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Am J Physiol Cell Physiol 290: C1092-C1099, 2006. First published November 16, 2005; doi:10.1152/ajpcell.00380.2005
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VASCULAR BIOLOGY

Heme oxygenase-1 mediates the anti-inflammatory actions of 2'-hydroxychalcone in RAW 264.7 murine macrophages

Hadil Abuarqoub, Roberta Foresti, Colin J. Green, and Roberto Motterlini

Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, United Kingdom

Submitted 28 July 2005 ; accepted in final form 7 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chalcones are a group of plant-derived polyphenolic compounds that belong to the flavonoids family, and possess a wide variety of cytoprotective and modulatory functions. Chalcones exert their cytoprotective actions via activation of specific transcriptional factors and upregulation of endogenous defensive pathways, such as phase II enzymes and the stress protein heme oxygenase-1 (HO-1). In this study, we investigated the anti-inflammatory action of 2'-hydroxychalcone (2-HC) in a model of lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages and examined the role of HO-1 in this process. Our results demonstrate that 2-HC potently induces HO-1 expression and markedly reduces LPS-mediated nitrite and TNF-{alpha} production. These effects are accompanied by inhibition of inducible nitric oxide synthase protein expression and abolished by blockade of heme oxygenase activity with either tin protoporphyrin IX or HO-1 small interfering RNA. By using a pharmacological approach and siRNA technology, we also found that phosphatidylinositol 3-kinase is a major cellular mediator in 2-HC-induced HO-1 expression. These findings strongly suggest that 2-HC exerts anti-inflammatory actions via activation of the HO-1 pathway and help to elucidate the mechanisms underlying the potential therapeutic value of chalcones.

lipopolysaccharide; inflammation; nitric oxide

THE USE OF NATURALLY OCCURRING compounds that possess intrinsic anti-inflammatory and antioxidant properties and can trigger the intracellular cascade of protective pathways offers a promising stratagem for therapeutic applications. An interesting class of agents that might fulfill these criteria is the phytochemicals chalcones, which have been shown to possess antioxidant, oxygen scavenging, and anti-inflammatory properties in a variety of experimental systems (4, 26). Chalcones are a group of phenolic compounds that belong to the flavonoids family and widely occur in nature as pigments; for example, they can be found in hops, plants, and broccoli (23, 33). Chalcones exhibit a specific chemical structure that renders them powerful inducers of the phase II family of detoxifying enzymes, which are involved in protection against cancer (9). Indeed, many studies (i.e., 37) have reported that pretreatment of animals with phase II enzymes inducers reduced the risk of developing chemically induced cancers. Interestingly, a daily dietary regimen of dried broccoli sprouts attenuated oxidative stress, hypertension, and inflammation (41) in rats, indicating the potency and multiplicity of the positive effects elicited by certain plant components.

The anti-inflammatory action of chalcone derivatives per se has been examined and appears to be associated with suppression of inflammatory mediators, such as nitric oxide (NO) and tumor necrosis factor-{alpha} (TNF-{alpha}), which are generated by macrophages stimulated with lipopolysaccharide (LPS) (6). This protective mechanism could derive from simultaneous inhibition of the production of various inflammatory mediators (16) and/or by a direct inhibitory action of the activation of transcription factors (NF-{kappa}B, AP-1) that regulate the inflammatory response (6, 20). The beneficial activity of chalcones may also originate from their ability to induce endogenous cytoprotective pathways such as heme oxygenase-1 (HO-1) (13), a potent antioxidant enzyme that protects against a variety of stressful insults (12, 14). Our group (5, 25, 35) has previously reported that polyphenolic compounds, such as curcumin and caffeic acid phenethyl ester, mediate cytoprotection through HO-1 induction and this effect requires the activation of the transcription factor Nrf2. The idea that naturally occurring compounds are activators of endogenous antioxidant and cytoprotective pathways is supported also by Alcaraz and coworkers (1), who used the synthetic chalcone 3',4',5',3,4,5-hexamethoxy-chalcone to demonstrate that HO-1 plays a crucial role in the control of inflammatory processes (40). Studies have shown that HO-1 and its product carbon monoxide (CO) can reduce edema, leukocyte adhesion, and migration and production of cytokines (29, 30). The HO-1 pathway also stimulates the anti-inflammatory molecule interleukin-10 (18), and the fact that inflammatory stimuli induce the expression of HO-1, suggests that this is an adaptive cellular response to inflammation (39).

