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eNOS Promoter Activation by Red Wine Polyphenols: an Interaction Study

eNOS Promoter Activation by Red Wine Polyphenols: an Interaction Study Keywords Kúcové slová: 1. Introduction A significantly reduced incidence of ischaemic heart-disease deaths despite a high saturated and monounsaturated fat intake in certain areas of France has led to the concept of the "French paradox". This phenomenon was attributed to a higher intake of alcohol and, in particular, of wine in France (St Leger et al., 1979). Moderate ethanol intake from any type of beverage improves lipoprotein metabolism and, lowers cardiovascular mortality risk, but wine, particularly red wine with its abundant content of phenolic acids, polyphenols, and flavonoids seems to confer additional health benefits. These include increase in high-density lipoprotein cholesterol levels and decreased oxidation of low-density lipoprotein (LDL) cholesterol, antioxidant activity, decreased platelet aggregation and adhesion, as well as improved endothelium-dependent vasodilatation. Many of these effects are compatible with the action of endotheliumderived nitric oxide (NO) (Wallerath et al., 2003). In the development of atherosclerosis, reduced bioavailability of NO, formed by endothelial nitric oxide synthase (eNOS) Acta Fac. Pharm. Univ. Comen. LX, 2013, (1), p. 27-33 precedes the appearance of visible vessel alterations. Thus, improved NO bioavailability would be a promising step in the therapy and prevention of cardiovascular disorders (Räthel et al., 2007), implying that NO may be a mediator of the cardiovascular protection provided by red wine. As the long-term treatment of cultured endothelial cells with red wine or red wine polyphenols induces eNOS expression and causes a sustained increase in endothelial NO production, upregulation of eNOS is probably based on synergistic mechanisms between the different polyphenolic components (Schmitt & Dirsch, 2009). The concentration of active ingredients in some herbs or dietary source is lower than therapeutic dosages, which has led to scepticism and suggestion that herbal therapeutic efficacies are because of placebo effects. By contrast, there have been reports of the total contents of herbal product showing a significantly better effect than an equivalent dose of a single isolated active ingredient (Ma et al., 2009). There is an increasing awareness that analyses of single components are not always adequate to clearly assess the health benefits of natural product mixtures from dietary sources, since they involve interaction effects (Kurin et al., 2012). Interactions are generally described as being synergistic or antagonistic. Synergy means, broadly, "working together" and antagonism means "working against each other", and these terms imply the existence of some intermediate, zero-interactive state in which agents do neither of the above (e.g. additivity) (Berenbaum, 1989). The aim of our study was an interaction study of four red wine polyphenols (resveratrol ­ R, quercetin ­ Q, kaempferol ­ KF, isorhamnetin ­ IR; Fig. 1) on eNOS promoter activation in endothelial EA.hy926 cells using median effect equations, where the effects of single compounds and their equimolar mixtures were determined and the interactions of combinations were evaluated according to Chou (2006). quercetin (98% purity) (Sigma-Aldrich, USA), kaempferol (99% purity) and isorhamnetin ( 99% purity) (Carl Roth, Germany) dissolved in dimethyl sulfoxide (DMSO). Phorbol12-myristate-13-acetate (Alexis Biochemicals, Austria) was used as a reference (positive control) and the final DMSO concentrations in all treatment did not exceed 0.1%. Control cells were always treated with an equal volume of solvent. The concentration of single polyphenols used was 3­100 µM and their equimolar combinations final mixture concentration were 1­30 µM, (e.g., the 30 µM final equimolar combination Q+R was composed of 15 µM of R and 15µM of Q). After 18 hours incubation with the respective compounds, the cells were washed with PBS and lysed with lysis buffer (Promega, Germany). To determine eNOS promoter activity, the luminescence generated from the luciferase activity was measured using Tecan Genios Pro (Tecan, Austria) plate reader. The values were then normalised to the protein level determined by the Bradford assay as described by Bradford with slight modifications (Bradford, 1976). Statistical and Interaction Analysis All data were obtained in three independent experiments performed in quadruplets. Data are expressed as mean ±SD. Differences between groups for statistical significance were evaluated by ANOVA with Bonferroni post hoc test using GraphPad Prism software (version 5.01, GraphPad Software, Inc., La Jolla, CA, USA). P-values < 0.05 were considered significant. The concentration of sample leading to 50% effect (EC50 ) was calculated from the dose­effect relationship of polyphenols effect on eNOS promoter activation using GraphPad Prism software. The interaction analysis evaluating synergy or antagonism of the combinations was done according to mass­ action law principle (Chou, 2006), described by Equation (1) for n-drug combination at x% inhibition, using combination index (CI) for interaction interpretation 2. Materials