Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 expression in RAW 264.7 macrophages through the inactivation of NF-kappaB

Abstract

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signalling pathway.

Figure 1

Chemical structure of ergolide isolated from Inula britannica.

Figure 2

Effects of ergolide on nitrite accumulation and iNOS activity in RAW 264.7 macrophages stimulated with LPS/IFN-γ. Ergolide was added to RAW 264.7 cells in 100 mm-culture dishes immediately prior to the addition of 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ, and the cells were incubated for 18 h. Nitrite accumulation (A) was determined by Griess reaction in culture medium, and iNOS activity (B) was measured by [³H]-L-citrulline formation with cell-free extracts. Data are expressed as mean±s.d. from three separate experiments in duplicate. Significant differences between control and ergolide-treated groups are indicated by *P<0.05.

Figure 3

Effects of ergolide on iNOS activity once expressed in RAW 264.7 macrophages pretreated with LPS/IFN-γ and on nNOS from rat brain. (A) Cell lysates were prepared from RAW 264.7 macrophages stimulated with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ for 18 h. iNOS activity in the lysates was measured using [³H]-L-citrulline formation with or without the addition of L-NMMA or ergolide. (B) nNOS from rat brain was prepared as described in ‘Methods.' nNOS activity was measured using L-[3H]-citrulline formation with or without the addition of L-NMMA or ergolide. Data are expressed as mean±s.d. from three separate experiments in duplicate. Significant differences between control and ergolide-treated groups are indicated by *P<0.05.

Figure 4

Effects of ergolide on the expression of iNOS. RAW 264.7 macrophages were stimulated with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ and incubated for 18 h in the presence or absence of ergolide. Western blot analysis (A) and Northern blot analysis (B) were carried out as described under ‘Methods'. Results show a representative blot and the mean±s.d. of the corresponding band intensities from three separate experiments. Significant differences between control and ergolide-treated groups are indicated by *P<0.05.

Figure 5

Effects of ergolide on COX-2 activity and COX-2 protein in RAW 264.7 macrophages treated with LPS/IFN-γ. (A) The cells were first treated with acetylsalicylic acid (500 μM) to inactivate COX followed by replacement of the medium with fresh medium together with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ. The cells were further incubated in the presence or absence of ergolide. Detailed procedures for cell culture and COX-2 assay are described under ‘Methods'. (B) To measure the level of COX-2 protein, the cells were stimulated with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ for 18 h in the presence or absence of ergolide and analysed by Western blot analysis as described under ‘Methods'. Results show a representative blot and the mean±s.d. of the corresponding band intensities from three separate experiments. Significant differences between control and ergolide-treated groups are indicated by *P<0.05.

Figure 6

Effects of ergolide on COX-2 activity once expressed by pretreatment with LPS/IFN-γ in RAW 264.7 macrophages. RAW 264.7 macrophages were stimulated with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ for 16 h. After washing and replacement with fresh medium, ergolide or indomethacin was added and equilibrated for 20 min. The cells were further incubated with 10 μM arachidonic acid for 13 min, and then PGE₂ content was determined in culture medium. Data are expressed as mean±s.d. from three separate experiments in duplicate. Significant differences between control and treated groups are indicated by *P<0.05.

Figure 7

Effects of ergolide on the activation of NF-κB in RAW 264.7 macrophages treated with LPS/IFN-γ. The cells were stimulated with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ for 1 h after 1 h-pretreatment of ergolide in the presence or absence of cysteine 1 mM. (A) Nuclear extracts were prepared as described under ‘Methods' and DNA-binding activity of NK-κB was determined by EMSA. (B) The nuclear extracts prepared from the experiments in (A) were analysed by Western blotting with an anti-p65 polyclonal antibody. Results show a representative blot and the mean±s.d. of the corresponding band intensities from three separate experiments. Significant differences between control and ergolide-treated groups are indicated by *P<0.05.

Figure 8

Effects of ergolide on the degradation of IκB-α in RAW 264.7 cells treated with LPS/IFN-γ. (A) The cells were stimulated with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ for indicated times. Cellular extracts were analysed by Western blotting with an anti-IκB-α polyclonal antibody. (B) After treating with ergolide for 1 h in the presence or absence of cysteine 1 mM, cells were stimulated with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ for 30 min. Cellular extracts were analysed by Western blotting with an anti-IκB-α polyclonal antibody. Results show a representative blot and the mean±s.d. of the corresponding band intensities from three separate experiments. Significant differences between control and ergolide-treated groups are indicated by *P<0.05.

Figure 9

Effects of ergolide on the DNA-binding of NF-κB in LPS/IFN-γ pretreated cells. RAW 264.7 macrophages were pretreated with 1 μg ml⁻¹ LPS/10 units ml⁻¹ IFN-γ for 30 min. Subsequently, 6 μM ergolide was added and nuclear extracts were prepared 20, 40, 60 and 90 min later, and DNA-binding activity of NK-κB was determined by EMSA. Results show a representative blot and the mean±s.d. of the corresponding band intensities from three separate experiments. Significant differences between LPS/IFN-γ-treatment group (60 min) and ergolide-treated groups are indicated by *P<0.05.