Sulforaphane reduces vascular inflammation in mice and prevents TNF-α-induced monocyte adhesion to primary endothelial cells through interfering with the NF-κB pathway - PubMed (original) (raw)

doi: 10.1016/j.jnutbio.2014.03.011. Epub 2014 Apr 4.

Hongwei Si 2, Pon Velayutham Anandh Babu 3, Dengke Pan 4, Yu Fu 5, Elizabeth A S Brooke 1, Halley Shah 1, Wei Zhen 5, Hong Zhu 6, Dongmin Liu 7, Yunbo Li 8, Zhenquan Jia 9

Affiliations

Sulforaphane reduces vascular inflammation in mice and prevents TNF-α-induced monocyte adhesion to primary endothelial cells through interfering with the NF-κB pathway

Palanisamy Nallasamy et al. J Nutr Biochem. 2014 Aug.

Abstract

Sulforaphane, a naturally occurring isothiocyanate present in cruciferous vegetables, has received wide attention for its potential to improve vascular function in vitro. However, its effect in vivo and the molecular mechanism of sulforaphane at physiological concentrations remain unclear. Here, we report that a sulforaphane concentration as low as 0.5 μM significantly inhibited tumor necrosis factor-α (TNF-α)-induced adhesion of monocytes to human umbilical vein endothelial cells, a key event in the pathogenesis of atherosclerosis both in static and under flow conditions. Such physiological concentrations of sulforaphane also significantly suppressed TNF-α-induced production of monocyte chemotactic protein-1 and adhesion molecules including soluble vascular adhesion molecule-1 and soluble E-selectin, key mediators in the regulation of enhanced endothelial cell-monocyte interaction. Furthermore, sulforaphane inhibited TNF-α-induced nuclear factor (NF)-κB transcriptional activity, Inhibitor of NF-κB alpha (IκBα) degradation and subsequent NF-κB p65 nuclear translocation in endothelial cells, suggesting that sulforaphane can inhibit inflammation by suppressing NF-κB signaling. In an animal study, sulforaphane (300 ppm) in a mouse diet significantly abolished TNF-α-increased ex vivo monocyte adhesion and circulating adhesion molecules and chemokines in C57BL/6 mice. Histology showed that sulforaphane treatment significantly prevented the eruption of endothelial lining in the intima layer of the aorta and preserved elastin fibers' delicate organization, as shown by Verhoeff-van Gieson staining. Immunohistochemistry studies showed that sulforaphane treatment also reduced vascular adhesion molecule-1 and monocyte-derived F4/80-positive macrophages in the aorta of TNF-α-treated mice. In conclusion, sulforaphane at physiological concentrations protects against TNF-α-induced vascular endothelial inflammation, in both in vitro and in vivo models. This anti-inflammatory effect of sulforaphane may be, at least in part, associated with interfering with the NF-κB pathway.

Keywords: In vivo; Physiological concentrations; Sulforaphane; TNF-α; Vascular inflammation.

Copyright © 2014 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Conflict of interest: None declared.

Figures

Fig. 1

Fig. 1. Sulforaphane inhibited TNF-α-stimulated monocyte adhesion to HUVECs in static and shear flow conditions

(A–C) Sulforaphane inhibited TNF-α-induced monocyte adhesion to ECs in static conditions. HUVECs were pre-treated with various concentrations of sulforaphane (S) for 1 h before addition of TNF-α (T 2 ng/mL) in the presence or absence of sulforaphane for 6 h. THP-1 cells were labeled with a fluorescence probe and the adhesion was determined using a Microplate Reader at excitation and emission wavelengths of 496 nm and 520 nm (A). Images were captured using a florescence microscope (B) or a Nikon Phase contrast microscope (D). Values are mean ± SEM, n=4. (D) TNF-α-induced monocyte adhesion to ECs under shear flow control. Values are mean ± SEM, n=3. *, p<0.05 vs. control; #, p<0.05 vs. TNF-α alone-treated cells.

Fig. 2

Fig. 2. Sulforaphane suppressed the production of MCP-1 (A), sVCAM-1 (B) and sE-Selectin (C) in HUVECs

HUVECs were pre-treated with various concentrations of sulforaphane (S) for 1 h before addition of TNF-α (T 2 ng/mL) in the presence or absence of sulforaphane for 6 h. MCP-1, sVCAM-1 and sE-Selectin were measured by ELISA. Data are expressed as mean ± SEM from three experiments. *, p<0.05 vs. control; #, p<0.05 vs. TNF-α alone-treated cells. MCP-1, monocyte chemoattractant protein -1; sVCAM-1, soluble vascular adhesion molecule-1; sE-Selectin, soluble E-Selectin.

Fig. 3

Fig. 3. Sulforaphane inhibited TNF-α-induced NF-κB signaling in ECs

(A) The effect of sulforaphane on TNF-α-induced NF-κB transcriptional activity. ECs were co-transfected with NF-κB promoter-luciferase vector and pRL reporter control plasmid. 24 h after transfection, ECs were treated with sulforaphane for 1 h before addition of TNF-α (2 ng/mL) for 6 h. Luciferase activity, normalized to pRL activity in the cell extracts, was determined. Values are mean ± SEM, n=3. (B) IκBα protein level was determined by Western blot analysis. β-actin was used as a loading control. Values are mean ± SE, n=3. (c) NF-κB p65 nuclear translocation was visualized using immunofluorescence staining. Representative sections are shown NF-κB p65, overlay and DAPI. *, p<0.05 vs. control; #, p<0.05 vs. TNF-α alone-treated cells.

Fig. 4

Fig. 4. Dietary sulforaphane reduced the monocyte binding to aortic endothelium (A–B), the secretion of serum chemokines (C–D) and adhesion molecules (E–G) in TNF-α treated mice

Values are mean ± SEM, n=8–10. *, p<0.05 vs. control; #, p<0.05 vs. TNF-α alone-treated mice. sICAM-1, soluble intercellular adhesion molecule-1; sVCAM-1, soluble vascular adhesion molecule-1; sE-Selectin, soluble E-Selectin; MCP-1/JE, mouse monocyte chemotactic protein 1/JE; TNF-α, Tumor necrosis factor-α; CXCL1/KC, Chemokine (C-X-C motif) ligand 1.

Fig 5

Fig 5. Immunohistochemical staining for adhesion molecule VCAM-1 and F4/80-positive monocytes-derived macrophages in aortic cross-sections

Representative photomicrographs of immunohistochemical staining for F4/80-positive monocytes-derived macrophages (A) and VCAM-1 (B). Quantitative analysis of VCAM-1 (C) and F4/80 (D). Arrows indicate typical positive-stained regions and original magnification is 40X. T, TNF-α; T +S, TNF-α + sulforaphane. Data are expressed as mean ± SEM, n=5, *, p<0.05 vs. control; #, p<0.05 vs. TNF-α alone-treated mice.

Fig. 6

Fig. 6. Dietary sulforaphane prevented TNF-α-induced aortic endothelial injury (A) and disruption of aortic elastin fiber (B) in aortic cross sections of TNF-α treated mice

Representative histological sections of aorta for hematoxylin and eosin staining (A) and Verhoeff-Van Gieson staining (B). Aortic sections were stained with hematoxylin and eosin and Verhoeff-Van Gieson staining as described in the Materials and Methods section.

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