Synergistic chondroprotective effects of curcumin and resveratrol in human articular chondrocytes: inhibition of IL-1beta-induced NF-kappaB-mediated inflammation and apoptosis - PubMed (original) (raw)
Synergistic chondroprotective effects of curcumin and resveratrol in human articular chondrocytes: inhibition of IL-1beta-induced NF-kappaB-mediated inflammation and apoptosis
Constanze Csaki et al. Arthritis Res Ther. 2009.
Abstract
Introduction: Currently available treatments for osteoarthritis (OA) are restricted to nonsteroidal anti-inflammatory drugs, which exhibit numerous side effects and are only temporarily effective. Thus novel, safe and more efficacious anti-inflammatory agents are needed for OA. Naturally occurring polyphenolic compounds, such as curcumin and resveratrol, are potent agents for modulating inflammation. Both compounds mediate their effects by targeting the NF-kappaB signalling pathway.
Methods: We have recently demonstrated that in chondrocytes resveratrol modulates the NF-kappaB pathway by inhibiting the proteasome, while curcumin modulates the activation of NF-kappaB by inhibiting upstream kinases (Akt). However, the combinational effects of these compounds in chondrocytes has not been studied and/or compared with their individual effects. The aim of this study was to investigate the potential synergistic effects of curcumin and resveratrol on IL-1beta-stimulated human chondrocytes in vitro using immunoblotting and electron microscopy.
Results: Treatment with curcumin and resveratrol suppressed NF-kappaB-regulated gene products involved in inflammation (cyclooxygenase-2, matrix metalloproteinase (MMP)-3, MMP-9, vascular endothelial growth factor), inhibited apoptosis (Bcl-2, Bcl-xL, and TNF-alpha receptor-associated factor 1) and prevented activation of caspase-3. IL-1beta-induced NF-kappaB activation was suppressed directly by cocktails of curcumin and resveratrol through inhibition of Ikappakappa and proteasome activation, inhibition of IkappaBalpha phosphorylation and degradation, and inhibition of nuclear translocation of NF-kappaB. The modulatory effects of curcumin and resveratrol on IL-1beta-induced expression of cartilage specific matrix and proinflammatory enzymes were mediated in part by the cartilage-specific transcription factor Sox-9.
Conclusions: We propose that combining these natural compounds may be a useful strategy in OA therapy as compared with separate treatment with each individual compound.
Figures
Figure 1
Chemical structures of resveratrol and curcumin. Curcumin is derived from the rhizomes of turmeric (Curcuma longa) and resveratrol is found in the skin of red grapes, red berries, peanuts, root extracts of the weed Polygonum cuspidatum and numerous other plants. As suggested by their chemical structure, both compounds are polyphenols and therefore they exhibit similar properties as anti-oxidative and anti-inflammatory agents and can act as free radical scavengers.
Figure 2
Effects of resveratrol and curcumin and IL-1β on the viability and proliferation of primary chondrocytes in vitro. To evaluate the effect of curcumin, resveratrol and/or IL-1β-induced cytotoxicity, primary chondrocytes were treated with 10 ng/ml IL-1β, 50 μM resveratrol, 50 μM curcumin, 50 μM resveratrol and 50 μM curcumin; alternatively they were pre-treated with 50 μM resveratrol, 50 μM curcumin, 50 μM resveratrol and 50 μM curcumin for 4 hours and then co-treated with 10 ng/ml IL-1β, or were left untreated and evaluated after 24 hours using the MTT method. In cells treated with either curcumin, resveratrol or a combination of both, the cytotoxic effects induced by IL-1β were significantly decreased (*) and cell viability was comparable with control cultures.
