A.F. Mendes

Differential roles of hydrogen peroxide and superoxide in mediating IL-1-induced NF-KB activation and iNOS expression in bovine articular chondrocytes

Our previous studies showed that reactive oxygen species (ROS) are required for the pro‐inflammatory cytokine interleukin‐1β (IL‐1) to induce the activity of the Nuclear transcription Factor‐kappaB (NF‐κB) and the expression of the inducible isoform of the nitric oxide synthase (iNOS) in bovine articular chondrocytes. This study aimed at elucidating the role of hydrogen peroxide (H2O2) and the superoxide radical, two major ROS, in mediating those IL‐1‐induced responses. The results obtained show that chondrocytes produce both H2O2 and superoxide radical in response to IL‐1. Treatment of the chondrocyte cultures with H2O2 alone did not induce NF‐κB activation or iNOS expression. Addition of H2O2 simultaneously with IL‐1 did neither enhance nor inhibit NF‐κB activation and iNOS expression, relatively to treatment with IL‐1 alone. Accordingly, treatment with catalase did not inhibit those IL‐1‐induced responses. Treatment with superoxide dismutase, however, effectively prevented IL‐1‐induced IκB‐α degradation and iNOS expression. Taken together, the results obtained indicate that superoxide mediates IL‐1‐induced IκB‐α degradation and the consequent NF‐κB activation and iNOS expression in chondrocytes, whereas H2O2 does not seem to participate in those IL‐1‐induced responses. In conclusion, the present study identifies the superoxide radical as the ROS involved in mediating the IL‐1‐induced signaling pathway that leads to NF‐κB activation and to the expression of NF‐κB‐dependent genes in bovine articular chondrocytes.

Role of glucose as a modulator of anabolic and catabolic gene expression in normal and osteoarthritic human chondrocytes.

Cartilage matrix homeostasis involves a dynamic balance between numerous signals that modulate chondrocyte functions. This study aimed at elucidating the role of the extracellular glucose concentration in modulating anabolic and catabolic gene expression in normal and osteoarthritic (OA) human chondrocytes and its ability to modify the gene expression responses induced by pro‐anabolic stimuli, namely Transforming Growth Factor‐β (TGF). For this, we analyzed by real time RT‐PCR the expression of articular cartilage matrix‐specific and non‐specific genes, namely collagen types II and I, respectively. The expression of the matrix metalloproteinases (MMPs)‐1 and ‐13, which plays a major role in cartilage degradation in arthritic conditions, and of their tissue inhibitors (TIMP) was also measured. The results showed that exposure to high glucose (30 mM) increased the mRNA levels of both MMPs in OA chondrocytes, whereas in normal ones only MMP‐1 increased. Collagen II mRNA was similarly increased in normal and OA chondrocytes, but the increase lasted longer in the later. Exposure to high glucose for 24 h prevented TGF‐induced downregulation of MMP‐13 gene expression in normal and OA chondrocytes, while the inhibitory effect of TGF on MMP‐1 expression was only partially reduced. Other responses were not significantly modified. In conclusion, exposure of human chondrocytes to high glucose, as occurs in vivo in diabetes mellitus patients and in vitro for the production of engineered cartilage, favors the chondrocyte catabolic program. This may promote articular cartilage degradation, facilitating OA development and/or progression, as well as compromise the quality and consequent in vivo efficacy of tissue engineered cartilage. J. Cell. Biochem. 112: 2813–2824, 2011.