and methods Cell Culture The human endothelial cell line EA.hy926 (Edgell et al., 1983), stably transfected with the plasmid p-eNOS-3500-Hu-Luc-neo (Li et al., 1998) containing 3600 base pairs of the human eNOS promoter driving a luciferase reporter gene (EA.hy926-heNOSLuc) were used for measuring the eNOS promoter activity. Luciferase Reporter Gene Assay Stably transfected EA.hy926-heNOS-Luc cells were grown in Dulbecco's modified Eagle's medium without phenol red supplemented with 584 mg/ml glutamine, 100 U/ml benzylpenicillin, 100 mg/ml, streptomycin (Lonza, Belgium), HAT supplement (100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine) (Biochrom, Germany) and 10% heat-inactivated foetal bovine serum (Gibco via Invitrogen, UK) until passage 15. For the experiments, the cells were seeded for 24 hours in 96-well plates at a density of 4 × 105 cells/well and were stimulated with polyphenols ­ resveratrol (99% purity), (CI)x Equation 1 n (CI)x is the sum of the dose of n drugs that exerts x% inhibition in a combination. In the denominator (Dx) is for D "alone" that inhibits a system x%. If CI value is =, > or < 1, an additive, synergistic or antagonistic effect is indicated. The dose-reduction index (DRI) means how many-fold the dose of each drug in a synergic combination could be reduced at a given effect level compared with the doses of each drug alone. The DRI value for each corresponding drug was given for n-drug combinations, Equation (2) (DRI)1 ( ) ( ) ( ) ( ) (DRI)2 ( ) ...etc. ( ) Equation 2 Resveratrol Quercetin Kaempferol Figure 1. Structures of resveratrol, quercetin, kaempferol and isorhamnetin. Value of DRI > 1 indicates a favourable dose reduction, and the higher DRI value indicates the higher dose reduction for a given therapeutic effect, but does not necessarily always indicate synergism. Both CI and DRI were calculated using a median-effect analysis by CompuSyn software (version 1.0.1, ComboSyn, Inc., Paramus, NJ, USA). Isorhamnetin 3. Results and discussion NO is one of the main mediators of vasodilatation, and decreased NO levels play a central role in endothelial dysfunction. In mammalians, endothelial NO is produced by the enzyme eNOS, which converts L-arginine in the presence of O2 and NADPH into L-citrulline and NO (Appeldoorn et al., 2009). The generation of NO plays a major role in maintaining cardiovascular homeostasis by governing blood pressure, improving endothelial function, suppressing vascular smooth muscle mitogenesis, inhibiting leukocyte adhesion and platelet aggregation. Dietary polyphenols are widely distributed in vegetables, fruits and beverages such as tea and wine. Several recent studies have demonstrated that polyphenols such as resveratrol, quercetin, epigallocatechin-3-gallate and delphinidin enhance NO output to improve endothelium-dependent vascular relaxation (Xu et al., 2004). As the moderate regular red wine consumption are associated with a reduced risk of cardiovascular diseases and are related with activation of eNOS system at different levels (Wallerath et al., 2005), in this study, we investigated the influence of resveratrol, quercetin, kaempferol and isorhamnetin, the polyphenols that are present in red wines, on the eNOS promoter activity. Both individual substance or in their equimolar mixtures were investigated. Further, we evaluated their interactions when used in combinations. As it was described in Materials and Methods, first of all we explored activity of single polyphenols on eNOS promoter activation in four different concentrations of polyphenols (3­10­30­100 µM) and from the dose­effect relationship the EC50 values using GraphPad Prism software were determined. PMA as a positive control activated eNOS promoter (Fig. 2). As it is seen in Table 1 and in supplementary information Table S1 (where the statistic evaluation of each sample is done), all polyphenols activated eNOS promoter, where the EC50 values were in micromolar concentration ranging from 3.44 µM (R2 = 0.96) for kaempferol to 9.89 µM for isorhamnetin (R2 = 0.94). Acta Fac. Pharm. Univ. Comen. LX, 2013, (1), p. 27-33 Table 1. Activation of eNOS promotor with single wine polyphenols Compound Resveratrol Quercetin Isorhamnetin Kaempferol EC50 (µM) 4.07 6.92 9.89 3.44 R2 0.93 0.93 0.94 0.96 EC50 (effective concentration) means the concentration (in µM) of the compound leading to the half maximal effect. EC50 and R2 (value quantifying the goodness of fit) were calculated using GraphPad Prism (version 5.01, USA). Wallerath et al. (2005) demonstrated that quercetin has no effect on eNOS promoter activity up to 33 µM. However, in our experiment, we found out that quercetin activated eNOS promoter (EC50 6.92 µM; R2 = 0.93). We also demonstrated that quercetin activates eNOS promoter not only alone but also in mixtures with other red wine polyphenols (Tables 1 and 2; Tables S2 and S3). It is known that kaempferol significantly induces NO production in endothelial cells (Chen et al., 2010) and isorhamnetin has shown inhibitory effect on ox-LDL induced eNOS downregulation (Bao & Lou, 2006), but for the first time we described that kaempferol and isorhamnetin activate eNOS promoter (Table 1). Resveratrol has been shown to enhance the expression of eNOS modulate the deacetylation of eNOS and increase