytes as an in vitro model of degenerative joint disease. MATERIALS AND METHODS Reagents. Fetal bovine serum (FBS), LPS (E coli serotype O55:B5), human transferrin, sodium selenite, MTT dye, and antibody against ␤-actin were purchased from Sigma. Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium, trypsin–EDTA, and antibiotics were purchased from Lonza. Antibodies against phospho–p38 kinase and p38 kinase were purchased from Millipore. Cell culture. The ATDC-5 murine chondrogenic cell line was purchased from RIKEN Cell Bank. Cells were cultured in DMEM/Ham’s F-12 medium supplemented with 5% FBS, 10 g/ml human transferrin, 3 ⫻ 10–8M sodium selenite, and antibiotics (50 units/ml penicillin and 50 g/ml streptomycin). Cells were used in the undifferentiated state in order to mimic the loss of differentiation observed in degenerative joint diseases such as OA (29). Oleocanthal and derivatives. Figure 1 shows the chemical structure of oleocanthal and the related synthetic derivatives. Cell treatments and nitrite assay. ATDC-5 cells (viability ⬎95% as evaluated by trypan blue exclusion) were plated at an initial density of 8 ⫻ 104/well in 24-well plates. After 6 hours of adherence, cells were preincubated for 12 hours with oleocanthal or oleocanthal synthetic derivatives (1–25 M) and then challenged for 24 or 48 hours with LPS (250 ng/ml) in the presence and absence of oleocanthal or derivatives in 5% FBS medium. All compounds were dissolved in DMSO and added directly to the culture media. Control cells were treated with DMSO alone, the final concentration of which (never exceeding 0.5%) had no noticeable effect on the growth of cells. Nitrite accumulation was measured in the culture medium using the Griess reaction, as previously described (29). Cell viability. Cell viability was examined using a colorimetric assay based on the MTT labeling reagent (30). Cells (8 ⫻ 103/well) were seeded in 96-well plates. Assays were performed according to the instructions and protocol provided by the manufacturer (Sigma-Aldrich). Briefly, cells were preincubated for 12 hours with oleocanthal or derivatives and then stimulated with oleocanthal or derivatives (1–25 M) alone or in combination with LPS (250 ng/ml) in 5% FBS medium for 24 hours at 37°C. After that, cells were incubated with 10 l of MTT (5 mg/ml) for 4 hours at 37°C. Then, after dissolving the formazan, the spectrophotometric absorbance was measured EFFECT OF OLEOCANTHAL AND ITS DERIVATIVES ON INFLAMMATION 1677 Cell protein extraction and Western blotting. ATDC-5 chondrogenic cells were seeded in P100 plates at an initial density of 1.5 ⫻ 106/plate. After 12 hours of preincubation with oleocanthal or derivative 231, cells were stimulated for 24 hours with oleocanthal or derivative 231 (1–25 M) alone or in combination with LPS (250 ng/ml) in 5% FBS medium. Cells were rapidly washed with ice-cold phosphate buffered saline and scraped in lysis buffer for protein extraction, as reported previously (29). Immunoblots were visualized with an Immobilon Western Detection kit (Millipore) using horseradish peroxidase–labeled secondary antibody. To confirm equal loading in each sample, the membranes were striped in glycine buffer at pH 2 and reblotted with anti–␤-actin antibody. The images were captured and analyzed with an EC3 imaging system (UVP). Statistical analysis. Data are reported as the mean ⫾ SEM of at least 3 independent experiments, each with at least 3 independent observations. Statistical analysis was performed using analysis of variance followed by the Student-NewmanKeuls test or Bonferroni multiple comparison test using the Prism computerized package (GraphPad Software). P values less than 0.05 were considered significant. RESULTS Figure 1. Chemical structure of oleocanthal and the related synthetic derivatives used in the study. using a microtiter enzyme-linked immunosorbent assay reader at 550 nm (Multiskan EX; Thermo Labsystems). Oleocanthal and related derivatives suppress NO production in the LPS-activated ATDC-5 cell line. In this series of experiments, ATDC-5 chondrogenic cells were stimulated with LPS (250 ng/ml) in the presence or absence of oleocanthal or derivatives to determine if these compounds modulate NO levels in culture supernatant. As shown in Figure 2, compared with control cells, ATDC-5 cells challenged with LPS