Anti-inflammatory and Chondroprotective Activity of (+)-α-Pinene: Structural and Enantiomeric Selectivity

Previous studies have suggested that α-pinene, a common volatile plant metabolite, may have anti-inflammatory effects in human chondrocytes, thus exhibiting potential antiosteoarthritic activity. The objective of this study was to further characterize the potential antiosteoarthritic activity of selected pinene derivatives by evaluating their ability to modulate inflammation and extracellular matrix remodeling in human chondrocytes and to correlate the biological and chemical properties by determining whether the effects are isomer- and/or enantiomer-selective. To further elucidate chemicopharmacological interactions, the activities of other naturally occurring monoterpenes with the pinane nucleus were also investigated. At noncytotoxic concentrations, (+)-α-pinene (1) elicited the most potent inhibition of the IL-1β-induced inflammatory and catabolic pathways, namely, NF-κB and JNK activation and the expression of the inflammatory (iNOS) and catabolic (MMP-1 and -13) genes. (-)-α-Pinene (2) was less active than the (+)-enantiomer (1), and β-pinene (3) was inactive. E-Pinane (4) and oxygenated pinane-derived compounds, pinocarveol (5), myrtenal (6), (E)-myrtanol (7), myrtenol (8), and (Z)-verbenol (9), were less effective or even completely inactive and more cytotoxic than the pinenes tested (1-3). The data obtained show isomer- and enantiomer-selective anti-inflammatory and anticatabolic effects of α-pinene in human chondrocytes, (+)-α-pinene (1) being the most promising for further studies to determine its potential value as an antiosteoarthritic drug.

Role of nitric oxide in the activation of NF-kappaB, AP-1 and NOS II expression in articular chondrocytes.

Abstract. Objective and design: Determine the sources of nitric oxide (NO) and evaluate its role in the activation of nuclear Factor-kappaB (NF-κB) and activator protein-1 (AP-1) and in the expression of NO synthase II (NOS II), induced by interleukin-1β (IL-1).¶Material or subjects: Primary cultures of bovine articular chondrocytes.¶Treatment: The cells were treated with IL-1, 5 ng/ml with or without the NO donor S-nitroso-N-acetylpenicillamine (SNAP), in concentrations ranging from 10 to 300 μM.¶Methods: NF-κB and AP-1 activation were evaluated by electrophoretic mobility shift assay. Northern blot was used to detect NOS II mRNA levels and western blot to evaluate IκB-α, NOS I and NOS II protein levels.¶Results: Under basal conditions, chondrocytes expressed NOS I, which was lost upon IL-1 treatment. SNAP inhibited IL-1-induced NF-κB activation and NOS II expression. When added alone, SNAP induced AP-1 activation to approximately the same extent as IL-1.¶Conclusions: These results suggest that, in chondrocytes, NO is a key regulator of the signaling pathways leading from IL-1 to NF-κB and AP-1 activation and to the expression of genes that are involved in the pathophysiology of arthritic diseases.

Evaluation of the anti-inflammatory, anti-catabolic and pro-anabolic effects of E-caryophyllene, myrcene and limonene in a cell model of osteoarthritis

Osteoarthritis is a progressive joint disease and a major cause of disability for which no curative therapies are yet available. To identify compounds with potential anti-osteoarthritic properties, in this study, we screened one sesquiterpene, E-caryophyllene, and two monoterpenes, myrcene and limonene, hydrocarbon compounds for anti-inflammatory, anti-catabolic and pro-anabolic activities in human chondrocytes. At non-cytotoxic concentrations, myrcene and limonene inhibited IL-1β-induced nitric oxide production (IC50=37.3μg/ml and 85.3µg/ml, respectively), but E-caryophyllene was inactive. Myrcene, and limonene to a lesser extent, also decreased IL-1β-induced NF-κB, JNK and p38 activation and the expression of inflammatory (iNOS) and catabolic (MMP-1 and MMP-13) genes, while increasing the expression of anti-catabolic genes (TIMP-1 and -3 by myrcene and TIMP-1 by limonene). Limonene increased ERK1/2 activation by 30%, while myrcene decreased it by 26%, relative to IL-1β-treated cells. None of the compounds tested was able to increase the expression of cartilage matrix-specific genes (collagen II and aggrecan), but both compounds prevented the increased expression of the non-cartilage specific, collagen I, induced by IL-1β. These data show that myrcene has significant anti-inflammatory and anti-catabolic effects in human chondrocytes and, thus, its ability to halt or, at least, slow down cartilage destruction and osteoarthritis progression warrants further investigation.