the plasma NO levels (Wang et al., 2012). The ability of resveratrol to activate eNOS promoter was described in the two previous studies (Wallerath et al., 2002; 2005) and our results (Table 1) are in accordance with them. of polyphenols combinations in the second step of our work. We prepared binary, tertiary and quaternary mixtures of tested polyphenols in four concentrations (1­3­10­30 µM), where the contribution of each part was always equimolar (e.g. the 30 µM final equimolar combination Q+R was composed of 15 µM of R and 15 µM of Q) and gave the same final molar concentration of the mixture as the single compound samples. As it is shown in Table 2, EC50 values of polyphenols mixtures ranged from 3.31 µM (R2 = 0.97) for R+Q to 11.09 µM (R2 = 0.95) for IR+KF. For Q+R and IR+KF mixture isobolographic analysis was performed as seen in Figures 3 and 4. The solid line in the isobologram is based on the individual median effects (e.g. EC50) of both compounds and represents the collection of concentration pairs which have zero interaction (e.g. they display additivity). From the definition, points under this solid line represent synergic composition (as they give the same effect with a smaller dose in total), whereas points above this solid line describe antagonistic ones (as they give the same effect with a higher dose). The point in the middle of the curved dashed line shows the experimentally determined position of 300 00 the EC50 value of the Q+R mixture (Fig. 3) or IR+KF mixture *** *** 250 00 (Fig. 4) and its position manifests the synergic or antagonistic interaction of the mixture. 200 00 *** When we used combination index analysis based on the ns 150 00 mass­action law for quantifying drug interactions, we were ns 100 00 able to determine not only binary mixtures interactions (as with isobologram) but also perform n>2-drug combinations 50 00 interactions analysis. The results of Q+R and IR+KF isobolo0 grams analysis were in concordance with the CI analysis m PMA R 30M R 10 0M R 3M M R 1M medium DMSO where the CI for Q+R was 0.65 (from the definition CI < 1 inFigure .2. PMA res resveratrol in eNOS activation activation. Figure 2. PMA vs. vs. sveratrol in eNOS promot promoter ter n dicates synergy) and CI for IR+KF was 2.17 (from the definition CI > 1 indicates antagonism). One can see in Table 2, that Positive control - orbol-12-myri ate P vs. (2 veratrol activ Positive The barspho­ phorbol-12-myristate-13-acetateresv M; PMA) in eNOS promoter controlepresent meanistate-13-aceta (2 M; PMA) ***P<0 againstvity treatm alone n. re ±SD, n=3; *P P<0.05, **P< <0.01, 0.001 ment activation DMSO four mixtures acted synergic (Q+R, Q+IR+KF, R+Q+KF and vs. resveratrol activity in eNOS promoter activation. The bars rep(ANOVA A/Bonferroni). . Q+R+IR+KF) where the CI vary from 0.65 to 0.87, one mixture resent mean ±SD, n=3; *P<0.05, **P<0.01, ***P<0.001 against effect was nearly additive R+IR+KF (CI = 1.08) and other six DMSO treatment alone (ANOVA/Bonferroni). mixtures were antagonistic with CI from 1.16 to 2.01. It is known that resveratrol has protective effects on multi- Interaction studies, which determine synergy or antagonism targets related to cardiovascular diseases. It seems that a of substances, are relatively well known for at least three drug targeting multiple points may exhibit better therapeu- decades amongst antioxidants. Examples are synergic eftic efficacy than one target blocking or activating in com- fects between vitamins E and C (Scarpa et al., 1984; Han et al., plex conditions. Common disorders such as cardiovascular 1991), vitamin E and -carotene (Palozza & Krinsky, 1992) or diseases tend to result from multiple molecular abnormali- vitamin E with flavan-3-ols (Zhou et al., 2005), with rootlets ties (Wang et al., 2012), thus multi-targeting drugs or com- extracts (Peyrat-Maillard et al., 2001) or with tea polyphebinations of drugs seem to bring much more efficiency into nols (Zhou et al., 2000). Nevertheless, there is no information therapy or prevention. We performed an interaction study about wine polyphenols interactions related to eNOS path- . m uimolar mixtu ure Figure 3. Isobologram of Q+R equ Table 2. EC50 , CI, and DRI values of polyphenol mixtures at 50% effect dose level Polyphenol mixture R+Q R+IR R+KF Q+IR Q+KF IR+KF R+Q+IR Q+IR+KF R+Q+KF R+IR+KF Q+R+IR+KF EC50 (µM) 3.31 (1.655 : 1.655) 6.76 (3.38 : 3.38) 4.54 (2.27 : 2.27) 11.48 (5.74 : 5.74) 5.43 (2.715 : 2.715) 11.09 (5.545 : 5.545) 7.31 (2.436 : 2.436 : 2.436) 4.89 (1.63 : 1.63 : 1.63) 3.50 (1.16 : 1.16 : 1.16) 5.06 (1.686 : 1.686 : 1.686) 3.65 (0.9125 : 0.9125 : 0.9125 : 0.9125) R2 0.97 0.99 0.95 0.96 0.89 0.95 0.99 0.95 0.97 0.97 0.97 CI 0.65 1.16 1.22 1.41 1.18 2.17 1.20 0.87 0.79 1.08 0.71 Interaction Synergy Slight antagonism Moderate antagonism Moderate antagonism Slight antagonism Antagonsim Slight antagonism Slight synergy Moderate synergy Additivity Moderate synergy DRI 2.5 : 4.2 1.2 : 3.0 1.8 : 1.5 1.2 : 1.7 2.6 : 1.3 1.8 : 0.6 1.7 : 2.8 : 4.1 4.2 : 6.1 : 2.1 3.5 : 5.9 : 2.9 2.4 : 5.9 : 2.0 7.6 : 4.5 : 10.8 : 3.8 Polyphenols equimolar mixtures: R ­ resveratrol, Q ­ quercetin, IR ­ isorhamnetin, KF ­ kaempferol. EC50 (effective concentration) means the concentration (in µM) of the compound leading to the half maximal effect. EC50 