Quercetin-3- O- β-D-Glucuronide Suppresses Lipopolysaccharide-Induced JNK and ERK Phosphorylation in LPS-Challenged RAW264.7 Cells

Abstract

Quercetin, a flavonol, has been reported to exhibit a wide range of biological properties including anti-oxidant and anti-inflammatory activities. However, pharmacological properties of quercetin-3-O-β-D-glucuronide (QG), a glycoside derivative of quercetin, have not been extensively examined. The objective of this study is to elucidate the anti-inflammatory property and underlying mechanism of QG in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells in comparison with quercetin. QG significantly suppressed LPS-induced extracellular secretion of pro-inflammatory mediators such as nitric oxide (NO) and PGE₂, and pro-inflammatory protein expressions of iNOS and COX-2. To elucidate the underlying mechanism of the anti-inflammatory property of QG, involvement of MAPK signaling pathways was examined. QG significantly attenuated LPS-induced activation of JNK and ERK in concentration-dependent manners with a negligible effect on p38. In conclusion, the present study demonstrates QG exerts anti-inflammatory activity through the suppression of JNK and ERK signaling pathways in LPS-challenged RAW264.7 macrophage cells.

Keywords: ERK; JNK; Lipopolysaccharide; Quercetin; Quercetin-3-O-β-D-glucuronide; RAW264.7 cells.

Fig. 1.

Chemical structure of quercetin-3-O-β-D-glucuronide.

Fig. 2.

Effects of QG on LPS-induced extracellular release of NO (A) and PGE₂. (B) in RAW264.7 macrophage cells. RAW264.7 cells were pretreated with indicated concentrations of QG for 1 hr before incubation with LPS (200 ng/ml) for 24 hrs. The level of nitrite and PGE₂ were measured using Griess reagent and ELISA assay, respectively. QG significantly suppressed LPS-induced extracellular secretion of NO and PGE₂. (C) Effect of QG on the viability of RAW264.7 cells. No significant cell death was observed with QG concentrations used in the present study. The data were obtained from three independent experiments and expressed as mean ± SD (n=3). ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatment with LPS alone. QC and Q stand for quercetin-3-O-β-D-glucuronide and quercetin, respectively. Quercetin was used as a reference. Examination of quercetin at 100 μM concentration was not done due to the decreased cell viability.

Fig. 3.

Effects of QG on LPS-induced expression of iNOS and COX-2 in RAW264.7 cells. (A) The cell lysates were subjected to SDSPAGE, and then protein levels of iNOS and COX-2 were determined by Western blot analysis. QG significantly attenuated LPS-induced overexpression of iNOS and COX-2. Images are representative of three independent experiments that shows reproducible results. (B, C) Quantitative analyses of immunoblots of iNOS and COX-2. QG significantly suppressed LPS-induced iNOS and COX-2 expression. The data were obtained from three independent experiments and expressed as mean ± SD (n=3). ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatment with LPS alone. QC and Q stand for quercetin-3-O-β -D-glucuronide and quercetin, respectively. Quercetin was used as a reference. Examination of quercetin at 100 μM concentration was not done due to the decreased cell viability.

Fig. 4.

Effect of QG on LPS-induced extracellular secretion of TNFalpha in RAW264.7 macrophage cells. RAW264.7 cells were pretreated with indicated concentrations of QG for 1 hr, then incubated with LPS (200 ng/ml) for 24 hrs. The concentration of TNFalpha in collected cell culture media was measured by ELISA assay as described in the methods. Although quercetin significantly suppressed LPS-induced TNFalpha cytokine in a concentration-dependent manner, QG showed a negligible effect in LPS-stimulated TNFalpha production. The values are expressed as mean ± SD for three independent experiments. ^**p<0.01 indicate statistically significant differences from treatments with LPS alone. QC and Q stand for quercetin-3-O-β-D-glucuronide and quercetin, respectively. Quercetin was used as a reference. Examination of quercetin at 100 μM concentration was not done due to the decreased cell viability.

Fig. 5.

Effect of QG on LPS-induced activation of MAPK signaling pathway in RAW264.7 macrophage cells. (A) Representative immunoblots; (B, C, D) quantitative analyses of immunoblots. Cells were challenged with 200 ng/ml LPS in the absence or presence of QG. LPS-induced phosphorylation of JNK (B) and ERK (C) was significantly attenuated with QG treatment with a minor effect on p38 (D) phosphorylation. Images are representative of three independent experiments that shows reproducible results. The values are expressed as mean ± SD for three independent experiments. ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatments with LPS alone. QC and Q stand for quercetin-3-O-β-D-glucuronide and quercetin, respectively. Quercetin was used as a reference. Examination of quercetin at 100 μM concentration was not done due to the decreased cell viability.