Effect of Ixeris dentata Nakai Extract on Nitric Oxide Production and Prostaglandin E2 Generation in LPS-stimulated RAW264.7 Cells

Abstract

Inflammation is the basis of severe acute and chronic diseases. This study investigated the anti-inflammatory property of a crude methanol extract (MeOH-ex) and the solvent fractions of Ixeris dentata Nakai (IDN) in LPS-stimulated murine macrophage-like cell line RAW264.7. Here, we showed that the ethyl acetate fraction (EtOAc-fr) had the most potent inhibitory activity on LPS-induced nitric oxide (NO) production among the tested samples, i.e., IDN MeOH-ex and the three different solvent fractions (chloroform, n-hexane, and EtOAc). We further found that the EtOAc-fr significantly inhibited LPS-induced prostaglandin PGE2 (PGE2) generation in RAW264.7 cells. Furthermore, the treatment with EtOAc-fr effectively suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). These results suggest that the EtOAc-fr of IDN MeOH-ex exhibits an anti-inflammatory activity in vitro by inhibiting LPS-induced NO production and PGE2 generation via suppression of iNOS and COX-2 expression.

Keywords: COX-2; Inflammation; Ixeris dentate Nakai; Nitric oxide; Prostaglandin E2; iNOS.

Figure 1. The effect of a crude methanol extract and solvent fractions of Ixeris dentata Nakai (IDN) on nitric oxide production in LPS-stimulated RAW264.7 cells. Cells were treated with a range of concentrations of (A) IDN crude methanol extract (MeOH-ex), (B) normal hexane fraction (n-hexane-fr), (C) chloroform fraction (CHCl₃-fr), and (D) ethyl acetate fraction (EtOAcfr) or vehicle (DMSO, white and black bars) for 24 h, as presented in the graphs. The production of nitric oxide (NO) in cells stimulated with 1 µg/mL of LPS was determined. Data are expressed as mean±SEM of three individual experiments with triplicate of each experiment. ^#p<0.05 vs. normal control, and ^*p<0.05 vs. vehicle-treated control.

Figure 2. The effect of a crude methanol extract and solvent fractions of Ixeris dentata Nakai (IDN) on prostaglandin E₂ generation in LPS-stimulated RAW264.7 cells. Cells were treated with a range of concentrations of (A) IDN crude methanol extract (MeOH-ex), (B) normal hexane fraction (n-hexane-fr), (C) chloroform fraction (CHCl₃-fr), and (D) ethyl acetate fraction (EtOAcfr) or vehicle (DMSO, white and black bars) for 24 h, as presented in the graphs. The generation of prostaglandin E₂ (PGE₂) in cells stimulated by 1 µg/mL of LPS was determined. Data are expressed as mean±SEM of three separated experiments with triplicate of each experiment. ^#p<0.05 vs. normal control, and ^*p<0.05 vs. vehicle-treated control.

Figure 3. The effect of ethyl acetate fraction (EtOAc-fr) of Ixeris dentata Nakai (IDN) crude methanol extract on the expression of iNOS and COX-2, and viability of RAW264.7 cells. Cells were treated with a range of concentrations of EtOAc-fr of MeOH-ex for 24 h. (A) The protein expression of iNOS and COX-2 were determined using western blot analysis. Blots were quantitated by Image J software. The graphs show the relative expression level of (B) iNOS and (C) COX-2 normalized to β-actin levels. Cell morphological changes (D) and viability (E) after treatment with EtOAc-fr or vehicle (DMSO) for 24 h were examined, and the results are expressed as percentages relative to the vehicle (DMSO)-treated control (white bar). Data are expressed as mean±SEM of three separated experiments with triplicate of each experiment. ^#p<0.05 vs. normal control, and ^*p<0.05 vs. vehicle-treated control.