Anti-inflammatory evaluation of the methanolic extract of Taraxacum officinale in LPS-stimulated human umbilical vein endothelial cells

Abstract

Background: Atherosclerosis is a chronic vascular inflammatory disease. Since even low-level endotoxemia constitutes a powerful and independent risk factor for the development of atherosclerosis, it is important to find therapies directed against the vascular effects of endotoxin to prevent atherosclerosis. Taraxacum officinale (TO) is used for medicinal purposes because of its choleretic, diuretic, antioxidative, anti-inflammatory, and anti-carcinogenic properties, but its anti-inflammatory effect on endothelial cells has not been established.

Methods: We evaluated the anti-inflammatory activity of TO filtered methanol extracts in LPS-stimulated human umbilical vein endothelial cells (HUVECs) by monocyte adhesion and western blot assays. HUVECs were pretreated with 100 μg/ml TO for 1 h and then incubated with 1 μg/ml LPS for 24 h. The mRNA and protein expression levels of the targets (pro-inflammatory cytokines and adhesion molecules) were analyzed by real-time PCR and western blot assays. We also preformed HPLC analysis to identify the components of the TO methanol extract.

Results: The TO filtered methanol extracts dramatically inhibited LPS-induced endothelial cell-monocyte interactions by reducing vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and pro-inflammatory cytokine expression. TO suppressed the LPS-induced nuclear translocation of NF-κB, whereas it did not affect MAPK activation.

Conclusions: Our findings demonstrated that methanol extracts of TO could attenuate LPS-induced endothelial cell activation by inhibiting the NF-κB pathway. These results indicate the potential clinical benefits and applications of TO for the prevention of vascular inflammation and atherosclerosis.

Keywords: Anti-inflammation; Endothelial cell; Endotoxin; NF-κb; Taraxacum officinale.

Fig. 1

Effect of the Taraxacum officinale (TO) methanol extract on the viability of HUVECs. Cells were treated with different concentrations of the TO extract for 24 h, and viability was determined by the MTS assay. The results of independent experiments were averaged as the percentage cell viability compared with untreated control. Results shown are the mean ± SE of triple determinations

Fig. 2

Effect of the Taraxacum officinale (TO) methanol extract on LPS-induced monocyte adhesion to HUVECs. HUVECs were pretreated with TO extract (100 μg/ml) for 1 h, and then stimulated with 1 μg/ml LPS for 24 h. Adhesion of fluorescent THP-1 monocytes was photographed by fluorescence microscopy (a) and analyzed (b). Scale bars: 1000 μm. Results shown are the mean ± SE of 3 independent experiments. **p < 0.01 vs LPS only treatment

Fig. 3

Effect of the Taraxacum officinale (TO) methanol extract on LPS-induced VCAM-1 and MCP-1 expression. HUVECs were pretreated with TO extract (100 μg/ml) for 1 h and then incubated with 1 μg/ml LPS for 24 h. a Total cell lysate was prepared, the proteins were separated by SDS-PAGE, and the VCAM-1 levels were assessed by western blot analysis. b The mRNA levels were normalized to that of a housekeeping gene (18S rRNA) as well as the vehicle-only-treated control, and the 2^-ΔΔCt for each mRNA is reported. Results shown are the mean ± SE of 3 independent experiments. **p < 0.01, ***p < 0.001 vs LPS only treatment

Fig. 4

Effect of the Taraxacum officinale (TO) methanol extract on LPS-induced pro-inflammatory cytokine expression. The pro-inflammatory cytokine mRNA levels were normalized to that of a housekeeping gene (18S rRNA) as well as the vehicle-only-treated control, and the 2^-ΔΔCt for each mRNA is reported. Results shown are the mean ± SE of 3 independent experiments (a, b, c). ***p < 0.001 vs LPS only treatment

Fig. 5

Effect of the Taraxacum officinale (TO) methanol extract on the LPS-induced nuclear translocation of NF-κB p65 in HUVECs. HUVECs were pretreated with TO extract (100 μg/ml) for 1 h and then stimulated with LPS (1 μg/ml) for 1 h. a Representative images of immunofluorescence staining showing NF-κB p65 (red) and cell nuclei stained with Hoechst 33,258 (blue). Scale bars: 200 μm. b Quantitation of NF-κB p65 nuclear translocation in the indicated groups. c IκBα phosphorylation assessed by western blot analysis. Results are shown as the mean ± SE (n = 3–4). *p < 0.05, **p < 0.01 vs LPS only treatment

Fig. 6

HPLC chromatogram of the Taraxacum officinale methanol extract. 1. Protocatechuic acid; 2. Chlorogenic acid; 3. Caffeic acid; 4. p-Coumaric acid; and 5. Ferulic acid. The other peaks were not identified owing to a lack of authentic compounds to compare with