Squamosamide derivative FLZ inhibits TNF-α-induced ICAM-1 expression via down-regulation of the NF-κB signaling pathway in ARPE-19 cells

Abstract

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). It has been reported that tumor necrosis factor-α (TNF-α) could induce intercellular adhesion molecule-1 (ICAM-1) expression in RPE cells. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant anti-inflammatory activity. However, the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells remain unknown. Therefore, in the present study, we evaluated the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells. We found that FLZ prevented TNF-α-induced ICAM-1 expression and the ability of monocytes to adhere to ARPE-19 cells induced by TNF-α. Furthermore, FLZ inhibited TNF-α-induced NF-κB p65 expression, as well as phosphorylation of IκBα in ARPE-19 cells. Taken together, these results suggest that FLZ inhibited TNF-α-induced ICAM-1 expression through blocking NF-κB signaling pathway in ARPE-19 cells. Thus, FLZ could be used for designing novel therapeutic agents against AMD.

Keywords: Squamosamide derivative FLZ; age-related macular degeneration (AMD); intercellular adhesion molecule-1 (ICAM-1); retinal pigment epithelium (RPE); tumor necrosis factor-α (TNF-α).

Figure 1

Effect of FLZ on ARPE-19 cell viability. ARPE-19 cells were treated with various concentrations (0, 10, 25 and 50 μg/ml) of FLZ for 24 h. The cytotoxicity was then measured by MTT assay. All experiments were repeated at least three times. Data are means ± SD.

Figure 2

Effect of FLZ on adhesion molecule expression in TNF-α-stimulated ARPE-19 cells. ARPE-19 cells were preincubated with various concentrations (0, 10, 25 and 50 μg/ml) of FLZ for 12 h and stimulated with TNF-α (10 ng/ml) for 6 h. A. ICAM-1 mRNA expression was determined by qRT-PCR. B. ICAM-1 protein expression was determined by Western blot. All experiments were repeated at least three times. Data are means ± SD. *P<0.05 vs. control group, #P<0.05 vs. TNF-α group.

Figure 3

Effects of FLZ on cell adhesion assays in vitro. ARPE-19 cells were preincubated with various concentrations (0, 10, 25 and 50 μg/ml) of FLZ for 12 h and subsequently stimulated with TNF-α for 6 h. Fluorescein-labeled THP-1 cells were added to cytokine treated or untreated monolayers of ARPE-19 cells. The numbers of fluorescein-labeled THP-1 cells were determined. FLZ inhibited TNF-α-increased the ability of monocytes to adhere to ARPE-19 cells. All experiments were repeated at least three times. Data are means ± SD. *P<0.05 vs. control group, #P<0.05 vs. TNF-α group.

Figure 4

Effects of FLZ on NF-κB signaling pathway in TNF-α-stimulated ARPE-19 cells. A. ARPE-19 cells were preincubated with or without various concentrations of FLZ for 12 h, then treated with TNF-α for 30 min. Then, lysates of cells were blotted with an antibody against the p65 subunit of NF-κB (nucleus) and an anti-phospho-IκBα antibody (cytoplasm). Lamin A and β-actin were used as loading controls for nuclear and cytosolic protein fractions, respectively. B and C. results were quantified with densitometry. All experiments were repeated at least three times. Data are means ± SD. *P<0.05 vs. control group, #P<0.05 vs. TNF-α group.