Figure 3
Effects of resveratrol and curcumin on IL-1β-induced mitochondrial changes and apoptosis in primary chondrocytes. (a) Transmission electron microscopy was performed to demonstrate the effects of resveratrol and curcumin on IL-1β-stimulated primary chondrocytes in monolayer culture at an ultrastructural level. Untreated control cultures consisted of vital, active chondrocytes containing mitochondria, rough endoplasmic reticulum and many other cell organelles (panel a). In contrast, stimulation of chondrocytes with 10 ng/ml IL-1β for 1, 12, 24, and 48 hours resulted in degenerative changes in the cells. After 1 hour, chondrocytes became rounded and the nucleus contained more condensed chromatin (panel b). After 12 hours, multiple vacuoles, swelling of rough endoplasmic reticulum and clustering of swollen mitochondria were visible (panel c). Inset: arrows demonstrate swollen mitochondria. Longer incubations of 24 to 48 hours led to the formation of apoptotic bodies and cell lysis (panels d to e). Treatment of IL-1β-stimulated primary chondrocytes with resveratrol and curcumin (both at 50 μM), however, inhibited the adverse effects of IL-1β (panels f-i), and after 48 hours of treatment (panel i) chondrocytes demonstrated large, flattened cells with numerous microvilli-like processes, mitochondria and endoplasmic reticulum comparable with control cultures. (b) To quantify apoptosis in these cultures, 100 cells from 20 microscopic fields were counted. The number of apoptotic cells was highest in cultures stimulated with IL-1β alone and rose steadily over the entire culture period. In contrast, treatment of IL-1β-stimulated cultures with resveratrol and/or curcumin inhibited the apoptotic effects of IL-1β and the number of apoptotic cells remained significantly lower over the entire culture period (*).
Figure 4
Effects of resveratrol and curcumin on IL-1β-induced apoptosis as demonstrated by poly(ADP-Ribose) polymerase cleavage in primary chondrocytes. As IL-1β-mediated, caspase-induced cleavage of the DNA repair enzyme poly(ADP-Ribose) polymerase (PARP) is a sign of apoptosis, primary chondrocyte cultures were treated with 10 ng/ml IL-1β, 50 μM resveratrol, 50 μM curcumin, 50 μM resveratrol and 50 μM curcumin, or pre-treated with 50 μM resveratrol, 50 μM curcumin, 50 μM resveratrol and 50 μM curcumin for 4 hours and then co-treated with 10 ng/ml IL-1β, or left untreated for 24 hours. Equal amounts (500 ng protein per lane) of total protein were separated by 7.5% SDS-PAGE and analysed by immunoblotting with anti-PARP antibody. Stimulation of chondrocytes with IL-1β alone induced PARP cleavage. Pre-treatment with either resveratrol, curcumin or a combination of both inhibited IL-1β-induced PARP cleavage, however, and levels seen were similar to control cultures. Synthesis of the housekeeping protein β-actin remained unaffected.
Figure 5
Effects of resveratrol and curcumin on IL-1β-induced NF-κB-dependent gene products in primary chondrocytes. (a) The effect of resveratrol/curcumin on IL-1β-induced NF-κB-dependent anti-apoptotic gene products in primary chondrocytes was studied. To determine whether resveratrol and curcumin treatment actively stimulates the production of anti-apoptotic gene products, primary chondrocyte cultures were either stimulated for 0, 12, 24, and 48 hours with 10 ng/ml IL-1β or pre-treated with resveratrol and curcumin (50/50 μM) followed by 0, 12, 24, and 48 hours stimulation with 10 ng/ml IL-1β. Equal amounts (500 ng protein per lane) of total proteins were separated by 10% SDS-PAGE and analysed by immunoblotting with anti-Bcl-2, anti-Bcl-xL and anti-TNF-α receptor-associated factor 1 (anti-TRAF1) antibodies. A time-dependent downregulation of the expression of Bcl-2, Bcl-xL and TRAF1 by IL-1β was observed. In contrast, pre-treatment with resveratrol and curcumin resulted in a time-dependent increase of these anti-apoptotic proteins. Synthesis of the housekeeping protein β-actin remained unaffected. (b) The effect of resveratrol/curcumin on IL-1β-induced NF-κB-dependent pro-apoptotic protein caspase-3 was also studied in primary chondrocytes. Whole cell lysates of primary chondrocyte cultures were either stimulated for 0, 12, 24, and 48 hours with 10 ng/ml IL-1β or pre-treated with resveratrol and curcumin (50/50 μM) followed by 0, 12, 24, and 48 hours of stimulation with 10 ng/ml IL-1β-, and evaluated with western blot analysis to examine the effect on the pro-apoptotic protein caspase-3. Equal amounts (500 ng protein per lane) of total proteins were separated by 12% SDS-PAGE and analysed by immunoblotting with an antibody against active caspase-3. Stimulation of the cultures with IL-1β resulted in a time-dependent activation of caspase-3. In contrast, combinational treatment of resveratrol and curcumin inhibited caspase-3 activation in a time-dependent manner. Synthesis of the housekeeping protein β-actin was not affected.