showed a high accumulation of NO (evaluated as nitrite) (mean ⫾ SEM 1.38 ⫾ 0.67 M versus 40.12 ⫾ 0.38 M at 24 hours and 1.31 ⫾ 0.28 M versus 62.78 ⫾ 2.31 M at 48 hours; P ⬍ 0.001 for each comparison). Oleocan- Figure 2. Oleocanthal (OC) suppresses lipopolysaccharide (LPS)–induced nitric oxide (NO) production. ATDC-5 cells (8 ⫻ 104) were pretreated with 1–25 M oleocanthal for 12 hours and then exposed to 250 ng/ml LPS for 24 hours (A) or 48 hours (B). Control cells received drug vehicle. The culture medium was subsequently separated and analyzed for nitrite levels. NO concentration was determined using the Griess reaction. Values are the mean and SEM of at least 3 independent experiments. ⴱⴱⴱ ⫽ P ⬍ 0.001 versus control cells. # ⫽ P ⬍ 0.05; ## ⫽ P ⬍ 0.01; ### ⫽ P ⬍ 0.001, versus 250 ng/ml LPS alone. 1678 IACONO ET AL Figure 3. Oleocanthal derivatives suppress LPS-induced NO production. ATDC-5 cells (8 ⫻ 104) were pretreated with 1–25 M oleocanthal derivatives for 12 hours and then exposed to 250 ng/ml LPS for 24 hours (A) or 48 hours (B). Control cells received drug vehicle. The culture medium was subsequently isolated and analyzed for nitrite levels. NO concentration was determined using the Griess reaction. Values are the mean and SEM of at least 3 independent experiments. ⴱⴱⴱ ⫽ P ⬍ 0.001 versus control cells. # ⫽ P ⬍ 0.05; ## ⫽ P ⬍ 0.01; ### ⫽ P ⬍ 0.001, versus 250 ng/ml LPS alone. See Figure 2 for definitions. thal by itself (1–25 M) did not affect basal NO production (data not shown). Pretreatment of ATDC-5 cells with oleocanthal significantly inhibited the LPS-induced NO production in a dose-dependent manner (Figures 2A and B). To examine the possibility that oleocanthal de- rivatives could inhibit LPS-induced NO production, we carried out experiments under the same conditions as described above for oleocanthal. As shown in Figure 3, LPS led to a significant increase in NO levels in the cell supernatants after 24 hours (Figure 3A) and after 48 hours (Figure 3B). Among all the compounds tested, EFFECT OF OLEOCANTHAL AND ITS DERIVATIVES ON INFLAMMATION 1679 Figure 4. Effect of oleocanthal and derivatives on ATDC-5 cell viability. ATDC-5 cells (8 ⫻ 103) were pretreated with 1–25 M oleocanthal (A) or derivatives (B) for 12 hours and then exposed to 250 ng/ml LPS for 24 hours. Cell viability was measured using the MTT assay as described in Materials and Methods. Control cells received drug vehicle. Values are the mean and SEM of at least 3 independent experiments. ⴱⴱⴱ ⫽ P ⬍ 0.001 versus control cells. # ⫽ P ⬍ 0.05; ## ⫽ P ⬍ 0.01; ### ⫽ P ⬍ 0.001, versus 250 ng/ml LPS alone. See Figure 2 for definitions. derivatives 127, 129, 139, and 231 inhibited NO generation in a concentration-dependent manner. However, derivatives 159 and 166 showed a trend toward inhibiting NO production after 24 and 48 hours of LPS challenge at the highest or next-highest concentration. Effect of oleocanthal and derivatives on ATDC-5 cell viability. Since NO production is proportional to cell number and cell vitality, we used the MTT assay to evaluate whether oleocanthal and the derivatives affected cell viability. Oleocanthal alone did not modify cell viability, except for a significant cytotoxic effect at the highest concentration. As shown in Figure 4A, LPS by itself decreased cell viability, whereas pretreatment of cells with oleocanthal did not significantly affect cell viability, except at the 25 M concentration. As shown in Figure 4B, chondrocytes stimulated with both LPS and oleocanthal derivatives (127, 129, 139, 159, or 166) for 24 hours displayed a significant decrease in cell viability compared with LPS alone, at all concentrations for derivative 159 and at the highest concentrations (25 M or 10 and 25 M) for derivatives 127, 129, 139, and 166. Interestingly, among all of the oleocanthal derivatives tested so far, only derivative 231 did not show any cytotoxic effect, suggesting that the pharmacologic activity was not related to any alteration in chondrocyte viability. Effect of oleocanthal on iNOS and p38 protein expression. To investigate whether the inhibitory effect of oleocanthal on NO production was related to NOS2 synthesis inhibition, we examined NOS2 protein expression using Western blot analysis. As shown in Figure 5, iNOS protein was markedly induced upon exposure to LPS for 24 