Hostname: page-component-78c5997874-s2hrs Total loading time: 0 Render date: 2024-11-05T04:02:58.007Z Has data issue: false hasContentIssue false

1-Phenyl-6,7-dihydroxy-isochroman suppresses lipopolysaccharide-induced pro-inflammatory mediator production in human monocytes

Published online by Cambridge University Press:  27 January 2011

Giuliana Trefiletti
Affiliation:
Department of Physiology and Pharmacology ‘Vittorio Erspamer’, ‘Sapienza’ University of Rome, P. le Aldo Moro 5, 00185, Rome, Italy
Anna Rita Togna
Affiliation:
Department of Physiology and Pharmacology ‘Vittorio Erspamer’, ‘Sapienza’ University of Rome, P. le Aldo Moro 5, 00185, Rome, Italy
Valentina Latina
Affiliation:
Department of Physiology and Pharmacology ‘Vittorio Erspamer’, ‘Sapienza’ University of Rome, P. le Aldo Moro 5, 00185, Rome, Italy
Carolina Marra
Affiliation:
Department of Chemistry, ‘Sapienza’ University of Rome, P. le Aldo Moro 5, 00185, Rome, Italy
Marcella Guiso
Affiliation:
Department of Chemistry, ‘Sapienza’ University of Rome, P. le Aldo Moro 5, 00185, Rome, Italy
Giuseppina I. Togna*
Affiliation:
Department of Physiology and Pharmacology ‘Vittorio Erspamer’, ‘Sapienza’ University of Rome, P. le Aldo Moro 5, 00185, Rome, Italy
*
*Corresponding author: G. I. Togna, fax +39 6 49912363, email [email protected]
Rights & Permissions [Opens in a new window]

Abstract

Extra-virgin olive oil is an integral ingredient of the Mediterranean diet, and it has been suggested that its high consumption has beneficial effects on human health. Its protective effect, in particular against the development of CVD, has been related not only to the high content of oleic acid, but also to the antioxidant and anti-inflammatory properties of polyphenols. In order to verify the anti-inflammatory and anti-atherogenic properties of hydroxy-isochromans, a class of ortho-diphenols present in extra-virgin olive oil, we investigated the potential ability of 1-phenyl-6,7-dihydroxy-isochroman (L137) to modulate the production of key inflammatory mediators by human monocytes, by evaluating its in vitro effects on prostanoid (thromboxane A2 and PGE2) and cytokine (TNF-α) production. Its effect on the protein expression of the inducible form of cyclo-oxygenase-2 (COX-2), a pro-inflammatory enzyme responsible for elevated prostanoid levels, was also explored. The results showed that L137 significantly inhibited both prostanoid and TNF-α production in lipopolysaccharide-primed human monocytes in a dose-dependent manner, by inhibiting the COX activity of COX-2. We also demonstrated that the effects of the isochroman are mediated, at least partly, through the suppression of NF-κB activation leading to the down-regulation of the synthesis of COX-2.

Type
Short Communication
Copyright
Copyright © The Authors 2011

The beneficial effects of the Mediterranean diet are well recognised(Reference Hu1Reference Estruch3). Olive oil is an integral ingredient of this diet, and it has been suggested that its regular consumption, as the main source of fat, exerts protective effects against the development of CVD(Reference Fung, Rexrode and Mantzoros2Reference López-Miranda, Pérez-Jiménez and Ros5). Converging evidence suggests that the cardioprotective effects of extra-virgin olive oil are related not only to its high content of oleic acid, which exerts anti-atherosclerotic and anti-inflammatory effects(Reference Harwood and Yaqoob6, Reference Massaro, Carluccio and De Caterina7), but also to the presence of antioxidants, including phenols, in the non-saponifiable fraction(Reference Bendini, Cerretani and Carrasco-Pancorbo8, Reference Cicerale, Lucas and Keast9).