Figure 6
Effects of resveratrol and curcumin on IL-1β-induced NF-κB-dependent proinflammatory and matrix-degrading gene products in primary chondrocytes. To evaluate whether resveratrol and curcumin exert time-dependent effects on IL-1β-induced NF-κB-dependent expression of proinflammatory and matrix-degrading gene products, primary chondrocyte cultures were either stimulated for 0, 12, 24, and 48 hours with 10 ng/ml IL-1β or pre-treated with resveratrol and curcumin (50 μM each) followed by 0, 12, 24, and 48 hours of stimulation with 10 ng/ml IL-1β; after extraction of whole cell lysates (500 ng protein per lane), they were probed for the expression of matrix metalloproteinase (MMP)-3, MMP-9, cylcooxygenase-2 (Cox-2) and vascular endothelial growth factor (VEGF) by western blot analysis. Stimulation of IL-1β alone consistently resulted in time-dependent production of MMP-3, MMP-9, Cox-2 and VEGF. In contrast, pre-treatment with resveratrol and curcumin downregulated MMP-3, MMP-9, Cox-2 and VEGF time dependently. Synthesis of the housekeeping protein β-actin was unaffected.
Figure 7
Effects of resveratrol and curcumin on IL-1β-induced inhibition of collagen type II and Sox-9 production in chondrocytes. To evaluate the effects of resveratrol and curcumin on IL-1β-stimulated chondrogenic inhibition in primary chondrocytes, whole cell lysates (500 ng protein per lane) were probed with antibodies to (a) collagen type II (panel I), as the most abundant cartilage-specific extracellular matrix protein, and (b) the chondrogenic-specific transcription factor Sox-9 (panel I). Cultures were treated with 10 ng/ml IL-1β, 50 μM resveratrol, 50 μM curcumin, 50 μM resveratrol and 50 μM curcumin, or were pre-treated with 50 μM resveratrol, 50 μM curcumin, 50 μM resveratrol and 50 μM curcumin for 4 hours and then co-treated with 10 ng/ml IL-1β, or left untreated for 24 hours. Untreated cultures had strong (a) collagen type II and (b) Sox-9 and stimulation with IL-1β alone greatly reduced collagen type II as well as Sox-9 production. However, pre-treatment of the cultures with resveratrol, curcumin or a combination of both inhibited the adverse effects of IL-1β and the chondrocytes produced large quantities of collagen type II and Sox-9 at levels similar to control cultures. This was confirmed by quantitative densitometry (a, panel II and b, panel II). The mean values and standard deviations from three independent experiments are shown. White, grey and solid bars represent different molecular forms of collagen type II. Synthesis of the housekeeping protein β-actin remained unaffected.
Figure 8
Inhibition of IL-1β-induced NF-κB activation and nuclear translocation by resveratrol and curcumin in primary chondrocytes using APAAP. (a) Human chondrocyte cultures either served as controls (panel A, not treated) or treated either with 10 ng/ml IL-1β 5, 15 and 30 minutes alone or pre-treated with 50 μM resveratrol and 50 μM curcumin for 4 hours and then co-treated with 10 ng/ml IL-1β for 5, 15 and 30 minutes, before immunolabelling with phospho-p65 antibodies. In control cells, anti-phospho-p65 labelling was restricted to the cytoplasm (panel A). Cells treated with IL-1β alone revealed nuclear translocation of phospho-p65 (panels B-D) that was inhibited by co-treatment with resveratrol and curcumin (panels E-G). Data shown are representative of three independent experiments. A-G: × 160, bar = 50 μm. (b) Panel I: western blot analysis with IL-1β-treated chondrocyte nuclear extracts. Serum-starved chondrocytes were pre-incubated with 50 μM resveratrol and 50 μM curcumin for 5, 10, 20, 30, 40 and 50 minutes, co-treated with 10 ng/ml IL-1β for 30 minutes, and then probed for phospho-p65 by western blot analysis using antibodies to phospho-specific p65 and poly(ADP-Ribose) polymerase (PARP) (control). Resveratrol and curcumin pre-treatment inhibited IL-1β-induced NF-κB activation in a time-dependent manner. NF-κB nuclear translocation was inhibited completely after 50 minutes of pre-treatment with resveratrol and curcumin. Panel II: serum-starved human chondrocytes were pre-incubated with resveratrol and curcumin at various concentrations (5 μM, 10 μM, 20 μM, 30 μM, 40 μM and 50 μM each) for 4 hours followed by 10 ng/ml IL-1β stimulation for 30 minutes. The nuclear extracts (500 ng protein per lane) were probed for phospho-p65 by western blot analysis using antibodies to phospho-specific p65 and PARP (control). A concentration-dependent inhibition of NF-κB nuclear translocation was observed. At a concentration of 40 μM resveratrol and 40 μM curcumin, NF-κB nuclear translocation was completely inhibited. The inhibition of NF-κB nuclear translocation by resveratrol and curcumin is therefore concentration as well as time dependent. Synthesis of PARP remained unaffected in nuclear extracts.