and R2 (value quantifying the goodness of fit) were calculated using GraphPad Prism (version 5.01., USA). CI ­ combination index, based on the mass­action law is quantifying drug interaction in terms of synergy, additivity or antagonism (CI <, = or >1). DRI represents the order of magnitude (fold) of dose reduction that is allowed in combination for a given degree of effect as compared with the dose of each drug alone. CI and DRI were calculated using CompuSyn software (version 1.0.1, USA). Interactions are determined according to Chou (2006). way. Räthel et al. investigated apart from resveratrol also red wine polyphenol extracts (RWPE) from 180 wine types. Using luciferase reporter gene expression as an indicator for eNOS promoter activity they found out that all RWPE under investigation increased eNOS promoter activity, but the biological activity was dependent to an individual polyphenol pattern. When they compared the RWPE results with resveratrol, they discovered that resveratrol mimics the effects of RWPE at concentrations higher than that calculated to be present in analysed wines and thus, resveratrol alone does not account for the observed effects of RWPE. Thus, synergy with other compounds in red wine is suggested (Räthel et al., 2007). Chan et al. have shown that the effects of ethanol on NO production and inducible nitric oxide synthase (iNOS) gene expression in murine macrophage cells (RAW 264.7) was synergistically increased when combined with quercetin and resveratrol in reducing NO production by both scavenging NO and reducing iNOS gene expression (Chan et al., 2000). We found out that quercetin with resveratrol act synergistically in eNOS promoter activation (Table 2 and Fig. 3). This is in accordance with our previous results with resveratrol and quercetin, where they synergistically inhibited vascular smooth muscle cell proliferation when used in a mixture (Kurin et al., 2012). Besides CI we determined the DRI as well. DRI represents the order of magnitude (fold) of dose reduction that is al- lowed in combination for a given degree of effect as compared with the dose of each drug alone, or in other words it indicates to what extent the concentration of drug can be reduced in a mixture in order to achieve a given effect level compared with a single drug treatment. DRI values higher than 1 are desirable, but they do not necessarily indicate synergy. As seen in Table 2, in the Q+R mixture are DRI values 2.5 for quercetin and 4.2 for resveratrol, what means that in Q+R mixture we needed 2.5 times lower dose of quercetin and 4.2 times lower dose of resveratrol to achieve the same effect that would be reached by the single compound treatment. Despite we are not able to explain the inner mechanism of interactions among tested red wine polyphenols in eNOS promoter activation, we take into account that as the eNOS promoter activation involves multiple processes, the interference with multiple different targets is needed. Herbal drugs as complexes of substances or prepared mixtures of natural compounds open the possibility of novel multicomponent treatment or prevention approach development through synergistic interactions, which could impact multiple targets simultaneously, thus being better suitable for controlling complex diseases or biochemical pathways such as eNOS (Zimmermann et al., 2007). In a small experimental model, we have shown that red wine polyphenols when used in mixtures are needed in a smaller (ANOVA A/Bonferroni). . Acta Fac. Pharm. Univ. Comen. LX, 2013, (1), p. 1-33 EC50 of Q+R mixture isobologram of resveratrol and quercetin . m u u Figure 3. Isobologram of Q+R equimolar mixtu re amount and reach many times higher effects that is single molecule able to. "French paradox" until today has not been explained by a single effective molecule, our work suggests that the positive effects of red wine on cardiovascular system should be explained by the synergy of polyphenols mixtures present in red wine, thus despite their low concentration, their effects could be given by their cooperation in multiple system. The interaction study of red wine polyphenols indicated that in eNOS promoter activation, the final effects of mixtures vary from synergistic to antagonistic. Currently, the mechanism of their interaction is not known. However, when they are used together in a quaternary mixture, the final effect is synergic. Figure 3. Isobologram of Q+R equimolar mixture. 4. Conclusion In conclusion, resveratrol, quercetin, kaempferol and isorhamnetin, the substances present in red wine, can activate eNOS promoter when used alone or in equimolar mixtures. Acknowledgements We thank Prof. Verena Dirsch, Department of Pharmacognosy, University of Vienna for providing laboratory space and equipment for the experiments. This work was partially supported by VEGA 2/0081/11 grant. EC50 of IR 10 EC50 of IR+KF mixture EC50of IR+KF mixture isobologram of isorhamnetin and kaempf erol isorhamnetin (µM) 8 6 4 2 0 -2 -1 0 1 2 3 4 5 6 EC50 of KF 1:1 mixture kaempferol (µM) Figure 4. Isobologram of IR+KF equimolar mixture Figure4. Isobologram of IR+KF equimolar mixture. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

eNOS Promoter Activation by Red Wine Polyphenols: an Interaction Study

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de Gruyter