To better understand the role of chalcones in inflammation, we investigated in the present study the effect of 2'-hydroxychalcone (2-HC) on HO-1 expression in RAW 264.7 macrophages and assessed whether the enzyme is involved in the anti-inflammatory action of 2-HC on stimulation of macrophages with LPS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. 2-HC was purchased from Indofine (Hillsborough, NJ). LPS (Escherichia Coli serotype 026:B6) was obtained from Sigma. Tin protoporphyrin IX (SnPPIX) was obtained from Porphyrin Products (Logan, UT). All other reagents were purchased from Sigma unless otherwise specified. HO-1 and phosphatidylinositol 3-kinase (PI3K) small interfering RNA (siRNA) and its transfection kit were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture. Murine 264.7 macrophages were purchased from the European Collection of Cell Cultures (Salisbury, UK) and cultured in Dulbecco's modified Eagle's medium containing 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% fetal bovine serum, as previously described (34). Cells were grown in a humidified atmosphere at 37°C and 5% CO2.

Experimental protocol. Macrophages were treated for 6 h in the presence of 2-HC (5, 15, and 30 µM) and heme oxygenase activity as well as HO-1 protein expression were determined. Heme oxygenase activity was also measured in cells exposed for 6 h to 2-HC and subsequently cultured in fresh medium alone for 12, 18, or 24 h. Furthermore, the activity was determined in cells treated for 24 h with LPS in the presence or absence of 2-HC. Cell viability was assessed by different methods at the end of the experiments. To examine the potential anti-inflammatory action of chalcones, macrophages were exposed for 24 h to LPS (1 µg/ml) in the presence or absence of 2-HC (5, 15, and 30 µM), and nitrite levels and inducible NO synthase (iNOS) protein expression were determined at the end of the incubation period. To investigate the involvement of heme oxygenase, experiments were conducted either in the presence of SnPPIX (10 µM), an inhibitor of heme oxygenase activity, or siRNA for HO-1 (31). In a similar set of experiments, cells were treated with 2-HC for 6 h before incubation with LPS and nitrite production was measured after 24 h. The levels of TNF-{alpha} were also determined in cells exposed for 12 h to 0.1 µg/ml LPS in the presence or absence of 2-HC. Similarly, TNF-{alpha} was measured in experiments conducted with 10 µM SnPPIX. The participation of the mitogen-activated protein kinase (MAPK) pathway in the increase of heme oxygenase activity and HO-1 expression by 2-HC was assessed with PD-098059 (ERK inhibitor, 25 µM), SB-203580 (p38 inhibitor, 5 µM), or SP-600125 (JNK inhibitor, 10 µM). An inhibitor of the phosphatidylinositol 3-kinase (PI3K) pathway (LY-294002, 25 µM) and siRNA for PI3K were also tested. To investigate the effect of 2-HC on the translocation of the transcription factor NF-{kappa}B to the nucleus, cells were preincubated with 2-HC for 30 min, followed by treatment with LPS 1 µg/ml for 30 and 60 min. At the end of the experimental protocol, the nuclear extraction was performed as mentioned previously (5). Furthermore, the effect of 2-HC on activation of Nrf2 in the nuclear extract was evaluated after incubation of macrophages with increasing concentrations of 2-HC (0–30 µM) for 60 min.

Determination of nitrite levels. Nitrite levels were determined with the use of the Griess method, as previously described by our group (34). Briefly, the medium from treated cells cultured in 24-well plates was removed and placed into a 96-well plate (50 µl per well). The Griess reagent was added to each well to begin the reaction, the plate was shaken for 10 min, and the absorbance was read at 550 nm on a Molecular Devices VERSAmax plate reader. The nitrite level in each sample was calculated from a standard curve generated with sodium nitrite (0–300 µM in cell culture medium).

