Suppression of nitric oxide production in mouse macrophages by soybean flavonoids accumulated in response to nitroprusside and fungal elicitation

Abstract

Background: The anti-inflammatory properties of some flavonoids have been attributed to their ability to inhibit the production of NO by activated macrophages. Soybean cotyledons accumulate certain flavonoids following elicitation with an extract of the fungal pathogen Diaporthe phaseolorum f. sp. meridionalis (Dpm). Sodium nitroprusside (SNP), a nitric oxide donor, can substitute for Dpm in inducing flavonoid production. In this study, we investigated the effect of flavonoid-containing diffusates obtained from Dpm- and SNP-elicited soybean cotyledons on NO production by lipopolysaccharide (LPS)- and LPS plus interferon-gamma (IFNgamma)-activated murine macrophages.

Results: Significant inhibition of NO production, measured as nitrite formation, was observed when macrophages were activated in the presence of soybean diffusates from Dpm- or SNP-elicited cotyledons. This inhibition was dependent on the duration of exposure to the elicitor. Daidzein, genistein, luteolin and apigenin, the main flavonoids present in diffusates of elicited cotyledons, suppressed the NO production by LPS + IFNgamma activated macrophages in a concentration-dependent manner, with IC50 values of 81.4 microM, 34.5 microM, 38.6 microM and 10.4 microM respectively. For macrophages activated with LPS alone, the IC50 values were 40.0 microM, 16.6 microM, 10.4 microM and 2.8 microM, respectively. Western blot analysis showed that iNOS expression was not affected by daidzein, was reduced by genistein, and was abolished by apigenin, luteolin and Dpm- and SNP-soybean diffusates at concentrations that significantly inhibited NO production by activated macrophages.

Conclusions: These results suggest that the suppressive effect of flavonoids on iNOS expression could account for the potent inhibitory effect of Dpm- and SNP-diffusates on NO production by activated macrophages. Since the physiological concentration of flavonoids in plants is normally low, the treatment of soybean tissues with SNP may provide a simple method for substantially increasing the concentration of metabolites that are beneficial for the treatment of chronic inflammatory diseases associated with NO production.

Figure 1

Concentration-dependent production of NO by macrophages stimulated with LPS and IFNγ. Macrophages were stimulated with LPS (A) or LPS (20 ng/mL) plus IFNγ (B) for 48 h, after which the cells were harvested and the NO released was measured as nitrite using the Griess reagent. The columns represent the means ± SE of three independent experiments, each done in quadruplicate. *P < 0.05 (by Student's t test).

Figure 2

Effect of diffusates from elicited cotyledons on NO production by stimulated macrophages. Cells were cultured for 48 h with LPS (20 ng/mL) (square symbols) or LPS (20 ng/mL) plus IFNγ (56 IU/mL) (circle symbols) in the presence of plant diffusates collected at different times after Dpm (A) or SNP (B) inoculation. The concentrations of NO were expressed as nitrite. The points are the means ± SE of three independent experiments, each done in quadruplicate.

Figure 3

Dose-dependent effect of the isoflavones daidzein (A) and genistein (B) and the flavones luteolin (C) and apigenin (D) on NO production by stimulated macrophages. The cells were cultured for 48 h with LPS (20 ng/mL) (square symbols) or LPS (20 ng/mL) plus IFNγ (56 IU/mL) (circle symbols) in the presence of the indicated concentrations of isoflavones and flavones. The amount of NO released into the culture supernatants was expressed as nitrite. The points are the means ± SE of three independent experiments, each done in quadruplicate.

Figure 4

Effect of soybean diffusates and flavonoids on iNOS expression in macrophages. Western blots were done using macrophage extracts obtained from cells activated with 20 ng/mL LPS plus 56 IU/mL IFNγ alone (Lane 1) or together with diffusates from soybean elicited with Dpm (Lane 2) or SNP (Lane 3) or with the flavonoids genistein (Lane 4), apigenin (Lane 5), luteolin (Lane 6) or daidzein (Lane 7). The arrow indicates a protein of ~130 kDa detected by the anti-mac NOS antibody.