Flavonoid fraction of Bergamot juice reduces LPS-induced inflammatory response through SIRT1-mediated NF-κB inhibition in THP-1 monocytes

Abstract

Plant polyphenols exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory genes. Recently, Citrus bergamia has been studied as a natural source of bioactive molecules with antioxidant activity, but few studies have focused on molecular mechanisms underlying their potential beneficial effects. Several findings have suggested that polyphenols could influence cellular function by acting as activators of SIRT1, a nuclear histone deacetylase, involved in the inhibition of NF-κB signaling. On the basis of these observations we studied the anti-inflammatory effects produced by the flavonoid fraction of the bergamot juice (BJe) in a model of LPS-stimulated THP-1 cell line, focusing on SIRT1-mediated NF-κB inhibition. We demonstrated that BJe inhibited both gene expression and secretion of LPS-induced pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) by a mechanism involving the inhibition of NF-κB activation. In addition, we showed that BJe treatment reversed the LPS-enhanced acetylation of p65 in THP-1 cells. Interestingly, increasing concentrations of Sirtinol were able to suppress the inhibitory effect of BJe via p65 acetylation, underscoring that NF-κB-mediated inflammatory cytokine production may be directly linked to SIRT1 activity. These results suggest that BJe may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process.

Figure 1. UHPLC chromatogram of BJe.

A representative chromatogram of the BJe flavonoid components is shown. The sample was run for five times. For peak identification see Table 1.

Figure 2. Effect of BJe on THP-1 cell viability in presence or absence of LPS.

Different concentrations of BJe (0.01, 0.05, 0.1 and 0.5 mg/ml) were added to the culture medium 30 min before LPS treatment (500 ng/ml for 3 hs) and then cell viability was assessed by the MTT test. Results are expressed as percentages of untreated cultures. Data are means ± SEM from three independent experiments performed in eightplicate.

Figure 3. Effects of BJe on cytokine gene expression in THP-1 cells stimulated with LPS.

THP-1 cells were treated with different concentrations of BJe (0.05–0.5 mg/ml for 30 min) before exposure to 500 ng/ml of LPS for 3 hs. Results from real-time PCR of IL-6 (A), IL-1β (B) and TNF-α (C) are expressed as a relative fold change compared to untreated cells, after normalization against 18S as endogenous control. Columns and bars represent means ± SEM from triplicate experiments. * p<0.05, ** p<0.01, *** p<0.001, significant values in comparison with control cells; ^§§§ p<0.001, significant values in comparison with LPS treated cells (ANOVA followed by Student-Newman Keuls multiple comparisons test).

Figure 4. BJe prevents the LPS-stimulated release of IL-1β and TNF-α in THP-1 monocytes.

The cells were treated with increasing concentrations of BJe (0.05–0.5 mg/ml for 30 min) prior to add LPS (500 ng/ml; 3 hs). Then, secretion of IL-6 (A), IL-1β (B) and TNF-α (C) in the media was evaluated by ELISA assay. Data are the mean ± SEM of three independent experiments performed in triplicate. *** p<0.001, significant differences vs untreated cultures; ^§ p<0.05 and ^§§§ p<0.001, significant differences vs LPS treated cells (ANOVA followed by Student-Newman Keuls multiple comparisons test).

Figure 5. Inibitory effect of BJe on LPS-induced NF-κB activation in THP-1 cells.

(A) The cells were exposed to 0.1 mg/ml BJe 30 min before LPS treatment (500 ng/ml for 3 hs), and then NF-κB activation was determined by the electrophoretic mobility shift assay (EMSA). A competition assay was performed using both biotin-labeled and unlabeled specific probe (cold probe, CP). (B) Densitometric analysis of three independent blots (mean ± SEM) is reported. * and *** p<0.05 and p<0.001 vs untreated cultures, respectively; ^§§§, p<0.001 vs LPS-treatment (ANOVA followed by Student-Newman Keuls multiple comparisons test).

Figure 6. Sirtinol reverts the inhibitory effect of BJe on LPS-induced activation of NF-κB.

(A) Exposure of THP-1cells to 0.1 mg/ml BJe reduced LPS-induced NF-κB activation; this effect was reverted by 10 µM Sirtinol, a SIRT1 inhibitor. EMSA analysis was performed using both biotin-labeled and unlabeled specific probe (cold probe). (B) Densitometric analysis of three independent blots (mean ± SEM) is presented. ** p<0.01 and *** p<0.001 vs controls; ^§§§, p<0.001 vs LPS treated cells. ^# p<0.05 vs BJe plus LPS treated cells (ANOVA followed by Student-Newman Keuls multiple comparisons test).

Figure 7. BJe treatment reverts the LPS-enhanced acetylation of p65 in THP-1 cells.

(A) After LPS treatment in presence or absence of BJe and/or Sirtinol, THP-1 cells were lysed and proteins were immunoprecipitated using an anti-p65 antibody. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with antibody against acetyl-lysine residues. Immunoprecipitation negative control was set by incubating cell lysates under similar conditions, but without the immunoprecipitating antibody. (B) Densitometric analysis of three independent blots (mean ± SEM) is presented. ** p<0.01 and *** p<0.001 vs control cultures; ^§§§ p<0.001 vs LPS treated cells; ^### p<0.001 vs LPS plus BJe treated cells (ANOVA followed by Student-Newman Keuls multiple comparisons test).