Heme oxygenase activity assay. Heme oxygenase activity was determined at the end of each treatment as described previously by our group (11). Briefly, microsomes from harvested cells were added to a reaction mixture containing NADPH, glucose-6-phosphate dehydrogenase, and rat liver cytosol as a source of biliverdin reductase, and the substrate hemin. The reaction mixture was incubated in the dark at 37°C for 1 h and was terminated by the addition of 1 ml of chloroform. After being vigorously vortexed and centrifuged, the extracted bilirubin in the chloroform layer was measured by the difference in absorbance between 464 and 530 nm ({epsilon} = 40 mM–1·cm–1).

Preparation of nuclear extract. Nuclear extraction was performed at the end of the experiment as described previously (5). Briefly, cells were washed twice with cold PBS and harvested by centrifugation at 3,000 rpm for 3 min at 4°C. Cells were carefully resuspended in a cold buffer (buffer A) containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 10% Nonidet P-40 and protease inhibitors (Roche), and incubated on ice for 15 min. The homogenate was then centrifuged at 3,000 rpm for 3 min, the pellet was resuspended, and then incubated for 15 min in buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, and 1 mM EGTA]. Finally, the samples were spun at 13,000 rpm for 5 min and the supernatant was stored at –80°C until needed.

Western blot analysis for HO-1, iNOS, NF-{kappa}B, and Nrf2. Samples of RAW 264.7 cells were analyzed by Western immunoblot technique, as already reported (5, 24, 27, 34). Briefly, an equal amount of proteins (30 µg) for each sample was separated by SDS-PAGE, transferred overnight to nitrocellulose membranes, and the nonspecific binding of antibodies was blocked with 3% nonfat dried milk in PBS. Membranes were then probed with a polyclonal rabbit anti-HO-1 antibody (Bioquote, York, UK) (1:1,000, dilution in Tris-buffered saline, pH 7.4), NF-{kappa}B (1:500 dilution), Nrf2 (1:500 dilution), or iNOS (1:1,000 dilution) antibodies (all purchased from Santa Cruz Biotechnology, Insight Biotechnology, Wembley, UK). After three washes with PBS containing 0.05% (vol/vol) Tween 20, blots were visualized with the use of an amplified alkaline phosphatase kit from Sigma (Extra-3A). For equal loading verification, the samples were also probed with beta-actin polyclonal antibodies (Abcam, Cambridge, UK).

HO-1 and PI3K siRNA transfection. RAW264.7 macrophages were grown in 6- or 12-well plates and transiently transfected with HO-1 or PI3K siRNA (Santa Cruz Biotechnology) mixed with siRNA transfection reagent (Santa Cruz Biotechnology) according to the manufacturer's instructions. After incubation at 37°C and 5% CO2 for 30 h, cells were treated with 2-HC and/or LPS, as described in the Experimental protocol. The samples were then prepared and analyzed for HO-1 and iNOS Western blot analysis as well as nitrite and TNF-{alpha} production.

Measurement of TNF-{alpha} production. The level of TNF-{alpha} present in each sample was determined with the use of a commercially available kit from R&D Systems (Abingdon, UK) (34). The assay was performed according to the manufacturer's instructions. Briefly, cell culture supernatants were collected immediately after the treatment and spun at 13,000 g for 2 min to remove any particulates. The medium was added to a 96-well plate precoated with affinity-purified polyclonal antibodies specific for mouse TNF-{alpha}. An enzyme-linked polyclonal antibody specific for mouse TNF-{alpha} was added to the wells and left to react for 2 h, followed by a final wash to remove any unbound antibody-enzyme reagent. The intensity of the color detected at 450 nm (correction wavelength 570 nm) was measured after addition of a substrate solution and was proportional to the amount of TNF-{alpha} produced.