hours. Pretreatment with oleocanthal resulted in a dose-dependent inhibition of LPS-induced iNOS protein. The effect at the highest dose (25 M) was related to a cytotoxic effect of oleocanthal. Indeed, ␤-actin expression was strongly reduced at this dose. In addition, oleocanthal was able to induce a strong phosphorylation of p38 kinase, which was associated with a decrease in p38 expression at the highest tested doses. Among the oleocanthal derivatives tested so far, we chose derivative 231, which had previously shown no 1680 IACONO ET AL of modification of protein expression, with either structural or signaling proteins. DISCUSSION Figure 5. Effect of oleocanthal alone or in combination with LPS on inducible NO synthase (iNOS) protein expression and on p38 protein expression and phosphorylation. ATDC-5 cells (1.5 ⫻ 106) were pretreated with 1–25 M oleocanthal for 12 hours and then exposed to 250 ng/ml LPS for 24 hours. Inducible NO synthase, phospho-p38, p38, and ␤-actin (an internal standard) were detected using Western blot analysis with specific antibodies. Results are representative of 3 separate experiments. CON ⫽ control cells (see Figure 2 for other definitions). effect on cell viability (Figure 4B). As shown in Figure 6, LPS-mediated iNOS expression was significantly blunted by this synthetic derivative in a clear dosedependent manner. It is noteworthy that compared with oleocanthal, derivative 231 had no side effects in terms Figure 6. Effect of derivative 231 on inducible NO synthase (iNOS) protein expression and on p38 protein expression and phosphorylation. ATDC-5 cells (1.5 ⫻ 106) were pretreated with 1–25 M derivative 231 for 12 hours and then exposed to 250 ng/ml LPS for 24 hours. Inducible NO synthase, phospho-p38, p38, and ␤-actin (an internal standard) were detected using Western blot analysis with specific antibodies. Results are representative of 3 separate experiments. CON ⫽ control cells (see Figure 2 for other definitions). In the present study, we have shown for the first time that oleocanthal, which is present in the phenolic fraction of virgin olive oil, and the related synthetic derivative 231 reduce LPS-induced iNOS expression and NO production in the ATDC-5 murine chondrogenic cell line in a dose-dependent manner. Under most conditions, NO is a highly reactive gas that is involved in the pathogenesis of arthritis. It is noteworthy that normal cartilage produces little NO (25), whereas chondrocytes and synovial cells from patients with OA and RA produce abundant NO, as do cytokine-challenged chondrocytes (31). In fact, activated articular chondrocytes produce more NO than any other cells, including synoviocytes, hepatocytes, and macrophages (32). In chondrocytes, iNOS expression is induced by mechanical and biochemical factors, including inflammatory mediators such as IL-1␤ (33), TNF␣ (34), and LPS (28). An excess of NO inhibits both proteoglycan and collagen synthesis (35), activates metalloproteinases (36), mediates chondrocyte apoptosis (37), and promotes chondrocyte inflammatory responses (38). Experiments performed in iNOSknockout mice have shown these mice to be resistant to experimental OA, demonstrating that NO generated from the up-regulation of iNOS plays a pivotal role in the catabolic events of OA (39,40). Current treatment options used to manage OA are not curative and fail to reverse the degenerative process of OA. Among the commonly used pharmacologic agents are NSAIDs, corticosteroids, and hyaluronan preparations (41). NSAIDs in particular are widely used, but their prolonged consumption is associated with serious adverse side effects such as gastrointestinal ulcerations. The need for effective treatment modalities with fewer side effects has prompted OA patients to consider complementary approaches to control pain as well as to improve function and quality of life. Vegetable oil used in the Mediterranean diet has clear beneficial effects, particularly in cardiovascular diseases. Olive oil, which is the main fat used in the Mediterranean diet, has demonstrated efficacy not only in several clinical trials, but also in experimental models of inflammation. Indeed, olive oil significantly reduced the incidence of experimental autoimmune encephalomyelitis in the guinea pig (42) and increased the survival rate of MRL/lpr mice, which are prone to