Recently, Bianco et al. (Reference Bianco, Coccioli and Guiso10) have identified in extra-virgin olive oil a new class of ortho-diphenols, 6,7-dihydroxy-isochromans: 1-phenyl-6,7-dihydroxy-isochroman (encoded L137) and 1-(3-methoxy-4-hydroxy-phenyl)-6,7-dihydroxy-isochroman. It has also been demonstrated that these compounds are not present in fresh olive fruits(Reference Guiso, Marra and Arcos11), but their synthesis may begin during the malaxation process and continue during the oil storage, in competition with the ortho-diphenol oxidative degradation, through the reaction between hydroxytyrosol and aldehydes and ketones, concurrently present in the olive oil(Reference Montedoro, Servili and Baldioli12, Reference Cartoni, Coccioli and Jasionowska13). The antioxidant power of the olive oil dihydroxy-isochromans and their ability to inhibit the human platelet response to agonists that induce reactive oxygen species-mediated platelet activation has been demonstrated in our previous study(Reference Togna, Togna and Franconi14).

On the basis of previous reports suggesting anti-atherogenic and anti-inflammatory properties of extra-virgin olive oil(Reference Carluccio, Siculella and Ancora15Reference Perona, Cabello-Moruno and Ruiz-Gutierrez17), the present study aims to investigate the potential of L137 to modulate the production of key inflammatory mediators by human monocytes, by evaluating its in vitro effects on prostanoid (thromboxane A2 and PGE2) and TNF-α production induced by lipopolysaccharide (LPS).

The effect of L137 on NF-κB-mediated expression of the inducible form of cyclo-oxygenase (COX-2), a pro-inflammatory enzyme responsible for elevated levels of prostanoids(Reference Raso, Meli and Di Carlo18), was also verified.

Methods

Preparation of human peripheral blood mononuclear cell cultures

Fresh EDTA-treated buffy coats from the blood of healthy volunteers were provided by the blood transfusion centre (‘Sapienza’ University of Rome). Peripheral blood mononuclear cells were isolated by centrifugation on a Ficoll–Metrizoate density gradient. The mononuclear cells at the interface were washed twice with Mg2+/Ca2+-free phosphate-buffered solution by re-suspension and centrifugation at 300 g at room temperature. Cells (>90 % monocytes, as determined by non-specific esterase staining) were subsequently tested for their viability by trypan blue exclusion and resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium, supplemented with 10 % heat-inactivated fetal bovine serum, 4 mm-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml). Approximately 107 mononuclear cells were plated in twelve-well tissue culture plates, and monocytes were obtained by selective adherence (120 min, 37°C, 5 % CO2). Non-adherent cells were removed and discarded, while the adherent cells were washed carefully twice with pre-warmed medium. Complete medium was then added to the plates, and the cells were cultured for 24 h before treatments.

Experimental procedure

L137 synthesised in our laboratory by the reaction between hydroxytyrosol and benzaldehyde under very mild conditions(Reference Guiso, Marra and Cavarischia19) was dissolved in Tris–HCl buffer solution (pH 7·8) and assayed at concentrations ranging from 0·5 to 100 μm. To investigate the effect of the tested compound on LPS-induced activation, L137 was added to monocyte cultures 30 min before the stimulus (LPS 50 ng/ml) for 24 h incubation. Then, the supernatants were collected for the measurement of prostanoid and cytokine contents and lactate dehydrogenase release, and the cells were used for subsequent protein measurement(Reference Bradford20), electrophoresis separation and the methylthiazoletrazolium test.

In addition, some experiments were conducted in cultured monocytes pre-treated with aspirin or NS-398 to block constitutive COX-1 or inducible COX-2, respectively. According to Demasi et al. (Reference Demasi, Caughey and James21), to block constitutive COX-1, monocytes were pre-treated with 0·05 mm-aspirin for 30 min, and then washed twice with pre-warmed medium. NS-398, a selective COX-2 inhibitor, was added to the culture medium at a concentration of 10 μm, 30 min before the treatment. Human monocytes were then incubated for 24 h in a complete medium and primed with LPS (50 ng/ml), in the presence or absence of L137. TXB2 (the stable breakdown product of TXA2) and PGE2 concentrations were determined by RIA. The least detectable concentration was 2 pg/ml for both assays.

In order to rule out the possible effect of L137 on the peroxidase activity of COX-2 (conversion of PGG2 to PGE2), the cells were pre-incubated with 5 mm-aspirin to block COX, but not peroxidase, activity of COX-2, and then treated with L137 (10 and 50 μm). PGG2 (5 μm) was used as the substrate to generate PGE2 bypassing the COX step(Reference Wang, Bai and Zhu22).

TNF-α immunoreactivity was measured using a specific human ELISA kit (sensitivity < 9 pg/ml). Cell viability was determined by the lactate dehydrogenase and methylthiazoletrazolium tests.

