Quercetin Inhibits the Production of IL-1β-Induced Inflammatory Cytokines and Chemokines in ARPE-19 Cells via the MAPK and NF-κB Signaling Pathways

Abstract

Quercetin, a bioflavonoid derived from vegetables and fruits, exerts anti-inflammatory effects in various diseases. Our previous study revealed that quercetin could suppress the expression of matrix metalloprotease-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1) to achieve anti-inflammatory effects in tumor necrosis factor-α (TNF-α)-stimulated human retinal pigment epithelial (ARPE-19) cells. The present study explored whether quercetin can inhibit the interleukin-1β (IL-1β)-induced production of inflammatory cytokines and chemokines in ARPE-19 cells. Prior to stimulation by IL-1β, ARPE-19 cells were pretreated with quercetin at various concentrations (2.5-20 µM). The results showed that quercetin could dose-dependently decrease the mRNA and protein levels of ICAM-1, IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1). It also attenuated the adherence of the human monocytic leukemia cell line THP-1 to IL-1β-stimulated ARPE-19 cells. We also demonstrated that quercetin inhibited signaling pathways related to the inflammatory process, including phosphorylation of mitogen-activated protein kinases (MAPKs), inhibitor of nuclear factor κ-B kinase (IKK)α/β, c-Jun, cAMP response element-binding protein (CREB), activating transcription factor 2 (ATF2) and nuclear factor (NF)-κB p65, and blocked the translocation of NF-κB p65 into the nucleus. Furthermore, MAPK inhibitors including an extracellular signal-regulated kinase (ERK) 1/2 inhibitor (U0126), a p38 inhibitor (SB202190) and a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) decreased the expression of soluble ICAM-1 (sICAM-1), but not ICAM-1. U0126 and SB202190 could inhibit the expression of IL-6, IL-8 and MCP-1, but SP600125 could not. An NF-κB inhibitor (Bay 11-7082) also reduced the expression of ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1. Taken together, these results provide evidence that quercetin protects ARPE-19 cells from the IL-1β-stimulated increase in ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1 production by blocking the activation of MAPK and NF-κB signaling pathways to ameliorate the inflammatory response.

Keywords: anti-inflammatory; chemokines; cytokines; quercetin; retinal pigment epithelial cells.

Figure 1

Interleukin-1β (IL-1β) induces the expression of intercellular adhesion molecule-1 (ICAM-1), soluble ICAM-1 (sICAM-1), IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in human retinal pigment epithelial (ARPE-19) cells. (A) IL-1β at concentrations of 0.1–2 ng/mL was used to stimulate ARPE-19 cells for the indicated times. The protein expression of ICAM-1 was analyzed by Western blotting (top panels) and quantified by Image Lab software (lower panels). (B) The levels of sICAM-1, (C) IL-6, (D) IL-8 and (E) MCP-1 in ARPE-19 cells were measured using Enzyme-Linked Immunosorbent Assay (ELISA) after stimulation with 1 ng/mL IL-1β for the indicated times. The data are expressed as mean ± SD of three independent experiments. * p < 0.05 compared with the basal level.

Figure 2

Quercetin attenuates the expression of ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1 in IL-1β-stimulated ARPE-19 cells. (A) Effects of quercetin on ARPE-19 cell viability. ARPE-19 cells were treated for 24 h with 2.5–40 µM quercetin and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the cell viability. (B) ICAM-1 protein level was evaluated by Western blotting and then quantified using Image Lab software. (C) The levels of sICAM-1, (D) IL-6, (E) IL-8 and (F) MCP-1 were assessed by ELISA after cells were incubated for 1 h with quercetin at the indicated doses and then activated with 1 ng/mL IL-1β for 24 h. The data are expressed as mean ± SD of three independent experiments. ^# p < 0.05 versus control cells. * p < 0.05 versus IL-1β-stimulated cells.

Figure 3

Quercetin attenuates the expression of ICAM-1, IL-6, IL-8 and MCP-1 mRNA in IL-1β-stimulated ARPE-19 cells. ARPE-19 cells were pretreated with 20 µM quercetin for 1 h before stimulation with 1 ng/mL IL-1β for 4 h. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) was used to determine the fold changes in (A) ICAM-1, (B) IL-6, (C) IL-8 and (D) MCP-1 gene expression with β-actin as an internal control. The data are expressed as mean ± SD of three independent experiments. ^# p < 0.05 versus control cells. * p < 0.05 versus IL-1β-stimulated cells.

