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

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-γ (IFNγ)-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 + IFNγ activated macrophages in a concentration-dependent manner, with IC(50 )values of 81.4 μM, 34.5 μM, 38.6 μM and 10.4 μM respectively. For macrophages activated with LPS alone, the IC(50 )values were 40.0 μM, 16.6 μM, 10.4 μM and 2.8 μM, 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

Síntese de óxido nítrico dependente da nitrato redutase na resposta de defesa de Arabidopsis thaliana contra Pseudomonas syringae

Nitrate reductase (NR) was recently shown to play an important role during phytopathogenic interactions by providing substrates for the synthesis of nitric oxide (NO), a key signal for plant defense responses. In order to give additional support to this hypothesis, we compared NO-mediated defense responses of wild-type and NR double-deficient (nia1 nia2) Arabidopsis thaliana plants inoculated with the IBSBF-1115 (ibs) strain of Pseudomonas syringae pv. maculicola (Psm) and with genetically characterized avirulent (avr) or virulent (vir) strains of Psm. Inoculation of wild-type leaves with avr or ibs, but not vir, stimulated NO emission, as measured by the indicator 4,5-diaminofluorescein. NO emission induced by avr was higher than that induced by ibs. Wild-type plants displayed the hypersensitive response (HR) when infiltrated with the strains avr or ibs, although a stronger HR was induced by avr. The vir strain did not induce HR in wild-type plants, and leaves developed severe infection symptoms. nia1 nia2 plants did not show significantly increased NO emission nor did they develop HR to any of the analyzed strains of Psm, but displayed clorotic lesions and higher bacterial growth in their leaves. Overall, these results highlight the importance of NR-dependent NO synthesis for plant defenses against pathogen attack.A nitrato redutase (NR) possui importante papel na interação fitopatogênica por prover os substratos para a síntese do óxido nítrico (NO), um sinalizador essencial para a defesa vegetal. A fim de fornecer suporte adicional a essa hipótese, neste trabalho foram comparadas as respostas de defesa mediadas por NO de plantas de Arabidopsis thaliana selvagens e deficientes para a NR (nia1 nia2) quando inoculadas com a linhagem IBSBF-1115 (ibs) de Pseudomonas syringae pv. maculicola (Psm) e com as linhagens geneticamente caracterizadas como avirulenta (avr) ou virulenta (vir) de Psm. A inoculação de folhas selvagens com avr ou ibs, mas não vir, estimulou a emissão de NO, medida pelo indicador 4,5-diaminofluoresceína. A emissão de NO induzida por avr foi maior que aquela induzida por ibs. Plantas selvagens apresentaram resposta hipersensitiva (RH) quando infiltradas com avr ou ibs, mas uma RH mais forte foi induzida por avr. A linhagem vir não induziu RH nas plantas selvagens, e as folhas desenvolveram sintomas severos da infecção. Plantas nia1 nia2 não tiveram um aumento significativo da emissão de NO nem desenvolveram RH após inoculação com as linhagens analisadas de Psm, apresentando intenso crescimento bacteriano e clorose foliar. Em suma, esses resultados evidenciam a importância da síntese de NO dependente da NR na defesa vegetal ao ataque de patógenos.104107Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq