Oleacein Attenuates Lipopolysaccharide-Induced Inflammation in THP-1-Derived Macrophages by the Inhibition of TLR4/MyD88/NF-κB Pathway

Abstract

It is known that plant phenolic compounds exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory factors. Recently, Olea europaea has been studied as a natural source of bioactive molecules; however, few studies have focused on the biological effect of oleacein (OLC), the most abundant secoiridoid. Therefore, the aim of this study was to investigate the potential anti-oxidant activity of OLC, as well as to study its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated THP-1-derived macrophages. LPS brought a dramatic increase of both release and gene expression of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α), as well as a decrease of anti-inflammatory ones (IL-10), the effects of which are reverted by OLC. Moreover, it reduced the levels of COX-2, NO and PGE₂ elicited by LPS exposure in THP-1 macrophages. Interestingly, OLC modulated inflammatory signaling pathways through the inhibition of CD14/TLR4/CD14/MyD88 axis and the activation of NF-κB. Finally, OLC showed relevant anti-oxidant capability, assessed by abiotic assays, and reduced the intracellular amount of ROS generated by LPS exposure in THP-1 macrophages. Overall, these results suggest that the anti-oxidant activity and anti-inflammatory effect of OLC may cooperate in its protective effect against inflammatory stressors, thus being a possible alternative pharmacological strategy aimed at reducing the inflammatory process.

Keywords: CD14; LPS; MyD88; NF-κB; Olea europea; ROS; THP-1 monocytes; TLR4; inflammation; oleacein.

Figure 1

Effect of OLC in THP-1-derived macrophages viability in presence or absence of LPS. Different concentrations of OLC (1, 2.5 and 5 μM) were added to culture medium 30 min before LPS treatment (100 ng/mL for 24 h), and then cell viability was assessed by the MTT test. The results are expressed as the percentage of viable cells in treated cultures compared to the percentage in untreated ones. Data are means ± SEM from three independent experiments performed in eight replicates.

Figure 2

OLC reduced the release of IL-1β, IL-6 and TNF-α and enhanced that of IL-10 in LPS-stimulated THP-1-derived macrophages. The cells were treated with increasing concentrations of OLC (1, 2.5 and 5 μM) for 30 min prior LPS (100 ng/mL) for 24 h. Then, secretion of IL-1β (A), IL-6 (B), TNF-α (C) and IL-10 (D) in the cultured medium was evaluated by ELISA assay. Data are the mean ± SEM of three independent experiments performed in triplicate. *** p < 0.001 vs. ctrl; ^§ p < 0.05 vs. LPS-treated cells; ^§§§ p < 0.001 vs. LPS-treated cells.

Figure 3

Effects of OLC on cytokine gene expression in THP-1-derived macrophages stimulated with LPS. THP-1 cells were treated with different concentrations of OLC (1, 2.5 and 5 μM) for 30 min prior LPS (100 ng/mL) for 24 h. The results from real-time PCR of IL-1β (A), IL-6 (B), TNF- α (C) and IL-10 (D) are expressed as a relative fold change compared to untreated cells, after normalization against β-actin as endogenous control. Data are the mean ± SEM of three independent experiments performed in triplicate. *** p < 0.001 vs. ctrl; ^§§ p < 0.01 vs. LPS-treated cells; ^§§§ p < 0.001 vs. LPS-treated cells.

Figure 4

Effects of OLC in LPS-induced production of pro-inflammatory mediators in THP-1-derived macrophages. THP-1-derived macrophages were treated with different concentrations of OLC (1, 2.5 and 5 μM) for 30 min prior LPS (100 ng/mL) for 24 h. The results from the Griess reaction of NO (A) and ELISA assay of PGE₂ (B) and COX-2 (C) are shown. Data are the mean ± SEM of three independent experiments performed in triplicate. *** p < 0.001 vs. ctrl; ^§ p < 0.05 vs. LPS-treated cells; ^§§ p < 0.01 vs. LPS-treated cells; ^§§§ p < 0.001 vs. LPS-treated cells.

Figure 5

Effects of OLC on the expression of TLR4 and CD14 in THP-1 differentiated cells exposed to LPS. The evaluation of the expression of the two membrane markers was performed by a two-staining flow cytometry method (PE-TLR4 and FITC-CD14) and real-time PCR. The dot plots (A) are representative of three separate experiments executed in triplicate (n = 9). The histograms (B) show the percentage ± SEM of cells positive for both TLR4 and CD14 staining. The results from the real-time PCR of TLR4 (C) and CD14 (D) are expressed as a relative fold change compared to untreated cells, after normalization against β-actin as endogenous control. *** p < 0.001 vs. ctrl; ^§§ p < 0.01 vs. LPS-treated cells; ^§§§ p < 0.001 vs. LPS-treated cells.

Figure 6

Modulation of LPS-induced activation of MyD88 and NF-κB in THP-1-derived macrophages by OLC. THP-1-derived macrophages were treated with different concentrations of OLC (1, 2.5 and 5 μM) for 30 min prior LPS (100 ng/mL) for 24 h. The results from MyD88 measured by ELISA assay (A) and real-time PCR (B) are shown along with results of DNA binding activity of NF-κB p65 (C) obtained by ELISA tests. Data are the mean ± SEM of three independent experiments performed in triplicate. *** p < 0.001 vs. ctrl; ^§ p < 0.05 vs. LPS-treated cells; ^§§ p < 0.01 vs. LPS-treated cells; ^§§§ p < 0.001 vs. LPS-treated cells.

Figure 7

Anti-oxidant effect of OLC in LPS-stressed THP-1-derived macrophages. Levels of intracellular ROS were measured by means of flow cytometry employing DCFH₂-DA as probe. The plots are representative of three independent experiments (A). The histograms (B) display the percentage ± SEM of healthy cells (non-fluorescent, M1) and the percentage with increased intracellular ROS (fluorescent, M2) of three separate experiments in triplicate (n = 9). *** p < 0.001 vs. ctrl; ^§ p < 0.05 vs. LPS-treated cells; ^§§ p < 0.01 vs. LPS-treated cells; ^§§§ p < 0.001 vs. LPS-treated cells.

Figure 8

Mechanisms underlying the protective effects of OLC.

Figure 9

PMA-induced differentiation in THP-1 monocytes. THP-1 cells were incubated for 48 h with or without PMA (100 ng/mL).