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0301-2298
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10.2478/afpuc-2013-0013
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Abstract

Keywords Kúcové slová: 1. Introduction A significantly reduced incidence of ischaemic heart-disease deaths despite a high saturated and monounsaturated fat intake in certain areas of France has led to the concept of the "French paradox". This phenomenon was attributed to a higher intake of alcohol and, in particular, of wine in France (St Leger et al., 1979). Moderate ethanol intake from any type of beverage improves lipoprotein metabolism and, lowers cardiovascular mortality risk, but wine, particularly red wine with its abundant content of phenolic acids, polyphenols, and flavonoids seems to confer additional health benefits. These include increase in high-density lipoprotein cholesterol levels and decreased oxidation of low-density lipoprotein (LDL) cholesterol, antioxidant activity, decreased platelet aggregation and adhesion, as well as improved endothelium-dependent vasodilatation. Many of these effects are compatible with the action of endotheliumderived nitric oxide (NO) (Wallerath et al., 2003). In the development of atherosclerosis, reduced bioavailability of NO, formed by endothelial nitric oxide synthase (eNOS) Acta Fac. Pharm. Univ. Comen. LX, 2013, (1), p. 27-33 precedes the appearance of visible vessel alterations. Thus, improved NO bioavailability would be a promising step in the therapy and prevention of cardiovascular disorders (Räthel et al., 2007), implying that NO may be a mediator of the cardiovascular protection provided by red wine. As the long-term treatment of cultured endothelial cells with red wine or red wine polyphenols induces eNOS expression and causes a sustained increase in endothelial NO production, upregulation of eNOS is probably based on synergistic mechanisms between the different polyphenolic components (Schmitt & Dirsch, 2009). The concentration of active ingredients in some herbs or dietary source is lower than therapeutic dosages, which has led to scepticism and suggestion that herbal therapeutic efficacies are because of placebo effects. By contrast, there have been reports of the total contents of herbal product showing a significantly better effect than an equivalent dose of a single isolated active ingredient (Ma et al., 2009). There is an increasing awareness that analyses of single components are not always adequate to clearly assess the health benefits of natural product mixtures from dietary sources, since they involve interaction effects (Kurin et al., 2012). Interactions are generally described as being synergistic or antagonistic. Synergy means, broadly, "working together" and antagonism means "working against each other", and these terms imply the existence of some intermediate, zero-interactive state in which agents do neither of the above (e.g. additivity) (Berenbaum, 1989). The aim of our study was an interaction study of four red wine polyphenols (resveratrol ­ R, quercetin ­ Q, kaempferol ­ KF, isorhamnetin ­ IR; Fig. 1) on eNOS promoter activation in endothelial EA.hy926 cells using median effect equations, where the effects of single compounds and their equimolar mixtures were determined and the interactions of combinations were evaluated according to Chou (2006). quercetin (98% purity) (Sigma-Aldrich, USA), kaempferol (99% purity) and isorhamnetin ( 99% purity) (Carl Roth, Germany) dissolved in dimethyl sulfoxide (DMSO). Phorbol12-myristate-13-acetate (Alexis Biochemicals, Austria) was used as a reference (positive control) and the final DMSO concentrations in all treatment did not exceed 0.1%. Control cells were always treated with an equal volume of solvent. The concentration of single polyphenols used was 3­100 µM and their equimolar combinations final mixture concentration were 1­30 µM, (e.g., the 30 µM final equimolar combination Q+R was composed of 15 µM of R and 15µM of Q). After 18 hours incubation with the respective compounds, the cells were washed with PBS and lysed with lysis buffer (Promega, Germany). To determine eNOS promoter activity, the luminescence generated from the luciferase activity was measured using Tecan Genios Pro (Tecan, Austria) plate reader. The values were then normalised to the protein level determined by the Bradford assay as described by Bradford with slight modifications (Bradford, 1976). Statistical and Interaction Analysis All data were obtained in three independent experiments performed in quadruplets. Data are expressed as mean ±SD. Differences between groups for statistical significance were evaluated by ANOVA with Bonferroni post hoc test using GraphPad Prism software (version 5.01, GraphPad Software, Inc., La Jolla, CA, USA). P-values < 0.05 were considered significant. The concentration of sample leading to 50% effect (EC50 ) was calculated from the dose­effect relationship of polyphenols effect on eNOS promoter activation using GraphPad Prism software. The interaction analysis evaluating synergy or antagonism of the combinations was done according to mass­ action law principle (Chou, 2006), described by Equation (1) for n-drug combination at x% inhibition, using combination index (CI) for