Figure 4

Quercetin inhibits the phosphorylation of mitogen-activated protein kinases (MAPKs) and inhibitor of nuclear factor κ-B kinase (IKK)α/β in IL-1β-stimulated ARPE-19 cells. ARPE-19 cells were treated with 20 µM quercetin for 1 h prior to the stimulation with 1 ng/mL IL-1β for the indicated time. Western blotting and Image Lab software were used to analyze and quantify the phosphorylation of (A) extracellular signal-regulated kinase (ERK) 1/2, (B) p38, (C) c-Jun N-terminal kinase (JNK) 1/2 and (D) IKKα/β. The data are expressed as mean ± SD of three independent experiments. ^# p < 0.05 versus control cells. * p < 0.05 versus IL-1β-stimulated cells.

Figure 5

Quercetin attenuates the phosphorylation of cAMP response element-binding protein (CREB), activating transcription factor 2 (ATF2), c-Jun and nuclear factor (NF)-κB p65 in IL-1β-stimulated ARPE-19 cells. ARPE-19 cells were treated with 20 µM quercetin for 1 h prior to stimulation with 1 ng/mL IL-1β for the indicated time. Western blotting and Image Lab software were used to analyze and quantify the phosphorylation of (A) CREB, (B) ATF2, (C) c-Jun and (D) Nuclear factor (NF)-κB p65. The data are expressed as mean ± SD of three independent experiments. ^# p < 0.05 versus control cells. * p < 0.05 versus IL-1β-stimulated cells.

Figure 6

Inhibitory effects of MAPKs and NF-κB inhibitors on the protein expression of ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1 in IL-1β-stimulated ARPE-19 cells. ARPE-19 cells were pretreated with 10 µM U0126 (U0), 10 µM SB202190 (SB), 10 µM SP600125 (SP) or 5 µM Bay11-7082 (Bay) for 1 h prior to stimulation with 1 ng/mL IL-1β for 24 h. (A) ICAM-1 protein expression was measured by Western blotting and quantified using Image Lab software. (B) The levels of sICAM-1, (C) IL-6, (D) IL-8 and (E) MCP-1 were detected by ELISA. The data are expressed as mean ± SD of three independent experiments. ^# p < 0.05 versus control cells. * p < 0.05 versus IL-1β-stimulated cells.

Figure 7

Inhibitory effects of MAPKs and NF-κB inhibitors on the expression of mRNA for ICAM-1, IL-6, IL-8 and MCP-1 in IL-1β-stimulated ARPE-19 cells. ARPE-19 cells were treated with 10 µM U0126 (U0), 10 µM SB202190 (SB), 10 µM SP600125 (SP) or 5 µM Bay11-7082 (Bay) for 1 h, followed by stimulation with 1 ng/mL IL-1β for 4 h. The fold changes in (A) ICAM-1, (B) IL-6, (C) IL-8 and (D) MCP-1 gene expression were analyzed using RT-qPCR with β-actin as an internal control. The data are expressed as mean ± SD of three independent experiments. ^# p < 0.05 versus control cells. * p < 0.05 versus IL-1β-stimulated cells.

Figure 8

Quercetin attenuates NF-κB p65 translocation in IL-1β-stimulated ARPE-19 cells. Immunofluorescence staining was used to evaluate NF-κB p65 translocation in (A) ARPE-19 cells stimulated with 1 ng/mL IL-1β for the indicated time, and (B) ARPE-19 cells pretreated with 20 µM quercetin or 5 µM Bay 11-7082 for 1 h prior to activation with 1 ng/mL IL-1β for 10 min. The image is representative of the results of four independent experiments. Green: the location of the p65 subunit; Blue: DAPI for nuclear staining.

Figure 9

Quercetin significantly suppresses THP-1 cell adherence to IL-1β-stimulated ARPE-19 cells. ARPE-19 cells were preincubated with quercetin (10, 20 µM), 10 µM U0126, 10 µM SB202190, 10 µM SP600125 or 5 µM Bay 11-7082 for 1 h and then stimulated with 1 ng/mL IL-1β for 24 h. (A,B) A THP-1 monocyte adhesion assay was used to evaluate the physiological function of ICAM-1. The fluorescence intensity represents THP-1 cell adhesion to IL-1β-stimulated ARPE-19 cells, which was quantified using Image J software. The data are expressed as mean ± SD of three independent experiments. ^# p < 0.05 versus control cells. * p < 0.05 versus IL-1β-stimulated cells.

Figure 10

Schematic diagram of the signaling pathways involved in attenuation of IL-1β-induced inflammation by quercetin via downregulation of ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1 expression in ARPE-19 cells. Quercetin attenuated ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1 expression via the MAPK or NF-κB pathways in IL-1β-stimulated ARPE-19 cells.