Extra Virgin Olive Oil Polyphenol-Enriched Extracts Exert Antioxidant and Anti-Inflammatory Effects on Peripheral Blood Mononuclear Cells from Rheumatoid Arthritis Patients

Abstract

Rheumatoid arthritis (RA) is a long-term systemic autoimmune disorder that causes joint inflammation, swelling, pain, bone erosion, and deformities. Recent findings emphasize the anti-inflammatory and antioxidant properties of bioactive natural compounds, such as polyphenols extracted from plants and fruits, and their possible synergistic effect when used in combination with current therapies to improve the prognosis and symptoms of inflammatory rheumatic diseases. Here, we report that Sicilian extra virgin olive oil polyphenol-enriched extracts (PE-EVOOs) reduce intracellular reactive oxygen species (ROS) and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β), in peripheral mononuclear cells (PBMCs) obtained from both RA patients and healthy subjects (HSs) treated with lipopolysaccharides (LPS) as a control. HPLC-ESI-MS analysis highlighted that PE-EVOOs are rich in different polyphenolic compounds responsible for many of the observed biological effects. At molecular levels, Western blotting analyses revealed that PE-EVOO treatment is associated with the downregulation of the phosphorylated and active form of the inflammatory transcription factor NF-κB and the pro-inflammatory enzyme cyclooxygenase 2 (COX2). In addition, PE-EVOOs upregulated the transcription factor Nrf2 and its target antioxidant enzyme catalase and manganese superoxide dismutase (MnSOD). Collectively, these results suggest a possible use of PE-EVOOs as potential adjuvants for the treatment of RA.

Keywords: IL-1β; Nrf2; PBMCs; TNF-α; extra virgin olive oil polyphenols; rheumatoid arthritis.

Figure 1

PE-EVOOs exert a high dose-dependent radical scavenging activity. The antioxidant activity of Sicilian PE-EVOOs was evaluated by a DPPH radical scavenging assay. Different concentrations of PE-EVOOs (from 0.5 to 50 µg/mL) were added to an ethanol DPPH• solution, and the relative absorption was measured at 517 nm spectrophotometrically. The bar graphs represent the mean of three independent experiments ± SD. p-value summary < 0.001 compared to the only vehicle control (ethanol).

Figure 2

PE-EVOO effects on PBMC viability. PBMCs (100 × 10³/100 µL/well) were incubated with different doses (0.5–50 µg/mL) of PE-EVOOs for 48 h. Then, the percentage of viable cells was assessed by an MTS assay. The values reported are the mean ± SD of three independent experiments. p-value summary < 0.05 with respect to cells cultured in medium alone (RPMI).

Figure 3

PE-EVOOs reduce the production of pro-inflammatory cytokines from RA patients and HS LPS-stimulated PBMCs. Cumulative data obtained from flow cytometric analyses of RA patients (A) and HS LPS-stimulated (B) PBMCs intracellularly stained for TNF-α and IL-1β show that both cytokines decreased after 48 h of 10 µg/mL PE-EVOO treatment. Histogram overlays of the production of TNF-α (C) and IL-1β (D) from an RA representative patient. ** p < 0.01 and *** p < 0.001 with respect to cells cultured in medium alone (RPMI) or with LPS (LPS).

Figure 4

PE-EVOOs reduce TNF-α production only from monocytes and not from T helper cells. Cumulative data obtained from flow cytometric analyses of RA patients (A) and HS LPS-stimulated (B) PBMCs intracellularly stained for TNF-α production from Th1 cells and monocytes show that only monocytes producing TNF-α decreased after 48 h of 10 µg/mL PE-EVOO treatment. ** p < 0.01 with respect to cells cultured in medium alone (RPMI) or with LPS.

Figure 5

PE-EVOOs exert anti-inflammatory effects by reducing the expression of the active form of NF-κB and COX2. Western blotting analysis of NF-κB, phosphorylated-NF-κB, and COX2 in RA patients and HS LPS-stimulated PBMCs treated for 48 h with 10 µg/mL of PE-EVOOs. Equal loading of proteins was verified by immunoblotting for GAPDH. The bar graphs represent the mean of three independent experiments. ** p < 0.01 and *** p < 0.001 with respect to cells cultured in medium alone (RPMI).

Figure 6

PE-EVOOs exert antioxidant effects in RA patients and HS LPS-stimulated PBMCs. Cumulative data obtained from flow cytometric analysis of RA patients (A) and HS LPS-stimulated (B) PBMCs treated for 48 h with 10 µg/mL of PE-EVOOs stained for intracellular ROS using the redox-sensitive fluorochrome H2-DCFDA, as reported in the Section 2. Histogram overlays of intracellular ROS production obtained from PBMCs of a representative RA patient (C) and HSs (D). * p < 0.05 and ** p < 0.01 with respect to cells cultured in medium alone (RPMI) or with LPS.

Figure 7

PE-EVOOs exert antioxidant effects upregulating the active form of Nrf2 and its transcriptional targets, catalase and MnSOD. Western blotting analysis of Nrf2, p-Nrf2 (Ser 40), catalase, and MnSOD in RA patients and HS LPS-stimulated PBMCs treated for 48 h with 10 µg/mL of PE-EVOOs. Equal loading of proteins was verified by immunoblotting for GAPDH. The bar graphs represent the mean of three independent experiments ± SD. ** p < 0.01 and *** p < 0.001 with respect to cells cultured in medium alone (RPMI).