interaction interpretation 2. Materials and methods Cell Culture The human endothelial cell line EA.hy926 (Edgell et al., 1983), stably transfected with the plasmid p-eNOS-3500-Hu-Luc-neo (Li et al., 1998) containing 3600 base pairs of the human eNOS promoter driving a luciferase reporter gene (EA.hy926-heNOSLuc) were used for measuring the eNOS promoter activity. Luciferase Reporter Gene Assay Stably transfected EA.hy926-heNOS-Luc cells were grown in Dulbecco's modified Eagle's medium without phenol red supplemented with 584 mg/ml glutamine, 100 U/ml benzylpenicillin, 100 mg/ml, streptomycin (Lonza, Belgium), HAT supplement (100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine) (Biochrom, Germany) and 10% heat-inactivated foetal bovine serum (Gibco via Invitrogen, UK) until passage 15. For the experiments, the cells were seeded for 24 hours in 96-well plates at a density of 4 × 105 cells/well and were stimulated with polyphenols ­ resveratrol (99% purity), (CI)x Equation 1 n (CI)x is the sum of the dose of n drugs that exerts x% inhibition in a combination. In the denominator (Dx) is for D "alone" that inhibits a system x%. If CI value is =, > or < 1, an additive, synergistic or antagonistic effect is indicated. The dose-reduction index (DRI) means how many-fold the dose of each drug in a synergic combination could be reduced at a given effect level compared with the doses of each drug alone. The DRI value for each corresponding drug was given for n-drug combinations, Equation (2) (DRI)1 ( ) ( ) ( ) ( ) (DRI)2 ( ) ...etc. ( ) Equation 2 Resveratrol Quercetin Kaempferol Figure 1. Structures of resveratrol, quercetin, kaempferol and isorhamnetin. Value of DRI > 1 indicates a favourable dose reduction, and the higher DRI value indicates the higher dose reduction for a given therapeutic effect, but does not necessarily always indicate synergism. Both CI and DRI were calculated using a median-effect analysis by CompuSyn software (version 1.0.1, ComboSyn, Inc., Paramus, NJ, USA). Isorhamnetin 3. Results and discussion NO is one of the main mediators of vasodilatation, and decreased NO levels play a central role in endothelial dysfunction. In mammalians, endothelial NO is produced by the enzyme eNOS, which converts L-arginine in the presence of O2 and NADPH into L-citrulline and NO (Appeldoorn et al., 2009). The generation of NO plays a major role in maintaining cardiovascular homeostasis by governing blood pressure, improving endothelial function, suppressing vascular smooth muscle mitogenesis, inhibiting leukocyte adhesion and platelet aggregation. Dietary polyphenols are widely distributed in vegetables, fruits and beverages such as tea and wine. Several recent studies have demonstrated that polyphenols such as resveratrol, quercetin, epigallocatechin-3-gallate and delphinidin enhance NO output to improve endothelium-dependent vascular relaxation (Xu et al., 2004). As the moderate regular red wine consumption are associated with a reduced risk of cardiovascular diseases and are related with activation of eNOS system at different levels (Wallerath et al., 2005), in this study, we investigated the influence of resveratrol, quercetin, kaempferol and isorhamnetin, the polyphenols that are present in red wines, on the eNOS promoter activity. Both individual substance or in their equimolar mixtures were investigated. Further, we evaluated their interactions when used in combinations. As it was described in Materials and Methods, first of all we explored activity of single polyphenols on eNOS promoter activation in four different concentrations of polyphenols (3­10­30­100 µM) and from the dose­effect relationship the EC50 values using GraphPad Prism software were determined. PMA as a positive control activated eNOS promoter (Fig. 2). As it is seen in Table 1 and in supplementary information Table S1 (where the statistic evaluation of each sample is done), all polyphenols activated eNOS promoter, where the EC50 values were in micromolar concentration ranging from 3.44 µM (R2 = 0.96) for kaempferol to 9.89 µM for isorhamnetin (R2 = 0.94). Acta Fac. Pharm. Univ. Comen. LX, 2013, (1), p. 27-33 Table 1. Activation of eNOS promotor with single wine polyphenols Compound Resveratrol Quercetin Isorhamnetin Kaempferol EC50 (µM) 4.07 6.92 9.89 3.44 R2 0.93 0.93 0.94 0.96 EC50 (effective concentration) means the concentration (in µM) of the compound leading to the half maximal effect. EC50 and R2 (value quantifying the goodness of fit) were calculated using GraphPad Prism (version 5.01, USA). Wallerath et al. (2005) demonstrated that quercetin has no effect on eNOS promoter activity up to 33 µM. However, in our experiment, we found out that quercetin activated eNOS promoter (EC50 6.92 µM; R2 = 0.93). We also demonstrated that quercetin activates eNOS promoter not only alone but also in mixtures with other red wine polyphenols (Tables 1 and 2; Tables S2 and S3). It is known that kaempferol significantly induces NO production in endothelial cells (Chen et al., 2010) and isorhamnetin has shown inhibitory effect on ox-LDL induced eNOS downregulation (Bao & Lou, 2006), but for the first time we described that kaempferol and isorhamnetin activate eNOS promoter (Table 1). Resveratrol has been shown to enhance the expression of eNOS modulate