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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

ANO inhibited IL-6 production by peritoneal macrophages stimulated with TLR agonists. Peritoneal macrophages (5 × 104/well) were stimulated with LPS (2 μg/mL) (A), Poly I:C (50 μg/mL) (B), Pam3CSK4 (5 μg/mL) (C) and zymosan A (100 μg/mL) (D) in the presence or absence of various concentrations of ANO at 37°C for 24 h. Levels of IL-6 in the culture supernatants were determined by ELISA. Growth of macrophages was assessed by adding 20 μl/well of alamarBlue™ dye for the last 2 to 3 h of the incubation period and expressed as FI values. Values represent the means ± S.D. of triplicate cultures. Results are representative of two separate experiments with similar results. *p < 0.05; **p < 0.01, significantly different when compared with control culture.
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Fig1: ANO inhibited IL-6 production by peritoneal macrophages stimulated with TLR agonists. Peritoneal macrophages (5 × 104/well) were stimulated with LPS (2 μg/mL) (A), Poly I:C (50 μg/mL) (B), Pam3CSK4 (5 μg/mL) (C) and zymosan A (100 μg/mL) (D) in the presence or absence of various concentrations of ANO at 37°C for 24 h. Levels of IL-6 in the culture supernatants were determined by ELISA. Growth of macrophages was assessed by adding 20 μl/well of alamarBlue™ dye for the last 2 to 3 h of the incubation period and expressed as FI values. Values represent the means ± S.D. of triplicate cultures. Results are representative of two separate experiments with similar results. *p < 0.05; **p < 0.01, significantly different when compared with control culture.

Mentions: During our screening program to identify substance(s) that inhibited secretion of pro-inflammatory cytokines, we isolated ANO from the low-molecular weight fraction of RJ. We then examined the effects of ANO on pro-inflammatory cytokine production by macrophages activated by their recognition of pathogen-associated molecular patterns (PAMPs) via Toll-like receptors (TLRs). Towards that end, the following activators were used: LPS (TLR4 agonist), poly I:C (TLR3 agonist), Pam3CSK4 (TLR 1/2 agonist) or zymosan A (TLR2 agonist). Exposure of murine peritoneal macrophages to these PAMPs induced secretion of substantial amounts of IL-6. When ANO was added to the culture in the presence of the bacterial and viral PAMPs, IL-6 secretion was significantly inhibited in a dose-dependent fashion (Figure 1). Since the macrophage growth curve was not parallel to the IL-6 inhibition curve, the inhibition was not due to decreases in macrophage growth (Figure 1).Figure 1


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)

ANO inhibited IL-6 production by peritoneal macrophages stimulated with TLR agonists. Peritoneal macrophages (5 × 104/well) were stimulated with LPS (2 μg/mL) (A), Poly I:C (50 μg/mL) (B), Pam3CSK4 (5 μg/mL) (C) and zymosan A (100 μg/mL) (D) in the presence or absence of various concentrations of ANO at 37°C for 24 h. Levels of IL-6 in the culture supernatants were determined by ELISA. Growth of macrophages was assessed by adding 20 μl/well of alamarBlue™ dye for the last 2 to 3 h of the incubation period and expressed as FI values. Values represent the means ± S.D. of triplicate cultures. Results are representative of two separate experiments with similar results. *p < 0.05; **p < 0.01, significantly different when compared with control culture.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4308844&req=5

Fig1: ANO inhibited IL-6 production by peritoneal macrophages stimulated with TLR agonists. Peritoneal macrophages (5 × 104/well) were stimulated with LPS (2 μg/mL) (A), Poly I:C (50 μg/mL) (B), Pam3CSK4 (5 μg/mL) (C) and zymosan A (100 μg/mL) (D) in the presence or absence of various concentrations of ANO at 37°C for 24 h. Levels of IL-6 in the culture supernatants were determined by ELISA. Growth of macrophages was assessed by adding 20 μl/well of alamarBlue™ dye for the last 2 to 3 h of the incubation period and expressed as FI values. Values represent the means ± S.D. of triplicate cultures. Results are representative of two separate experiments with similar results. *p < 0.05; **p < 0.01, significantly different when compared with control culture.
Mentions: During our screening program to identify substance(s) that inhibited secretion of pro-inflammatory cytokines, we isolated ANO from the low-molecular weight fraction of RJ. We then examined the effects of ANO on pro-inflammatory cytokine production by macrophages activated by their recognition of pathogen-associated molecular patterns (PAMPs) via Toll-like receptors (TLRs). Towards that end, the following activators were used: LPS (TLR4 agonist), poly I:C (TLR3 agonist), Pam3CSK4 (TLR 1/2 agonist) or zymosan A (TLR2 agonist). Exposure of murine peritoneal macrophages to these PAMPs induced secretion of substantial amounts of IL-6. When ANO was added to the culture in the presence of the bacterial and viral PAMPs, IL-6 secretion was significantly inhibited in a dose-dependent fashion (Figure 1). Since the macrophage growth curve was not parallel to the IL-6 inhibition curve, the inhibition was not due to decreases in macrophage growth (Figure 1).Figure 1

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