Cell viability. Cell viability was determined using an Alamar Blue assay kit (Serotec) and carried out according to the manufacturer's instructions, as we previously reported (13, 34). The assay is based on the detection of metabolic activity of living cells by using a redox indicator, which changes from an oxidized (blue) form to a reduced (red) form. The intensity of the red color is proportional to the metabolism of the cells, which is calculated as the difference in absorbance between 570 and 600 nm, and expressed as a percentage of control. An assay for the release lactate dehydrogenase (LDH) activity was also performed with the use of a cytotoxicity detection kit according to manufacturer's instructions (Roche). Briefly, at the end of the incubation period, cell supernatants were collected, and any cell residue was removed by centrifugation at 250 g. The reaction mixture (which is composed of the catalyst and the dye solution) was then added to the cell-free supernatant, incubated at room temperature for 15 min, after which the absorbance was measured at 490 and 690 nm. LDH activity was expressed as percentage of maximal LDH release, which was obtained by lysis of cells with Triton X-100 (1% in DMEM at 25°C).

Statistical analysis. Differences among the groups were analyzed using one-way ANOVA and Student's t-test. Values were expressed as means ± SE and differences between groups were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
2'-HC induces heme oxygenase activity and HO-1 expression in RAW 264.7 macrophages. We have reported previously that 2-HC induces heme oxygenase activity and HO-1 expression in bovine aortic endothelial cells (13) and we examined here whether a similar effect could be reproduced in RAW 264.7 macrophages. As shown in Fig. 1, A and B, exposure of cells for 6 h to 2-HC resulted in a concentration-dependent increase in heme oxygenase activity and HO-1 protein expression, respectively. We also assessed whether this was a long-lasting effect, and for this purpose, heme oxygenase activity was measured in macrophages incubated with 2-HC for 6 h, followed by 12-h exposure to medium alone. Interestingly, heme oxygenase activity remained elevated even after chalcone was removed (Fig. 1C), suggesting that macrophages need a long time to metabolize the compound. This possibility was further sustained by the fact that the extent of heme oxygenase activity was similar between cells incubated for 6 h with 2-HC and cells exposed for 6 h to 2-HC, followed by 12-h incubation in medium alone. However, the activity declined to near control values at the subsequent time points analyzed (18 and 24 h, data not shown). It is known that macrophages respond to LPS challenge by upregulating many endogenous protective pathways, including HO-1 (7). Therefore, we determined the activity of heme oxygenase in RAW 264.7 cells exposed for 24 h to LPS alone or in combination with 2-HC. Our results showed that cells incubated with LPS alone exhibited a marked rise in heme oxygenase activity, which reached very high levels when LPS and 2-HC were used simultaneously (Fig. 2A). As observed with 2-HC alone, the increase in activity elicited by LPS plus 2-HC was dependent on the concentration of 2-HC.


Figure 1
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  		Fig. 1. The effect of 2'-hydroxychalcone (2-HC) on heme oxygenase (HO) activity and HO-1 expression in RAW 264.7 macrophages. Cells were exposed to various concentrations (5–30 µM) of 2-HC for 6 h; heme oxygenase activity (A) and HO-1 protein expression (B) were determined at the end of the incubation. Cells treated with medium alone represent the control group (0 µM). The Western blot is representative of 3 independent experiments. C: cells were exposed to various concentrations (5–30 µM) of 2-HC for 6 h, followed by 12-h incubation in fresh medium alone. Heme oxygenase activity was measured at the end of the incubation (6+12 h), as described in MATERIALS AND METHODS. Cells treated with medium alone represent the control group (0 µM). Bars represent the means ± SE of 5–6 independent experiments per group. *P < 0.001 vs. 0 µM (control).

 

Figure 2
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  		Fig. 2. 2-HC modulates lipopolysaccharide (LPS)-stimulated nitrite production and inducible nitric oxide synthase (iNOS) expression in macrophages. A: cells were exposed to various concentrations (5–30 µM) of 2-HC in the presence of LPS 1 µg/ml for 24 h and heme oxygenase activity was measured at the end of the incubation period. Cells treated with medium alone represent the control group (open bars). B: RAW264.7 macrophages were exposed to LPS (1 µg/ml) in the presence or absence of 2-HC and nitrite production was determined at 24 h. Bars represent the means ± SE of 5–6 independent experiments, *P < 0.001 vs. control; {dagger}P < 0.001 vs. LPS. C: cells were exposed to LPS (1 µg/ml) in the presence or absence of 2-HC and iNOS expression was determined at 24 h. Western blot is representative of 3 independent experiments. beta-Actin was used as an internal control for equal loading.