the deacetylation of eNOS and increase the plasma NO levels (Wang et al., 2012). The ability of resveratrol to activate eNOS promoter was described in the two previous studies (Wallerath et al., 2002; 2005) and our results (Table 1) are in accordance with them. of polyphenols combinations in the second step of our work. We prepared binary, tertiary and quaternary mixtures of tested polyphenols in four concentrations (1­3­10­30 µM), where the contribution of each part was always equimolar (e.g. the 30 µM final equimolar combination Q+R was composed of 15 µM of R and 15 µM of Q) and gave the same final molar concentration of the mixture as the single compound samples. As it is shown in Table 2, EC50 values of polyphenols mixtures ranged from 3.31 µM (R2 = 0.97) for R+Q to 11.09 µM (R2 = 0.95) for IR+KF. For Q+R and IR+KF mixture isobolographic analysis was performed as seen in Figures 3 and 4. The solid line in the isobologram is based on the individual median effects (e.g. EC50) of both compounds and represents the collection of concentration pairs which have zero interaction (e.g. they display additivity). From the definition, points under this solid line represent synergic composition (as they give the same effect with a smaller dose in total), whereas points above this solid line describe antagonistic ones (as they give the same effect with a higher dose). The point in the middle of the curved dashed line shows the experimentally determined position of 300 00 the EC50 value of the Q+R mixture (Fig. 3) or IR+KF mixture *** *** 250 00 (Fig. 4) and its position manifests the synergic or antagonistic interaction of the mixture. 200 00 *** When we used combination index analysis based on the ns 150 00 mass­action law for quantifying drug interactions, we were ns 100 00 able to determine not only binary mixtures interactions (as with isobologram) but also perform n>2-drug combinations 50 00 interactions analysis. The results of Q+R and IR+KF isobolo0 grams analysis were in concordance with the CI analysis m PMA R 30M R 10 0M R 3M M R 1M medium DMSO where the CI for Q+R was 0.65 (from the definition CI < 1 inFigure .2. PMA res resveratrol in eNOS activation activation. Figure 2. PMA vs. vs. sveratrol in eNOS promot promoter ter n dicates synergy) and CI for IR+KF was 2.17 (from the definition CI > 1 indicates antagonism). One can see in Table 2, that Positive control - orbol-12-myri ate P vs. (2 veratrol activ Positive The barspho­ phorbol-12-myristate-13-acetateresv M; PMA) in eNOS promoter controlepresent meanistate-13-aceta (2 M; PMA) ***P<0 againstvity treatm alone n. re ±SD, n=3; *P P<0.05, **P< <0.01, 0.001 ment activation DMSO four mixtures acted synergic (Q+R, Q+IR+KF, R+Q+KF and vs. resveratrol activity in eNOS promoter activation. The bars rep(ANOVA A/Bonferroni). . Q+R+IR+KF) where the CI vary from 0.65 to 0.87, one mixture resent mean ±SD, n=3; *P<0.05, **P<0.01, ***P<0.001 against effect was nearly additive R+IR+KF (CI = 1.08) and other six DMSO treatment alone (ANOVA/Bonferroni). mixtures were antagonistic with CI from 1.16 to 2.01. It is known that resveratrol has protective effects on multi- Interaction studies, which determine synergy or antagonism targets related to cardiovascular diseases. It seems that a of substances, are relatively well known for at least three drug targeting multiple points may exhibit better therapeu- decades amongst antioxidants. Examples are synergic eftic efficacy than one target blocking or activating in com- fects between vitamins E and C (Scarpa et al., 1984; Han et al., plex conditions. Common disorders such as cardiovascular 1991), vitamin E and -carotene (Palozza & Krinsky, 1992) or diseases tend to result from multiple molecular abnormali- vitamin E with flavan-3-ols (Zhou et al., 2005), with rootlets ties (Wang et al., 2012), thus multi-targeting drugs or com- extracts (Peyrat-Maillard et al., 2001) or with tea polyphebinations of drugs seem to bring much more efficiency into nols (Zhou et al., 2000). Nevertheless, there is no information therapy or prevention. We performed an interaction study about wine polyphenols interactions related to eNOS path- . m uimolar mixtu ure Figure 3. Isobologram of Q+R equ Table 2. EC50 , CI, and DRI values of polyphenol mixtures at 50% effect dose level Polyphenol mixture R+Q R+IR R+KF Q+IR Q+KF IR+KF R+Q+IR Q+IR+KF R+Q+KF R+IR+KF Q+R+IR+KF EC50 (µM) 3.31 (1.655 : 1.655) 6.76 (3.38 : 3.38) 4.54 (2.27 : 2.27) 11.48 (5.74 : 5.74) 5.43 (2.715 : 2.715) 11.09 (5.545 : 5.545) 7.31 (2.436 : 2.436 : 2.436) 4.89 (1.63 : 1.63 : 1.63) 3.50 (1.16 : 1.16 : 1.16) 5.06 (1.686 : 1.686 : 1.686) 3.65 (0.9125 : 0.9125 : 0.9125 : 0.9125) R2 0.97 0.99 0.95 0.96 0.89 0.95 0.99 0.95 0.97 0.97 0.97 CI 0.65 1.16 1.22 1.41 1.18 2.17 1.20 0.87 0.79 1.08 0.71 Interaction Synergy Slight antagonism Moderate antagonism Moderate antagonism Slight antagonism Antagonsim Slight antagonism Slight synergy Moderate synergy Additivity Moderate synergy DRI 2.5 : 4.2 1.2 : 3.0 1.8 : 1.5 1.2 : 1.7 2.6 : 1.3 1.8 : 0.6 1.7 : 2.8 : 4.1 4.2 : 6.1 : 2.1 3.5 : 5.9 : 2.9 2.4 : 5.9 : 2.0 7.6 : 4.5 : 10.8 : 3.8 Polyphenols equimolar mixtures: R ­ resveratrol, Q ­ quercetin, IR ­ isorhamnetin, KF ­ kaempferol. EC50 (effective concentration) means the concentration (in µM) of the compound