Inhibition of LPS-induced inducible NO synthase and cyclooxygenase-2 protein by EAAT
The effect of EAAT on inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression was studied using immunoblotting to investigate whether the inhibition of NO production was due to decreased iNOS and COX-2 protein levels. The results demonstrated that incubation with EAAT (125 μg/mL, 250 μg/mL, and 500 μg/mL) in the presence of LPS for 24 hours inhibited iNOS and COX-2 protein expression in mouse macrophage RAW264.7 Go 6976 in a dose-dependent manner (Figure 2A). The detection of β-actin was also performed in the same blot as an internal control.
Inhibition of (A) iNOS, COX-2 and (B) NF-κB, and (C) inhibition of ...
Inhibition of (A) iNOS, COX-2 and (B) NF-κB, and (C) inhibition of phosphorylation of mitogen-activated protein kinases (MAPKs) expressions by EAAT in LPS-stimulated RAW264.7 cells. Cells were incubated for 24 hours with 100 ng/mL LPS in the absence or the presence of EAAT (0 μg/mL, 125 μg/mL, 250 μg/mL, and 500 μg/mL). EAAT was added 1 hour before incubation with LPS. Lysed cells were then prepared and subjected to Western blotting using an antibody specific for iNOS, COX-2, IκB-α, p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-JNK, and JNK. β-actin was used as an internal control. A representative Western blot from two separate experiments is shown. COX-2 = cyclooxygenase-2; EAAT = ethyl acetate fraction of Acanthopanax trifoliatus; ERK = extracellular signal-regulated kinase; iNOS = inducible nitric oxide synthase; JNK = c-Jun N-terminal kinase; LPS = lipopolysaccharide.