Figure 9
Effects of resveratrol and curcumin treatment on IL-1β-induced IκBα degradation. Serum-starved human chondrocytes were pre-treated with 10 ng/ml IL-1β alone for 4 hours, or with 100 μM _N_-Ac-Leu-Leu-norleucinal (ALLN) for 30 minutes, or pre-treated with 50 μM resveratrol, 50 μM curcumin or 100 μM ALLN alone for 4 hours and then co-treated with IL-1β (10 ng/ml) for 8 hours. Other serum-starved chondrocytes were pre-treated with 50 μM resveratrol or 50 μM curcumin alone for 4 hours and then co-treated with IL-1β (10 ng/ml) for 8 hours. Some cultures were left untreated and evaluated after 12 hours. Cytoplasmic extracts (500 ng protein per lane) were fractionated and then subjected to western blotting with phosphospecific IκBα antibody. The data demonstrate that resveratrol (but not curcumin) inhibits IL-1β-induced IκBα degradation. Data shown are representative of three independent experiments. The same membrane was re-blotted with antibodies to β-actin.
Figure 10
Effects of resveratrol and curcumin treatment on IL-1β-induced IκBα ubiquitination. Serum-starved human chondrocytes were pre-treated with 10 ng/ml IL-1β alone for 4 hours, or 100 μM _N_-Ac-Leu-Leu-norleucinal (ALLN) for 30 minutes, or pre-treated with 50 μM resveratrol, 50 μM curcumin or 100 μM ALLN alone for 4 hours and then co-treated with IL-1β (10 ng/ml) for 8 hours. Other serum-starved chondrocytes were pre-treated with 50 μM resveratrol or 50 μM curcumin alone for 4 hours and then co-treated with IL-1β (10 ng/ml) for 8 hours. Some cultures were left untreated and evaluated after 12 hours. Cytoplasmic extracts were immunoprecipitated with an antibody against IκBα and subjected to western blot analysis using a monoclonal anti-ubiquitin antibody. Resveratrol (but not curcumin), stabilized IL-1β-induced ubiquitination of IκBα. Data shown are representative of three independent experiments.
Figure 11
Effects of resveratrol and curcumin treatment on IL-1β-induced IκB kinase activation. Serum-starved primary human chondrocytes were pre-treated with (a) 50 μM resveratrol or (b) 50 μM curcumin for 4 hours and then co-treated with IL-1β (10 ng/ml) for the indicated times. Whole cell extracts were immunoprecipitated with an antibody against IκB kinase (IKK)-α and analysed by an immune complex kinase assay. To examine the effect of curcumin on the expression level of IKK proteins, whole cell extracts (500 ng protein per lane) were fractionated by SDS-PAGE and examined using western blot analysis with anti-IKK-α and anti-IKK-β antibodies. Data shown are representative of three independent experiments. The results demonstrate that resveratrol does not affect IL-1β-induced IKK activation.
Figure 12
Inhibitory effects of resveratrol and curcumin on IL-1β-induced NF-κB activation and apoptosis in primary human chondrocytes in vitro. IL-1β stimulates the IL-1β receptor, intiating an intracellular signal transduction cascade, which activates the cytoplasmic IκBα kinases (IKK)-α, IKK-β, and IKK-γ. These kinases phosphoryle inactive IκBα. Phosphorylated IκBα is then ubiquitinated and degraded by the proteasome and active NF-κB is released. NF-κB translocates to the nucleus, where it activates proinflammatory and pro-apoptotic gene production. In chondrocytes, resveratrol and curcumin both inhibit the NF-κB signal transduction pathway but in different ways: resveratrol inhibits ubiquitination of phosphorylated IκBα, and blocks translocation of the activated NF-κB to the nucleus. In a similar fashion to resveratrol, curcumin also inhibits translocation of the activated NF-κB to the nucleus. However in contrast to resveratrol, curcumin does not have a degradation inhibiting effect on phosphorylated IκBα. Instead, curcumin inhibits IL-1β signalling at an earlier point by inhibition of IKK-α, IKK-β, and IKK-γ.
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