Chrysin Attenuates VCAM-1 Expression and Monocyte Adhesion in Lipopolysaccharide-Stimulated Brain Endothelial Cells by Preventing NF-κB Signaling

Abstract

Adhesion of leukocytes to endothelial cells plays an important role in neuroinflammation. Therefore, suppression of the expression of adhesion molecules in brain endothelial cells may inhibit neuroinflammation. Chrysin (5,7-dihydroxyflavone) is a flavonoid component of propolis, blue passion flowers, and fruits. In the present study, we examined the effects of chrysin on lipopolysaccharide (LPS)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in mouse cerebral vascular endothelial (bEnd.3) cells. In bEnd.3 cells, LPS increased mRNA expression of VCAM-1 in a time-dependent manner, and chrysin significantly decreased LPS-induced mRNA expression of VCAM-1. Chrysin also reduced VCAM-1 protein expression in a concentration-dependent manner. Furthermore, chrysin blocked adhesion of monocytes to bEnd.3 cells exposed to LPS. Nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase, which are all activated by LPS, were significantly inhibited by chrysin. These results indicate that chrysin inhibits the expression of VCAM-1 in brain endothelial cells by inhibiting NF-κB translocation and MAPK signaling, resulting in the attenuation of leukocyte adhesion to endothelial cells. The anti-inflammatory effects of chrysin suggest a possible therapeutic application of this agent to neurodegenerative diseases, such as multiple sclerosis, septic encephalopathy, and allergic encephalomyelitis.

Keywords: blood-brain barrier; brain endothelial cell; chrysin; lipopolysaccharide; monocyte adhesion; neuroinflammation; vascular cell adhesion molecule-1 (VCAM-1).

Figure 1

The effect of chrysin on LPS-induced adhesion molecule mRNA and protein expression in bEnd.3 cells. (a) RT-PCR of adhesion molecules after 10 μg/mL LPS treatment. bEnd.3 cells were treated with 10 μg/mL LPS for 4, 8, or 12 h; (b) RT-PCR of adhesion molecules after LPS and chrysin treatment. Cells were pretreated with chrysin (10–100 μM) for 30 min and co-stimulated with 10 μg/mL LPS for 8 h; (c) the levels of ICAM-1, VCAM-1, and E-selectin mRNA were evaluated by RT-PCR and quantified by densitometry; (d) Western blotting of adhesion molecules after 10 μg/mL LPS treatment. bEnd.3 cells were treated with 10 μg/mL LPS for 12 or 24 h; (e) Western blotting of adhesion molecules after LPS and chrysin treatment. Cell were pretreated with chrysin (10–100 μM) for 30 min and co-stimulated with 10 μg/mL LPS for 24 h; and (f) the levels of ICAM-1, VCAM-1, and E-selectin protein were evaluated by Western blotting and quantified by densitometry. β-actin was used for normalization. Mean ± SEM, * p < 0.05 vs. control and # p < 0.05 vs. 10 μg/mL LPS alone, n = 3.

Figure 2

The effect of chrysin on LPS-induced VCAM-1 protein expression and U937 cells attachment to bEnd.3 cells. (a) Immunocytochemistry after LPS and chrysin treatment. Inset: bEnd.3 cells were exposed to 10 μg/mL LPS for the indicated times (0–24 h). Cells were pretreated with chrysin (10–100 μM) for 30 min and co-stimulated with 10 μg/mL LPS for 24 h. VCAM-1 (green) was detected by immunofluorescence using anti-VCAM-1 antibody. Nuclei were visualized by Hoechst staining (blue). Representative images and quantitative results are shown in the top and bottom panels, respectively. Scale bar, 20 μm; (b) after treatment with 10 μg/mL LPS for 24 h in the presence or absence of chrysin, bEnd.3 cells were incubated with U937 monocytes (green) for 2 h. Representative images and quantitative results are shown in the top and bottom panels, respectively. Scale bar: 40 μm; and (c) the effect of chrysin alone on cell viability in bEnd.3 and U937 cells. After the treatment with various concentrations of chrysin for 24 h, the cytotoxicity was determined by MTT assay. Mean ± SEM, * p < 0.05 vs. control and # p < 0.05 vs. vehicle, n = 3.

Figure 3

The effect of chrysin on LPS-induced translocation of NF-κB in bEnd.3 cells. (a) Cells were pretreated with SN50 for 30 min and co-stimulated with LPS (10 μg/mL) for 8 h. The levels of VCAM-1 mRNA were evaluated by RT-PCR and quantified by densitometry; (b) cells were pretreated with chrysin (10–100 μM) for 30 min and then stimulated with LPS (10 μg/mL) for 8 h. Whole cell and nuclear extracts were prepared and analyzed by Western blot analysis with anti-IκBα antibody. The levels of protein were normalized with β-actin; (c) cells were pretreated with chrysin (10–100 μM) for 30 min and then co-stimulated with LPS (10 μg/mL) for 8 h. NF-κB p65 (green) was detected by immunofluorescence using anti-p65 antibody. Nuclei were visualized by Hoechst staining (blue). Scale bar: 20 μm; and (d) Western blots of p65 subunit of NF-κB in nucleus and cytosolic fraction of bEnd.3 cells treated with LPS (10 μg/mL) and chrysin for 8 h. Actin was used as internal standard of cytosol fraction and histone 1 (H1) of the nuclear fraction. * p < 0.05 vs. control without LPS and # p < 0.05 vs. vehicle without SN50, n = 3.

Figure 4

Effect of MAPK on LPS-induced VCAM-1 expression and of chrysin on p38 and JNK activation in bEnd.3 cells. (a) Cells were pretreated with SB202190 (SB, 10 μM), SP600125 (SP, 10 μM), and PD98059 (PD, 10 μM) for 30 min and then co-stimulated with LPS (10 μM) for 8 h. The levels of VCAM-1 mRNA were evaluated by RT-PCR and quantified by densitometry; and (b,c) cells were pretreated with chrysin (10–100 μM) for 30 min and then co-stimulated with LPS (10 μg/mL) for 1 h. Whole cell extracts were prepared and analyzed by Western blot with anti-phospho p38, anti-phospho JNK, p38 MAPK antibody, or JNK antibody. * p < 0.05 vs. control and # p < 0.05 vs. vehicle, n = 3.

Figure 5

The therapeutic effect of chrysin on LPS-induced adhesion molecule mRNA expression in bEnd.3 cells. (a) Cells were co-treated with chrysin (10–100 μM) and 10 μg/mL LPS for 8 h; (b) the levels of ICAM-1, VCAM-1, and E-selectin mRNA were evaluated by RT-PCR and quantified by densitometry; (c) cell were post-treated with chrysin (10–100 μM) at 4 h after administration of 10 μg/mL LPS; and (d) the levels of ICAM-1, VCAM-1, and E-selectin mRNA were evaluated by RT-PCR and quantified by densitometry. β-actin was used for normalization. Mean ± SEM, * p < 0.05 vs. control and # p < 0.05 vs. 10 μg/mL LPS alone, n = 3.