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Dietary blue pigments derived from genipin, attenuate inflammation by inhibiting LPS-induced iNOS and COX-2 expression via the NF-κB inactivation.

Wang QS, Xiang Y, Cui YL, Lin KM, Zhang XF - PLoS ONE (2012)

Bottom Line: The anti-inflammatory effect of blue pigments in vivo was studied in carrageenan-induced paw edema and LPS-injecting ICR mice.Finally, blue pigments significantly inhibited paw swelling and reduced plasma TNF-α and IL-6 production in vivo.These results suggest that the anti-inflammatory properties of blue pigments might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1β, and TNF-α expression through the down-regulation of NF-κB activation, which will provide strong scientific evidence for the edible blue pigments to be developed as a new health-enhancing nutritional food for the prevention and treatment of inflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, People's Republic of China.

ABSTRACT

Background and purpose: The edible blue pigments produced by gardenia fruits have been used as value-added colorants for foods in East Asia for 20 years. However, the biological activity of the blue pigments derived from genipin has not been reported.

Methodology/principal findings: The anti-inflammatory effect of blue pigments was studied in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage in vitro. The secretions of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were inhibited in concentration-dependent manner by blue pigments. Real-time reverse-transcription polymerase chain reaction (Real-time RT-PCR) analyses demonstrated that the mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-α) was inhibited, moreover, ELISA results showed that the productions of IL-6 and TNF-α were inhibited. Cell-based ELISA revealed the COX-2 protein expression was inhibited. The proteome profiler array showed that 12 cytokines and chemokines involved in the inflammatory process were down-regulated by blue pigments. Blue pigments inhibited the nuclear transcription factor kappa-B (NF-κB) activation induced by LPS, and this was associated with decreasing the DNA-binding activity of p65 and p50. Furthermore, blue pigments suppressed the degradation of inhibitor of κB (IκB) α, Inhibitor of NF-κB Kinase (IKK) α, IKK-β, and phosphorylation of IκB-α. The anti-inflammatory effect of blue pigments in vivo was studied in carrageenan-induced paw edema and LPS-injecting ICR mice. Finally, blue pigments significantly inhibited paw swelling and reduced plasma TNF-α and IL-6 production in vivo.

Conclusions and implications: These results suggest that the anti-inflammatory properties of blue pigments might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1β, and TNF-α expression through the down-regulation of NF-κB activation, which will provide strong scientific evidence for the edible blue pigments to be developed as a new health-enhancing nutritional food for the prevention and treatment of inflammatory diseases.

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Effects of blue pigments on the degradation of IκB-α, IKK-α, IKK-β and phosphorylation of IκB-α.RAW 264.7 cells were pre-incubated with indicated concentrations of blue pigments for 2 h and stimulated with LPS (0.2 µg/mL) for 30 min. Total cellular proteins were then prepared and Western blotted for IκB-α, IKK-α, IKK-β and p-IκB-α using specific IκB-α, IKK-α, IKK-β and p-IκB-α antibodies. The β-actin protein was used as internal controls. NF-κB inhibitor of BAY 11-7082 (10 µM) was used as positive control.
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pone-0034122-g007: Effects of blue pigments on the degradation of IκB-α, IKK-α, IKK-β and phosphorylation of IκB-α.RAW 264.7 cells were pre-incubated with indicated concentrations of blue pigments for 2 h and stimulated with LPS (0.2 µg/mL) for 30 min. Total cellular proteins were then prepared and Western blotted for IκB-α, IKK-α, IKK-β and p-IκB-α using specific IκB-α, IKK-α, IKK-β and p-IκB-α antibodies. The β-actin protein was used as internal controls. NF-κB inhibitor of BAY 11-7082 (10 µM) was used as positive control.