Western blotting analysis of cyclo-oxygenase-2 and NF-κB expression

Proteins (30 μg) from each sample were denatured in boiling Laemmli buffer for 5 min and resolved by SDS-PAGE on a polyacrylamide gel consisting of a 4 % stacking and a 10 % resolving layer using a Mini-PROTEAN II apparatus (BioRad, Hercules, CA, USA). After electrophoresis, proteins were transferred to nitrocellulose membranes; the membranes were blocked for 1 h at room temperature in Tris-buffered saline–0·1 % Tween 20 (supplemented with 1 % fat-free dried milk and 1 % bovine serum albumin) incubated with primary antibodies, with horseradish peroxidase-conjugated secondary antibodies, and then detected by the enhanced chemiluminescence detection system.

Statistical analysis

Data are presented as means with their standard errors. For statistical analysis, data were analysed by a one-way ANOVA, followed by Bonferroni's post hoc test. A significant difference was defined as a P value < 0·05.

Results and discussion

In the present study, the potential of L137 to inhibit pro-inflammatory mediator production was investigated in vitro by using LPS-stimulated adherent human monocytes. LPS treatment caused a strong increase in prostanoid production, and L137 significantly inhibited the production of PGE2 and TXA2 starting from 1 and 10 μm, respectively (Table 1). To confirm that the inhibiting effect of L137 is not due to cytoxicity, we used lactate dehydrogenase and methylthiazoletrazolium tests. Results showed that at the employed concentrations, cell viability was not affected by L137 (data not shown).

Table 1 Inhibiting effect of 1-phenyl-6,7-dihydroxy-isochroman (L137) on prostanoid production induced by lipopolysaccharide (LPS) in human monocytes pre-treated or not with aspirin (ASA)

(Mean values with their standard errors)

TX, thromboxane.

Mean values were significantly different from the control: *P < 0·01, **P < 0·001.

Performed in triplicate on cells from four different donors.

Monocytes were treated with L137 or buffer solution (control) 30 min before LPS (50 ng/ml).

§ Monocytes were pre-treated with ASA (0·05 mm) for 30 min, then washed twice and stimulated with LPS (50 ng/ml) in the presence or absence of indicated concentrations of L137.

After 24 h incubation, the medium was removed, and prostanoid concentration (ng/mg protein) was determined by RIA.

As has been clearly described(Reference Brock, McNish and Peters-Golden23), synthesis of some prostanoid products depends on different COX isoforms: monocyte TXA2 on both COX-1 and COX-2 and PGE2 mainly on COX-2 activity, respectively. In order to quantify the inhibitory potential of L137 on COX-2 activity, experiments were carried out on monocytes pre-treated with aspirin so as to block the prostanoid production derived from the constitutive isoform of COX (COX-1). Aspirin pre-treatment, in fact, followed by several washings is able to inhibit COX-1 activity, but does not significantly affect COX-2-derived PGE2 and TXB2 production induced by LPS(Reference Demasi, Caughey and James21). As reported in Table 1, the inhibitory effect of L137 on COX-2-derived prostanoid production reached statistical significance even at the lowest concentration assayed (1 μm), suggesting that its inhibitory effect is mainly referred to COX-2 activity. Results obtained with NS-398, the specific inhibitor of COX-2 activity, confirmed this possibility: L137 did not modify the COX-1-derived TXB2 production (3·52 (se 0·36) in control v. 4·60 (se 0·88) in 100 μm-L137-treated monocytes).

COX-2 peroxidase activity was not affected by the treatment with the isochroman. Indeed, L137 did not modify the PGE2 production when PGG2 was used as the substrate (10·8 (se 2·2) ng/ml in controls v. 12·07 (se 1·4) and 13·87 (se 2·5) in human monocytes treated with L137 at 10 and 50 μm, respectively).

The production of TNF-α, one of the major pro-inflammatory cytokines involved in the pathogenesis of chronic inflammatory diseases and modulated by oxidative stress(Reference Calamia24), was also impaired by L137. The amount of TNF-α produced over 24 h by LPS-stimulated, but untreated, cells was 0·8–5·2 ng/ml, and L137 treatment at 0·5, 10 and 100 μm decreased the TNF-α production by about 30, 60 and 80 %, respectively (P < 0·01).