 
2'-HC reduces LPS-stimulated nitrite production and iNOS expression: involvement of heme oxygenase pathway. We next assessed the anti-inflammatory action of 2-HC by measuring nitrite produced by macrophages challenged with LPS in the presence or absence of the compound. As shown in Fig. 2B, 2-HC effectively and in a concentration-dependent manner reduced the increase in nitrite levels elicited by LPS treatment; nitrite production was decreased to control values when 15 or 30 µM 2-HC was used. In addition, 2-HC decreased LPS-stimulated iNOS expression in a concentration-dependent manner (Fig. 2C). Similarly, but less potently, nitrite generation was diminished in macrophages preincubated with 2-HC for 6 h, followed by 24-h exposure to LPS alone (Fig. 3A). Because 2-HC upregulated HO-1 protein, we examined whether the heme oxygenase pathway played a role in the observed reduction of nitrite by using an inhibitor (SnPPIX, 10 µM) of heme oxygenase activity during the incubation of cells with LPS and 2-HC. The results of Fig. 3B indicate that SnPPIX completely blocked 2-HC-mediated decrease in nitrite production, sustaining an active involvement of heme oxygenase in this process.


Figure 3
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  		Fig. 3. HO mediates the decrease in nitrite production elicited by 2-HC. A: cells were exposed to 2-HC (5–30 µM) for 6 h, followed by 24-h incubation in medium containing LPS (1 µg/ml). Nitrite production was measured at the end of the incubation (6+24 h), as mentioned in MATERIALS AND METHODS. B: cells were exposed to LPS (1 µg/ml) for 24 h in the presence of 2-HC (5–30 µM) and tin protoporphyrin IX (SnPPIX; 10 µM) and nitrite production was determined as described before. Cells treated with medium alone represent the control group (open bars). Bars represent the means ± SE of 5–6 independent experiments per group. *P < 0.001 vs. control; {dagger}P < 0.05 vs. LPS alone.

 
Effects of 2'-HC and LPS on cell viability. Measurements of LDH release, as an index of cell damage, showed that incubation of macrophages for 24 h with increasing concentrations of 2-HC did not cause cytotoxicity compared with control (Fig. 4A). However, exposure to LPS alone resulted in increased cell injury and the presence of 2-HC markedly reversed this effect (Fig. 4A). Interestingly, cell viability assessed with a metabolic assay indicated that 30 µM 2-HC, but not 5 or 15 µM, decreased the cellular metabolic activity (Fig. 4B). A similar reduction was observed with LPS alone or LPS in the presence of 5 or 15 µM 2-HC, whereas coincubation of LPS with 30 µM 2-HC decreased cell viability by >65% (Fig. 4B). Therefore, although 2-HC does not cause rupture of the cell membrane and consequent release of LDH, these data indicate that the plant constituent affects the metabolism of macrophages when used at concentrations ≥30 µM.


Figure 4
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  		Fig. 4. Viability of cells exposed to 2-HC and LPS. Cells were exposed to LPS (1 µg/ml) for 24 h in the presence of 2-HC (5–30 µM) and the viability was determined using lactate dehydrogenase (LDH) release (A) and a metabolic assay (B), as described in MATERIALS AND METHODS. Cells treated with medium alone represent the control group (open bars). Bars represent the means ± SE of 5–6 independent experiments; *P < 0.001 vs. control.

 
2'-HC attenuates LPS-stimulated TNF-{alpha} production. Because TNF-{alpha} is a major cytokine involved in the inflammatory response triggered by LPS (38), we tested the effect of 2-HC on LPS-induced TNF-{alpha} production. Consistent with the results obtained with nitrite levels, 2-HC markedly decreased TNF-{alpha} production by LPS in a concentration-dependent manner (Fig. 5A) and inhibition of the heme oxygenase pathway with SnPPIX significantly prevented this effect (Fig. 5B).


