The transfected cells were subjected to pretreatment with experimental compounds for 1 h, followed by cytokine treatment for 24 h

The transfected cells were subjected to pretreatment with experimental compounds for 1 h, followed by cytokine treatment for 24 h. kinase (MAPK) activation inhibited CRP expression, implicating the involvement of both pathways in cytokine-induced CRP expression. These data revealed a previously unrecognized role of the p44/42 MAPK signaling pathway in CRP expression. Wine polyphenolics or the synthetic compounds of resveratrol did not affect cytokine-activated phosphorylation of these MAPKs. Conclusions Wine phenolics inhibit CRP expression; however, to do so, they do not utilize the MAPK pathways. and studies strongly suggest that CRP acts as a proatherogenic factor and promotes atherothrombosis [8,9]. CRP is shown to promote endothelial cell activation and dysfunction [10,11], affect vascular smooth muscle cell migration and proliferation [7,12,13], induce changes in matrix biology [14], and promote coagulation [15]. If CRP plays a role in pathogenesis of atherosclerosis, then the blockade of CRP synthesis or its actions would be beneficial in CP-724714 inhibiting the development of atherosclerosis. Overwhelming epidemiological evidence suggests that moderate consumption of alcoholic beverages, particularly red wine, lowers mortality rates from coronary heart diseases [16C21]. Cardiovascular benefits associated with moderate wine consumption have been thought to stem, at least partly, from antioxidant [22C24], anti-inflammatory [25C27], antiplatelet [28C30] and anticoagulant [31,32] activities of wine phenolics, particularly resveratrol. Resveratrol is shown to mimic calorie restriction by stimulating Sir2 (sirtuin 2, a histonedeacetylase), increasing DNA stability and extending lifespan of yeast by 70% [33]. Recent studies showed that resveratrol improves health and survival of mice in a high calorie diet by producing changes associated with longer lifespan, such as increased insulin sensitivity, reduced insulin-like growth factor-1 levels, and increased mitochondrial numbers [34]. Recent epidemiological studies found an inverse/U-shaped relation between alcoholic beverage consumption and plasma concentration of CRP expression [35]. In age-adjusted analyses, wine consumption appears to be more effective in reducing CRP levels compared with other alcoholic beverages. However, this difference disappeared when BMI was taken into account. In the present study, we investigated the effect of wine phenolics on cytokine-induced CRP expression in a cell model system. Further, we also tested whether chemically-modified derivatives of resveratrol could inhibit cytokine-induced CRP expression much more effectively than resveratrol. CRP is produced primarily in liver [36] and can be experimentally induced in human hepatoma Hep3B cells by treatment with proinflammatory cytokines [37]. Therefore, cytokine-induced CRP expression in Hep3B cells was chosen as a model system to investigate the effect of wine phenolics. Materials and methods Reagents Wine phenolics (resveratrol, quercetin, rutin, catechin and epicatechin) and 2,2-azino-bis(3-ethyl) benzthiazoline-6-sulfonic acid were obtained from Sigma (St Louis, MO, USA). Resveratrol derivatives were synthesized as described earlier [38,39] and were a kind gift from Dr M. Roberti, Universit di SKP1 Bologna, Italy, and Dr F. Raul, Laboratory of Nutritional Cancer Prevention, Strasbourg, France. They were stored as lyophilized powders in dark glass vials wrapped with aluminum foil until they were reconstituted on the day of the experiment. IL-1 and IL-6 were obtained from R&D CP-724714 Systems (Minneapolis, MN, USA) and chemiluminescence reagent was from PerkinElmer Life Sciences Inc. (Boston, MA, USA). Cell culture media and reagents were obtained from Invitrogen (Carlsbad, CA, USA). Phosphospecific antibodies and relevant control antibodies were from Cell Signaling Technology (Beverly, MA, USA). Polyclonal rabbit antihuman CRP antibodies were obtained from Sigma and monoclonal anti-CRP (HD 2.4) was from ATCC (Rockville, MD, USA). Cell culture Hep3B cells were obtained from ATCC (Rockville, MD, USA) and cultured to confluence at 37 C under 5%CO2 in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin and glutamine. CRP induction and treatment with phenolic compounds Monolayers of Hep3B cells were serum-starved overnight by replacing the serum-containing medium with serum-free DMEM. The medium was replaced with fresh serum-free medium and stabilized the cells for 2 h before starting experimental treatments. Hep3B cells were pretreated with wine phenolics, resveratrol CP-724714 derivatives or control vehicle for 1 h before the cells were stimulated with IL-1 (10 ng mL?1) and IL-6 (20 ng mL?1) for 24 h to induce CRP expression. All wine phenolics and resveratrol derivatives were dissolved in DMSO before they were diluted in serum-free medium to treat.