The effect of L137 on LPS-induced COX-2 protein expression was examined by Western blotting. Furthermore, since COX-2 is a NF-κB-regulated gene, we investigated whether L137 is able to suppress LPS-induced NF-κB activation. Human monocytes both untreated and pre-treated with L137 were primed with LPS. Whole-cell extracts were prepared and analysed by Western blotting. As shown in Fig. 1(A), L137 only at 100 μm significantly decreased LPS-induced COX-2 expression through the suppression of NF-κB activation (Fig. 1(B)). Because the inhibitory effect on COX-2-mediated prostanoid release was recorded even at the lowest concentration (1 μm), whereas only at 100 μm, COX-2 expression was significantly reduced, the effect of L137 seems to depend mainly on a direct inhibition of the COX activity of COX-2 and to involve a decrease in COX-2 synthesis only at the highest concentration.

Fig. 1 Effect of 1-phenyl-6,7-dihydroxy-isochroman (L137) on cyclo-oxygenase-2 (COX-2) (A) and NF-κB (B) protein expression in human monocytes stimulated with lipopolysaccharide (LPS; 50 ng/ml). The densitometric data were calculated as the fold decrease of the value for the LPS-stimulated group. Values are means from three different experiments, with standard errors represented by vertical bars. * Significant inhibition v. LPS-stimulated cells (P < 0·001). a,b Mean values with unlike letters were significantly different (P < 0·01).

In conclusion, in the present study, we demonstrated that L137 suppresses LPS-induced pro-inflammatory mediator production and COX-2 expression by inhibiting the activation of the NF-κB signal transduction pathway. This effect, in addition to the reported antioxidant and anti-platelet activity(Reference Togna, Togna and Franconi14, Reference Lorenz, Zeh and Martens-Lobenhoffer25), indicates that the isochroman compounds may also contribute to the anti-atherogenic and anti-inflammatory properties of the extra-virgin olive oil.

Acknowledgements

We gratefully acknowledge Paola Patrignani for providing specific PGE2 antiserum. The present study was partially supported by a grant to G. I. T. from the ‘Sapienza’ University of Rome. None of the authors has any conflicts of interest. The contribution of the authors was as follows: G. T., A. R. T. and G. I. T. designed the study; G. T., A. R. T. and V. L. contributed to the execution of the experimental work; M. G. and C. M. synthesised, purified and characterised the isochroman. All authors contributed to and approved the final manuscript.