Regulation of catecholamine release and tyrosine hydroxylase in human adrenal chromaffin cells by interleukin-1β : role of neuropeptide Y and nitric oxide

J. Neurochem. (2009) 109, 911–922.

Expression and function of the insulin receptor in normal and osteoarthritic human chondrocytes: modulation of anabolic gene expression, glucose transport and GLUT-1 content by insulin

OBJECTIVE Chondrocytes respond to insulin, but the presence and role of the specific high affinity insulin receptor (InsR) has never been demonstrated. This study determined whether human chondrocytes express the InsR and compared its abundance and function in normal and osteoarthritis (OA) human chondrocytes. DESIGN Cartilage sections were immunostained for detection of the InsR. Non-proliferating chondrocyte cultures from normal and OA human cartilage were treated with 1nM or 10nM insulin for various periods. InsR, insulin-like growth factor receptor (IGFR), aggrecan and collagen II mRNA levels were assessed by real time RT-PCR. InsR, glucose transporter (GLUT)-1, phospho-InsRbeta and phospho-Akt were evaluated by western blot and immunofluorescence. Glucose transport was measured as the uptake of [3H]-2-Deoxy-d-Glucose (2-DG). RESULTS Chondrocytes staining positively for the InsR were scattered throughout the articular cartilage. The mRNA and protein levels of the InsR in OA chondrocytes were approximately 33% and 45%, respectively, of those found in normal chondrocytes. Insulin induced the phosphorylation of the InsRbeta subunit. Akt phosphorylation and 2-DG uptake increased more intensely in normal than OA chondrocytes. Collagen II mRNA expression increased similarly in normal and OA chondrocytes while aggrecan expression remained unchanged. The Phosphoinositol-3 Kinase (PI3K)/Akt pathway was required for both basal and insulin-induced collagen II expression. CONCLUSIONS Human chondrocytes express functional InsR that respond to physiologic insulin concentrations. The InsR seems to be more abundant in normal than in OA chondrocytes, but these still respond to physiologic insulin concentrations, although some responses are impaired while others appear fully activated. Understanding the mechanisms that regulate the expression and function of the InsR in normal and OA chondrocytes can disclose new targets for the development of innovative therapies for OA.

Screening of Five Essential Oils for Identification of Potential Inhibitors of IL-1-induced Nf-κB Activation and NO Production in Human Chondrocytes: Characterization of the Inhibitory Activity of α-Pinene

Nuclear factor-kappaB is a key transcription factor activated by pro-inflammatory signals, like interleukin-1beta (IL-1), being required for the expression of many inflammatory and catabolic mediators, such as nitric oxide (NO), that play an important role in arthritic diseases. This work aimed at screening and identifying natural inhibitors of IL-induced NF-kappaB activation and NO production in human articular chondrocytes. Five essential oils obtained from four plants of the Iberian flora, Mentha x piperita L. (Lamiaceae), Origanum virens L. (Lamiaceae), Lavandula luiseri L. (Lamiaceae), and Juniperus oxycedrus L. subsp. oxycedrus (Cupressaceae), were screened for their ability to prevent IL-1-induced NO production. The oil showing higher inhibitory activity was fractionated, concentrated, analyzed for composition elucidation and prepared for further assays. For this purpose, the human chondrocytic cell line C-28/I2 was used to evaluate NF-kappaB activation by determining the cytoplasmic levels of the total and phosphorylated forms of the inhibitory protein, I kappaB-alpha, and the NF-kappaB-DNA binding activity. The essential oil from the leaves of J. oxycedrus in a concentration of 0.02 % (v/v) achieved the greatest inhibition (80 +/- 8%) of IL-1-induced NO production. Chemical analysis showed that this essential oil is predominantly composed of monoterpene hydrocabons, being alpha-pinene [2,6,6-trimethyl-bicyclo(3.1.1)hept-3-ene] the major constituent (76 %). Similarly to the effect of the whole oil, a fraction containing 93% alpha-pinene reduced significantly IL-1-induced I kappaB-alpha degradation. Moreover, alpha-pinene also decreased I kappaB-alpha phosphorylation, NF-kappaB-DNA binding activity, and NO production. Another fraction containing oxygenated mono- and sesquiterpenes was nearly as effective as alpha-pinene. The ability of the alpha-pinene-containing fraction to reduce IL-1-induced NF-kappaB activation and NO production warrants further studies to demonstrate the usefulness of alpha-pinene in the treatment of arthritic diseases and other conditions in which NF-kappaB and NO play pathological roles.