leading to the half maximal effect. EC50 and R2 (value quantifying the goodness of fit) were calculated using GraphPad Prism (version 5.01., USA). CI ­ combination index, based on the mass­action law is quantifying drug interaction in terms of synergy, additivity or antagonism (CI <, = or >1). DRI represents the order of magnitude (fold) of dose reduction that is allowed in combination for a given degree of effect as compared with the dose of each drug alone. CI and DRI were calculated using CompuSyn software (version 1.0.1, USA). Interactions are determined according to Chou (2006). way. Räthel et al. investigated apart from resveratrol also red wine polyphenol extracts (RWPE) from 180 wine types. Using luciferase reporter gene expression as an indicator for eNOS promoter activity they found out that all RWPE under investigation increased eNOS promoter activity, but the biological activity was dependent to an individual polyphenol pattern. When they compared the RWPE results with resveratrol, they discovered that resveratrol mimics the effects of RWPE at concentrations higher than that calculated to be present in analysed wines and thus, resveratrol alone does not account for the observed effects of RWPE. Thus, synergy with other compounds in red wine is suggested (Räthel et al., 2007). Chan et al. have shown that the effects of ethanol on NO production and inducible nitric oxide synthase (iNOS) gene expression in murine macrophage cells (RAW 264.7) was synergistically increased when combined with quercetin and resveratrol in reducing NO production by both scavenging NO and reducing iNOS gene expression (Chan et al., 2000). We found out that quercetin with resveratrol act synergistically in eNOS promoter activation (Table 2 and Fig. 3). This is in accordance with our previous results with resveratrol and quercetin, where they synergistically inhibited vascular smooth muscle cell proliferation when used in a mixture (Kurin et al., 2012). Besides CI we determined the DRI as well. DRI represents the order of magnitude (fold) of dose reduction that is al- lowed in combination for a given degree of effect as compared with the dose of each drug alone, or in other words it indicates to what extent the concentration of drug can be reduced in a mixture in order to achieve a given effect level compared with a single drug treatment. DRI values higher than 1 are desirable, but they do not necessarily indicate synergy. As seen in Table 2, in the Q+R mixture are DRI values 2.5 for quercetin and 4.2 for resveratrol, what means that in Q+R mixture we needed 2.5 times lower dose of quercetin and 4.2 times lower dose of resveratrol to achieve the same effect that would be reached by the single compound treatment. Despite we are not able to explain the inner mechanism of interactions among tested red wine polyphenols in eNOS promoter activation, we take into account that as the eNOS promoter activation involves multiple processes, the interference with multiple different targets is needed. Herbal drugs as complexes of substances or prepared mixtures of natural compounds open the possibility of novel multicomponent treatment or prevention approach development through synergistic interactions, which could impact multiple targets simultaneously, thus being better suitable for controlling complex diseases or biochemical pathways such as eNOS (Zimmermann et al., 2007). In a small experimental model, we have shown that red wine polyphenols when used in mixtures are needed in a smaller (ANOVA A/Bonferroni). . Acta Fac. Pharm. Univ. Comen. LX, 2013, (1), p. 1-33 EC50 of Q+R mixture isobologram of resveratrol and quercetin . m u u Figure 3. Isobologram of Q+R equimolar mixtu re amount and reach many times higher effects that is single molecule able to. "French paradox" until today has not been explained by a single effective molecule, our work suggests that the positive effects of red wine on cardiovascular system should be explained by the synergy of polyphenols mixtures present in red wine, thus despite their low concentration, their effects could be given by their cooperation in multiple system. The interaction study of red wine polyphenols indicated that in eNOS promoter activation, the final effects of mixtures vary from synergistic to antagonistic. Currently, the mechanism of their interaction is not known. However, when they are used together in a quaternary mixture, the final effect is synergic. Figure 3. Isobologram of Q+R equimolar mixture. 4. Conclusion In conclusion, resveratrol, quercetin, kaempferol and isorhamnetin, the substances present in red wine, can activate eNOS promoter when used alone or in equimolar mixtures. Acknowledgements We thank Prof. Verena Dirsch, Department of Pharmacognosy, University of Vienna for providing laboratory space and equipment for the experiments. This work was partially supported by VEGA 2/0081/11 grant. EC50 of IR 10 EC50 of IR+KF mixture EC50of IR+KF mixture isobologram of isorhamnetin and kaempf erol isorhamnetin (µM) 8 6 4 2 0 -2 -1 0 1 2 3 4 5 6 EC50 of KF 1:1 mixture kaempferol (µM) Figure 4. Isobologram of IR+KF equimolar mixture Figure4. Isobologram of IR+KF equimolar mixture.

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Jun 27, 2013

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