Mentions: As the activation of NF-κB is critically required for the activations of iNOS, COX-2, TNF-α, PEG2 and IL-6 by LPS, we determined the DNA-binding activity of NF-κB subunits p50 and p65 using the Universal EZ-TFA transcription factor colorimetric assay, which instead of the DNA-binding principle of the electrophoretic mobility shift assay with the 96-well format of an enzymelinked immunosorbent assay. Accordingly, a NF-κB DNA binding assay was carried out using nuclear extracts from RAW 264.7 cells stimulated with LPS in the presence or absence of blue pigments. Treatment of RAW 264.7 cells with LPS (0.2 µg/mL) was found to increase the expression of NF-κB subunits p50 and p65, however, the expressions of p50 (Figure 6A) and p65 (Figure 6B) pretreated these cells with blue pigments prior to LPS were reduced in a concentration-dependent manner when compared with the single LPS stimulation group (P<0.01). Taken together, the above findings showed that blue pigments suppressed iNOS, COX-2, TNF-α, PEG2 and IL-6 expression at least in part via an NF-κB-dependent mechanism. We also explored whether blue pigments inhibited the LPS-stimulated degradation of IκB-α in RAW 264.7 cells by Western blotting with anti-IκB-α antibody. Figure 7 shows that LPS-induced IκB-α degradation was significantly blocked by blue pigments pretreatment. Furthermore, to determine whether this IκB-α degradation was related to IκB-α phosphorylation, we examined the effect of blue pigments on the LPS-induced p-IκB-α by Western blotting, and found that blue pigments also significantly reduced LPS-induced IκB-α phosphorylation. The β-actin protein was used as internal controls. Since IKK-α and -β are upstream kinases of IκB in the NF-κB signal pathway [17], we examined the effects of blue pigments on LPS induced IKK-α, -β activation by immunoblotting using IKK-α, -β antibodies. Blue pigments (100 µM) inhibited the expression of IKK-α and IKK-β. The β-actin protein was used as internal control. (The western blot bands were quantified by densitometry and the data are presented in Figure S2).


Dietary blue pigments derived from genipin, attenuate inflammation by inhibiting LPS-induced iNOS and COX-2 expression via the NF-κB inactivation.

Wang QS, Xiang Y, Cui YL, Lin KM, Zhang XF - PLoS ONE (2012)

Effects of blue pigments on the degradation of IκB-α, IKK-α, IKK-β and phosphorylation of IκB-α.RAW 264.7 cells were pre-incubated with indicated concentrations of blue pigments for 2 h and stimulated with LPS (0.2 µg/mL) for 30 min. Total cellular proteins were then prepared and Western blotted for IκB-α, IKK-α, IKK-β and p-IκB-α using specific IκB-α, IKK-α, IKK-β and p-IκB-α antibodies. The β-actin protein was used as internal controls. NF-κB inhibitor of BAY 11-7082 (10 µM) was used as positive control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316609&req=5

