Phenolic Compounds of Red Wine Aglianico del Vulture Modulate the Functional Activity of Macrophages via Inhibition of NF-κB and the Citrate Pathway

Figure 6

Effect of RWP on ACLY. (a) HEK293 cells were transiently transfected with pGL3 basic-LUC vectors containing the −3116/−20 bp full-length region of the ACLY gene promoter (3000) or a truncated version of this region (1000). Then, cells were triggered with LPS in the absence (LPS) or in the presence of RWP 20 μg/mL or RWP 200 μg/mL. Unstimulated cells were used as a negative control. The luciferase gene reporter activity was assessed after 24 hours. Primary human monocytes, preincubated for 1 hour with RWP, were activated to macrophages with LPS, and protein levels of ACLY (b) and acetylated H3 and total H3 (d) were evaluated. In (b, d) ACLY, acetylated H3, total H3, and β-actin proteins were immunodecorated with specific antibodies. The intensities of immunolabeled protein bands were measured by using a quantitative software and normalized to β-actin: values obtained are reported under western blot images. Protein expression levels in control sample were taken as 1, and other samples were expressed in the proportion of the control. (c) In cells treated as in (b, d) ACLY enzymatic activity was quantified. In (a) and (c), values represent of three experiments with three replicates in each. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test for multiple comparisons. Different letters indicate significant differences at . C: control; L: LPS; R20: RWP 20 μg/mL; R200: RWP 200 μg/mL.

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