Allium hookeri root extract exerts anti-inflammatory effects by nuclear factor-κB down-regulation in lipopolysaccharide-induced RAW264.7 cells

Abstract

Background: Allium hookeri (AH) is widely consumed as a vegetable and herbal medicine in southeastern Asia. AH has been reported antioxidant, antimicrobial, improvement of bone health and antidiabetic effects. In the present study, we investigated the inhibitory effect of a methanol extract of AH root (AHE) on inflammatory response in lipopolysaccharide (LPS)-induced RAW264.7 cells.

Methods: Initially, characterization of organic sulfur compounds in AHE was determined using high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). Cells were incubated with LPS and AHE for 24 h. The productions of nitric oxide (NO), reactive oxygen species (ROS), and inflammation-related cytokines were examined. Gene and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were assessed by polymerase chain reaction and Western blotting. Key factor, nuclear factor kappa B (NF-κB) was also determined.

Results: AHE contained organosulfur compounds such as alliin and S-allylcysteine by HPLC-ESI-MS. AHE significantly inhibited NO, ROS, and cytokines production in LPS-induced RAW264.7 cells. In addition, AHE treatment inhibited iNOS and COX-2 mRNA and protein levels, leading to a decrease in iNOS-derived NO level. Furthermore, NF-κB activation was, at least in part, suppressed by AHE treatment.

Conclusion: Our data suggest that AHE treatment inhibits the inflammation condition through suppression of iNOS and COX-2 expression via NF-κB down-regulation.

Keywords: Allium hookeri; COX-2; Inflammation; NF-κB; iNOS.

Fig. 1

Total ion chromatogram of alliin and SAC with quasi-molecular ion peaks

Fig. 2

Total ion chromatogram of alliin and SAC from the methanol extract of Allium hookeri root (AHE)

Fig. 3

AHE decreased NO and ROS production in LPS-induced RAW264.7 cells. Cells were incubated with AHE (100, 200 and 300 μg/ml), ADS (1 ng/ml) and LPS (2 μg/ml) for 24 h. a NO and b ROS assay was performed. Data were expressed as means ± SD of triplicate tests. *p < 0.05 indicate statistically significant differences compared to LPS-treatment alone

Fig. 4

AHE decreased IL6 and TNF-α production in LPS-induced RAW264.7 cells. Cells were incubated with AHE (100, 200 and 300 μg/ml), ADS (1 ng/ml) and LPS (2 μg/ml) for 24 h. a IL6 and b TNF-α levels were measured by ELISA kit. Data were expressed as means ± SD of triplicate tests. *p < 0.05 indicate statistically significant differences compared to LPS-treatment alone

Fig. 5

AHE inhibited the mRNA and protein level of iNOS and COX-2 in LPS-induced RAW264.7 cells. Cells were incubated with AHE (100, 200 and 300 μg/ml), ADS (1 ng/ml) and LPS (2 μg/ml) for 24 h. a RT-PCR and b western blot was performed. *p < 0.05 indicate statistically significant differences compared to LPS-treatment alone

Fig. 6

AHE suppressed p65 translocation and NF-κB and IκBα activation in LPS-induced RAW264.7 cells. Cells were incubated with AHE (100, 200 and 300 μg/ml), ADS (1 ng/ml) and LPS (2 μg/ml) for 24 h. a Cytosolic IκBα and b nuclear NF-κB were analyzed by western blot. *p < 0.05 indicate statistically significant differences compared to LPS-treatment alone