Resveratrol, a constituent of red wine, which has long been suspected to have cardioprotective effects [20
], has received much attention in recent years for its therapeutic potential for many diseases and potential for prolonging the lifespan [48
]. Although the mechanism by which resveratrol exerts a wide range of beneficial effects across disease models is unclear, multiple direct targets for this compound have been identified [48
]. The protective effects of resveratrol against heart diseases are thought to stem from its ability to decrease platelet aggregation [29
], promote vasorelaxation [55
], reduce lipid peroxidation [22
], improve serum cholesterol and triglyceride concentrations [57
], and reduce procoagulant tissue factor [31
]. The beneficial effects of red wine are possibly not only mediated through the phenolics, but also ethanol, as it was shown to modulate various biological activities including inhibition of platelet aggregation [59
] and tissue factor expression in monocytes [60
]. In the present study, we identified an additional potential mechanism by which resveratrol and other wine phenolics could provide protection against atherosclerosis. The data presented herein show that wine phenolics (resveratrol and quercetin) suppress the expression of CRP, a protein that is believed to play a key role in the pathogenesis of atherosclerosis [8
A half maximal concentration of resveratrol and quercetin required to inhibit cytokine-induced expression of CRP in the present study is similar to the reported concentration of these compounds required to modulate various biological activities in in vitro
]. Based on in vivo
pharmacokinetics of these compounds [48
], it is unlikely that such high concentrations of resveratrol and quercetin are ever achieved in vivo
either after daily intake of two glasses of wine or even after administration of pharmacological doses. However, despite skepticism concerning the bioavailability, a growing body of recent in vivo
studies provides evidence that resveratrol has protective effects in rodent models of stress and disease [48
]. It has been shown that resveratrol or its metabolites may accumulate at much higher concentrations in tissues than in blood [62
]. This, coupled with potential synergistic interaction of resveratrol and quercetin with other constituents of the diet, may explain how a relatively low dose of resveratrol obtained from red wine could produce measurable health benefits [48
]. In this context, it may be pertinent to note that quercetin, whose concentration in red wine is 10 or more times higher than resveratrol, also suppressed CRP expression as effectively as resveratrol. This suggests that quercetin may be the principal active ingredient in red wine that is capable of suppressing CRP induction.
Despite initial doubts regarding the physiological relevance of in vitro
studies in which typically high concentrations of resveratrol were used, recent studies suggest the therapeutic potential for resveratrol [34
]. This stirred interest in developing more potent resveratrol mimetics. Chemical structural alterations of the stilbene motif of resveratrol were shown to yield extremely effective analogs [38
]. A cis-
form of methyl derivative of resveratrol (R3) was shown to be hundredfold more potent than resveratrol in inhibiting cell proliferation by arresting the cell cycle at the G2/M phase transition in Caco-2 cells [38
]. Similarly, resveratrol analogs bearing the 3,5-dimethoxy motif at the A phenyl ring with amino, methoxy and hydroxyl moieties at the 3′- and 4′-positions (7b, 8b, 11b, 12b and 12c) were found to be more effective than resveratrol as apoptosis-inducing agents [39
]. In our recent studies, these derivatives were shown to be 2- to twenty-fivefold more effective, compared with resveratrol, in inhibiting TF expression in monocytes [65
]. In the present study we found that a number of these derivatives, particularly R3 and 7b, suppressed CRP induction in Hep3B cells very effectively when compared with resveratrol. For example, 50 μM of resveratrol inhibited CRP induction by about 50%, whereas a similar inhibition was attained with 1 μM of R3 and 7b compounds. Thus, resveratrol derivatives may have potential in becoming viable therapeutic agents in the future. It would be interesting to examine whether quercetin derivatives would have much higher inhibitory activity than the derivatives of resveratrol, but unavailability of chemically synthesized quercetin derivatives prevented us from investigating this possibility.
Quercetin, resveratrol or its derivatives suppressed the induction of CRP expression primarily by inhibiting transcriptional activation of the CRP gene. Suppression of CRP antigen levels in cells treated with these compounds is associated with the lower accumulation of CRP mRNA. Consistent with these data, these phenolic compounds were shown to inhibit cytokine-induced CRP promoter activity.
Our data provide additional information on the mechanism of transcriptional induction of IL-1β and IL-6-induced CRP expression in Hep3B cells. Previously, it has been shown that the transcription factors C/EBPβ [66
], STAT3 [69
] and NF-κB [40
] participate in IL-1β and IL-6-induced CRP expression. It was shown that overexpressed NF-κB induced CRP expression [44
]; however, it was not clear whether IL-1β worked through the activation of the NF-κB pathway in synergizing the effects of IL-6 on CRP expression [71
]. Mutation of the κB site on the CRP proximal promoter did not abolish the synergistic effect of IL-1β on IL-6 [71
]. Our results showing that the inhibitors of the NF-κB pathway failed to suppress IL-6 and IL-1β-induced-CRP expression support the view that the sole mechanism of IL-1β action on the CRP promoter is not just NF-κB. However, it is possible that polyphenols may be inhibiting CRP transcription partially through the short promoter by perturbing the interaction of NF-κB and C/EBPβ on the short promoter as NF-κB was shown to act synergistically with C/EBPβ on the 157 bp CRP proximal promoter region to regulate CRP gene expression [71
]. Here it may pertinent to note that wine phenolics inhibited only partly the cytokine-induced 157 bp promoter activity whereas they completely attenuated the cytokine-induced CRP mRNA and protein expression. These data suggest that that polyphenols may additionally regulate CRP expression through the promoter regions located outside the 157 bp promoter. In addition to the known transcription factors acting on the CRP proximal promoter, it has been recently reported that the p38 MAPK pathway also contributes to cytokine-induced CRP expression [46
]. Our studies reported herein reveal that IL-6 and 1 L-1β-induced expression of CRP in Hep3B cells also involves the participation of p44/42 MAPK activation. IL-6 and IL-1β treatment induced the activation of p38 and p44/42 MAPK, and the specific inhibitors of p38 and p44/42 MAPK fully attenuated the cytokine-induced expression of CRP.
Surprisingly, we found that none of these mechanisms known to participate in inducing CRP expression was involved in the inhibition of CRP expression by resveratrol and its derivatives. Two other substances, nitric oxide donors and cholesterol-lowering agents statins, have also been reported to inhibit cytokine-induced CRP expression [70
]. Probably, the mechanism of inhibition of CRP expression by these substances also did not involve the known pathways required for the induction of CRP expression. Combined data indicate that there is a common pathway, as yet unidentified, that mediates the inhibition of cytokine-induced CRP expression by nitric oxide, statins, resveratrol and its derivatives.
In summary, the data presented in the manuscript provide convincing evidence that resveratrol and quercetin suppress cytokine-induced CRP expression and illustrate the feasibility of developing effective drugs to suppress aberrant expression of CRP. As these drugs not only suppress CRP but many other mediators that contribute to atherosclerosis, they will have a good potential to become an effective treatment for atherosclerosis.