ABSTRACT
Aim To investigate the anti-inflammatory effects of Aesculetin from Viola tianshanica Maxim in LPS-stimulated RAW 264.7 cells and the underlying mechanism.Methods RAW 264.7 cells were divided into control group, model group( LPS), 0.16, 0.8, 4, 20 μmol·L-1 AESN groups( different concentrations of AESN + LPS)and positive control group(10 μmol·L-1 Indomethacin+LPS).LPS(1 mg·L-1)was used to stimulate RAW 264.7 cells for 24 h to establish inflammatory model.MTS assay was used to detemine cytotoxicity of Aesculetin in RAW 264.7 cells.Griess method was used to detect NO secretion in LPS-stimulated RAW 264.7 cells.ELISA was applied to determine the contents of TNF-α, IL-6 and IL-1β in cell culture supernatant.qRT-PCR was employed to detect the mRNA expression levels of TNF-α, IL-6, IL-1β and iNOS.Immunofluorescence assay was used to evaluated the protein expressions of iNOS, p-NF-κB p65, IκBα, p-p38 and p-ERK1/2.Enzyme assay was used to detect the inhibition activity of Aesculetin on cyclooxygenase 1/2(COX 1/2).Results Aesculetin significantly inhibited the secretion of inflammatory mediator NO, mRNA and protein expression of iNOS in LPS-induced RAW 264.7 at 0.16, 0.8, 4 and 20 μmol·L-1.The contents of TNF-α, IL-6 and IL-1β in supernatant significantly decreased, and the mRNA expression levels of TNF-α, IL-6 and IL-1β were also reduced by Aesculetin.Aesculetin also obviously inhibited the protein degradation of IκBα and inhibited the nuclear translocation of p-NF-κB p65, p-p38, p-ERK1/2.In addition, Aesculetin had significant inhibitory activities on COX-1 and COX-2, and the IC50 was 28.1 μmol·L-1, 2.3 μmol·L-1, respectively.Conclusions AESN has good anti-inflammatory effect, and its mechanism is closely related to the inhibition of NF-κB and MAPK signaling pathways