pone-0034122-g007: Effects of blue pigments on the degradation of IκB-α, IKK-α, IKK-β and phosphorylation of IκB-α.RAW 264.7 cells were pre-incubated with indicated concentrations of blue pigments for 2 h and stimulated with LPS (0.2 µg/mL) for 30 min. Total cellular proteins were then prepared and Western blotted for IκB-α, IKK-α, IKK-β and p-IκB-α using specific IκB-α, IKK-α, IKK-β and p-IκB-α antibodies. The β-actin protein was used as internal controls. NF-κB inhibitor of BAY 11-7082 (10 µM) was used as positive control.
Mentions: As the activation of NF-κB is critically required for the activations of iNOS, COX-2, TNF-α, PEG2 and IL-6 by LPS, we determined the DNA-binding activity of NF-κB subunits p50 and p65 using the Universal EZ-TFA transcription factor colorimetric assay, which instead of the DNA-binding principle of the electrophoretic mobility shift assay with the 96-well format of an enzymelinked immunosorbent assay. Accordingly, a NF-κB DNA binding assay was carried out using nuclear extracts from RAW 264.7 cells stimulated with LPS in the presence or absence of blue pigments. Treatment of RAW 264.7 cells with LPS (0.2 µg/mL) was found to increase the expression of NF-κB subunits p50 and p65, however, the expressions of p50 (Figure 6A) and p65 (Figure 6B) pretreated these cells with blue pigments prior to LPS were reduced in a concentration-dependent manner when compared with the single LPS stimulation group (P<0.01). Taken together, the above findings showed that blue pigments suppressed iNOS, COX-2, TNF-α, PEG2 and IL-6 expression at least in part via an NF-κB-dependent mechanism. We also explored whether blue pigments inhibited the LPS-stimulated degradation of IκB-α in RAW 264.7 cells by Western blotting with anti-IκB-α antibody. Figure 7 shows that LPS-induced IκB-α degradation was significantly blocked by blue pigments pretreatment. Furthermore, to determine whether this IκB-α degradation was related to IκB-α phosphorylation, we examined the effect of blue pigments on the LPS-induced p-IκB-α by Western blotting, and found that blue pigments also significantly reduced LPS-induced IκB-α phosphorylation. The β-actin protein was used as internal controls. Since IKK-α and -β are upstream kinases of IκB in the NF-κB signal pathway [17], we examined the effects of blue pigments on LPS induced IKK-α, -β activation by immunoblotting using IKK-α, -β antibodies. Blue pigments (100 µM) inhibited the expression of IKK-α and IKK-β. The β-actin protein was used as internal control. (The western blot bands were quantified by densitometry and the data are presented in Figure S2).

Bottom Line: The anti-inflammatory effect of blue pigments in vivo was studied in carrageenan-induced paw edema and LPS-injecting ICR mice.Finally, blue pigments significantly inhibited paw swelling and reduced plasma TNF-α and IL-6 production in vivo.These results suggest that the anti-inflammatory properties of blue pigments might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1β, and TNF-α expression through the down-regulation of NF-κB activation, which will provide strong scientific evidence for the edible blue pigments to be developed as a new health-enhancing nutritional food for the prevention and treatment of inflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, People's Republic of China.

ABSTRACT

Background and purpose: The edible blue pigments produced by gardenia fruits have been used as value-added colorants for foods in East Asia for 20 years. However, the biological activity of the blue pigments derived from genipin has not been reported.

Methodology/principal findings: The anti-inflammatory effect of blue pigments was studied in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage in vitro. The secretions of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were inhibited in concentration-dependent manner by blue pigments. Real-time reverse-transcription polymerase chain reaction (Real-time RT-PCR) analyses demonstrated that the mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-α) was inhibited, moreover, ELISA results showed that the productions of IL-6 and TNF-α were inhibited. Cell-based ELISA revealed the COX-2 protein expression was inhibited. The proteome profiler array showed that 12 cytokines and chemokines involved in the inflammatory process were down-regulated by blue pigments. Blue pigments inhibited the nuclear transcription factor kappa-B (NF-κB) activation induced by LPS, and this was associated with decreasing the DNA-binding activity of p65 and p50. Furthermore, blue pigments suppressed the degradation of inhibitor of κB (IκB) α, Inhibitor of NF-κB Kinase (IKK) α, IKK-β, and phosphorylation of IκB-α. The anti-inflammatory effect of blue pigments in vivo was studied in carrageenan-induced paw edema and LPS-injecting ICR mice. Finally, blue pigments significantly inhibited paw swelling and reduced plasma TNF-α and IL-6 production in vivo.

Conclusions and implications: These results suggest that the anti-inflammatory properties of blue pigments might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1β, and TNF-α expression through the down-regulation of NF-κB activation, which will provide strong scientific evidence for the edible blue pigments to be developed as a new health-enhancing nutritional food for the prevention and treatment of inflammatory diseases.

Show MeSH
Related in: MedlinePlus