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Anti-inflammatory effects of adenosine N1-oxide.

Kohno K, Ohashi E, Sano O, Kusano H, Kunikata T, Arai N, Hanaya T, Kawata T, Nishimoto T, Fukuda S - J Inflamm (Lond) (2015)

Bottom Line: Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly.We found that it is refractory to adenosine deaminase-mediated conversion to inosine.Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock.

View Article: PubMed Central - PubMed

Affiliation: Core Technology Division, Research and Development Center, Hayashibara Co., Ltd, Okayama, Japan.

ABSTRACT

Background: Adenosine is a potent endogenous anti-inflammatory and immunoregulatory molecule. Despite its promise, adenosine's extremely short half-life in blood limits its clinical application. Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly. ANO is an oxidized product of adenosine at the N1 position of the adenine base moiety. We found that it is refractory to adenosine deaminase-mediated conversion to inosine. We further examined the anti-inflammatory activities of ANO in vitro and in vivo.

Methods: The effect of ANO on pro-inflammatory cytokine secretion was examined in mouse peritoneal macrophages and the human monocytic cell line THP-1, and compared with that of adenosine, synthetic adenosine receptor (AR)-selective agonists and dipotassium glycyrrhizate (GK2). The anti-inflammatory activity of ANO in vivo was examined in an LPS-induced endotoxin shock model in mice.

Results: ANO inhibited secretion of inflammatory mediators at much lower concentrations than adenosine and GK2 when used with peritoneal macrophages and THP-1 cells that were stimulated by LPS plus IFN-γ. The potent anti-inflammatory activity of ANO could not be solely accounted for by its refractoriness to adenosine deaminase. ANO was superior to the synthetic A1 AR-selective agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), A2A AR-selective agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamideadenosine hydrochloride (CGS21680), and A3 AR-selective agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), in suppressing the secretion of a broad spectrum of pro-inflammatory cytokines by peritoneal macrophages. The capacities of ANO to inhibit pro-inflammatory cytokine production by THP-1 cells were comparable with those of CCPA and IB-MECA. Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock. A significant increase in survival rate was also observed by oral administration of ANO. Mechanistic analysis suggested that the up-regulation of the anti-inflammatory transcription factor c-Fos was, at least in part, involved in the ANO-induced suppression of pro-inflammatory cytokine secretion.

Conclusions: Our data suggest that ANO, a naturally occurring molecule that is structurally close to adenosine but is functionally more potent, presents potential strategies for the treatment of inflammatory disorders.

No MeSH data available.


Related in: MedlinePlus

Effect of H-89 or Wartmannin on ANO-induced inhibition of TNF-α production. Peritoneal macrophages were incubated with various concentrations of H-89 (A) or Wartmannin (B) for 30 min before stimulation with LPS (2 μg/mL) in the presence of absence of ANO (1 μM) or adenosine (2.5 μM). After 24 h, levels of TNF-α in the culture supernatants were determined by ELISA. Values represent the means ± S.D. of quadruplicate cultures. Results are expressed as the percentage of TNF-α released from macrophages in response to LPS and are representative of two separate experiments with similar results. ƒƒ, p < 0.01, significantly different when compared with LPS stimulation in the absence of adenosine or ANO. *p < 0.05; **p < 0.01, significantly different when compared with vehicle control culture in the presence of adenosine. #, p < 0.05; ##, p < 0.01, significantly different when compared with vehicle control culture in the presence of ANO.
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Fig9: Effect of H-89 or Wartmannin on ANO-induced inhibition of TNF-α production. Peritoneal macrophages were incubated with various concentrations of H-89 (A) or Wartmannin (B) for 30 min before stimulation with LPS (2 μg/mL) in the presence of absence of ANO (1 μM) or adenosine (2.5 μM). After 24 h, levels of TNF-α in the culture supernatants were determined by ELISA. Values represent the means ± S.D. of quadruplicate cultures. Results are expressed as the percentage of TNF-α released from macrophages in response to LPS and are representative of two separate experiments with similar results. ƒƒ, p < 0.01, significantly different when compared with LPS stimulation in the absence of adenosine or ANO. *p < 0.05; **p < 0.01, significantly different when compared with vehicle control culture in the presence of adenosine. #, p < 0.05; ##, p < 0.01, significantly different when compared with vehicle control culture in the presence of ANO.

Mentions: We found that ANO inhibited TNF-α secretion by LPS/huIFN-γ-stimulated THP-1 cells partially through A2A AR (Figure 7). Classical signaling through A2A AR depends on its coupling to the heterotrimeric Gs protein and stimulation of adenylyl cyclase, resulting in elevation of intracellular levels of cyclic AMP (cAMP) [6-8]. We observed that ANO increased cAMP levels in both peritoneal macrophages and a murine macrophage cell line, RAW264.7 cells (data not shown). Elevation of cAMP in RAW264.7 cells inhibited LPS-induced TNF-α production via the protein kinase A (PKA)-dependent pathway [26]. Therefore, we examined the effect of a PKA inhibitor (H-89) on adenosine- or ANO-induced inhibition of TNF-α secretion by LPS-stimulated peritoneal macrophages. In this experiment, to exclude extraneous signaling events exerted by muIFN-γ, macrophages were stimulated with LPS only. As shown in Figure 9A, adenosine-induced inhibition of TNF-α secretion was recovered in a dose-dependent manner by pre-incubation with H-89, and complete recovery was observed with 5 μM H-89. However, H-89 had no effect on the ANO-induced inhibition of TNF-α secretion.Figure 9


Anti-inflammatory effects of adenosine N1-oxide.