References

1 Hu, FB (2003) The Mediterranean diet and mortality – olive oil and beyond. N Engl J Med 348, 25952596.CrossRefGoogle ScholarPubMed
2 Fung, TT, Rexrode, KM, Mantzoros, CS, et al. (2009) Mediterranean diet and incidence of and mortality from coronary heart disease and stroke in women. Circulation 119, 10931100.CrossRefGoogle ScholarPubMed
3 Estruch, R (2010) Anti-inflammatory effects of the Mediterranean diet: the experience of the PREDIMED study. Proc Nutr Soc 69, 333340.CrossRefGoogle ScholarPubMed
4 Covas, MI, Konstantinidou, V & Fitó, M (2009) Olive oil and cardiovascular health. J Cardiovasc Pharmacol 54, 477482.CrossRefGoogle ScholarPubMed
5 López-Miranda, J, Pérez-Jiménez, F, Ros, E, et al. (2010) Olive oil and health: summary of the II international conference on olive oil and health consensus report, Jaén and Córdoba (Spain) 2008. Nutr Metab Cardiovasc Dis 20, 284294.CrossRefGoogle ScholarPubMed
6 Harwood, JL & Yaqoob, P (2002) Nutritional and health aspects of olive oil. Eur J Lipid Sci Technol 104, 685697.3.0.CO;2-Q>CrossRefGoogle Scholar
7 Massaro, M, Carluccio, MA & De Caterina, R (1999) Direct vascular antiatherogenic effects of oleic acid: a clue to the cardioprotective effects of the Mediterranean diet. Cardiologia 44, 507513.Google Scholar
8 Bendini, A, Cerretani, L, Carrasco-Pancorbo, A, et al. (2007) Phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules 12, 16791719.CrossRefGoogle ScholarPubMed
9 Cicerale, S, Lucas, L & Keast, R (2010) Biological activities of phenolic compounds present in virgin olive oil. Int J Mol Sci 11, 458479.CrossRefGoogle ScholarPubMed
10 Bianco, A, Coccioli, F, Guiso, M, et al. (2001) The occurrance in olive oil of a new class of phenolic compounds: hydroxy-isochromans. Food Chem 77, 405411.CrossRefGoogle Scholar
11 Guiso, M, Marra, C & Arcos, RR (2008) An investigation on dihydroxy-isochroman in extra virgin olive oil. Nat Prod Res 22, 14031409.CrossRefGoogle ScholarPubMed
12 Montedoro, G, Servili, M, Baldioli, M, et al. (1992) Simple and hydrolyzable phenolic compounds in virgin olive oil: their extraction separation and quantitative and semi-quantitative evaluation by HPLC. J Agric Food Chem 40, 15711576.CrossRefGoogle Scholar
13 Cartoni, GP, Coccioli, F, Jasionowska, R, et al. (2000) HPLC analysis of the benzoic and cinnamic acids in edible vegetable oils. Ital J Food Sci 12, 163167.Google Scholar
14 Togna, GI, Togna, AR, Franconi, M, et al. (2003) Olive oil isochromans inhibit human platelet reactivity. J Nutr 133, 25322536.CrossRefGoogle ScholarPubMed
15 Carluccio, MA, Siculella, L, Ancora, MA, et al. (2003) Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscler Thromb Vasc Biol 23, 622629.CrossRefGoogle ScholarPubMed
16 Miles, EA, Zoubouli, P & Calder, PC (2005) Differential anti-inflammatory effects of phenolic compounds from extra virgin olive oil identified in human whole blood cultures. Nutrition 21, 389394.CrossRefGoogle ScholarPubMed
17 Perona, JS, Cabello-Moruno, R & Ruiz-Gutierrez, V (2006) The role of virgin olive oil components in the modulation of endothelial function. J Nutr Biochem 17, 429445.CrossRefGoogle ScholarPubMed
18 Raso, GM, Meli, R, Di Carlo, G, et al. (2001) Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1. Life Sci 68, 921931.CrossRefGoogle ScholarPubMed
19 Guiso, M, Marra, C & Cavarischia, C (2001) Isochromans from 2-(3′,4′-dihydroxy) phenylethanol. Tetrahedron Lett 42, 65316534.CrossRefGoogle Scholar
20 Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248254.CrossRefGoogle ScholarPubMed
21 Demasi, M, Caughey, GE, James, MJ, et al. (2000) Assay of cyclooxygenase-1 and 2 in human monocytes. Inflamm Res 49, 737743.CrossRefGoogle ScholarPubMed
22 Wang, P, Bai, HW & Zhu, BT (2010) Structural basis for certain naturally occurring bioflavonoids to function as reducing co-substrates of cyclooxygenase I and II. PLoS One 5, e12316.CrossRefGoogle ScholarPubMed
23 Brock, TG, McNish, RW & Peters-Golden, M (1999) Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2. J Biol Chem 274, 1166011666.CrossRefGoogle ScholarPubMed
24 Calamia, KT (2003) Current and future use of anti-TNF agents in the treatment of autoimmune, inflammatory disorders. Adv Exp Med Biol 528, 545549.CrossRefGoogle ScholarPubMed
25 Lorenz, P, Zeh, M, Martens-Lobenhoffer, J, et al. (2005) Natural and newly synthesized hydroxy-1-aryl-isochromans: a class of potential antioxidants and radical scavengers. Free Radic Res 39, 535545.CrossRefGoogle ScholarPubMed
Figure 0

Table 1 Inhibiting effect of 1-phenyl-6,7-dihydroxy-isochroman (L137) on prostanoid production induced by lipopolysaccharide (LPS) in human monocytes pre-treated or not with aspirin (ASA)(Mean values with their standard errors)†

Figure 1

Fig. 1 Effect of 1-phenyl-6,7-dihydroxy-isochroman (L137) on cyclo-oxygenase-2 (COX-2) (A) and NF-κB (B) protein expression in human monocytes stimulated with lipopolysaccharide (LPS; 50 ng/ml). The densitometric data were calculated as the fold decrease of the value for the LPS-stimulated group. Values are means from three different experiments, with standard errors represented by vertical bars. * Significant inhibition v. LPS-stimulated cells (P < 0·001). a,b Mean values with unlike letters were significantly different (P < 0·01).