Figure 5
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  		Fig. 5. 2-HC modulates LPS-stimulated TNF-{alpha} production. A: cells were exposed to LPS (0.1 µg/ml) for 12 h in the presence or absence of 2-HC (5–30 µM) and TNF-{alpha} production was determined as described. B: cells were exposed to LPS for 12 h in the presence or absence of 2-HC and SnPPIX (10 µM) and TNF-{alpha} production was determined as described in MATERIALS AND METHODS. Cells treated with medium alone represent the control group (open bars). Bars represent the means ± SE of 5–6 independent experiments, *P < 0.001 vs. control; {dagger}P < 0.05 vs. LPS alone.

 
PI3K-mediated induction of heme oxygenase activity and HO-1 expression by 2-HC is required to attenuate nitrite and TNF-{alpha} production in LPS-stimulated macrophages. To investigate the signaling cascade that mediates the increase in heme oxygenase activity and HO-1 expression by 2-HC, we employed pharmacological antagonists of different MAPK family and PI3K pathways. As observed previously with endothelial cells (13), inhibition of the ERK, p38, or JNK slightly diminished the induction of HO-1 by 2-HC (Fig. 6, A and B). However, blockade of PI3K completely prevented the 2-HC-mediated upregulation of HO-1 (Fig. 6B). To further support a role of PI3K-mediated induction of HO-1 by 2-HC and to confirm the obligatory role of HO-1 in modulating its anti-inflammatory activity in LPS-stimulated macrophages, experiments were performed using siRNA for both PI3K and HO-1. As shown in Fig. 7A, 2-HC failed to induce HO-1 expression in macrophages treated with either HO-1 or PI3K siRNA. Notably, cells treated with HO-1 siRNA did not change iNOS expression in nonstimulated macrophages (Fig. 7A). Intriguingly, the increase in nitrite levels and TNF-{alpha} production in LPS-stimulated macrophages was not prevented by 2-HC after blockade of HO-1 with siRNA (Fig. 7, B and C, respectively). It is interesting to note that in macrophages treated with HO-1 siRNA the levels of nitrite and TNF-{alpha} production after treatment with LPS was 4–5 times higher than in cells with normal HO-1 expression (compare Fig. 2B with 7B and Fig. 5A with 7C).


Figure 6
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  		Fig. 6. Effect of MAPK inhibitors on the on the increase of heme oxygenase activity and HO-1 expression mediated by 2-HC. A: cells were pretreated for 30 min with PD-098059 (ERK inhibitor, 25 µM) or SP-600125 (JNK inhibitor, 10 µM) in serum free-medium before exposure to 15 µM 2-HC for 6 h. Heme oxygenase activity and HO-1 expression were determined at the end of the incubation. B: cells were pretreated for 30 min LY-294002 [phosphatidyinositol 3-kinase (PI3K) pathway inhibitor, 25 µM] or SB-203580 (p38 inhibitor, 5 µM) in serum-free medium before exposure to 15 µM 2-HC for 6 h. Heme oxygenase activity and HO-1 expression were determined at the end of the incubation. Western blots are representative of 3 independent experiments. beta-actin was used as an internal control for equal loading. Cells treated with medium alone represent the control group (open bars). Bars represent the means ± SE of 5–6 independent experiments, *P < 0.001 vs. control; {dagger}P < 0.001 vs. 2-HC alone.

 

Figure 7
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  		Fig. 7. HO-1 expression is required for the anti-inflammatory actions of 2-HC. A: cells were transfected with HO-1 or PI3K small interfering RNA (siRNA) and exposed to various concentrations (5–30 µM) of 2-HC for 6 h and HO-1 protein expression was determined. Cells transfected with HO-1 siRNA were also exposed to LPS in the presence or absence of 2-HC for 24 h and iNOS expression determined. B: cells transfected with HO-1 siRNA were exposed to LPS (1 µg/ml) in the presence or absence of 2-HC and nitrite production was determined after 24 h. C: cells transfected with HO-1 siRNA were exposed to LPS (0.1 µg/ml) for 12 h in the presence or absence of 2-HC and TNF-{alpha} production was determined as described in MATERIALS AND METHODS. Cells treated with medium alone represent the control group (0 µM). Western blots are representative of 3 independent experiments. Bars represent the means ± SE of 5–6 independent experiments per group. Scr, scrambled siRNA. *P < 0.001 vs. 0 µM (control); {dagger}P < 0.05 vs. siRNA.