Dexamethasone prevents interleukin-1beta-induced nuclear factor-kappaB activation by upregulating IkappaB-alpha synthesis, in lymphoblastic cells.

AIMS: Glucocorticoids (GCs) exert some of their anti-inflammatory actions by preventing the activation of the transcription factor nuclear factor (NF)-kappaB. The GC-dependent inhibition of NF-kappaB may occur at different levels, but the mechanisms involved are still incompletely understood. In this work, we investigated whether the synthetic GC, dexamethasone (Dex), modulates the activity of NF-kappaB in the lymphoblastic CCRF-CEM cell line. We also evaluated the ability of Dex to prevent the activation of NF-kappaB in response to the potent proinflammatory cytokine, interleukin (IL)-1beta. RESULTS: Exposure of the cells to Dex (1 microM) induced the rapid degradation of IkappaB-alpha, leading to the transient translocation of the NF-kappaB family members p65 and p50 from the cytoplasm to the nucleus, as evaluated by western blot. Electrophoretic mobility shift assays revealed that, in the nucleus, these NF-kappaB proteins formed protein-DNA complexes, indicating a transient activation of NF-kappaB. Additionally, Dex also induced de novo synthesis of IkappaB-alpha, following its degradation. Finally, when the cells were exposed to Dex (1 microM) prior to stimulation with IL-1beta (20 ng/ml), Dex was efficient in preventing IL-1beta-induced NF-kappaB activation. The GC antagonist, RU 486 (10 microM), did not prevent any of the effects of Dex reported here. CONCLUSION: Our results indicate that, in CCRF-CEM cells, Dex prevents NF-kappaB activation, induced by IL-1beta, by a mechanism that involves the upregulation of IkappaB-alpha synthesis, and that depends on the early and transient activation of NF-kappaB.

Resveratrol Modulates Cytokine-Induced JAK/STAT Activation More Efficiently than 5-Aminosalicylic Acid: An In Vitro Approach

Background Many advances have been recently made focused on the valuable help of dietary polyphenols in chronic inflammatory diseases. On the other hand, current treatment options for intestinal bowel disease patients are unsatisfying and, for this reason, it is estimated that many patients use dietary supplements to achieve extra benefits. Aim The aim of this work was to analyze under a mechanistic perspective the anti-inflammatory potential of resveratrol, a natural polyphenolic compound, and to compare it with a pharmaceutical agent, 5-aminosalicylic acid, using the intestinal HT-29 cell line, as a cellular model. Methodology and Principal Findings In the present study, HT-29 colon epithelial cells were pre-treated with 25 µM resveratrol and/or 500 µM 5-aminosalicylic acid and then exposed to a combination of cytokines (IL-1α, TNF-α, IFN-γ) for a certain period of time. Our data showed that resveratrol, used in a concentration 20 times lower than 5-aminosalicylic acid, was able to significantly reduce NO and PGE2 production, iNOS and COX-2 expression and reactive oxidant species formation induced by the cytokine challenge. However, as already verified with 5-aminosalicylic acid, in spite of not exhibiting any effect on IkB-α degradation, resveratrol down-regulated JAK-STAT pathway, decreasing the levels of activated STAT1 in the nucleus. Additionally, resveratrol decreased the cytokine-stimulated activation of SAPK/JNK pathway but did not counteract the cytokine-triggered negative feedback mechanism of STAT1, through p38 MAPK. Conclusion/Significance Taken together, our results show that resveratrol may be considered a future nutraceutical approach, promoting remission periods, limiting the inflammatory process and preventing colorectal cancer, which is common in these patients.