autoimmune EFFECT OF OLEOCANTHAL AND ITS DERIVATIVES ON INFLAMMATION disease (43). In comparing fish oil and olive oil supplements in patients with RA in a double-blind, noncrossover study, Cleland et al (44) found improvements in the painful joint score and grip strength at 12 weeks in those taking fish oil, while morning stiffness and the analog pain score improved in both groups. This result was significant only in those taking olive oil, consistent with an earlier report by Brzeski et al (45). The beneficial effect of olive oil was also reported by Darlington and Stone (46), who found reduced levels of C-reactive protein (an acute-phase protein which correlates with disease activity in RA) with olive oil treatment. The phenolic fraction of virgin olive oil has generated much interest in its health-promoting properties (47). It is known that the phenolic fraction of virgin olive oil is directly related to the intensity of throat irritation. The intensity of throat irritation is dependent on the concentration of oleocanthal. Indeed, oleocanthal, which has not been identified in any other vegetable oil, is responsible for the stinging sensation localized to the posterior oropharyngeal region upon consumption of virgin olive oil (10). This sensation has been described as a peppery bite at the back of the throat and is similar to that caused by the NSAID ibuprofen. The latter finding provoked the hypothesis that oleocanthal might possess pharmacologic properties similar to those of ibuprofen and several other NSAIDs. Furthermore, it is worth noting that long-term ingestion of small doses of oleocanthal via consumption of virgin olive oil may be responsible in part for the low incidence of heart disease, certain cancers, and other degenerative diseases associated with the Mediterranean diet (10). More recently, oleocanthal has been described to have potential pharmacologic properties that may be useful for treating patients with neurodegenerative diseases, since it is able to inhibit fibrillization of tau protein, a key protein involved in the pathogenesis of Alzheimer’s disease and other tauopathies (48). In summary, our study shows for the first time that oleocanthal and its derivative 231 down-regulate iNOS protein expression in LPS-challenged chondrocytes, resulting in a reduction of nitrite production in the cellular supernatant. This effect is related to a specific antiinflammatory pharmacologic property of these drugs, since the effect is apparently independent of cytotoxic effects, particularly for the 231 derivative. Indeed, a low cytotoxic effect of the natural compound oleocanthal has been observed in our experiments. Presumably, the cytotoxic activity of oleocanthal is linked to the strong increase in phosphorylation levels of p38 kinase (and to a decrease in its expression), whereas the 1681 saturated derivative 231 is completely devoid of this side effect. In principle, activation of the p38 signaling pathway during toxic aggression may aim at initiating either a defense or a homeostatic mechanism and therefore contribute to cell survival or, alternatively, to the signaling or execution of some of the apoptotic events (49). There is some evidence, none of which can be ruled out, for a role of p38 in both directions. Intriguingly, other drugs of the aryl-propionic family of NSAIDs such as carprofen can induce early p38 activation driving nuclear fragmentation in prostate cancer cells (50). The fact that derivative 231 is completely devoid of this effect makes this analog a suitable and potential pharmacologic weapon for the modulation of NO production in chondrocytes. Further studies remain to be conducted, including in vivo experiments to investigate the mechanisms of action of oleocanthal and its derivatives and to examine their pharmacologic activity using animal arthritis models. ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Miss Veronica Lopez and Miss Beatriz Malvar. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Gualillo had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Meli, Smith, Gualillo. Acquisition of data. Iacono, Gómez, Sperry, Conde, Bianco. Analysis and interpretation of data. Meli, Gómez-Reino, Gualillo. REFERENCES 1. Rashad S, Low F, Revell P, Hemingway A, Rainsford K, Walker F. 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