 
Effect of 2-HC on NF-{kappa}B and Nrf2 activation. The nuclear factor-{kappa}B (NF-{kappa}B) is a transcriptional factor playing a central role in the regulation of many immune and inflammatory responses (42). Therefore, in nuclear extracts collected from our experiments, we determined by Western blot analysis whether 2-HC could modulate the translocation of the P65 subunit of NF-{kappa}B elicited by LPS. Although LPS caused a marked increase of NF-{kappa}B after 30 and 60 min of incubation (Fig. 8A), 2-HC did not effect this response, suggesting that the anti-inflammatory action of this chalcone occurs via different mechanisms. Because Nrf2 activation has been reported to be crucial for HO-1 induction in cells treated with polyphenolic compounds such as curcumin and caffeic acid phenethyl ester (5), we investigated whether the levels of this transcription factor would be affected by 2-HC. As shown in Fig. 8B, nuclear extracts from macrophages treated with 2-HC displayed an increased expression of Nrf2.


Figure 8
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  		Fig. 8. Effect of 2-HC on NF-{kappa}B and Nrf2 activation. A: cells were pretreated with 15 µM 2-HC for 30 min, followed by an additional 30- or 60-min incubation with LPS (1 µg/ml). The translocation of NF-{kappa}B to the nucleus was determined in nuclear extracts by Western blot analysis. B: cells were treated for 60 min with increasing concentrations of 2-HC (0–30 µM) and nuclear extract prepared for determination of Nrf2 expression. In both graphs, Western blots are representative of 3 independent experiments. beta-Actin was used as an internal control for equal loading.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Oxidative stress and inflammation are the major underlying contributors to the development of many pathological states, including cancer, cardiovascular disease, and diabetes, and it is remarkable that a diet rich in antioxidants can effectively reduce the risk of developing these diseases (37). In particular, food that contains inducers of phase II proteins appears to provide significant protection and benefit when consumed on a regular basis. Thus, vegetables belonging to the cruciferous family (broccoli, cauliflower, and brussels sprouts), fruit, berries, and some spices, such as turmeric, contain flavonoids, polyphenols (e.g., curcumin and chalcones), and a variety of phytochemicals that activate protective mechanisms (10). A series of plant-derived chemicals have been reported to induce HO-1 in different cell types, including curcumin (5, 25, 35), caffeic acid phenethyl ester (35), carnosol (21), resveratrol (17), chalcones and rosolic acid (13), and in some of these studies, HO-1 was shown to mediate the antioxidant properties of the compounds (13, 25). In the present study, we examined the potential involvement of HO-1 in the anti-inflammatory activity elicited by 2-HC. We found that 2-HC potently induced heme oxygenase activity and HO-1 expression in RAW 264.7 macrophages and that this induction correlated with a decrease in iNOS expression and production of NO as well as TNF-{alpha} release by LPS-stimulated macrophages. Inhibition of heme oxygenase activity by SnPPIX or siRNA for HO-1 reversed these effects, suggesting that the anti-inflammatory properties of 2-HC involve the dynamic action of this cytoprotective protein. By using pharmacological agents and the siRNA technology, we also identified PI3K as the most likely pathway controlling the induction of HO-1 by 2-HC.