Kohno K, Ohashi E, Sano O, Kusano H, Kunikata T, Arai N, Hanaya T, Kawata T, Nishimoto T, Fukuda S - J Inflamm (Lond) (2015)

Effect of H-89 or Wartmannin on ANO-induced inhibition of TNF-α production. Peritoneal macrophages were incubated with various concentrations of H-89 (A) or Wartmannin (B) for 30 min before stimulation with LPS (2 μg/mL) in the presence of absence of ANO (1 μM) or adenosine (2.5 μM). After 24 h, levels of TNF-α in the culture supernatants were determined by ELISA. Values represent the means ± S.D. of quadruplicate cultures. Results are expressed as the percentage of TNF-α released from macrophages in response to LPS and are representative of two separate experiments with similar results. ƒƒ, p < 0.01, significantly different when compared with LPS stimulation in the absence of adenosine or ANO. *p < 0.05; **p < 0.01, significantly different when compared with vehicle control culture in the presence of adenosine. #, p < 0.05; ##, p < 0.01, significantly different when compared with vehicle control culture in the presence of ANO.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig9: Effect of H-89 or Wartmannin on ANO-induced inhibition of TNF-α production. Peritoneal macrophages were incubated with various concentrations of H-89 (A) or Wartmannin (B) for 30 min before stimulation with LPS (2 μg/mL) in the presence of absence of ANO (1 μM) or adenosine (2.5 μM). After 24 h, levels of TNF-α in the culture supernatants were determined by ELISA. Values represent the means ± S.D. of quadruplicate cultures. Results are expressed as the percentage of TNF-α released from macrophages in response to LPS and are representative of two separate experiments with similar results. ƒƒ, p < 0.01, significantly different when compared with LPS stimulation in the absence of adenosine or ANO. *p < 0.05; **p < 0.01, significantly different when compared with vehicle control culture in the presence of adenosine. #, p < 0.05; ##, p < 0.01, significantly different when compared with vehicle control culture in the presence of ANO.
Mentions: We found that ANO inhibited TNF-α secretion by LPS/huIFN-γ-stimulated THP-1 cells partially through A2A AR (Figure 7). Classical signaling through A2A AR depends on its coupling to the heterotrimeric Gs protein and stimulation of adenylyl cyclase, resulting in elevation of intracellular levels of cyclic AMP (cAMP) [6-8]. We observed that ANO increased cAMP levels in both peritoneal macrophages and a murine macrophage cell line, RAW264.7 cells (data not shown). Elevation of cAMP in RAW264.7 cells inhibited LPS-induced TNF-α production via the protein kinase A (PKA)-dependent pathway [26]. Therefore, we examined the effect of a PKA inhibitor (H-89) on adenosine- or ANO-induced inhibition of TNF-α secretion by LPS-stimulated peritoneal macrophages. In this experiment, to exclude extraneous signaling events exerted by muIFN-γ, macrophages were stimulated with LPS only. As shown in Figure 9A, adenosine-induced inhibition of TNF-α secretion was recovered in a dose-dependent manner by pre-incubation with H-89, and complete recovery was observed with 5 μM H-89. However, H-89 had no effect on the ANO-induced inhibition of TNF-α secretion.Figure 9

Bottom Line: Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly.We found that it is refractory to adenosine deaminase-mediated conversion to inosine.Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock.

View Article: PubMed Central - PubMed

Affiliation: Core Technology Division, Research and Development Center, Hayashibara Co., Ltd, Okayama, Japan.

ABSTRACT

Background: Adenosine is a potent endogenous anti-inflammatory and immunoregulatory molecule. Despite its promise, adenosine's extremely short half-life in blood limits its clinical application. Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly. ANO is an oxidized product of adenosine at the N1 position of the adenine base moiety. We found that it is refractory to adenosine deaminase-mediated conversion to inosine. We further examined the anti-inflammatory activities of ANO in vitro and in vivo.

Methods: The effect of ANO on pro-inflammatory cytokine secretion was examined in mouse peritoneal macrophages and the human monocytic cell line THP-1, and compared with that of adenosine, synthetic adenosine receptor (AR)-selective agonists and dipotassium glycyrrhizate (GK2). The anti-inflammatory activity of ANO in vivo was examined in an LPS-induced endotoxin shock model in mice.

Results: ANO inhibited secretion of inflammatory mediators at much lower concentrations than adenosine and GK2 when used with peritoneal macrophages and THP-1 cells that were stimulated by LPS plus IFN-γ. The potent anti-inflammatory activity of ANO could not be solely accounted for by its refractoriness to adenosine deaminase. ANO was superior to the synthetic A1 AR-selective agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), A2A AR-selective agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamideadenosine hydrochloride (CGS21680), and A3 AR-selective agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), in suppressing the secretion of a broad spectrum of pro-inflammatory cytokines by peritoneal macrophages. The capacities of ANO to inhibit pro-inflammatory cytokine production by THP-1 cells were comparable with those of CCPA and IB-MECA. Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock. A significant increase in survival rate was also observed by oral administration of ANO. Mechanistic analysis suggested that the up-regulation of the anti-inflammatory transcription factor c-Fos was, at least in part, involved in the ANO-induced suppression of pro-inflammatory cytokine secretion.

Conclusions: Our data suggest that ANO, a naturally occurring molecule that is structurally close to adenosine but is functionally more potent, presents potential strategies for the treatment of inflammatory disorders.

No MeSH data available.


Related in: MedlinePlus