We have recently reported that 2-HC and other similar chalcones can induce HO-1 in endothelial cells (13) and here we extend our previous findings by showing a similar effect in macrophages. In endothelial cells, preincubation with 2-HC decreased the damage caused by subsequent challenge with hydrogen peroxide (13). In the present study, we emphasize that the strong anti-inflammatory activity of this particular chalcone is mediated by HO-1. Because a decrease in nitrite production was observed also when macrophages were preincubated with 2-HC before LPS challenge, we can exclude that this effect was due to a direct interaction of 2-HC with LPS. The persistent HO-1 induction observed after removal of 2-HC from the culture media may be due to the ability of chalcones and polyphenolic compounds to coordinate to thiols and glutathione, which is a crucial modulator of the expression of antioxidant genes, including HO-1 (35). In a finding similar to ours, Alcaraz et al. (1) have recently demonstrated that another chalcone derivative known to elicit anti-inflammatory actions was able to induce HO-1 and simultaneously downregulate an inflammatory pathway (NF-{kappa}B), strongly sustaining HO-1 as an important component of chalcones' mechanisms of action. Although the contribution of HO-1 products (i.e., CO, biliverdin, and iron) has not been examined, several studies (2, 3, 8, 28, 29, 36) point to HO-1-derived CO as the potential metabolite to combat inflammation, which is the common denominator of cardiovascular and neurodegenerative diseases. In addition, we have recently investigated the anti-inflammatory characteristics of CO-releasing molecules (CORM-2 and CORM-3) in macrophages stimulated with LPS and found that CO liberated by these agents significantly reduced nitrite production (34); similar results on nitrite and TNF-{alpha} release were obtained also with the use of other water-soluble CO-releasing molecules synthesized in our laboratories (Abuarqoub H and Motterlini R, unpublished observations). In contrast, biliverdin and bilirubin, the other products of heme degradation by heme oxygenase, did not affect nitrite levels (34). It was interesting to note that while CORM-3 influenced only nitrite production (34), implying inhibition of NOS activity by CO, 2-HC decreased both nitrite and the expression of iNOS, suggesting that the plant-derived compound might act via multiple cellular mechanisms. It is important to point out that 30 µM 2-HC caused at 24 h a decrease in cell metabolism, which was further reduced when the compound was used in combination with LPS. Therefore, the inhibition of nitrite production and iNOS expression observed with 2-HC at this particular concentration could be partially explained by an increased cytotoxic effect. On the other hand, 2-HC protected macrophages from the release of LDH caused by LPS, underlying the ability of the chalcone to protect against specific types of cellular damage. Consistent with previous reports (6) using derivatives of 2-HC, LPS-stimulated TNF-{alpha} production was significantly diminished by 2-HC, further sustaining the role of chalcones as potent inhibitors of proinflammatory mediators induced by endotoxin. Notably, both inhibition of heme oxygenase activity by SnPPIX and blockade of HO-1 expression using siRNA technique completely abolished this effect, indicating that the heme oxygenase pathway contributes to 2-HC-mediated anti-inflammatory action by modulating crucial steps of the inflammatory response.

Previous studies (15, 19) have shown the involvement of MAPK pathways in the biological actions of chalcones; however, our results show that MAPK inhibitors only slightly decrease the induction of HO-1 and heme oxygenase activity by 2-HC (see also Ref. 13). Conversely, blockade of the signaling pathway PI3K, which is a crucial element for ther determination of cell survival and death (22), completely suppressed HO-1 induction, suggesting that 2-HC requires PI3K to upregulate HO-1 expression in macrophages. This is in line with findings published in relation to heme oxygenase and carnosol, a phenol derived from the herb rosemary (21). We also examined whether 2-HC affected the activation of the transcription factor NF-{kappa}B elicited by LPS, but found that the plant constituent did not suppress the LPS-induced nuclear translocation of the p65 subunit of NF-{kappa}B.

In conclusion, this study shows that 2-HC downregulates the inflammatory response in an LPS-stimulated macrophage model and that the HO-1 pathway is strongly involved in this effect. Considering that a diverse group of plant-derived compounds possessing anti-inflammatory and antioxidant properties also increase the expression of HO-1 (13, 25, 35), and that polyphenolics present in food possess positive pharmacological effects (32), we suggest that many of the beneficial actions exerted by certain natural compounds are ultimately linked to heme oxygenase function as a crucial defensive and detoxifying cellular system (25).


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This work was supported in part by a Fellowship grant from the Dunhill Medical Trust (to R. Foresti and R. Motterlini).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Motterlini, Vascular Biology Unit, Dept. of Surgical Research, Northwick Park Institute for Medical Research, Watford Rd., Harrow, Middlesex, HA1 3UJ, UK (e-mail: r.motterlini{at}imperial.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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